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00_AACR_2015_Supp_Cover_Layout 1 4/2/15 7:41 AM Page 1 PROCEEDINGS Part 2: Clinical Trials and Late-Breaking Abstracts Continuing Medical Education ActivityAMA PRA Category 1 Credits™ available APRIL 18-22, 2015 • PENNSYLVANIA CONVENTION CENTER • PHILADELPHIA, PA • AACR.ORG • #AACR15 01_AACR_2015_LBA_FM_ppi_ii_Layout 1 4/2/15 4:51 PM Page i Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts AACR ANNUAL MEETING 2015 April 18-22, 2015 • Pennsylvania Convention Center • Philadelphia, PA Continuing Medical Education (CME) Information After participating in this CME activity, participants will be able to: ACCREDITATION STATEMENT Identify technological advances and tools to accelerate progress in cancer research, improve early detection, and early intervention, with the ultimate goal of extending patients’ lives and improving their quality of life. The American Association for Cancer Research (AACR) is accredited by the Accreditation Council of Continuing Medical Education (ACCME) to provide continuing medical education (CME) activities for physicians. CREDIT DESIGNATION STATEMENT The AACR has designated this live activity for a maximum of 45.5 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. Credit certification for individual sessions may vary, dependent upon compliance with the ACCME Accreditation Criteria. The final number of credits may vary from the maximum number indicated above. Explain the integration of information from basic and translational sciences to drug development, clinical research, and application of new findings. Integrate the use of biomarkers and other indicators to improve patient selection (or stratification) for clinical trials. Incorporate the latest research findings regarding therapies and treatment options including immunotherapy in a variety of cancer types in order to improve patient outcomes. Formulate new strategies that will further scientific and clinical research efforts towards the prevention and early detection of cancer. CLAIMING CME CREDIT Physicians and other healthcare professionals seeking AMA PRA Category 1 Credits™ for this continuing medical education activity must complete the online CME Request for Credit Survey available at www.aacr.org/am15cme by Wednesday, June 3, 2015. Information about CME accredited sessions and how to access the Request for Credit Survey will be available at the AACR CME Booth in the registration area of the Pennsylvania Convention Center. STATEMENT OF EDUCATIONAL NEED, TARGET AUDIENCE, AND LEARNING OBJECTIVES New technologies, scientific advances, and exponential developments in the field of translational cancer medicine have led to changes in oncology practice and significant patient benefit. By bridging the gap between what physicians understand about cancer biology and the clinical applications, this meeting aids basic researchers, physicians, and clinician-scientists in obtaining, synthesizing, and integrating the most cutting-edge research. This exposure is essential for the implementation of best practices, such as the most current molecular-based tests to aid in the diagnosis, treatment, and prevention of cancer. Through the active participation of clinical investigators and physicians in the meeting, laboratory researchers will obtain a better understanding of the wider context of their research in the “bench-to-bedside” continuum. Develop collaborations amongst physicians, researchers, and clinicianscientists to advance the cause of treating and preventing cancer. DISCLOSURE STATEMENT It is the policy of the AACR that the information presented at CME activities will be unbiased and based on scientific evidence. To help participants make judgments about the presence of bias, the AACR has provided information that Program Committee members, speakers, and abstract presenters have disclosed about financial relationships they have with commercial entities that produce or market products or services related to the content of this CME activity. As part of AACR's policy on full disclosure of relevant financial relationships, the disclosure information is available in the Annual Meeting Proceedings Supplement. ACKNOWLEDGMENT OF FINANCIAL OR OTHER SUPPORT This activity is supported by educational grants that will be disclosed at the activity. QUESTIONS ABOUT CME? Please contact the AACR Office of CME at (215) 440-9300 or [email protected]. Next Annual Meeting: April 16-20, 2016, New Orleans, LA American Association for Cancer Research • AACR ANNUAL MEETING 2015 i 01_AACR_2015_LBA_FM_ppi_ii_Layout 1 4/2/15 4:51 PM Page ii Table of Contents Continuing Medical Education (CME) Information ...........................................i Late-Breaking Abstracts Clinical Trials Sunday, April 19 Sunday, April 19 Opening Plenary Session: 9:30 AM-12:00 PM The Genome and Beyond .................................................................1 Clinical Trials Plenary Session: 12:45 PM-2:55 PM Promising Trials in Immunotherapy ..................................................1 Late-Breaking Poster Sessions: 1:00 PM-5:00 PM Late-Breaking Research: Experimental and Molecular Therapeutics 1 ...............................45 Late-Breaking Research: Molecular and Cellular Biology 1 ................................................52 Late-Breaking Minisymposium: 3:15 PM-5:15 PM Minisymposium: Late-Breaking Research.......................................60 Poster Session: 1:00 PM-5:00 PM Phase II, III, and Special Population Clinical Trials .............................2 Monday, April 20 Clinical Trials Plenary Session: 3:15 PM-5:15 PM Clinical Trials of Combinations of Molecularly Targeted and Non-targeted Therapeutic Agents..............................13 Late-Breaking Poster Sessions: 8:00 AM-12:00 PM Late-Breaking Research: Molecular and Cellular Biology 2 .............63 Late-Breaking Research: Carcinogenesis .......................................72 Late-Breaking Research: Cancer Chemistry ...................................75 Monday, April 20 Poster Session: 8:00 AM-12:00 PM Clinical Trials in Progress ...............................................................15 Clinical Trials Symposium: 10:30 AM-12:20 PM Clinical Trials of New Drugs in Breast Cancer .................................28 Clinical Trials Minisymposium: 3:00 PM-5:00 PM Clinical Trials of Novel Therapeutics ...............................................29 Late-Breaking Poster Sessions: 1:00 PM-5:00 PM Late-Breaking Research: Clinical Research / Endocrinology............79 Late-Breaking Research: Tumor Biology 1 ......................................87 Tuesday, April 21 Late-Breaking Poster Sessions: 8:00 AM-12:00 PM Late-Breaking Research: Molecular and Cellular Biology 3 ...............94 Late-Breaking Research: Epidemiology ........................................105 Late-Breaking Research: Tumor Biology 2 ....................................112 Tuesday, April 21 Poster Session: 8:00 AM-12:00 PM Phase I Clinical Trials......................................................................32 Late-Breaking Poster Sessions: 1:00 PM-5:00 PM Late-Breaking Research: Immunology..........................................119 Late-Breaking Research: Experimental and Molecular Therapeutics 2 .............................127 Clinical Trials Plenary Session: 10:30 AM-12:40 PM Clinical Trials Using PARP Inhibitors................................................41 Wednesday, April 9 Clinical Trials Minisymposium: 3:00 PM-5:00 PM Clinical Trials of Agents Targeting Breast and Prostate Cancers ......42 Late-Breaking Poster Sessions: 8:00 AM-12:00 PM Late-Breaking Research: Prevention Research .............................135 Late-Breaking Research: Molecular and Cellular Biology 4 ...........141 Advocates Poster Sessions ..........................................................................151 Author Index .................................................................................................158 Disclosure of Financial Relationships .........................................................176 ii Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 1 Clinical Trials: Opening Plenary Session Opening Plenary Session Sunday, April 19, 2015 9:30 AM-12:00 PM Hall A (200 Level), Pennsylvania Convention Center Clinical Trials Plenary Session Sunday, April 19, 2015 12:45 PM-2:55 PM Terrace Ballroom I (400 Level), Pennsylvania Convention Center The Genome and Beyond Promising Trials in Immunotherapy Chairperson: Lewis C. Cantley, Sandra and Edward Meyer Cancer Center at Weill Cornell Medical College, New York, NY The complete text of abstract CT101 will be posted to the online Proceedings after presentation. 9:30 AM 9:55 AM Insights from cancer genomes into the mutational processes underlying cancer development Michael R. Stratton, Wellcome Trust Sanger Institute, Cambridge, United Kingdom Engineering the cancer genome Tyler Jacks, David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 10:20 AM Above the genome: The epigenome and its biology and translational potential Stephen B. Baylin, Johns Hopkins University School of Medicine, Baltimore, MD 10:45 AM Using genomics to personalize cancer immunotherapy Robert D. Schreiber, Washington University School of Medicine, St. Louis, MO 11:10 AM CT101 Phase III study of pembrolizumab (MK-3475) versus ipilimumab in patients with ipilimumab-naive advanced melanoma. Antoni Ribas1, Jacob Schachter2, Georgina V. Long3, Ana Arance4, Jean Jacques Grob5, Laurent Mortier6, Adil Daud7, Matteo S. Carlino8, Catriona McNeil9, Michal Lotem10, James Larkin11, Paul Lorigan12, Bart Neyns13, Christian U. Blank14, Omid Hamid15, Michele Kosh16, Honghong Zhou16, Nageatte Ibrahim16, Scot Ebbinghaus16, Caroline Robert17. 1UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA; 2Ella Lemelbaum Institute for Melanoma, Sheba Medical Center, Tel Hashomer, Israel; 3Melanoma Institute Australia and The University of Sydney, North Sydney, Australia; 4Hospital Clinic and Translational Genomics and Targeted Therapeutics in Solid Tumors (IDIBAPS), Barcelona, Spain; 5 Hôpital de la Timone, Marseille, France; 6Université Lille, CHRU LILLE, Lille, France; 7University of California, San Francisco, San Francisco, CA; 8Westmead and Blacktown Hospitals, The University of Sydney, and Melanoma Institute Australia, Sydney, Australia; 99Chris O’Brien Lifehouse, Royal Prince Alfred Hospital, and Melanoma Institute Australia, Camperdown, Australia; 1010Sharett Institute of Oncology, Hadassah Hebrew University Medical Center, Jerusalem, Israel; 11The Royal Marsden Hospital, London, United Kingdom; 12University of Manchester and The Christie NHS Foundation Trust, Manchester, United Kingdom; 1313Universitair Ziekenhuis Brussel, Brussels, United Kingdom; 14The Netherlands Cancer Institute, Amsterdam, Netherlands; 1515The Angeles Clinic and Research Institute, Los Angeles, CA; 16Merck & Co., Inc., Kenilworth, NJ; 17Institute Gustave Levy, Villejuif, France. 11:30 AM Discussant Jedd D. Wolchok, Memorial Sloan Kettering Cancer Center, New York, NY 11:40 AM Dual targeting of BCR-ABL with ABL001: A novel potent allosteric ABL kinase inhibitor in combination with nilotinib suppresses the emergence of disease resistance in models of CML William R. Sellers, Novartis Institutes for BioMedical Research, Cambridge, MA Co-Chairpersons: Michael Sadelain, Memorial Sloan Kettering Cancer Center, New York, NY; D. Ross Camidge, University of Colorado Denver, Aurora, CO The complete text of the abstracts in this session will be posted to the online Proceedings after presentation. 12:45 PM CT103 Clinical safety and efficacy of pembrolizumab (MK-3475) in patients with malignant pleural mesothelioma: Preliminary results from KEYNOTE-028. Evan W. Alley,1 L. Rhoda Molife,2 Armando Santoro,3 Kim Beckey,4 Sammy Yuan,4 Jonathan D. Cheng,4 Bilal Piperdi,4 Johannes H.M. Schellens5. 1University of Pennsylvania, Philadelphia, PA; 2The Royal Marsden/Institute of Cancer Research, Sutton, United Kingdom; 3Humanitas Research Hospital-Humanitas Cancer Center, Rozzano, Italy; 4Merck & Co., Inc., Kenilworth, NJ; 5The Netherlands Cancer Institute, Amsterdam, Netherlands. 1:05 PM CT104 Efficacy of pembrolizumab (MK-3475) and relationship with PD-L1 expression in patients with nonsmall cell lung cancer: Findings from KEYNOTE-001. Edward B. Garon,1 Naiyer Rizvi,2 Rina Hui,3 Natasha B. Leighl,4 Ani S. Balmanoukian,5 Joseph P. Eder,6 Amita Patnaik,7 Charu Aggarwal,8 Matthew A. Gubens,9 Leora Horn,10 Enric Carcereny,11 Myung-Ju Ahn,12 Enriqueta Felip,13 Jong-Seok Lee,14 Jin Zhang,15 Reshma A. Rangwala,15 Gregory M. Lubiniecki,15 Charlotte M. Roach,16 Kenneth Emancipator,15 Leena Gandhi17. 1David Geffen School of Medicine at UCLA, Los Angeles, CA; 2 Columbia University, New York, NY; 3Westmead Hospital, University of Sydney, Westmead, Australia; 4 Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 5The Angeles Clinic and Research Institute, Los Angeles, CA; 6Yale University Cancer Center, New Haven, CT; 7South Texas Accelerated Research Therapeutics (START), San Antonio, TX; 8University of Pennsylvania, Philadelphia, PA; 9University of California, San Francisco, San Francisco, CA; 10Vanderbilt Ingram Cancer Center, Nashville, TN; 11Catalan Institute of Oncology-Badalona, Badalona, Spain; 12Samsung Medical Center, Seoul, Republic of Korea; 13Vall d’Hebron University Hospital, Barcelona, Spain; 14Seoul National University, Bundang Hospital, Seoul, Republic of Korea; 15Merck & Co., Inc., Kenilworth, NJ; 16Dako, North America, Carpinteria, CA; 17Dana-Farber Cancer Institute, Boston, MA. 1:35 PM Discussant D. Ross Camidge, University of Colorado Denver, Aurora, CO 1:35 PM CT105 Safety and feasibility of chimeric antigen receptor modified T cells directed against mesothelin (CART-meso) in patients with mesothelin expressing cancers. Janos L. Tanyi, Andrew R. Haas, Gregory L. Beatty, Mark A. Morgan, Caitlin J. Stashwick, Mark H. O’Hara, David L. Porter, Marcela V. Maus, Bruce L. Levine, Simon F. Lacey, Anne Marie Nelson, Maureen McGarvey, Naseem DS Kerr, Gabriela Plesa, Carl H. June. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA. American Association for Cancer Research • AACR ANNUAL MEETING 2015 1 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 2 Clinical Trials Plenary Session: Promising Trials in Immunotherapy 1:55 PM Discussant Michael Sadelain, Memorial Sloan Kettering Cancer Center, New York, NY 2:05 PM CT106 A phase I/IIa study of IMCgp100: Partial and complete durable responses with a novel first-inclass immunotherapy for advanced melanoma. Mark R. Middleton,1 Pippa Corrie,2 Mario Sznol,3 Jeffrey Infante,4 Clive Mulatero,5 Jeff Evans,6 Neil Steven,7 David Krige,8 William H. Shingler,8 Yvonne McGrath,8 Namir J. Hassan,8 Bent K. Jakobsen8. 1University of Oxford, Oxford, United Kingdom; 2Addenbrooke’s Hospital, Cambridge, United Kingdom; 3Yale Cancer Center, New Haven, CT; 4Sarah Cannon Research Institute, Nashville, TN; 5St James Hospital, Leeds, United Kingdom; 6 University of Glasgow, Glasgow, United Kingdom; 7 Queen Elizabeth Hospital, Birmingham, United Kingdom; 8Immunocore Ltd, Abingdon, United Kingdom. 2:25 PM CT107 Epstein-Barr virus-specific cytotoxic T lymphocytes for treatment of rituximab-refractory EBV-associated lymphoproliferative disorder. Susan E. Prockop, Ekaterina Doubrovina, Karim Baroudy, Farid Boulad, Ramzi Khalaf, Esperanza B. Papadopoulos, Craig Sauter, Victoria Szenes, Stephanie Suser, Gloria Wasilewski, Julianna Ruggierio, Richard J. O’Reilly. Memorial Sloan Kettering Cancer Center, New York, NY. 2:45 p.m Discussant Antoni Ribas, UCLA Medical Center, Los Angeles, CA 2 Clinical Trials Poster Session Sunday, April 19, 2015 1:00 PM-5:00 PM Poster Section 22 Halls B-E (Level 200), Pennsylvania Convention Center Phase II, III, and Special Population Clinical Trials Poster Section 22 Poster Board 1 CT110 Randomized phase II study of duligotuzumab + FOLFIRI versus cetuximab + FOLFIRI in 2nd-line patients with KRAS wild-type (wt) metastatic colorectal cancer (mCRC). Andrew G. Hill,1 Michael Findlay,2 Matthew Burge,3 Christopher Jackson,4 Pilar Garcia Alfonso,5 Leslie Samuel,6 Vinod Ganju,7 Meinolf Karthaus,8 Alessio Amatu,9 Mark Jeffery,10 Maria DiBartolomeo,11 John Bridgewater,12 Andrew Coveler,13 Manuel Hidalgo,14 Amy V. Kapp,15 Roxana Sufan,16 Bruce McCall,16 Elicia Penuel,16 Andrea Pirzkall,16 Josep Tabernero17. 1Tasman Oncology Research, Southport, Queensland, Australia; 2Auckland UniServices Limited, Auckland, New Zealand; 3Royal Brisbane and Women’s Hospital, Herston, Australia; 4Department of Medicine, Dunedin School of Medicine, University of Otago, Otago, New Zealand; 5 Gregorio Marañón Hospital, Madrid, Spain; 6Aberdeen Royal Infirmary, Aberdeen, United Kingdom; 7Peninsula Oncology Centre, Frankston, Australia; 8Staedtisches Klinikum Muenchen GmbH Klinikum Neuperlach, Munich, Germany; 9Niguarda Cancer Center, Ospedale Niguarda Ca’ Granda, Milan, Italy; 10Canterbury Regional Cancer and Haematology Service, Christchurch, New Zealand; 11 Fondazione IRCCS Instituto Nazionale dei Tumori, Milan, Italy; 12 University College London Cancer Institute, London, United Kingdom; 13Seattle Cancer Care Alliance, Seattle, WA; 14Centro Integral Oncologico Clara Campal (CIOCC), Madrid, Spain; 15 Genentech, Inc., South San Francisco, CA; 16Genentech, Inc, South San Francisco, CA; 17Vall d’Hebron Institute of Oncology, Barcelona, CA. Background: Duligotuzumab (MEHD, MEHD7945A) is a novel dual-action humanized IgG1 antibody that blocks EGFR and HER3 binding, inhibiting all major ligand-dependent HER complex signaling. MEHD is active in multiple tumor models, including models resistant to anti-EGFR or anti-HER3. Emerging data in CRC suggest a role for HER3 in de novo and acquired resistance to antiEGFR therapy. Methods: This open-label, randomized Phase II study enrolled patients (pts) with KRAS exon 2 wt mCRC who progressed on/after oxaliplatin-containing chemotherapy. Pts received a combination of MEHD (1100 mg IV, q2w) or cetuximab (400 mg/m2 load, 250 mg/m2 IV, q1w) + FOLFIRI (q2w) until progression or intolerable toxicity. Endpoints included progression-free survival (PFS), and objective response rate (ORR), overall survival (OS), and adverse events (AEs). Tumor samples were mandatory and underwent biomarker analysis for ERBB3, NRG1 and EGFR ligand expression by qRTPCR, and ERBB3 by IHC. The primary efficacy analysis was conducted in patients with RAS wt tumors (no mutations detected in KRAS or NRAS exons 2, 3; exon 4 mutations pending). Results: Of 134 randomized patients, 98 were RAS ex2/3 wt (53 MEHD); median age 63 years, ECOG 0-1. As of 21Aug14, 11 pts remain active. Efficacy results (Table) show no benefit of MEHD + FOLFIRI; ORR was lower in the MEDH arm. No relationship was seen between PFS or ORR and mRNA expression for ERBB3 or NRG1, or ERB3 expression by IHC. There were fewer rash events of any grade in the MEHD arm (79% and 93%) but more diarrhea (89% and 66%). Incidence of Grade ≥ 3 AEs was similar between arms (87% and 89%); however, the frequency of SAEs was higher in the MEHD arm (55% and 48%). Cumulative dose intensity and duration of treatment with FOLFIRI were lower in the MEHD arm. Conclusions: MEHD + FOLFIRI did not improve outcomes of pts with RAS ex2/3 wt mCRC compared to cetuximab + FOLFIRI. Updated efficacy, safety and biomarker data will be presented. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 3 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials comparison P= 0.049). Statistical analyses are ongoing to examine the potential modifying effects of COMT genotype on other urinary estrogen metabolites. Conclusion: Daily intake of high-dose green tea extract for 12 months exerts significant effects on urinary excretion of estrogen metabolites, and these effects may be modified by COMT polymorphisms. The potential beneficial effects of green tea extract on estrogen metabolism support the further development of green tea as a potential chemopreventive agents for estrogen-related disease prevention. Funding source: NIH/NCI (R01 CA127236). CI=confidence interval. NE=not estimated Poster Section 22 Poster Board 2 CT111 Green tea extract supplementation, estrogen metabolism and breast cancer risk in postmenopausal women at high risk of breast cancer. Hamed Samavat,1 Renwei Wang,2 Anna Wu,3 Jian-Min Yuan,4 Mindy Kurzer1. 1University of Minnesota, St. Paul, MN; 2Division of Cancer Control and Population Sciences- University of Pittsburgh Cancer Institute, Pittsburgh, PA; 3Department of Preventive Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA; 4Division of Cancer Control and Population Sciences; Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA. Background: Green tea intake may be associated with reduced risk of several cancers including breast cancer. One of the proposed mechanisms by which green tea may modify breast cancer risk is through effects on estrogen metabolism. Genetic variation in catechol-O-methyltransferase (COMT), an enzyme involved in the metabolism of both estrogens and tea catechins, may also have a role. Few studies have examined the effects of green tea intake on circulating and urinary estrogens and estrogen metabolites, and even less is known about the potential modifying influence of polymorphisms in the COMT gene. Objectives: To investigate the effects of daily intake of a highly concentrated green tea extract (GTE) for one year on circulating and urinary estrogens and their metabolites in postmenopausal women with different COMT genotypes. Methods: The Minnesota Green Tea Trial (MGTT), a randomized, double-blind, placebo-controlled, phase II clinical trial, enrolled 1075 healthy postmenopausal women with heterogeneously or extremely dense breast tissue (age = 59.78 ± 5.02 years; body mass index= 25.70 ± 8.21 kg/m2). Participants were 93% non-Hispanic white and non-current hormone users. Half of the participants (n=538) were randomly assigned to the GTE group and were given 4 capsules a day, each containing 200 mg epigallocatechin gallate (EGCG) and the others (n=537) to the placebo group. Twenty-four hour urine samples were collected at month 0 and at the end of the study, and fasting blood samples were drawn at months 0, 6 and 12. Circulating and urinary estrogens, as well as urinary estrogen metabolites were quantified by liquid chromatography-tandem mass spectrometry. Results: GTE supplementation was associated with reduced urinary estriol (-10.7%, P = 0.08) and 2-methoxy estrone (-12.2%, P =0.014) compared with baseline, and these changes were significantly greater than the corresponding changes in the placebo group (P= 0.003, and P= 0.02, respectively). GTE supplementation had no significant effects on serum or urinary estrone and estradiol. Among women with the low activity COMT genotype, GTE supplementation reduced urinary estrone (-5.3%) compared with women with high activity COMT (+2.1%) (between groups Poster Section 22 Poster Board 3 CT112 Implementation of CLIA enabled integrated whole genome (WGS)/exome (WES)/transcriptome (RNAseq) next-gen sequencing to identify therapeutically relevant targets in advanced cancer patients. Mitesh J. Borad,1 Jan Egan,1 Mia Champion,1 Katherine Hunt,1 Robert McWilliams,2 Ann McCullough,1 Jessica Aldrich,3 Sara Nasser,3 Winnie Liang,3 Michael Barrett,1 David Craig,3 Ramesh Ramanathan,1 John Carpten,3 A. Keith Stewart,1 Alan Bryce1. 1Mayo Clinic Arizona, Scottsdale, AZ; 2Mayo Clinic Rochester, Rochester, MN; 3Translational Genomics Research Institute, Phoenix, AZ. Background: Genomic assessment of cancer has been revolutionized by Next-Generation sequencing and is increasingly being applied to guide clinical prognostic and therapeutic decisionmaking. Initial clinical applications have been limited to gene panels and whole exome based strategies due to challenges with time to reporting of results, specimen quantity, analyte quality, and ethicallegal-social issues (ELSI). Methods: Excisional or core tumor biopsies or bone marrow biopsies were obtained from consenting participants. Nucleic acid was extracted from fresh or frozen samples followed by WGS/WES/RNASeq on the Illumina HiSeq2000 or HiSeq2500 and bioinformatics analysis. Therapeutic targets were then prioritized by a multi-disciplinary Genomic Tumor Board (GTB) and CLIA validated using Sanger sequencing, RT-qPCR, FISH and/or IHC. Treatment was delivered using on/off-label FDA approved drugs, available clinical trials and single patient INDs. Results: We consented 64 and enrolled 35 patients with advanced, treatment-refractory cancers. Median age was 59 (range 27-91) with 62% male. The majority had ECOG scores of 1 (94%). A median of 79 (range 28-8891) potentially functional somatic point mutations were identified in each case. One to two mutations/case were further identified as therapeutically targetable in 60% of the cases. The median time from tissue acquisition to CLIA validated results was 116 days (range 42-282) with CLIA validation of targets achieved in 21 of 22 patients. Genomic, target directed treatment was ultimately instituted in 13 patients utilizing: on/off label FDA approved drugs (n=9), clinical trial (n=3) and single patient IND (n=1). Preliminary clinical efficacy was noted in 5 patients (2 PR, 3 SD). Integration of WES, long-insert WGS and RNA-Seq identified a case with an ERRFI1 mutant allele present in only 11% of the DNA reads while 82% of the RNA-Seq reads had the mutant transcript. The patient was treated with erlotinib and achieved a partial response by RECIST criteria. In another case, a fusion between FGFR2-MGEA5 was observed in WES and RNA-Seq data leading to prioritization as a drug target. Reasons for patients not receiving targeted therapy included: inability to access treatment (n=1), death prior to intended treatment (n=3), results returned after death (n=3), no targets identified (n=2) and decision to pursue alternate therapy (n=5). Conclusions: Integrating whole genome analysis in a CLIA setting is not only feasible, but also valuable, in the prioritization and selection of potential targeted therapies for patients with advanced tumors. Continued barriers to broad application include the need for shorter time to reporting as well as broad availability of therapies through basket studies. American Association for Cancer Research • AACR ANNUAL MEETING 2015 3 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 4 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials Poster Section 22 Poster Board 4 CT113 A phase III, multicenter, randomized study evaluating the efficacy and safety and efficacy of erythrocyte encapsulated l-asparaginase (Ery001) versus native lasparaginase (L-asp) in combination with COOPRALL regimen in patients with first relapse of acute lymphoblastic leukemia. Yves Bertrand,1 Andre Baruchel,2 Xavier Thomas,3 Nicolas Blin,4 Emmanuelle Tavernier,5 Yves Perel,6 Norbert Vey,7 Virginie Gandemer,8 Iman ElHariry,9 Yann Godfrin9. 1Pediatric Hematology, Hospices Civils de Lyon, Lyon, France; 2Robert Debré Hospital APHP and University Paris Diderot, Paris, France; 3Centre Hospitalier Lyon Sud, Pierre-Bénite, France; 4Nantes University Hospital, Nantes, France; 5Institut de Cancérologie de la Loire, Saint Priez en Jarez, France; 6Hôpital des Enfants - Hôpital Pellegrin, Bordeaux, France; 7Institut Paoli Calmettes, Marseille, France; 8 Centre Hospitalier Universitaire de Rennes, Rennes, France; 9 Erytech Pharma, Lyon, France. Asparaginases are a cornerstone in the treatment of ALL, but their utility is limited by toxicities including hypersensitivity. ERY001 improves pharmacokinetics and tolerability, while maintaining circulating asparaginase (ASPA) activity due to the protective barrier of the erythrocyte membrane. This phase III study enrolled pts with primary relapsed/refractory Ph- ALL. The co-primary endpoints were the duration of ASPA activity equal or greater than 100IU/L and the incidence of ASPA hypersensitivity during induction. Patients, aged 1-55 years, without prior ASPA hypersensitivity were randomized to ERY001 (150 IU/kg) or L-ASP(10.000IU/m²) in combination with COOPRALL. The patients with prior ASPA hypersensitivity received ERY001. ERY001 significantly reduced the incidence of ASPA hypersensitivity (0% vs 43%; p100IU/l significantly longer than LASP (20.5±5.2 vs 9.2±7.5 days; p<0.001). CR and clotting parameters were also significantly improved in the ERY001 arm. Other adverse events were generally similar in. One-year OS was 77% in the ERY001 arm vs 68% in L-ASP arm. ASPA activity was not reduced in hypersensitive patients. These highly encouraging results show that ERY001 is a suitable option for patients with relapsed ALL maintaining ASPA efficacy with improved tolerability. Poster Section 22 Poster Board 5 CT114 A phase II trial of exemestane and ruxolitinib for aIresistant ER+ breast cancer: Interim safety, efficacy, and biomarker analysis. Angela M. DeMichele,1 Christopher B. Colameco,1 Anna Kalota,1 Andrea B. Troxel,1 Robin Holmes,1 Rebecca Cimildoro,1 Kelly Zafman,1 Kevin R. Fox,1 Susan M. Domchek,1 Keerthi Gogineni,1 Angela R. Bradbury,1 Jennifer M. Matro,1 Natalie Shih,1 Michael D. Feldman,1 Amy S. Clark,1 Elizabeth O. Hexner,1 Jacqueline F. Bromberg2. 1University of Pennsylvania, Philadelphia, PA; 2Memorial Sloan Kettering Cancer Center, New York, NY. Resistance to aromatase inhibitors in ER+ breast cancer leads to recurrence and progression in the metastatic setting. JAK/STAT pathway activation is a resistance mechanism that could potentially be overcome with the use of JAK inhibitor therapy. Methods: We performed a phase II trial of exemestane, 25 mg daily, and ruxolitinib, 25 mg BID, in postmenopausal women with advanced, ER+ breast cancer who had progressed on a nonsteroidal aromatase inhibitor and had either measureable or boneonly disease. A Simon 2-stage design was employed. A “go” decision to second stage would occur if fewer than 5/15 patients experienced any grade 3/4 toxicity requiring discontinuation from the study within the first treatment cycle. Results: Fifteen patients were enrolled; during cycle 1, no patient discontinued for toxicity and 1 patient went off study for progression of bone disease. 36 grade 3 events occurred; anemia was most common (n=5), requiring transfusion in all patients. 47% required dose reduction. No partial or complete responses occurred; 3/15 (20%) had stable disease >6 months (clinical benefit, 4 CB). Baseline CRP >8 was significantly associated with CB (3/3 CB vs. 1/11 non-CB; p=0.011); other markers, including baseline ESR, IL-6 genotype status and primary tumor phosphoSTAT3 expression were not associated with CB in this small sample, though high tumoral pSTAT3 was seen in 66% of CB and 33% of non-CB. A novel pharmacodynamic (PD) assay to assess STAT3 phosphorylation in peripheral blood mononuclear cells after ruxolitinib exposure demonstrated differential effects in patients with CB vs. those without CB. Conclusions: The combination of exemestane and the JAK2 inhibitor ruxolitinib met safety criteria for continued enrollment. Anemia, an expected toxicity of R, was common and the high rate of severe anemia and need for dose reductions has led to a decision to reduce the starting dose of ruxolitinib to 15 mg BID moving forward. Promising predictive markers, including CRP, tumor pSTAT3 and a novel PD assay for pSTAT3 will be further evaluated. Poster Section 22 Poster Board 6 CT115 Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients. Yu Sawada,1 Toshiaki Yoshikawa,2 Kazuya Ofuji,2 Mayuko Yoshimura,2 Nobuhiro Tsuchiya,1 Mari Takahashi,2 Daisuke Nobuoka,2 Shoichi Mizuno,2 Itaru Endo,3 Tetsuya Nakatsura2. 1 Yokohama City University, National Cancer Center, Japan; 2 National Cancer Center, Japan; 3Yokohama City University, Japan. Background and purpose: In a phase I trial for advanced hepatocellular carcinoma (HCC), glypican-3(GPC3)-derived peptide vaccination was well-tolerated, and immune responses and antitumor efficacy were noted. The recurrence rates of HCC are still high and immunotherapy, as an adjuvant therapy, is expected. Therefore, we have conducted a phase II study of GPC3 peptide vaccine as an adjuvant therapy for HCC patients. Study Design: Forty one patients with initial HCC, who had undergone surgery or radiofrequency ablation (RFA), were analyzed in this phase II, open-label, single-arm trial.Ten vaccinations were performed for one year after curative treatment. The primary endpoints were 1- and 2-year recurrence rates. The secondary endpoints were safety and immune responses. We have evaluated GPC3-specific CTL response in PBMCs by IFN-γ enzyme-linked immunospot (ELISPOT) assay. We have evaluated the expression of GPC3 in the 33 primary tumors and 11 recurrence tumors, that could be obtained, by immunohistochemical analysis, and the phenotype of CTLs in recurrence tumor and vaccine site by flow cytometry. Results: GPC3 peptide vaccine showed no severe adverse events. 1-year and 2-year recurrence rates of the 41 patients treated with the vaccination were 24.4% and 53.7%. In this study, 35 patients had received surgery and 6 patients RFA therapy. We analyzed the case-control study to evaluate the reduction in the risk of post-operative recurrence by vaccination. Thirty three patients with initial HCC who underwent curative resection in the same period were selected as control group. The recurrence rate tended to be lower in 35 patients treated with surgery and the vaccination than in 33 patients with surgery alone (28.6% and 54.3% vs 39.4% and 54.5% at 1 year and 2 year, p=0.346, 0.983). In the patients with GPC3 positive tumor, the recurrence rate was significantly lower in 25 patients treated with surgery and the vaccination than in 21 patients with surgery alone (24% and 48% vs 52.4% and 61.9% at 1 year and 2 year, p=0.047, 0.387), and there was no significant difference in clinical background. There were not the correlation between the relapse free survival and the antigen-specific immune response as measured by IFN-γ ELISPOT assay. Two of 11 case appeared to lack GPC3 expression in the recurrence tumor, although GPC3 was expressed in the primary HCC tissue before GPC3 peptide vaccine. In the other case, GPC3 peptide specific CTLs had infiltrated into recurrence tumor, and the expression of PD-1 among CD8 positive T cells, was higher in recurrence tumor and vaccine site than in PBMCs. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 5 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials Conclusions: GPC3 peptide vaccine for patient with GPC3 positive tumor could improve 1-year recurrence rates. This study showed GPC3 expression of the primary tumor could be the biomarker for a larger randomized clinical trial to determine the efficacy of the GPC3-derived peptide vaccine. Poster Section 22 Poster Board 8 CT117 Evaluation of the effect of crizotinib (CRZ) on the QT interval in patients with ALK-positive non-small cell lung cancer (NSCLC). Weiwei Tan,1 Keith D. Wilner,1 Silvana Lanzalone,2 Anna Polli,2 Matthew L. Zierhut,3 Dana Nickens,3 Kenneth J. O’Byrne,4 Fiona H. Blackhall,5 Alice T. Shaw,6 Ravi Salgia,7 Jesus Correl Jaime,8 DongWan Kim9. 1Oncology Business Unit, Pfizer, Inc., San Diego, CA; 2 Oncology Business Unit, Pfizer, Inc., Milan, Italy; 3 Pharmacometrics, Global Clinical Pharmacology, Pfizer, Inc., San Diego, CA; 4Department of Medical Oncology, St. James’s Hospital, Dublin, Ireland; 5Christie Hospital NHS Trust, Department of Medical Oncology, Manchester, United Kingdom; 6Massachussetts General Hospital, Boston, MA; 7University of Chicago Medical Center, Chicago, IL; 8Hospital Universitario Virgen del Rocio, Servicio de Oncologia. Edificio del Hospital General, Sevilla, Spain; 9Seoul National University Hospital/Department of Internal Medicine, Seoul, Republic of Korea. Background: CRZ is an oral tyrosine kinase inhibitor approved for the treatment of ALK-positive NSCLC at a starting dose of 250 mg BID. An ECG substudy was conducted to evaluate and quantitate any potential impact of CRZ on corrected QT interval (QTc). This ECG substudy enrolled patients with ALK-positive advanced NSCLC at selected sites from the phase III trial PROFILE 1007 and the phase II trial PROFILE 1005. Methods: All patients enrolled in the ECG substudy received CRZ 250 mg BID orally on a continuous basis. Triplicate 12-lead ECG measurements, approximately 2 minutes apart, were collected after a fast of at least 1 hour at predose, 4 and 8 hours following morning CRZ dosing on Day 1 of Cycle 1, and predose, 2, 4, 6 and 8 hours following morning CRZ dosing on Day 1 of Cycle 2 (1 Cycle = 21 days). All ECG tracings from this substudy were sent to an independent ECG laboratory for blinded manual interval measurements. Plasma PK samples for CRZ and its metabolite, PF06260182, were collected at respective times immediately following each ECG measurement. ECG endpoints included QT interval corrected for heart rate (HR) using Fridericia’s (QTcF) (primary endpoint) and Bazett’s (QTcB) formulas and a model-based studyspecific correction factor (QTcS), PR interval, RR interval, and QRS complex. Categorical analysis was conducted for all QTc endpoints. ANOVA was performed for QTcF and QTcS, the QTc with the most appropriate HR correction. These analyses were performed using the ECG Evaluable Population (EEP), defined as all treated patients with an adequate baseline assessment and at least 1 adequate ECG measurement on Cycle 2 Day 1 (at CRZ steady state). A pharmacokinetic/pharmacodynamic analysis was performed using the ECG-PK matched dataset. Results: A total of 65 patients were enrolled in the ECG study, and 52 were in the EEP. ANOVA revealed that all upper limits of the 90% confidence intervals for the least squares mean changes from baseline in QTcF at all Cycle 2 Day 1 time points were <20 ms. The largest LS mean change from baseline was an increase of 12.3 ms, reported at 6 hours post morning dose on Cycle 2 Day 1. No patients had a maximum QTc ≥500 ms and 1 (1.9%) patient had a QTc increase from baseline of ≥60 ms. Consistently with previous findings, mean steady state predose (Ctrough) and maximum plasma concentrations (Cmax) of CRZ were 271 ng/mL and 345 ng/mL, respectively, and mean steady state Ctrough and Cmax of metabolite PF-06260812 were 86.7 ng/mL and 107 ng/mL, respectively. A relationship was observed between CRZ plasma concentration and QTc. HR decreased with increasing CRZ plasma concentration, as reported previously in other studies. Conclusions: Based on ANOVA results from this substudy, a large QTc effect (≥ 20 ms) by CRZ can be excluded however a smaller increase in QTc cannot be ruled out. Periodic ECGs monitoring should be considered for patients who have a history of QTc prolongation or who are taking medications that prolong QT. Poster Section 22 Poster Board 9 CT118 A randomized phase II study of the CSF-470 therapeutic vaccine plus BCG plus rhGM-CSF versus IFN-α2b in cutaneous melanoma patients stages IIB, IIC and III. José Mordoh,1 María Betina Pampena,2 Mariana Aris,2 Paula Blanco,2 Alicia I. Bravo,3 Juan Manuel O´Connor,4 Julio Kaplan,4 Franco Ramello,5 Estrella M. Levy,2 María M. Barrio2. 1Centro de Investigaciones Oncológicas - Fundación Cáncer - Instituto Alexander Fleming - IIB-BA Fundación Instituto Leloir, Buenos Aires, Argentina; 2Centro de Investigaciones Oncológicas Fundación Cáncer, Buenos Aires, Argentina; 3HIGA Eva Perón, Buenos Aires, Argentina; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Centro Médico San Lucas, Gualeguaychú - Entre Ríos, Argentina. Adjuvant treatment of high-risk cutaneous melanoma (CM) patients (pts) is still an unsolved issue, since the cost-benefit ratio of high-dose IFN-α2b is under discussion. The CSF-470 therapeutic vaccine, a mini-allograft of four lethally-irradiated allogeneic CM cell lines, with BCG and rhGM-CSF as adjuvants, is currently being tested in post-surgical adjuvancy vs medium-dose IFN-2b in stage IIB-III CM pts (phase II/III trial CASVAC-0401, NCT01729663).We present here the results of the phase II part of the study. A total of 31 pts (stage IIC=2; stage III: 29) were enrolled: 20 pts were randomized to receive CSF-470 vaccine and 11 pts to IFN-α2b. Pts assigned to the vaccine arm received i.d. 1.6x107 CSF-470 irradiated cells plus 106cfu BCG and 100μg rhGM-CSF (first day); 100 μg rhGM-CSF/day/3days were injected consecutively i.d. at the vaccination site. During the two-year treatment, pts in the vaccine arm received a total of 13 vaccinations. Pts assigned to the IFNα2b arm received 10 MU/day/5 days a week for 4 weeks; then 5 MU 3/week for 23 months. Imaging studies were performed to follow the clinical evolution of the disease. Analysis of blood chemistry and differential white blood cell counts were performed to monitor systemic toxicity. Immune monitoring was performed at baseline and at 6, 12 and 24 months from protocol start. Also, pts were evaluated by Quality of Life Questionnaires (QOL) along the study. After including 20 pts who received a total of 176 vaccinations, we conclude that: CSF-470 vaccine was well tolerated; the main toxicity was a grade 2 reaction at the injection site; 3/20 pts presented grade 3 allergic reactions that were easily handled with anti-histamines and corticosteroids. Pts in the IFNα2b arm presented grade 2-3 hematologic toxicity; 9 pts developed adverse events that forced treatment discontinuation provisionally or permanently. With a mean follow-up of 28 months and a maximum follow-up of 67 months, a significant benefit in the distant metastasis-free survival (DMFS) for CSF-470 was observed: 14/20 pts (70%) immunized with CSF-470 vaccine and only 4/11 pts (36.4 %) in the IFN-α2b arm remain without distant metastases (p=0.032). No significant differences in OS were yet observed. QOL was significantly superior for CSF-470 vaccine as compared to IFN-α2b treatment (p<0.0002). DMF pts developed a significantly higher DTH reaction after the 7th and 12th vaccine as compared to progressing pts. These results demonstrate a clear superiority of CSF-470 vaccine plus BCG plus GM-CSF vs IFN-α2b in the adjuvant setting in pts with high-risk CM. Poster Section 22 Poster Board 10 CT119 Early results from a phase II study of RRx-001, a novel, triple epigenetic inhibitor, showing resensitization to irinotecan in colorectal cancer. Tony Reid,1 George Fisher,2 Corey Carter,3 Cheryl Cho-Phan,4 Pamela Kunz,5 Bryan Oronsky,6 Gary R. Fanger,6 Scott Caroen,6 Christopher Parker,6 Jan Scicinski6. 1UC San Diego Moores Cancer Center, La Jolla, CA; 2Stanford Cancer Center, Stanford, CA; 3Walter American Association for Cancer Research • AACR ANNUAL MEETING 2015 5 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 6 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials Reed National Military Medical Center, Bethesda, MD; 4Stanford School of Medicine, Stanford, CA; 5Stanford University Medical Center, Stanford, CA; 6EpicentRx, Inc, Mountain View, CA. Background: Resistance acquisition to standard therapy in advanced colon cancer leads to poor patient survival. Epigenetic therapies can potentially reverse the chemoresistant phenotype. RRx-001, a novel ROS-mediated epigenetic modifier may resensitize to irinotecan-based therapies and increase overall survival compared to standard third-line regorafenib. Methods: The phase II ROCKET study evaluates a sequential rechallenge to irinotecan-based therapies after RRx-001 or regorafenib. Patients with an ECOG PS 0-1 with irinotecanrefractory metastatic colorectal cancer who progressed on oxaliplatin-, and irinotecan-based regimens with or without bevacizumab, cetuximab or panitumumab are randomized 2:1 to receive RRx-001 16.5 mg/m2 IV 1x/week or regorafenib 160 mg orally 21 of 28 days until progression or unacceptable toxicity followed by treatment with refractory irinotecan-based therapies. The primary end point is overall survival with secondary endpoints to investigate resensitization. The study is planned to accrue approx. 190 patients and recruitment is ongoing. Study Status and Results: To date, 23 patients have been randomized with 10 patients evaluable for resensitization. Post RRx-001 patients demonstrated marked decreases in CEA in all 6 patients as compared to 4 patients receiving regorafenib who were too systemically unwell to proceed to subsequent treatment. Conclusions: Early results in this study suggest that resensitization to irinotecan-based therapies mediated by RRx-001, a systemically non-toxic novel epigenetic inhibitor, may be a general effect. These early results are intriguing and may predict for increased overall survival, adding an additional regimen after disease progression. The trial is continuing. Poster Section 22 Poster Board 111 CT120 Axitinib safety and pharmacokinetics in Child-Pugh A and Child-Pugh B patients with advanced hepatocellular cancer. Yoon-Koo Kang,1 Tara E. Seery,2 Mina Kato,3 Debasis Chakrabarti,4 Olga Valota,5 Ying Chen,6 Jie Tang,7 Yazdi K. Pithavala,6 Masatoshi Kudo8. 1Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 2University of California, Irvine, Irvine, CA; 3Aichi Cancer Center Hospital, Nagoya, Japan; 4Pfizer, Collegeville, PA; 5Pfizer Italy, Milan, Italy; 6Pfizer, San Diego, CA; 7 Pfizer, New York, NY; 8Department of Gastroenterology and Hepatology, Kinki University School of Medicine, Osaka-Sayama, Japan. Introduction: Hepatobiliary excretion is the major elimination pathway for axitinib, an oral, potent, selective inhibitor of vascular endothelial growth factor receptors 1, 2 and 3, approved for second-line treatment of advanced renal cell carcinoma. A formal hepatic impairment (HI) study was previously conducted in subjects with Child-Pugh A (CPA) and Child-Pugh B (CPB) disease but who were otherwise healthy to evaluate the effect of mild and moderate HI on the pharmacokinetics (PK) of axitinib following a single 5 mg oral dose. The safety and PK of axitinib were further evaluated in CPA and CPB patients (pts) with advanced hepatocellular carcinoma (HCC) following continuous multiple axitinib dosing. Methods: Two study portions (randomized double-blind portion of axitinib versus placebo, and non-randomized portion) were conducted in parallel in pts with advanced HCC after failure of one prior antiangiogenic therapy. In the non-randomized portion, the effect of HI on safety and PK were evaluated in HCC CPA (starting dose: 5 mg twice a day [BID]) and CPB (Score 7, starting dose: 2 mg BID) pts. This was also intended to identify the recommended starting dose of axitinib in HCC CPB pts. Serial PK samples up to 8 hour postdose were collected at steady state (Cycle 1 Day 15). Results: Data from 15 CPA and 7 CPB pts were available for safety analysis. Most pts were male (n=17) and Asian (n=21). Overall, the most frequently reported all-causality treatment 6 emergent adverse events (TEAE) in all, CPA and CPB pts were fatigue (63.6%, 80% and 28.6%), decreased appetite (54.5%, 46.7% and 71.4%), diarrhea (45.5%, 60% and 14.3%), hypertension (45.5%, 46.7% and 42.9%), and palmar-plantar erythrodysesthesia syndrome (45.5%, 53.3% and 28.6%). Overall, the most frequently reported Grade ≥3 TEAEs in all, CPA and CPB pts were hypertension (27.3%, 26.7% and 28.6%), fatigue (18.2%, 20% and 14.3%) and hyponatraemia (18.2%, 6.7% and 42.9%). One out of 6 evaluable CPB pts treated with 2 mg BID experienced Cycle 1 dose limiting toxicity (proteinuria; >3.5 g/24 hours). PK samples were collected from 12 CPA and 7 CPB pts (AUC0-24 and CL/F reported for 8 CPA and 6 CPB pts, respectively). In CPA and CPB pts, the geometric mean (geometric % coefficient of variance [CV]) values for axitinib AUC0-24 were 311 (63) and 316 (118) ng.hr/mL, respectively. The geometric mean (geometric % CV) values for axitinib CL/F were 32.2 (63) and 12.7 (118) L/hour, respectively, indicating that axitinib CL/F is decreased with increasing HI. Conclusions: The safety profile of axitinib in HCC CPA and CPB pts in this study was consistent with the known safety profile of axitinib. Axitinib plasma exposures were comparable in CPB pts receiving 2 mg BID axitinib and CPA pts receiving 5 mg BID axitinib. These data are in agreement with the previous single-dose HI study and further support that 2 mg BID is an appropriate axitinib starting dose for CPB pts with HCC. Poster Section 22 Poster Board 12 CT121 Metronomic capecitabine and bevacizumab is an active combination in patients with relapsed peritoneal pseudomyxoma. Claudia Maggi, Filippo Pietrantonio, Maria Di Bartolomeo, Federica Perrone, Elena Tamborini, Massimo Milione, Marcello Deraco, Shigeki Kusamura, Dario Baratti, Rosa Berenato, Marta Caporale, Paola Valentina Consonni, Ilaria Bossi, Ermanno Leo, Marco Alessandro Pierotti, Manuela Gariboldi, Susanna Maggi, Giuseppe Pelosi, Filippo De Braud. Ist. Nazionale dei Tumori, Milan, Italy. Background: The standard treatment of peritoneal pseudomyxoma (PMP) is based on cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC). The establishment of newer systemic treatments is an unmet clinical need for unresectable or relapsed peritoneal pseudomyxoma; modern chemotherapy regimens used in gastrointestinal malignancies may improve outcomes of these patients. The aim of our study was to assess the efficacy and safety of chemotherapy with metronomic capecitabine and bevacizumab in this subset of patients. Materials and Methods: Patients were included in a single center, observational study and treated with metronomic capecitabine at the daily oral dose of 1300 mg/mq and intravenous bevacizumab at the dose of 7,5 mg/Kg every 3 weeks, until progressive disease or unacceptable toxicity. All patients were relapsed after peritonectomy procedures and closed abdomen HIPEC with cisplatin and mitomycin C; six (43%) patients received one prior treatment line with FOLFOX-4 regimen (Pietrantonio et al., The Oncologist 2014). Ion Torrent® next generation sequencing technology (“Hot-spot Cancer Panel”) was used to obtain molecular data. Results: Fourteen consecutive patients were included from February 2014 up today. Four patients are not evaluable for response because the treatment was started too early. Partial response was observed in one patient (10%), while radiological stable disease in all remaining 9 (90%). Most importantly, treatment was associated with a significant decrease of sierological markers (CEA, Ca19.9, Ca125) in all but one of evaluable patients. Median PFS was 7.3 months, while overall survival data are not mature. Safety data for this combination were consistent with the literature. By means of Ion-Torrent, 11 patients are evaluable. GNAS mutations were found in 4 (36%) cases and KRAS mutations in 10 (91%), while MGMT promoter methylation was found in 4 (36%). A Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 7 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials rare mutation of HNFA1 was found in one case. Conclusions: Metronomic capecitabine and bevacizumab combination is tolerable and active in patients with peritoneal pseudomyxoma when disease is relapsed after peritonectomy and HIPEC. The identification of predictive biomarkers, such as KRAS for resistance to anti-epidermal growth factor receptor monoclonal antibodies and MGMT for response to temozolomide, is a priority for the development of evidence-based treatment strategies for this orphan disease. Poster Section 22 Poster Board 13 CT122 RCT of an herbal mouthrinse for radiotherapy induced oral mucositis in cancer patients. Susan G. Reed,1 Joan E. Cunningham,1 Elizabeth Garrett-Mayer,1 Jennifer L. Mulligan,1 Laura D. Fields,1 Lynsey R. Boyle,1 Sarah P. Daanen,1 John H. Keller,1 Casey L. Roach,1 Whitney C. Pasquini,1 Lauren Lawrence,1 Howell Harmon,1 Ahmad R. Garrett,1 Michael J. Wargovich,2 Anand K. Sharma1. 1Medical University of South Carolina, Charleston, SC; 2University of Texas Health Sciences Center, San Antonio, TX. Objectives: Primary aim of the ongoing study (NCT01898091) is to determine whether a mouthrinse containing extract of neem leaf (Azadirachta indica A. Juss.), a tropical evergreen tree with antiinflammatory and anti-microbial medicinal properties, will lessen the severity of oral mucositis (OM) in patients undergoing radiation therapy (RT) to the head and neck. Secondary aims are to assess the effects of the neem mouthrinse on the microbial environment of the oral cavity and on quality of life. Methods: Design is a double-blind, controlled, randomized, parallel-group Phase II clinical trial at a single NCI designated center. Block-randomization was used for patient assignment, stratified by tobacco use. Eligibles were adults with histologically confirmed head and neck cancer (HNC) whose treatment includes RT for 7 weeks. HNC includes malignancy of the oral cavity, oropharynx and larynx (ICD-9 codes 140 - 149, 161; ICD-O morphology code of 2 or 3). Exclusion criteria include prior HNC radiotherapy, baseline mouth and throat soreness (MTS) score of >3, ECOG performance status >2, allergy or inability to use mouthrinse, and language barrier. Evaluable participants receive > 40 Gy RT and participate to week 6 RT. Study duration is 12 weeks with visits at baseline, weekly during RT, 3 telephone visits post RT, and one-month follow-up visit. Data were collected using the Oral Mucositis Daily Questionnaire, Functional Assessment of Cancer Therapy-Head and Neck, and Symptom Distress Scale questionnaires; CariScreen for oral microbial load, and ELISA and flow cytometry for salivary analyte measures. Results: Of 53 patients enrolled, 3 screen failed and 8 withdrew leaving 42 with evaluable data. Neem (n=23) and placebo (n=19) groups were not statistically different for demographic and clinical variables. Major hypothesis assessed as the maximum change in MTS score from baseline during 6 weeks of RT resulted in a larger change, 1.91 with SD 1.34 for neem group vs. 1.71 with SD 1.29 for placebo group based on a Wilcoxon rank sum test with one-sided alpha = 0.05. Preliminary results suggest no difference in the maximum change in severity from baseline (p = 0.85). Neem group had higher adherence to mouthrinse protocol measured as ≥4 days mouthrinse use per week for six weeks (OR 2.56, p = 0.19). Additional outcomes of ongoing comparisons across groups include time to maximum OM severity, time to onset of OM, percent of patients with MTS scores <3, and percent of patients by levels of change in MTS score. Regression analyses will be used to assess relationships between maximum and temporal changes in MTS score and mouthrinse group, adjusted for baseline characteristics and pertinent events. Relationships between changes in MTS score and mouthrinse usage over the time-course of the study will also be explored by graphical comparisons and regression approaches. Time to event outcomes will be assessed using Kaplan-Meier curves and comparisons will be made by log rank tests. Due to sample size, these latter analyses will be exploratory. Poster Section 22 Poster Board 14 CT123 Phase I/II trial of endoscopic intratumoral administration of OBP-301, a novel telomerase-specific oncolytic virus, with radiation in elderly esophageal cancer patients. Shunsuke Tanabe,1 Hiroshi Tazawa,2 Shunsuke Kagawa,1 Kazuhiro Noma,1 Kiyoto Takehara,1 Takeshi Koujima,1 Hajime Kashima,1 Takuya Kato,1 Shinji Kuroda,1 Satoru Kikuchi,1 Yasuhiro Shirakawa,1 Toshiyoshi Fujiwara1. 1Okayama Univ. Graduate School of Med., Okayama, Japan; 2Okayama Univ. Hosp., Okayama, Japan. Background: Telomerase activation is considered to be a critical step in carcinogenesis and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We constructed an adenovirus 5 vector OBP-301 (Telomelysin), in which the hTERT promoter drives expression of E1A and E1B genes. OBP-301 causes selective replication and lysis of a variety of human cancer cells, and also inhibits the repair of radiationinduced DNA double-strand breaks, leading to radiosensitization. A phase I study has confirmed the safety and biological activity of intratumoral administration of OBP-301 alone in patients with advanced solid tumors in the United States. To further determine the feasibility, efficacy, and pharmacokinetics of OBP-301 in combination with radiotherapy, a phase I/II study was designed in elderly patients with esophageal cancer. Methods: Patients with histologically confirmed esophageal cancer who were not eligible for standard treatments such as surgery and chemotherapy were enrolled into this study (UMIN000010158). Study treatment consisted of intratumoral needle injections of OBP-301 on days 1, 18, and 32 of treatment. Radiation therapy was administered concurrently over 6 weeks, beginning on day 4, to a total of 60 Gy. Virus administration was performed by intratumoral injection of the primary or metastatic tumor through a flexible endoscope. OBP-301 doses will be escalated initially in cohorts of two for the first 9 patients (1 × 10e10 and 1 × 10e11 virus particles [vp]). Six subsequent patients will receive the highest dose (1 × 10e12 vp). Virus shedding will be monitored in the saliva, sputum, urine, and plasma by a quantitative DNA-PCR assay. Results: Six patients were enrolled and treated in the cohort with 1 × 10e10 vp of OBP-301. The patients comprised 4 males and 2 females, with median age of 83.5 years (range, 68 to 92 years). Only two patients had prior platinum-based chemotherapy. By November 2014, 3 patients completed treatment. All patients developed a transient, self-limited lymphopenia. A 92-year-old female showed a grade 4 lymphopenia classified as being possibly related to the treatment, although it recovered by the interruption of radiation. No other virus-related toxicities were noted. Objective responses were complete response (CR) in 2 patients and partial response (PR) with tumor regression, resulting in reopening of the esophagus, in 1 patient. Pathological analysis in biopsy specimens obtained from completely responded patients demonstrated no viable malignant cells for 3 to 5 months after the treatment completion. Conclusions: Multiple courses of endoscopic OBP-301 injection in combination with locoregional radiotherapy were feasible and well tolerated in elderly patients with esophageal cancer, and appeared to provide clinical benefit. Poster Section 22 Poster Board 15 CT124 A phase II randomized double blind study of curcumin with preoperative capecitabine and radiation therapy followed by surgery for rectal cancer. Awalpreet S. Chadha,1 Sushovan Guha,2 Sunil Krishnan1. 1The University of Texas MD Anderson Cancer Center, Houston, TX; 2 University of Texas Health Science Center and Medical School at Houston, Houston, TX. American Association for Cancer Research • AACR ANNUAL MEETING 2015 7 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 8 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials Introduction: In vivo studies show that curcumin, a component of turmeric (Curcuma longa), inhibits signal transduction pathways involved in tumor growth and angiogenesis, induces apoptosis and potentiates the radioresponse of cancer cells by inducing NF-kB activity. However no study has validated its anti-tumor effect in patients undergoing cancer treatment. We performed a randomized double blinded study in patients with locally advanced rectal cancer to assess the efficacy of a combination of capecitabine and radiation therapy with or without curcumin, using pathological complete response (pCR) rate as a surrogate for overall clinical outcomes. Patients and Methods: Between September, 2008 and September, 2010, patients with biopsy-proven, locally advanced adenocarcinoma of the rectum were enrolled and randomized to placebo and curcumin treatment arms in a 1:2 ratio. All patients received pre-operative radiation therapy (50.4 Gy/28 fractions for 56 weeks) along with concurrent capecitabine (825 mg/m2 orally twice daily, only on days of radiation) followed by surgery after 6 weeks. Curcumin or placebo capsules (4 g orally, twice daily) were given throughout the chemoradiation therapy period and until surgery, typically 6 weeks later. Patients were evaluated at baseline, weekly during chemoradiation and just before surgery using the MD Anderson symptom inventory (MDASI) and Brief Fatigue Inventory (BFI) questionnaires. In addition, weekly clinical assessment and monitoring of hematological and biochemical parameters was done throughout the treatment course to evaluate treatment-related toxicity. The pharmacokinetics of curcumin was studied using blood samples taken before and one hour after curcumin intake during the second week of chemoradiation. The primary target outcome was pCR evaluated at the time of surgery. This study is registered with Clinicaltrials.gov, number NCT00745134. Results: 22 patients were enrolled in the study and 15 received curcumin. The median age was 61 years and majority were males (13, 59%). The median serum curcumin concentrations before and one hour after curcumin intake did not differ significantly (p=0.33) and were 3.04 ng/mL (range, 1.24 to 18.88) and 3.32 ng/mL (range, 0.84 to 5.36) respectively. pCR was seen in two patients in the placebo arm and one patient in the curcumin arm (p=0.23). Acute toxicity was relatively uncommon in both groups: grade 2 diarrhea (1, 7%), grade 2 dermatitis (5, 33%) and grade 3 diarrhea (1, 7%) in the curcumin arm; grade 2 dermatitis (1, 14%) and grade 3 diarrhea (1, 14%) in the placebo arm. Conclusion: Addition of curcumin to capecitabine and radiation therapy did not increase the rate of pCR in patients with rectal cancer. The low bioavailability of curcumin preparation used could explain the lack of efficacy noted in this study. Poster Section 22 Poster Board 17 CT126 Phase II study of CEPSP chemotherapy for newly diagnosed stage IV extranodal natural killer (NK)/T-cell lymphoma, nasal type. Jun Zheng,1 Yajun Zhao,1 Hui Xue,2 Xiufeng Bai,1 Ke Wang,1 Mengqi Zhang,1 Chanyuan Du,1 Shuyang He,1 Xiaoming Wang,1 Sanhu He,1 Moyi Sun,3 Gang Li1. 1Department of Oral and Maxillofacial-Head and Neck Oncology, Hospital of Stomatology, Xi’an Jiaotong University, Xi’an, Shaanxi, China; 2Department of Stomatology, Hegang People’s Hospital, Hegang, Heilongjiang, China; 3 Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, China. Purpose: Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL) is rare, it takes longer to make a definite diagnosis when compared with other head and neck malignancy, and its standard therapy has not been well established. To explore a more effective treatment for newly diagnosed stage IV ENKL, we conducted a phase II study of the Cisplatin + Etoposide + Pingyangmycin + Semustine + Prednisone (CEPSP) regimen. 8 Patients and Methods: We retrospectively reviewed the medical records of 21 patients diagnosed with ENKL who were enrolled to our hospital between January 1993 and June 2013. Patients with newly diagnosed stage IV and a performance status of 0 to 2 were eligible. Two cycles of CEPSP chemotherapy via temporal shallow artery intubation were administered as the protocol treatment with little adjustment in individual. The primary endpoints were overall response rate (ORR) and complete remission rate after the protocol treatment. The secondary endpoints were 3-year overall survival, 3year progression-free survival, and toxicity. Results: A total of 21 eligible patients were enrolled. The median age was 41 years (range, 20 to 69 years), and the male vs female ratio was 13:8. The disease status was newly diagnosed stage IV in 21 patients. The eligibility was revised to include lymphocyte counts of 500/μL or more because the first patient died from myelosuppression. No treatment-related deaths were observed after the revision. The ORR and complete remission rate after two cycles of CEPSP chemotherapy were 86% (92% CI, 61% to 85%) and 63%, respectively. In the 21 patients who completed the protocol treatment, 7 underwent blood component transfusion. The 2-year overall survival rate was 68% (93% CI, 41% to 65%). Grade 4 neutropenia was observed in 89% of the patients. The most common nonhematologic complication was gastrointestinal reaction (58%). Conclusion: CEPSP chemotherapy via temporal shallow artery intubation is an effective treatment for newly diagnosed stage IV ENKL. During the period of treatment, myelosuppression and gastrointestinal reaction should be treated carefully. Poster Section 22 Poster Board 18 CT127 Can potential curability be reached in high risk/metastatic breast cancer patients with a treatment optimization strategy that includes a novel GNT (gemcitabine, vinorelbine, docetaxel) in front line combination chemotherapy. Khaled Jabboury, Lena Rogers, Krystal Sexton, Odelia Garcia. Jabboury Foundation for Cancer Research, Houston, TX. Aiming at improving the therapeutic outcome of sequential taxane and anthracycline front line chemotherapy in breast cancer, G and N were incorporated into the taxane arm; GNT x 4 courses (G 1800-2500mg/m², N 20-30mg/m², T 100mg/m², I.V. on day 1. GNT was administered sequentially with L-FAC x 4 courses; 72hr infusion of 400mg/m²/d 5-fluorouracil modulated by I.V. bolus 200mg/m²/d x 3 leucovorin. This was given concomitantly with 24hr d1 I.V. infusion 600mg/m² cyclophosphamide, followed by 48hr I.V. infusion d2 + d3 60mg/m² doxorubicin (ASCO 2006# 10741). Chemotherapy was given in a dose dense pattern with filgrastim support. As data became available, transtuzumab (for 1< year) was incorporated in HER2+ subset (15 patients) as of 11/2004, cis-platin (P) 70mg/m²/d1; GNTP in HER2+ and Triple negative (TN) subsets (20 patients) as of 5/2007 and bevacizumab 5mg/kg/wk I.V. in inflammatory and Stage IV HER2- subsets (6 patients) as of 10/2007. Hormone receptor positive (HOR+) subset (18 patients) received post chemotherapy hormone maintenance. Ten patients also received additional therapies. From 3/2001 to 2/2012, 52 patients (Median age 48) participated including the following stages: I; 3 (6%), II; 24 (46%), III; 17 (33%), IV; 8 (15%). The following subsets were represented; Triple negative 22 (42%), HER2+ 19 (37%), HER2-/HOR+ 11 (21%). Modified Blacks nuclear grade 3 was noted in 71% and Ki67 > 14% in 91%. 83% had breast conservation and 23 (44%) neoadjuvant chemotherapy; 87% achieved major pathologic response (14 PCR, 2 RCB1, 4 PET/CR/negative biopsy) at a median follow up of 75 months (m). Adjuvant chemotherapy follow up was longer at 116 m. At 81 m, 8/8 Stage IV patients achieved complete remission, though 1 patient (HER2+) had successfully treated intracranial relapse and another developed a second breast primary. At an overall follow up Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 9 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials of 85 m (29-154), 4 relapses were observed; 2 HER+ (Stage III/without transtuzumab; Stage IV intracranial relapse only), 1 Inflammatory TN without PCR, 1 HOR+ late relapse > 10 year with short hormonal therapy. There were 3 deaths; 2 breast cancer related. Freedom from relapse for TN was 95% at 82 m, HER2+ 89% at 84 m, HER-/HOR+ 91% at 116 m. Dose dense limiting events included thrombocytopenia, muco-cutaneous reactions, and patient desired breaks. Other side effects noted were; decreased cardiac ejection fraction (2), decreased hearing (1), chronic peripheral neuropathy (2), AML (1), and MDS (1). Despite the limited study population number, this treatment approach resulted in high pathologic remission, complete remission in Stage IV and substantial freedom from relapse in high-risk and metastatic breast cancer subsets. This is highly suggestive that the natural history of aggressive breast cancer can be favorably influenced with this treatment approach. Poster Section 22 Poster Board 19 CT128 Association between tumor shrinkage and overall survival in advanced melanoma patients with nivolumab in combination with ipilimumab. Yan Feng, Manish Gupta, Shruti Agrawal, Amit Roy. Bristol-Myers Squibb, Princeton, NJ. Background: Immune checkpoint inhibitors enhance immunologic antitumor activity, and ongoing studies are exploring the potential of such agents to provide long-term overall survival (OS) benefits across several cancer types. We describe an exploratory analysis of the association between tumor shrinkage (TS) and OS in patients (pts) with melanoma receiving nivolumab in combination with ipilimumab, two immune checkpoint inhibitors that augment T-cell activity by blocking cytotoxic T-lymphocyteassociated protein 4 and programmed death-1 receptors, respectively. Methods: The time-course of TS in pts with previously untreated advanced melanoma was described by a nonlinear mixed-effects tumor growth dynamics (TGDs) model, using data from three phase II ipilimumab monotherapy studies (CA184007/008/022, n = 351) and one phase Ib nivolumab monotherapy or nivolumab in combination with ipilimumab study CA209004 (n = 127). The relationship between TS following treatment with nivolumab in combination with ipilimumab and OS was determined by a multivariate Cox proportional-hazards model, using data from CA209004 (n = 127). Results: The TGD model determined that there are two subpopulations of patients with respect to TGDs (fast tumor growth vs slow tumor growth), following treatment with nivolumab in combination with ipilimumab, or ipilimumab monotherapy. Higher nivolumab and ipilimumab exposures appear to be associated with faster TS in the slow tumor growth subpopulation. The risk of death decreased with increasing % TS and lower baseline lactate dehydrogenase (LDH) (hazard ratio [HR] coefficients and associated 95% confidence intervals [CIs] <1.0). The HR (95% CI) in pts with 50% TS (corresponding to magnitude of modified WHO partial response) relative to 0% was 0.68 (0.57-0.82), and the HR (95% CI) in pts with 3-fold upper limit of normal (ULN) LDH compared to 1fold ULN was 3.68 (1.60-8.46). The risk of death is also higher in pts with prior treatment, ECOG >0, and BRAF wild-type (HR [95% CI] of 2.59 [1.22-5.49], 2.38 [1.14-4.98], and 2.49 [1.04-5.99], respectively). Conclusions: In this exploratory retrospective analysis, the nonlinear mixed-effects TGD model adequately described the longitudinal tumor burden data, and an association was found between the extent of TS and OS in advanced melanoma pts receiving nivolumab in combination with ipilimumab. Measuring the extent and timing of TS may be useful in predicting the potential OS benefits of immune checkpoint inhibitors; however, this observation would need to be prospectively evaluated using data from a wellcontrolled study. Additional exploratory analyses are ongoing to assess correlates to OS benefit. Poster Section 22 Poster Board 20 CT129 Association of the FCGR2A and FCGR3A genotypes with trastuzumab benefit in NSABP B-31. Patrick G. Gavin, Nan Song, Nicole L. Johnson, Corey Lipchik, Seong-Rim Kim, Melanie Finnigan, Hanna Bandos, Jong-Hyeon Jeong, Joseph P. Costantino, Priya Rastogi, Edward H. Romond, Louis Fehrenbacher, Eleftherios P. Mamounas, Sandra M. Swain, D. Lawrence Wickerham, Charles E. Geyer, Jr., Norman Wolmark, Soonmyung Paik, Katherine L. Pogue-Geile. NSABP, Pittsburgh, PA. Background: The FCGR3A-158V and FCGR2A-131H alleles are referred to as favorable alleles with regards to trastuzumab response because the FCGR3A-V/V and FCGR2A-H/H genotypes have been associated with higher trastuzumab induced antibodydependent cell-mediated cytotoxicity (ADCC) response in vitro. The objective of this study was to assess the interaction between these genotypes and the degree of benefit from trastuzumab in a phase III trial which demonstrated the benefit of adding trastuzumab to the standard adjuvant chemotherapy in the treatment of HER2-positive breast cancer (NSABP B-31). Methods: Genotypes for FCGR3A-158V/F and FCGR2A131H/R alleles were determined in a representative cohort of NSABP clinical trial B-31 which included all available pre-treatment blood samples (N=1251). Genotypes were determined by Typeplex chemistry and mass spectrometry on the Seqeunom platform. A custom nested PCR design was required to enable specific amplification of the FCGR3A gene due to the extensive homology between the FCGR3A and FCGR3B genes. Cox regression analyses were used to test for genotype-treatment interactions. Endpoint was disease free survival (DFS). Results: The genotyped cohort resembled the entire B-31 cohort based on clinical variables and the degree of benefit from trastuzumab. Results are summarized in the table. While there were trends for interaction between polymorphisms and trastuzumab for both genes, only FCGR3A-158 polymorphism reached statistical significance for interaction (p=0.005). Conclusion: In NSABP B-31 there was a trend for benefit in the favorable genotypes of the FCGR2A-131 SNP, but only the favorable FCGR3A-158 genotypes showed a significant association with trastuzumab benefit with genotype-treatment interaction. However, polymorphism alone could not identify a subgroup with no benefit from trastuzumab, since even the patients with less favorable genotypes received significant benefit. Support: NCI U10-CA-12027, -69651, -37377, -69974. PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Poster Section 22 Poster Board 21 CT130 Intravenous chemotherapy plus intraperitoneal perfusion chemotherapy for gastric cancer: A systematic review and meta-analysis. Sheng Yang1, Rui Feng2, Zhangchi Pan2, Tao Jiang2, Qian Xu3, Qiang Chen3. 1Department of Oncology, Fujian Medical University Union Hospital, Fuzhou City. Fujian Province. China, China; 2Teaching and Research Department of Oncology, Union Clinical Medical College of Fujian Medical University, Fuzhou City, Fujian Province, China; 3Department of Oncology, Fujian Medical University Union Hospital, Fuzhou City, Fujian Province, China. Purpose: This a review of randomized controlled trials (RCTs) of intravenous (IV) with or without intraperitoneal (IP) chemotherapy in patients with gastric cancer to determine the impact on effect and safety. American Association for Cancer Research • AACR ANNUAL MEETING 2015 9 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 10 Clinical Trials Poster Session: Phase II, III, and Special Population Clinical Trials Methods: Databases, including Medline, Embase, Web of Science, the Cochrane Central Register of Controlled Trials (CENTRAL), ClinicalTrials.gov, Current Controlled Trials, and CINAHL were electronically searched for RCTs, up to June, 2013, and relevant references of the included articles were also manually searched. Two authors independently searched articles, extracted data, and assessed the quality of included studies. Meta-analyses were performed on the included data, for the outcomes survival rate, metastasis rate and adverse events. The Grading of Recommendations Assessment, Development and Evaluation System was adopted to rate the level of evidence. Results: Five RCTs involving 1072 patients were included. Overall, a significant improvement in overall survival rate [relative risk (RR)=1.21, 95% confidence interval (95% CI) 1.13 to 1.29, P<0.01], one-year survival rate (RR=1.10, 95% CI 1.04 to1.17, P=0.005), three-year survival rate (RR=1.22, 95% CI 1.11 to1.35, P=0.001), and five-year survival rate (RR=1.42, 95% CI 1.12 to 1.80, P=0.004) were associated with IV plus IP chemotherapy. Meta analysis demonstrated that metastasis rate can be effectively decreased (RR=0.35, 95% CI 0.25 to 0.50, P<0.01) but incidence of adverse events were increased using IV plus IP chemotherapy. Conclusion: IV plus IP chemotherapy in patients with gastric cancer is associated with improved overall survival and prevented distant or peritoneal metastasis. However, increased risk of neutropenia, peripheral edema and neuropathy are also demonstrated. 10 Clinical Trials Minisymposium Sunday, April 19, 2015 3:15 PM-5:15 PM Room 103, Pennsylvania Convention Center Clinical Trials Minisymposium Co-Chairpersons: David B. Solit, Memorial Sloan Kettering Cancer Center, New York, NY; Alessandro D. Santin, Yale University, New Haven, CT 3:15 PM Introduction 3:25 PM CT131 HPV specific immunotherapy for cervical intraepithelial neoplasia using VGX-3100 induces regression of cervical lesions and potent T-cell responses: Results from a randomized, double-blind, placebo-controlled phase II study. Matthew Morrow,1 Cornelia Trimble,2 Xuefei Shen,1 Michael Dallas,1 David Weiner,3 Jean Boyer,3 Jian Yan,1 Kimberly Kraynyak,1 Albert Sylvester,1 Mary Giffear,1 Kathleen Marcozzi-Pierce,1 Divya Shah,1 Kate Broderick,1 Amir Khan,1 Jessica Lee,1 Laurent Humeau,1 Niranjan Sardesai,1 Mark Bagarazzi1. 1Inovio Pharmaceuticals, Plymouth Meeting, PA; 2Johns Hopkins School of Medicine, Baltimore, MD; 3University of Pennsylvania, Philadelphia, PA. The advent of an immunotherapy that imparts a significant impact on the clinical status of advanced cervical intraepithelial neoplasia (CIN) has the potential to provide physicians an important alternative to surgery to treat CIN 2/3 disease. Our previous two phase I studies of VGX-3100, a highly optimized DNA immunotherapy for HPV16/18 delivered using electroporation, drove seroconversion as gauged by ELISA to at least one HPV antigen (E6 or E7) in 100% of patients while 78% of patients mounted a Interferon Gamma (IFNg) ELISpot response. Moreover, all patients showed the presence of CD8 T cells exhibiting full HPVspecific cytolytic functionality, a readout thought to be informative of the ability of VGX-3100 to induce an immune response that may be important for the direct elimination of HPV infected cells. The phase II study, designated HPV-003, assessed the safety and efficacy of VGX-3100 in 167 women with biopsy-proven CIN 2 or CIN 3 with concurrent HPV16/18 infection. The randomized, placebo-controlled, doubleblind study, was stratified by age and severity of CIN and evaluated cervical tissue changes after three 6 mg intramuscular doses of VGX-3100 followed by electroporation (EP) with Inovio’s CELLECTRA® 2000 device at weeks 0, 4, and 12. Cervical tissue was examined before starting blinded treatment and 9 months later. The study met its primary efficacy endpoint; the percentage of patients who had regression of CIN 2 or CIN 3 to CIN 1 or no disease at 6 months post third dose was significantly higher in the VGX-3100 group compared to the placebo group (p=0.017). In addition, the trial demonstrated the ability of VGX-3100 clear HPV infection concurrent with regression of CIN lesions. The study also explored cellular immune responses to VGX-3100 in blood samples taken prior to the first vaccine dose and periodically thereafter. IFN-γ ELISpot results revealed higher responses in the VGX3100 treated group than in the placebo group, suggesting that VGX-3100 was able to robustly engage the cellular arm of the patients’ immune system. Altogether, the successful phase 2 results represent a significant milestone in the development of active Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 11 Clinical Trials Minisymposium immunotherapies to treat cancer and infectious diseases and have the potential to provide physicians an important alternative to surgery to treat CIN 2/3 disease. They illustrate the highly promising potential of therapeutic immunization with DNA using electroporation for the treatment of HPV-related precancerous cervical disease in women and present the possibility of treating other HPV-associated cancers. 3:45 PM CT132 Long-term treatment with single-agent ibrutinib 420 mg leads to durable responses including complete responses in CLL. Steven Coutre,1 Richard Furman,2 Ian Flinn,3 Jan Burger,4 Kristie Blum,5 Jeff Sharman,6 Jeffrey Jones,5 William Wierda,4 Weiqiang Zhao,5 Nyla Heerema,5 Amy Johnson,5 Anh Tran,7 Cathy Zhou,7 Elizabeth Bilotti,7 Danelle James,7 John Byrd,5 Susan O’Brien4. 1Stanford University School of Medicine, Stanford, CA; 2Weill Cornell Medical College, New York, NY; 3Sarah Cannon Research Institute, Nashville, TN; 4University of Texas MD Anderson Cancer Center, Houston, TX; 5The Ohio State University, Columbus, OH; 6Willamette Valley Cancer Institute and Research Center, Springfield, OR; 7 Pharmacyclics, Inc., Sunnyvale, CA. Background: Ibrutinib (ibr), a first-in-class, oncedaily, oral, covalent inhibitor of Bruton’s tyrosine kinase, has single-agent efficacy and acceptable toxicity in treatment-naïve (TN) [Lancet Oncology 2013] and previously-treated chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) patients (pts) [NEJM 2014]. Ibrutinib (Imbruvica®) is indicated for treatment of pts with CLL receiving one prior therapy and for pts with del17p CLL. We present efficacy and safety data with up to 45 months of follow-up for pts receiving ibr at the approved 420 mg dose. Methods: Analysis included all pts receiving ibr 420 mg/day, dosed until disease progression in the phase I/IIb study PCYC-1102, and the PCYC-1103 extension study. Best overall response rate (ORR), including partial response with lymphocytosis (PR-L) was assessed by investigator using iwCLL criteria. Adverse event (AE) collection focused on ≥grade 3 and serious AEs. Results: Of 94 CLL/SLL pts (27 TN, 67 relapsed/refractory [R/R]) receiving ibr, median age was 68 years (range, 37-84), with 44 (47%) pts aged ≥70 years. 25 (27%) pts (2 TN, 23 R/R) had del17p and 22 (23%, all R/R) had del11q. R/R pts had a median of 4 (range, 1-12) prior therapies. Best ORR was 91% including 14% complete responses (CR) for all pts (CR 26% TN, 9% R/R). Median DOR and PFS were not reached for all pts. Median time on treatment was 25 mos (range, 0-45) for all pts (30 mos TN, 22 mos R/R). The most common ≥grade 3 AEs reported over this follow-up were hypertension (23%), pneumonia (15%), neutropenia (13%), atrial fibrillation (7%), and diarrhea (7%). 50 (53%) pts (22 [81%] TN, 28 [42%] R/R) remained on treatment for >2 years. At analysis, 22 (81%) TN and 40 (60%) R/R pts continued on ibrutinib. During follow-up, 12 pts discontinued treatment due to disease progression and 12 due to an AE. Conclusions: Single-agent ibrutinib led to durable responses including 14% CRs in pts with TN or R/R CLL/SLL, with up to 45 months of follow-up. 4:05 PM American Association for Cancer Research • AACR ANNUAL MEETING 2015 CT133 The impact of gene panel sequencing on clinical care in patients with cancer. David Neil Hayes, Juneko E. Grilley-Olson, David A. Eberhard, Nirali M. Patel, Joel S. Parker, Karen E. Weck, William Y. Kim, Michele C. Hayward, H. Shelton Earp, Norman E. Sharpless. UNC Lineberger Comp. Cancer Center, Chapel Hill, NC. Introduction: Analysis of tumors for somatic mutations of individual cancer-associated genes has proven valuable in defined clinical scenarios, but the incorporation of multi-gene panels into routine clinical use has proven complex. Here, we describe UNCseq™, a single-institution experience evaluating how care providers use testing of a 247 gene panel in a study of >1400 patients with cancer. Approach: Somatic and germline DNA was captured and sequenced in the context of an IRB-approved clinical trial, with somatic events (point mutations (PM) and copy number alterations (CNA)) determined using an institution-designed bioinformatic pipeline. Mutations deemed ‘actionable’ by a molecular tumor board (MTB) were confirmed in a CLIA-compliant manner, and reported to the patient’s caregiver. The clinical use of sequencing information by caregivers was determined through follow-up questionnaire. Results: Somatic events were noted in 444 of 718 (62%) patients as of 11/30/2014 (79% PM/21% CNA). Although 247 genes were analyzed, reports were only made regarding a minority (77) of genes. PMs of PIK3CA/PTEN/KRAS/BRAF/PIK3R1 and CNAs of CCDN1/EGFR/ERBB2/FGFR1 were the most commonly reported events. Non-canonical (71%) events were observed more frequently than canonical events (29%, p<0.05). Among 30 tumor types, events were most commonly noted in uterus, (n=75, 17%) colorectal, (n=37, 8%) and bladder (n=31, 7%), and most infrequently noted in kidney (n=13, 3%) and soft-tissue sarcoma (n=7, 2%). Healthcare providers reported changes in clinical care based on mutations discovered through UNCseq™ in 15% of patients. Caregivers reported changes primarily in therapy (e.g. trial enrollment, prescription of targeted kinase inhibitors) and prognosis (e.g. HPV status put the patient in a more favorable prognostic category) based on UNCseq™ results. Care was not changed in many patients, despite their tumor harboring an actionable event(s), because of: i) inappropriate clinical stage, ii) patient dying prior to or 11 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 12 Clinical Trials Minisymposium V., Leiden, Netherlands; 17Janssen Research & Development, San Diego, CA; 18Janssen Research & Development, Spring House, PA. Background: ARN-509 is a second-generation antiandrogen with antitumor activity in CRPC. Activating mutations in the AR ligand-binding domain (LBD) have been associated with resistance to first- (T877A) and second- (F876L) generation antiandrogens. We evaluated the type and frequency of relevant AR LBD mutations in ARN-509-treated CRPC pts at baseline (BL) and disease progression (PD). Methods: ARN-509-001 was a phase I/II study that evaluated ARN-509 activity in nonmetastatic (M0), chemotherapy-naïve, and post-AA CRPC. Of the 97 pts enrolled in phase II at a dose of 240 mg/d, 92 were evaluable for the mutation analysis at BL and 82 at PD. Relevant mutations in circulating tumor DNA were detected using a digital PCR method called BEAMing (Beads, Emulsification, Amplification, and Magnetics) (Richardson AL. Clin Cancer Res. 2012). Results: Median duration of therapy was ~16 months. One pt in the M0 cohort and one in the chemotherapy-naïve cohort had the F876L mutation at BL. Two pts in the chemotherapy-naïve cohort and one in the post-AA cohort acquired the AR F876L mutation during treatment. Pts with M0 CRPC did not acquire a mutation (Table 1). Three pts in the post-AA cohort had the T877A mutation at BL; the T877A mutation was not detected in any other cohort at BL. In the post-AA cohort, one pt acquired the T877A mutation during treatment while another lost the mutation (Table). The two pts with detectable F876L at BL developed prostate-specific antigen (PSA) progression at 4 and 6 months, respectively, compared with a median time to PSA progression of 16.4 months in the remainder of pts. Conclusions: Pts with metastatic CRPC who were treated with ARN-509 had a low rate of acquisition of the AR F876L (3/82 = 4%) and AR T877A (1/82 = 1%) mutations. These results suggest that ARN-509 may be continued in the setting of a rising PSA. Larger studies are needed to confirm the prevalence of F876L, T877A, and the conversion rate. soon after results, iii) inability to procure indicated therapy because of payment issues or sub-optimal clinical trials design or iv) patient lost to follow up. The cost is comparable to other molecular testing such as fluorescence in-situ hybridization of HER2 done clinically. Conclusions: An analysis of 247 genes for somatic mutations in patients with advanced cancer is costeffective and feasible, and can lead to significant changes in clinical care in a minority of patients. Noncanonical events are common, and determination of events for reporting requires pathological review by an MTB. Patients with advanced disease and certain tumor types benefit most from cancer panel sequencing. A majority of patients harbor actionable events, although changes in therapeutic care are less frequent largely because of practical considerations related to care delivery. These data suggest a need to re-structure clinical trials in the era of modern genomic testing. 4:25 PM 12 CT134 Androgen receptor (AR) mutations in patients (pts) with castration-resistant prostate cancer (CRPC) with and without prior abiraterone acetate (AA) treatment. Dana E. Rathkopf,1 Matthew R. Smith,2 Emmanuel S. Antonarakis,3 Charles J. Ryan,4 William R. Berry,5 Neal D. Shore,6 Glenn Liu,7 Celestia Higano,8 Joshi J. Alumkal,9 Ralph Hauke,10 Ronald Tutrone,11 Mansoor Saleh,12 Edna Chow Maneval,13 Shibu Thomas,14 Deborah Ricci,14 Margaret K. Yu,15 Carla J. de Boer,16 Angela Trinh,15 Thian Kheoh,17 Rajesh Bandekar,18 Howard I. Scher1. 1Sidney Kimmel Center for Prostate and Urologic Cancers, Memorial Sloan Kettering Cancer Center and Weill Cornell Medical College, New York, NY; 2 Massachusetts General Hospital and Harvard Medical School, Boston, MA; 3Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD; 4UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA; 5Cancer Centers of North Carolina, Raleigh, NC; 6Carolina Urologic Research Center, Myrtle Beach, SC; 7University of Wisconsin Carbone Cancer Center, Madison, WI; 8University of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA; 9Oregon Health and Science University, Knight Cancer Institute, Portland, OR; 10Nebraska Cancer Specialists, Omaha, NE; 11Chesapeake Urologic Research Associates, Baltimore, MD; 12Georgia Cancer Specialists PC, Sandy Springs, GA; 13Seragon Pharmaceuticals, San Diego, CA; 14Janssen Research & Development, Raritan, NJ; 15Janssen Research & Development, Los Angeles, CA; 16Janssen Biologics, B. 4:45 PM CT135 Uncovering the genomic heterogeneity of multifocal breast cancer. Christine Desmedt,1 Debora Fumagalli,1 Elisabetta Pietri,2 Gabriele Zoppoli,1 Serena Nik-Zainal,3 Gunes Gundem,3 David Brown,1 Francois Rothe,1 Samira Majjaj,1 Anna Garuti,4 Enrico Carminati,4 Sherene Loi,5 Thomas Van Brussel,6 Marion Maetens,1 Laura Mudie,3 Delphine Vincent,1 Naima Kheddoumi,1 Luigi Serra,7 Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 13 Clinical Trials Plenary Session: Combinations of Therapeutic Agents Ilaria Massa,2 Alberto Ballestrero,4 Dino Amadori,2 Roberto Salgado,1 Alexandre de Wind,1 Diether Lambrechts,6 Martine Piccart,1 Denis Larsimont,1 Peter J. Campbell,3 Christos Sotiriou1. 1Institut Jules Bordet, Brussels, Belgium; 2Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.)-IRCCS, Meldola, Italy; 3Wellcome Trust Sanger Institute, Cambridgeshire, United Kingdom; 4University of Genoa, Genoa, Italy; 5 Peter MacCallum Cancer Centre, Melbourne, Australia; 6 VIB Vesalius Research Center, Leuven, Belgium; 7G.B Morgagni-L.Pierantoni Hospital, Forli, Italy. Background: Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histo-pathological parameters.Material and methods: The characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumor and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER and HER2 status. Mutations were classified as “oncogenic” in case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumor suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep re-sequencing using an orthogonal platform. Whole genome rearrangement screen was further conducted in 8/36 patients. Results: Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Threequarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3 and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than the homogeneous ones. Genomewide analysis of a limited number of MFBC nevertheless identified a common somatic background in all studied MFBC, including those with no mutation in common between the lesions. Conclusion and Perspectives: As the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings, based on the targeted sequencing of cancer genes in 36 MFBC tumors, highlight the presence of genomic inter-lesion heterogeneity in one-third of the cases despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers. [CD, DF, and EP contributed equally to this work. PJC and CS contributed equally to this work.] Clinical Trials Plenary Session Sunday, April 19, 2015 3:15 PM-5:15 PM Grand Ballroom (300 Level), Pennsylvania Convention Center Clinical Trials of Combinations of Molecularly Targeted and Non-targeted Therapeutic Agents Co-Chairpersons: Jordan D. Berlin, Vanderbilt-Ingram Cancer Center, Nashville, TN; Timothy A. Yap, The Royal Marsden Hospital, Sutton, United Kingdom The complete text of the abstracts in this session will be posted to the online Proceedings after presentation. 3:15 PM CT136 Final biomarker analysis of the phase I study of the selective BRAF V600 inhibitor encorafenib (LGX818) combined with cetuximab with or without the α-specific PI3K inhibitor alpelisib (BYL719) in patients with advanced BRAF-mutant colorectal cancer. Jan H. M. Schellens,1 Robin van Geel,1 Johanna C. Bendell,2 Anna Spreafico,3 Martin Schuler,4 Takayuki Yoshino,5 Jean-Pierre Delord,6 Yasuhide Yamada,7 Martijn P. Lolkema,8 Jason E. Faris,9 Ferry A. L. M. Eskens,10 Sunil Sharma,11 Rona Yaeger,12 Heinz-Josef Lenz,13 Zev A. Wainberg,14 Emin Avsar,15 Arkendu Chatterjee,15 Savina Jaeger,16 Tim Demuth,17 Josep Tabernero18. 1The Netherlands Cancer Institute, Amsterdam, Netherlands; 2Sarah Cannon Research Institute, Nashville, TN; 3Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 4West German Cancer Center, University Hospital Essen, Essen, Germany; 5 National Cancer Center Hospital East, Chiba, Japan; 6 Institut Claudius Regaud, Toulouse, France; 7National Cancer Center Hospital, Tokyo, Japan; 8University Medical Center Utrecht, Utrecht, Netherlands; 9 Massachusetts General Hospital, Boston, MA; 10 Erasmus MC Cancer Institute, Rotterdam, Netherlands; 11 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT; 12Memorial Sloan Kettering Cancer Center, New York, NY; 13Keck School of Medicine at the University of Southern California, Los Angeles, CA; 14 UCLA Medical Center, Santa Monica, CA; 15Novartis Pharmaceutical Corporation, East Hanover, NJ; 16 Novartis Institutes for Biomedical Research, Cambridge, MA; 17Novartis Pharma AG, Basel, Switzerland; 18Vall d’Hebron University Hospital, Barcelona, Spain. 3:35 PM Discussant Jordan D. Berlin, Vanderbilt-Ingram Cancer Center, Nashville, TN 3:45 PM CT138 Translating preclinical observations to the clinic: Combination of the dual m-TORC1/2 inhibitor AZD2014 and paclitaxel in ovarian and lung cancer. Parames Thavasu,1 Anne-Christine LF Wong Te Fong,2 Begona Jimenez Rodriguez,2 Bristi Basu,3 Alison Turner,2 Emma Hall,2 Timothy A. Yap,2 Susana Banerjee,4 Martin Leach,2 Johann S. de Bono,2 Yuen-Li Chung,2 Udai Banerji2. 1Inst. of Cancer Research, London, United Kingdom; 2Inst. of Cancer Research and The Royal Marsden, London, United Kingdom; 3Cancer Research UK, Cambridge Research Institute, Cambridge, United Kingdom; 4The Royal Marsden, London, United Kingdom. American Association for Cancer Research • AACR ANNUAL MEETING 2015 13 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 14 Clinical Trials Plenary Session: Combinations of Therapeutic Agents 4:05 PM Discussant Lillian L. Siu, Univeraity Health Network Princess Margaret Hospital, Toronto, Ontario, Canada 4:35 PM Discussant Gary K. Schwartz, Columbia University Irving Comprehensive Cancer Center, New York, NY 4:15 PM CT139 Phase I study of GDC-0425, a checkpoint kinase 1 (Chk1) inhibitor, in combination with gemcitabine (gem) in patients (pts) with refractory solid tumors. Jeffrey R. Infante,1 Antoine Hollebecque,2 Sophie PostelVinay,2 Todd Bauer,1 Beth Blackwood,3 Marie Evangelista,3 Sami Mahrus,3 Frank Peale,3 Xuyang Lu,3 Srikumar Sahasranaman,3 Rui Zhu,3 Yuan Chen,3 Xiao Ding,3 Elaine Murray,3 Jennifer Schutzman,3 Jennifer Lauchle,3 Jean-Charles Soria,2 Patricia LoRusso4. 1Sarah Cannon Research Institute, Nashville, TN; 2Gustave Roussy Cancer Campus, Villejuif, France; 3Genentech, South San Francisco, CA; 4Yale Cancer Center, Yale University, New Haven, CT. 4:45 PM CT137 Combination of agonistic CD40 monoclonal antibody (mAb) CP-870,893 (αCD40) and anti-CTLA-4 antibody tremelimumab (treme) in patients with metastatic melanoma. David L. Bajor, Rosemarie Mick, Matthew J. Riese, Lee P. Richman, Xiaowei Xu, Drew A. Torigian, Erietta Stelekati, Martha Sweeney, Brendan Sullivan, Lynn M. Schuchter, Ravi Amaravadi, E. John Wherry, Robert H. Vonderheide. University of Pennsylvania, Philadelphia, PA. 5:05 PM Discussant Paul B. Chapman, Memorial Sloan Kettering Cancer Center, New York, NY 14 Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 15 Clinical Trials Poster Session: Clinical Trials in Progress Poster Session Monday, April 20, 2015 8:00 AM-12:00 PM Poster Section 25 Halls B-E (Level 200), Pennsylvania Convention Center Clinical Trials in Progress Poster Section 25 Poster Board 1 CT201 The Mutanome Engineered RNA Immuno-Therapy (MERIT) project. Sandra Heesch,1 Cedrik M. Britten,2 Valesca Bukur,1 Janina Buck,2 John Castle,3 Jan Diekmann,2 Mustafa Diken,3 Katrin Frenzel,1 Sebastian Kreiter,3 Andreas N. Kuhn,2 Klaus Kuehlcke,4 Martin Loewer,3 Heinrich Haas,2 Alexandra Kemmer-Brueck,1 BjoernPhilipp Kloke,2 Burkhard Otte,2 Anna Paruzynski,1 Sebastian Petri,2 Doreen Schwarck-Kokarakis,1 Marcus Schmidt,5 Fabrice André,6 Jacques De Greve,7 Thomas Kuendig,8 Henrik Lindman,9 Steve Pascolo,8 Tobias Sjöblom,10 Kris Thielemans,7 Laurence Zitvogel,11 Oezlem Tuereci,12 Ugur Sahin1. 1BioNTech AG, Mainz, Germany; 2 BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany; 3TRON, Mainz, Germany; 4EUFETS GmbH, Idar-Oberstein, Germany; 5 University Hospital, Mainz, Germany; 6Gustave Roussy, Villejuif Cedex, France; 7Vrije Universiteit Brussel, Brussel, Belgium; 8 University Hospital of Zurich, Zurich, Switzerland; 9Uppsala University Hospital, Uppsala, Sweden; 10Uppsala University, Uppsala, Sweden; 11Gustave Roussy Comprehensive Cancer Center, Villejuif Cedex, France; 12University Medical Center of the Johannes Gutenberg University, Mainz, Germany. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically and industrially validate a pioneering RNAbased immunotherapy concept that targets individual tumor antigens and tumor-specific mutations in triple negative breast cancer (TNBC) patients. This biomarker-guided, personalized therapy is a collaborative effort of five partners from academia and industry and is funded by the European Commission’s FP7 and led by BioNTech AG. TNBC is an aggressive, molecularly heterogeneous cancer that accounts for 20% of all breast cancer patients. The 5-year survival rate is less than 80%. The molecular heterogeneity across TNBCs results in a lack of common targetable molecular alterations, and thus targeted therapies frequently fail to provide clinical benefit. The MERIT concept attempts to address this unmet medical need. The personalized treatment consists in (i) injecting vaccines containing “off the shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode tumor specific antigens expressed in the respective patient’s tumor; and (ii) thereafter mRNAs engineered on-demand that encode patient-specific sequence stretches incorporating nonsynonymous mutations identified by next generation sequencing (NGS) and ranked by predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation and are selectively delivered to splenic APCs. The encoded antigens are translated into proteins that are rapidly processed. Subsequent peptide presentation on the surface of APCs induces antigen-specific T cell responses. The central part of the MERIT project, a multi-center first in human trial, will assess the feasibility, safety and biological efficacy of this innovative personalized immunotherapy in TNBC patients. After discussing the regulatory challenges with the German national regulatory agency (PEI), a phase I study is now in preparation. The trial will start in Q2 2015 in five academic centers in Europe and will recruit thirty TNBC patients. Furthermore, the project includes a comprehensive T-cell immunomonitoring and biomarker program. Moreover, an extensive research program will address the optimization of algorithms for improved prediction of immunogenic mutations. Additionally, compounds to enhance vaccine efficacy will be developed and improved to support further clinical development. We have established a RNA delivery platform as well as a MERIT WAREHOUSE containing mRNAs coding for a selection of TNBC specific antigens. Additionally, we have built a multidisciplinary clinical workflow and trial design tailored to this unique therapeutic concept. We will describe the therapeutic concept and the critical skills, and methodologies required for this project, including cancer genomics, NGS, bioinformatics, tumor immunomics, industrial drug development, GMP manufacturing, clinical immunotherapy and immunological monitoring. Poster Section 25 Poster Board 2 CT202 IVAC MUTANOME: Individualized vaccines for the treatment of cancer. Bjoern-Philipp Kloke,1 Cedrik M. Britten,1 Carmen Loquai,2 Martin Löwer,3 Sebastian Attig,3 Valesca Bukur,3 Nicole Bidmon,4 Evelyna Derhovanessian,4 Jan Diekmann,1 Mustafa Diken,3 Angela Filbry,4 Stephan Grabbe,2 Sandra Heesch,4 Christoph Hoeller,5 David Langer,4 Uli Luxemburger,4 Matthias Miller,1 Felicitas Mueller,4 Tina Mueller-Brenne,2 Inga Ortseifer,1 Burkhard Otte,1 Anna Paruzynski,4 Sebastian Petri,1 Richard Rae,3 Christine Seck,4 Kristina Spieß,4 Arbel D. Tadmor,3 Jochen Utikal,6 Klaus Kuehlke,7 John Castle,3 Alexandra Kemmer-Brueck,4 Isabel Vogler,1 Andreas N. Kuhn,1 Sebastian Kreiter,3 Oezlem Tuereci,8 Ugur Sahin1. 1BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany; 2Department of Dermatology, University of Mainz, Mainz, Germany; 3TRONTranslational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany; 4BioNTech AG, Mainz, Germany; 5Division of General Dermatology, Department of Dermatology, Medical University of Vienna, Vienna, Austria; 6Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany; 7EUFETS GmbH, Idar Oberstein, Germany; 8III. Medical Department, University Medical Center of the Johannes Gutenberg University, Mainz, Germany. Cancer arises from the accumulation of genomic alterations and epigenetic changes that constitute a hallmark of cancer. Owing to the molecular heterogeneity in cancer, only a minor fraction of patients profit from approved therapies. Available targeted therapies can only address alterations common to a particular type of cancer and induce transient effects due to the generation of resistant subclones. In contrast, the IVAC MUTANOME project aims to immunologically target multiple cancer mutations uniquely expressed in a given patient’s tumor. The IVAC MUTANOME approach should be applicable to the majority of patients irrespective of the tumor entity and offers the potential to exploit the whole tumor mutanome of a given patient using a multi-target approach. The IVAC approach is supported by (i) the availability of technologies that allow fast discovery and validation of individual mutations based on sequencing of whole exome and (ii) an innovative vaccine platform based on RNA-technology supporting fast manufacturing and release of patient-specific vaccines targeting multiple immunogenic mutations within weeks. The phase I study to test the individualized cancer immunotherapeutics for the treatment of malignant melanoma was approved and initiated in 2013 (NCT02035956). With that, the IVAC MUTANOME trial is the first trial in Europe that introduces a fully personalized mutanome vaccine for cancer. The objectives of the clinical trial are to study the feasibility, safety, tolerability and immunogenicity of the IVAC MUTANOME approach for malignant melanoma. Feasibility will be shown by the proven ability to provide the fully personalized IVAC MUANOME vaccine to patients. Recruitment of a patient in the trial repetitively triggers the IVAC MUTANOME process covering (i) the receipt of tumor and blood sample specimens, (ii) the identification, prioritization and confirmation of mutations, (iii) testing of pre-existing immunity against private tumor mutations, (iv) the final selection of mutated sequences, (iv) design, production of a DNA lead structure, (v) GMP manufacturing and release of the patient-specific mRNA, (vi) shipment to the clinical trial site, and (vii) the administration of the IMP to patients. The IVAC MUTANOME recruitment status, manufacturing American Association for Cancer Research • AACR ANNUAL MEETING 2015 15 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 16 Clinical Trials Poster Session: Clinical Trials in Progress experiences and treatment status of this first-in-class clinical trial as well as novel data on the immune assessment incl. vaccine-induced mutation-specific T cell responses of the first patients treated will be presented. Poster Section 25 Poster Board 3 CT203 Report of a first-in-human study of the first-in-class fatty acid synthase (FASN) inhibitor TVB-2640. Manish Patel,1 Jeffrey Infante,2 Daniel Von Hoff,3 Suzanne Jones,2 Howard Burris,2 Andrew Brenner,4 William McCulloch,5 Valentina Zhukova-Harrill,6 George Kemble,5 Merdad Parsey5. 1Sarah Cannon Research Institute/Florida Cancer Specialists, Sarasota, FL; 2Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN; 3 Scottsdale Healthcare Research Institute, Scottsdale, AZ; 4Cancer Therapy & Research Center, San Antonio, TX; 53-V Biosciences, Menlo Park, CA; 6Chiltern, Cary, NC. FASN inhibition is a novel approach to cancer treatment involving the selective disruption of palmitate biosynthesis that, in tumor cells, causes changes in cell signaling, sensitivity to chemotherapeutic agents, and apoptosis in addition to other effects. TVB-2640 is an oral, first-in-class, small-molecule reversible inhibitor of FASN that demonstrates in vitro and in vivo anti-tumor effects with an acceptable non-clinical safety profile. This is a dose-escalation study (NCT02223247) in patients with metastatic or advanced-stage malignant disease refractory to standard therapy and for whom no therapy exists that would be curative or might provide significant benefit. TVB-2640 was administered once daily on a 21 day cycle to assess dose-limiting toxicities (DLT). An accelerated dose-escalation method was used with single-patient cohorts with 100% dose escalations until NCICTCAE toxicity ≥ Grade 2 was encountered. Eligibility included adult patients with adequate bone marrow, hepatic and renal function. Patients with significant cardiovascular or ophthalmological disease and any conditions that might interfere with oral absorption were excluded. In addition to standard safety and PK assessments, specialist ophthalmological examinations and 24-hour Holter monitoring for QTc assessments were required. Blood, serum and tumor tissue (archival and/or fresh) for pharmacodynamic assessments, including mRNA expression profiles, were obtained. Twenty patients were enrolled across 4 dose levels, 60 mg/m2 (n=7), 80 mg/m2 (n=3), 120 mg/m2 (n=6), and 240 mg/m2 (n=4). DLTs of reversible corneal edema and iritis, believed to be a consequence of disrupted tear film lipid metabolism, were observed in two patients at 240 mg/m2, leading to further exploration of lower dose levels. Other toxicities have included palmar-plantar erythrodysaesthesia and/or peeling with minor GI symptoms and alopecia. No QTc prolongation has been observed to date. One patient had a paradoxical increase in triglycerides. TVB-2640 has shown excellent oral bioavailability PK in man; plasma exposure generally increases with increasing dose. A preliminary mean halflife of approximately 16 hrs at steady state has been observed, consistent with once a day administration. mRNA changes in whole blood are consistent with target engagement. In addition, key FASN-dependent signaling pathways were inhibited in the tumor tissue of one patient following dosing. Additional IHC and gene expression analysis of pre and post-dose tumor biopsies is ongoing. In this ongoing first in human trial we defined the PK and safety profile of the novel FASN inhibitor TVB-2640. Human exposures at all dose levels exceed those predicted to be efficacious in mouse preclinical models. 120 mg/m2 exceeds the MTD and the recommended continuous daily dose for future studies remains under study. Combination with chemotherapeutic agents is being examined. Poster Section 25 Poster Board 4 CT204 Preliminary results from PiSARRO, a phase Ib/II study of APR-246, a mutant p53 reactivating small molecule, in 16 combination with standard chemotherapy in platinum-sensitive ovarian cancer. Mikael von Euler,1 Klas G. Wiman,2 Hani Gabra,3 James D. Brenton,4 Bristi Basu,4 Ignace Vergote,5 Charlie Gourley,6 Austin Smith,7 Jessica Alfredsson,1 Nina Mohell,1 John A. Green8. 1Aprea, Solna, Sweden; 2Karolinska Institutet, Stockholm, Sweden; 3Imperial College London, London, United Kingdom; 4University of Cambridge, Cambridge, United Kingdom; 5University of Leuven, Leuven, Belgium; 6University of Edinburgh, Edinburgh, United Kingdom; 7Theradex Ltd., Crawley, United Kingdom; 8University of Liverpool, Liverpool, United Kingdom. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53. This phase Ib part of a proof of concept study aims to determine the recommended phase II dose (RP2D) of APR-246 in combination with carboplatin and pegylated liposomal doxorubicin (PLD) in platinum sensitive High Grade Serous Ovarian Cancer (HGSOC). Despite high response rates from carboplatin in combination with paclitaxel in first-line treatment of ovarian cancer, most patients relapse and develop resistance. Partially platinum sensitive patients relapse between 6 and 24 months and are commonly treated with second-line carboplatin and PLD (Pujade-Lauraine et al. JCO, 2010). The mechanisms of platinum resistance are multifactorial; two of the main causes are mutations in p53 and increased levels of intracellular glutathione. Like the analog PRIMA-1, APR-246 is a pro-drug that is converted to the active form MQ, which restores wild type conformation to mutant p53 (Lambert et al. Cancer Cell, 2009). In addition, APR-246 has been shown in vitro to reduce glutathione levels, resensitize cancer cells to platinum drugs, and induce ROS levels and ER stress (Mohell et al. Abstract #1801, AACR 2014; Lambert et al. Oncogene, 2010). In the first-in-human phase Ia study, APR-246 monotherapy was found to have a satisfactory safety and pharmacokinetic profile allowing it to be combined with full dose chemotherapy (Lehmann et al., JCO, 2012). The ongoing phase Ib/II study is enrolling patients with recurrent platinum sensitive HGSOC with positive p53 staining on immunohistochemistry. The phase Ib study has a 3+3 dose escalation design with 3 planned dose levels. APR-246 is administered as a 6h i.v. infusion on 4 consecutive days every 4 weeks. On day 4, APR-246 is given concomitantly with carboplatin AUC 5 and PLD 30 mg/m2. In the phase II part, 164 patients will be randomized to standard chemotherapy with or without APR-246. To date patients have been enrolled to all 3 dose cohorts. One DLT of ruptured diverticulum occurred at the 2nd dose level. No new safety concerns have emerged. The pharmacokinetic profile has not indicated any interaction between APR-246 and the chemotherapy. The first 3 patients have completed their therapy and are now in follow up. All 3 had partial response (PR) by RECIST 1.1 and 2/2 evaluable also had PR by GCIC. In conclusion, early results from the ongoing clinical study are encouraging and support the continued development of APR-246 in the phase II part of the study comparing platinum based standard chemotherapy with or without APR-246 in patients with HGSOC with mutant p53. Preliminary results from all three dose levels and the RP2D will be presented at the meeting. Poster Section 25 Poster Board 5 CT205 Intravenous delivery of a novel oncolytic immunotherapy agent, CAVATAK, in advanced cancer patients. Hardev Pandha,1 Kevin Harrington,2 Cristy Ralph,3 Alan Melcher,4 Darren R. Shafren5. 1Royal Surrey Hospital, Surrey, United Kingdom; 2 Royal Marsden Hospital, London, United Kingdom; 3St James Hospital,, Leeds, United Kingdom; 4St James Hospital, Leeds, United Kingdom; 5Viralytics Limited, Sydney, Australia. Background: Coxsackievirus A21 (CVA21) is a naturally occurring “common cold” intercellular adhesion molecule-1 (ICAM1)-targeted RNA virus. Surface ICAM-1 is up-regulated on a number of cancers including melanoma, non-small cell lung, bladder and prostate cancers. CAVATAK is a novel bio-selected formulation of Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 17 Clinical Trials Poster Session: Clinical Trials in Progress CVA21, which displays potent oncolytic activity against in vitro cultures of cancer cells and in vivo xenografts of a number of cancers. In this phase I/II study advanced cancer patients received multiple intravenous (IV) doses of CAVATAK to assess treatment tolerance, levels of viral replication and viral-induced immune activation within the tumor micro-environment. Methods: The phase I/II STORM (Systemic Treatment Of Resistant Malignancies: NCT02043665) study is investigating the tolerance of multiple escalating IV doses of CVA21 in approximately 30 advanced cancer patients. In cohort 1 (n=3), patients were infused with CVA21 at a dose of 1 x 108 TCID50, in cohort 2 patients (n=3) were infused with CVA21 at a dose of 3 x 108 TCID50 and treatment of patients in Cohort 3 (n=12-18) with CVA21 at a dose of 1 x 109 TCID50 has commenced. Tumor biopsies at 8 days following the initial CVA21 infusion are being monitored for levels of virus and markers of potential immune activation. Sequential serum samples are being analyzed for viral loads, kinetics of anti-CVA21 neutralizing antibody (nAb) development and immune system activation via relative serum levels of a panel of immune inflammatory cytokines /immune cell subsets. Results: To date, multiple CVA21 infusions of patients in Cohorts 1 and 2 have been generally well tolerated. Preliminary data indicate that the prolonged presence of serum CVA21 RNA in some, but not all, patients at times (up to 4 days post-infusion), when complete decay of the administered viral dose was expected; this may indicate possible viral replication within tumor. Such replication in pre-clinical xenograft models was potentially immunogenic, as evidenced by gene expression increases of CXCL-10 and PD-L1. Of particular interest was the finding of comparable kinetics of anti-CVA21 nAb development in patients receiving multiple infusions relative to those administered a single CVA21 infusion during a previous phase I dose-ranging study (NCT00636558). The interim data highlight a robust “multi-dosingwindow” in the absence of significant levels of nAb for approximately 7 days post initial viral infusion. Conclusion: To date, multiple IV infusions in advanced cancer patients have been generally well tolerated. Initial serum viral load data indicate potential tumor-specific CVA21 replication in some patients. Overall, the preliminary data offer an exciting possibly that tumor targeting, infection and immune activation mediated by IV CVA21 may lead to increases in anti-tumor activity, particularly when in future used in combination with immune checkpoint blockade. Poster Section 25 Poster Board 6 CT206 PFK-158, first-in-man and first-in-class inhibitor of PFKFB3/ glycolysis: A phase I, dose escalation, multi-center study in patients with advanced solid malignancies. Rebecca Redman,1 Paula Pohlmann,2 Michael Kurman,3 Gilles H. Tapolsky,3 Jason Chesney1. 1Brown Cancer Center, University of Louisville, Louisville, KY; 2Lombardi Cancer Center, Georgetown University, Washington, DC; 3Advanced Cancer Therapeutics, Louisville, KY. Metabolic alterations in cancer have been recognized as important novel targets, especially glycolysis. Over-expression of HIF-1α, activation of Ras, AKT or PI3K, loss of p53 or PTEN are associated with the development of human cancers and converge on glycolysis by activating PFKFB3, a bifunctional enzyme. This enzyme interconverts fructose-6-phosphate (F6P) to fructose-2,6bisphosphate (F2,6BP) and F2,6BP is an allosteric activator of 6phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and control point in the glycolytic pathway. PFKFB3 is of particular interest since it controls the intracellular concentration of F2,6BP, is required for tumorigenic growth and it has been found to be activated in human cancer cell lines and tumors, to be increased by hypoxic exposure via HIF-1α and by several oncogenes and mutations. PFK-158 is a potent and selective inhibitor of PFKFB3 that is currently being investigated in a phase I study in patients with advanced solid malignancies. PFK-158 is: (i) is a nanomolar inhibitor of recombinant PFKFB3; (ii) inhibits PFKFB3 activity and glycolysis in cancer cells; (ii) is well tolerated in vivo; and (iv) is very effective in multiple preclinical mouse models of human-derived tumors and syngeneic murine models. IND-enabling safety and toxicity studies demonstrated that PFK158 is well tolerated in rats and dogs and supported the initiation of a phase I trial that is now underway. The primary objective of the study is to describe the dose limiting toxicity and to determine either the maximum tolerated dose or biological effective dose of PFK-158 in a “3+3” cohortbased dose escalation design that follows a modified Fibonacci scheme. The pharmacokinetic profile of PFK-158 will also be determined. Multiple secondary endpoints have been incorporated to assess the effects of PFK-158 on peripheral blood mononuclear cell (F6P; F2,6BP; 14C-2-dexoyglucose uptake) and on glucose uptake using FDG-PET imaging. This trial, opened at two US sites, is currently enrolling Cohort 3 (96 mg/m2). Prior cohorts (24 and 48 mg/m2) have been completed without dose-limiting toxicities or drug-related significant adverse events. Of the patients enrolled to date, a patient of Cohort 1 was on study for 6 cycles during which his overall hepatic metastatic tumor burden decreased. In addition, numerous publications demonstrated that overexpression of HIF-1α, activation of Ras, AKT or PI3K, loss of p53 or PTEN converge on glycolysis by activating PFKFB3. Results of preclinical studies show that combination of PFK-158 with targeted agents, which indirectly activate glycolysis, leads to clear therapeutic benefits. In conclusion, PFK158 is the first-in-man and first-in-class PFKFB3 inhibitor to be examined in a phase I trial and may have significant clinical utility either as a monotherapy or when combined with targeted agents. Poster Section 25 Poster Board 7 CT207 Phase I dose escalation and pharmokinetic study of 14O-phosphonooxymethyltriptolide. Edward Greeno,1 Erkut Borazanci,2 Jon Gockerman,3 Ronald Korn,4 Ashok Saluja,1 Daniel Von Hoff2. 1University of Minnesota, Minneapolis, MN; 2Scottsdale Healthcare, Scottsdale, AZ; 3Novella Clinical, Morristown, NC; 4Imaging Endpoints, Scottsdale, AZ. Introduction: We are conducting a phase I, first-in-human trial of 14-O-phosphonooxymethyltriptolide (Minnelide,) a water-soluble prodrug of triptolide, a diterpene derived from the thunder god vine (tripterygium wilfordii). Triptolide is a potent inhibitor of heat shock protein 70 (HSP70) and pancreatic ductal adenocarcinoma overexpresses HSP70 as a protective mechanism. We have previously shown Minnelide to be effective and well tolerated in preclinical models of pancreatic carcinoma. Methods: The study uses a 3+3 dose escalation scheme, enrolling subjects with gastrointestinal malignancies refractory to standard therapies. The drug is administered as a daily, brief IV infusion for 21 days out of a 28 day cycle. The primary endpoint is toxicity, with secondary endpoints of pharmokinetics and response. Results: To date we have enrolled 27 subjects (17 pancreas, 7 colorectal, 3 other GI) at doses from 0.16 to 0.8 mg/m2, with 24 evaluable for toxicity. The therapy has been generally well tolerated with the only common toxicity being hematologic, but one patient experienced reversible cerebellar dysfunction at the highest dose. Pharmokinetics (n=21) indicate rapid conversion of Minnelide to triptolide, with all Minnelide cleared within 30 minutes and peak triptolide levels achieved within 5 minutes of completion of infusion. Triptolide was rapidly cleared with a half-life of less than 30 minutes, and complete clearance by 6 hours, except in the patient with cerebellar toxicity who demonstrated delayed clearance of the drug. All subjects, except those in the lowest dose cohort, have had a reduction in HSP70 levels. The 9 patients in the first 3 dose cohorts all progressed by the end of cycle two. Tumor response by PET after cycle 1 (n=19), shows a partial metabolic response in 36%, and stable metabolic disease in 52%. Response after 2 cycles by RECIST criteria (n=10) shows a 10% PR (gastric) and American Association for Cancer Research • AACR ANNUAL MEETING 2015 17 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 18 Clinical Trials Poster Session: Clinical Trials in Progress 60% SD (5 pancreas, 1 rectal) rate with disease control for up to 6 months. 5 patients remain on study, with ongoing enrollment planned. Conclusions: We have seen promising evidence of significant clinical activity in this group of heavily pretreated patients with refractory GI malignancies. The toxicity profile and optimal dosing of Minnelide are being defined. Poster Section 25 Poster Board 8 CT208 Preliminary results of a phase II trial of an adenovirus/PSA vaccine in men with recurrent prostate cancer. David M. Lubaroff, Daniel Vaena, James Brown, Kenneth Nepple, Pamela Zehr, Karen Griffith, Erica Brown, Julie Eastman, Gideon Zamba, Richard Williams. University of Iowa, Iowa City, IA. Introduction and Objectives: A phase II clinical trial of an adenovirus/PSA (Ad/PSA) vaccine was carried out in men with recurrent prostate cancer using two protocols. The objectives of the study were to determine whether the vaccine produced beneficial immune and clinical responses in men with a low burden of tumor. Methods: In Protocol #1 men with recurrent prostate cancer following definitive initial treatment for their disease were placed in one of two arms: Arm A; men received the vaccine alone at days 0, 30, and 60; Arm B; men received the vaccine 14 days after the initiation of androgen deprivation therapy (ADT). In Protocol #2 men with hormone refractory disease received the vaccine alone using the same 3 injection schedule. Each injection consists of 108 pfu of the Ad/PSA vaccine suspended in a collagen matrix. All patients returned at regular intervals for physical, chemical, radiologic, and immunologic evaluations. Results: We enrolled all of our targeted 82 patients, 50 in protocol 1 (25 Arm A, 25 Arm B) and 32 in protocol 2. All patients have been followed a minimum of 12 months with the longest being over 6 years. Eighty nine percent (89%) of all patients produced anti-PSA T cell immune responses; 71% in protocol 1 Arm A, 100% in protocol 1 Arm B, and 100% in protocol 2. Clinically, 58% of patients in protocol 1 Arm A and 75% of patients in protocol 2 demonstrated a decrease in serum PSA or an increase in PSA doubling times (PSADT). As of the submission date the PSA levels of 2 patients declined to 0.07 ng/ml and undetectable (<0.03). Conclusions: Preliminary results of patients thus far studied in a phase II clinical trial of the Ad/PSA vaccine are very encouraging, demonstrating both immunologic and clinical responses to the 3vaccination therapy. PSA responses indicate at least a decline in the growth and perhaps a destruction of recurrent prostate tumors in men immunized in two separate protocols. Data collection continues that include completion of immune assays on all 82 patients as well as monitoring PSA levels, PSADT, and radiologic changes. Poster Section 25 Poster Board 9 CT209 Correlation of robust local reactions prompting GMCSF dose reduction to clinical response in a phase II trial of the AE37+GM-CSF HER2 peptide vaccine. Julia M. Greene,1 Erika J. Schneble,1 Jennifer K. Litton,2 Jonathon Martin,1 Alfred F. Trappey,1 John S. Berry,1 Timothy J. Vreeland,1 Diane F. Hale,1 Guy T. Clifton,2 Alexandros Ardavannis,3 Michael Papamichail,3 Sonia Perez,3 Sathibalan Ponniah,4 Elizabeth A. Mittendorf,2 George E. Peoples5. 1San Antonio Military Medical Center, San Antonio, TX; 2MD Anderson Cancer Center, San Antonio, TX; 3Cancer Immunology Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece; 4Uniformed Services University of the Health Sciences, Bethesda, MD; 5Cancer Vaccine Development Program, San Antonio, TX. Background: We are conducting a phase II clinical trial of the HER2 peptide vaccine AE37+GM-CSF for prevention of breast cancer recurrence in disease-free, node-positive or high-risk nodenegative patients (pts), who have completed standard of care therapy. AE37, an Ii-Key hybrid of the HER2-derived peptide AE36 (aa:776-790), is an MHC Class II epitope capable of stimulating 18 tumor reactive CD4+ helper T-cells. Previous investigation of intradermal inoculation with E75 (HER2 aa:369-377) +GM-CSF vaccine in phase I/II trials showed no recurrences at 24 months among the 18% of pts requiring a GM-CSF dose reduction due to robust local reactions (LR). Phase I/II studies have shown the safety, immunogenicity, and potential clinical benefit of AE37 + GM-CSF. Here, we examine the relationship between GM-CSF dose reduction and clinical recurrence rate (RR) in pts receiving AE37+GM-CSF. Methods: Patients with any level of HER2 expression (IHC1-3+) were enrolled and randomized to receive 6 monthly intradermal inoculations of AE37+GM-CSF or GM-CSF alone (controls) during the primary vaccination series, then four booster vaccinations administered every 6 months. Enrollment has been completed and pts continue with their vaccination series. LRs are measured after each inoculation, and pts with a LR measuring ≥ 100 x 100mm have their GM-CSF dose reduced on subsequent inoculations. Local and systemic toxicity are being monitored and clinical recurrences documented. Proportional RR are compared using a chi-square or Fisher exact test as appropriate. Results: Of 301 enrolled pts, 154 were randomized to the vaccinated arm (VG) and 147 randomized to the control arm (CG). The groups are well-matched for clinicopathologic characteristics. Toxicities have been almost exclusively grade 1 and 2. Study-wide, 18.9% of pts required dose reduction (CG 15.6%, VG 22.1%; p=0.19). In vaccinated pts, the RR in the dose reduced group (DR) was 5.9% (2/34) versus the non-dose reduced group (NDR) RR of 14.2% (17/120) (p=0.25). Among controls, the DR RR was 17.4% (4/23) vs the NDR RR of 12.9% (16/124) (p=0.74). Comparing all DR pts, the relative risk of recurrence was reduced by 66% in the VG DR compared to the CG DR (5.9% versus 17.4%; p=0.21). Conclusions: In a randomized phase II trial of the AE37+GMCSF vaccine, pts who required a dose reduction of GM-CSF due to robust LR trend toward a lower recurrence rate. The overall dose reduction rate is similar to that in E75 trials (18.9% vs 17.9%) and occurred more frequently in the VG than CG. While dose reduction is seen in both the VG and the CG, the clinical benefit is only seen in the VG suggesting that while the LR may be a result of the GMCSF, the specificity provided by the peptide is required for clinical benefit. Furthermore, these data suggest that GM-CSF should be dosed to produce large LR to enhance the benefit of peptide vaccines in the adjuvant setting. Poster Section 25 Poster Board 10 CT210 Precision medicine for advanced pancreas cancer: the individualized molecular pancreatic cancer therapy (IMPaCT) trial. Lorraine Chantrill,1 Skye Simpson,1 Amber Johns,1 Mona MartynSmith,1 Angela Chou,1 Clare Watson,1 Adnan Nagrial,1 Venessa Chin,1 Lucille Sebastian,2 Sonia Yip,2 John Simes,2 Nick Pavlakis,3 Peter Grimison,4 Ray Asghari,5 Sandra Harvey,5 Andrew Biankin1. 1 Garvan Institute of Medical Research, Sydney, Australia; 2Clinical Trials Centre, University of Sydney, Sydney, Australia; 3Northern Cancer Institute, Sydney, Australia; 4Chris OBrien Lifehouse at RPA Cancer Centre, Sydney, Australia; 5Bankstown and Lidcombe Hospital, Sydney, Australia. Background: The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial is designed to exploit results from whole genome sequencing of pancreatic cancer collected under the auspices of the ICGC in Australia. Results showed that small subsets of patients had actionable changes in their tumor genome that could be druggable with currently available therapies. Only 7% of cases were found to be KRAS wildtype, and this phenotype may enrich for susceptibility to EGFR inhibition. Her2 positivity occurs in 2% and may confer sensitivity to Her inhibition. Tumors displaying defects in the DNA damage repair pathway (~5%) respond to DNA damaging chemotherapy. Trial Design: The IMPaCT trial has recently been amended to a single arm pilot study of first line molecularly guided therapy for Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 19 Clinical Trials Poster Session: Clinical Trials in Progress advanced pancreas cancer. Patients are permitted to begin their first cycle of chemotherapy with gemcitabine with or without nabpaclitaxel while awaiting molecular results. We screen potential patients for the three molecular targets: Her2 amplification: trastuzumab + gemcitabine; KRAS wildtype: erlotinib + gemcitabine; and DNA damage: platinum-based chemotherapy. In our initial cohort of patients who underwent resection with curative intent, 70% recurred. Recurrence occurred 16m after initial surgery. Collection of tissue commenced in 2009. The first site to open was in April 2013 by which time, only 8 patients for whom we had complete sequence data and actionable mutations were still alive, so we changed the trial to screen de novo metastatic patients. Using the WGS data, we constructed a custom sequencing panel to use DNA extracted from FFPE core biopsies to screen in real time for mutations in KRAS, BRCA2, BRCA1, PALB2 and ATM. Her2 screening is undertaken with IHC and FISH. We have screened 89 cases in 18m, 8 have relevant molecular targets. The average time from biopsy to delivery of results is 21d. 2 of the 8 eligible cases have commenced precision therapy on trial. Poster Section 25 Poster Board 11 CT211 Biomarker search using gene expression databases in a phase III controlled clinical trial of postoperative adjuvant chemotherapy for stage III colon cancer (ACTS-CC): correlation between clinicopathological factors and gene expression level. Hiroyuki Uetake,1 Toshiaki Ishikawa,1 Megimi Ishiguro,1 Shigeyuki Matsui2. 1Tokyo Medical & Dental Univ., Tokyo, Japan; 2Nagoya University, Nagoya, Japan. Background: The ACTS-CC trial is a randomized, controlled phase III study designed to validate the noninferiority of S-1 to UFT/leucovorin as adjuvant chemotherapy for stage III colon cancer and rectosigmoid cancer. A prospective biomarker search was performed as an auxiliary study of the ACTS-CC trial, using formalin-fixed, paraffin-embedded (FFPE) specimens obtained from 892 patients. The gene expression levels of 5-FU metabolizing enzymes and folate metabolizing enzymes and alterations of genome-wide copy numbers in tumor have been reported (2012, 2013 and 2014 Annual Meetings of AACR). In the present study, we performed correlation analyses of clinicopathological feature and gene expression level. Methods: Blocks of resected tumor specimens for this biomarker study were obtained after receiving informed consent from 892 of 1535 patients enrolled between April 2009 and January 2010. Macro-dissections were manually performed to extract total RNA and genomic DNA from 10-μm-thick FFPE specimens of colon cancer. Gene expression levels of 11 enzymes (TS, DPD, TP, OPRT, FPGS, GGH, DHFR, MTHFR, MTHFD, FOLRA, GART) related to 5FU and folic acid metabolism were studied according to the Danenberg Tumor ProfileTM method (Shirota, Y. JCO 2001). Results: Gene expression levels were successfully estimated in 521-808 samples (84.0-90.0%, median 89.4%). Among 11 genes, GGH most strongly correlated with tumor locations: expression level of GGH in the distal colon was 1.8 times higher than those in the proximal colon. Gene expressions of DPD and GGH were significantly influenced by differentiation of tumor. DPD and GGH mRNA levels in poorly differentiated tumors were 2.0 or 2.5 times higher than those in well differentiated tumors, respectively. Likewise, TS most strongly correlated with tumor depth: expression level in T1 or T2 tumor was 1.3 times higher than those in T4 tumor. Conclusions: Location, differentiation and depth of the tumor correlated with each gene expressions. Our findings will facilitate understanding the characteristics of colon cancer. Further investigation on gene expressions including genome-wide copy number analysis may contribute to the exploration of valid biomarkers. Poster Section 25 Poster Board 12 CT212 CHRONOS-2: A randomized, double-blind phase III study of phosphatidylinositol-3 kinase alpha/delta inhibitor copanlisib versus placebo in patients with rituximab-refractory indolent non-Hodgkin’s lymphoma (iNHL). Grzegorz S. Nowakowski,1 Igor Gorbatchevsky,2 Florian Hiemeyer,3 Lisa Cupit,2 Barrett H. Childs2. 1Mayo Clinic, Rochester, MN; 2Bayer HealthCare Pharmaceuticals, Whippany, NJ; 3Bayer Pharma AG, Berlin, Germany. Background: Copanlisib is a novel pan-Class I phosphatidylinositol-3-kinase (PI3K) inhibitor with potent preclinical inhibitory activity against both PI3K-δ and PI3K-α isoforms. A phase II study of copanlisib in patients with relapsed/refractory indolent or aggressive lymphoma reported a promising overall response rate of up to 53% for patients in the indolent NHL group (Dreyling et al., ENA 2014). The objective of this study is to evaluate the efficacy and safety of copanlisib in patients with indolent B-cell NHL relapsed after or refractory to standard therapy. Methods: In this study, patients meeting the following criteria will be eligible for enrollment: histologically confirmed diagnosis of indolent B-cell NHL, with follicular lymphoma (FL) grade 1-2-3a, marginal zone lymphoma (MZL; splenic, nodal, or extra-nodal), small lymphocytic lymphoma (SLL) with absolute lymphocyte count < 5 x 109/L at the time of diagnosis and at study entry, or lymphoplasmacytoid lymphoma/Waldenström macroglobulinemia (LPL/WM), and who have previously received ≥ 2 prior lines of therapy with rituximab and an alkylating agent and must be refractory to the last treatment with rituximab (refractory defined as not responding or progressing within 6 months of the last course of treatment). Patients will be randomized in 2:1 manner to receive 60 mg of copanlisib administered intravenously on days 1, 8 and 15 of a 28-day cycle or placebo administered on the same schedule. Patients will be treated until disease progression or intolerable toxicity. Patients in the placebo arm will be allowed to cross-over to receive follow-up treatment with copanlisib, after confirmed disease progression. Dose reductions due to toxicities to 45 mg and 30 mg will be allowed. Radiologic tumor assessment will be performed every 12, 16 or 24 weeks for years 1, 2, and 3, respectively. The primary endpoint will be progression-free survival (PFS). Secondary objectives include overall objective response rate (ORR), duration of response (DOR), time to progression (TTP), complete response rate (CRR), overall survival (OS), time to deterioration as well as time to improvement in disease-related symptoms. Exploratory objectives will include secondary PFS after first progression in placebo-treated patients who cross over to receive copanlisib and time to improvement and the time to deterioration in disease-related symptoms-physical (DRS-P) of at least 3 points as measured by the FLymSI-18 questionnaire. Approximately 189 patients who meet the eligibility criteria will be randomized 2:1, with approximately 126 patients in the copanlisib monotherapy arm and approximately 63 patients in placebo arm. The study is planned to detect a 132% increase in median PFS in copanlisib versus placebo (i.e. to detect a hazard ratio of 0.43), using a stratified log-rank test. Poster Section 25 Poster Board 13 CT213 Clinical effects of a ketogenic diet on brain tumor patients: tumor growth and quality of life. Leonora Renda,1 Norissa Honea,2 Christopher Dardis,2 Lynn S. Ashby,2 Adrienne C. Scheck2. 1University of Arizona Cancer Center, SJHMC, Phoenix, AZ; 2Barrow Neurological Institute, Phoenix, AZ. Glioblastoma (GB) is the most malignant brain cancer, with few patients surviving beyond 2 years despite treatment including surgical resection, radiation, chemotherapy and new experimental therapies. Improvements in survival require the design of novel therapies that take advantage of phenotypic traits common in tumor cells. One such trait is aberrant metabolism - tumors rely heavily on glucose and glutamine as energy sources and are unable American Association for Cancer Research • AACR ANNUAL MEETING 2015 19 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 20 Clinical Trials Poster Session: Clinical Trials in Progress to use other sources of energy. We and others have suggested the exploitation of this through the use of the ketogenic diet (KD), a high fat, low carbohydrate and protein diet (4:1 fat:protein plus carbohydrate) that causes the body to shift metabolically from glucose to ketones as the primary source of energy. In addition to reducing available glucose, in vitro and in vivo preclinical studies have shown that increasing ketones appears to have additional anti-tumor effects, even in the presence of normal glucose levels. We and others have shown that the KD improves survival in mouse models of malignant brain tumors, and we have shown that it potentiates the effects of radiation and chemotherapy in vitro and in vivo. Anecdotal evidence and published case reports suggest that it may also have efficacy in human glioma patients, thus warranting further investigation in a clinical setting. Here, we report results to date from our ongoing, phase I/II single-arm clinical trial of this intervention (clinicaltrials.gov NCT02046187). Patients are eligible to enroll if they have newly diagnosed GB and at least a subtotal surgical resection. The KD is used in addition to standard care (radiation and temozolomide). It is already known to be safe and effective in the treatment of refractory epilepsy. Patients follow a 4:1 ketogenic diet during concurrent radiation and chemotherapy; they then change to a more moderate 1:1 diet during adjuvant chemotherapy. Blood glucose and ketone measurements are recorded daily, with target levels of glucose at ~70ml/dl and ketones at ~4mmol/l. To date, our preliminary study suggests that the KD is generally well tolerated and steroid use does not preclude the patient’s ability to reduce glucose levels below 80ml/ml. Overall health and therapyrelated quality of life measurements decline from baseline to the end of RT as is typical for patients undergoing radiation therapy, but appears to recover back to baseline over time. This suggests that following the ketogenic diet probably does not adversely affect long-term quality of life. Additional patient followup data will be presented. Poster Section 25 Poster Board 14 CT214 Phase II CALM study: Changes in the tumor microenvironment induced by the immunotherapeutic agent coxsackievirus A21 delivered intratumorally in patients with advanced melanoma. Robert Andtbacka,1 Brendan Curti,2 Sigrun Hallmeyer,3 Darren R. Shafren4. 1Huntsman Cancer Institute, Salt Lake City, UT; 2 Providence Cancer Center, Portland, OR; 3Oncology Specialists S.C, Chicago, IL; 4Viralytics Limited, New Lambton, Australia. Background: CAVATAK, an oncolytic immunotherapy, is a bioselected oncolytic strain of coxsackievirus A21 (CVA21). Following intratumoral (IT) injection, CVA21 preferentially infects ICAM-1 expressing tumor cells, resulting in viral replication, cell lysis, and a systemic antitumor immune response. The phase II CALM study investigated the efficacy and safety of IT CVA21 in pts with advanced melanoma. The primary endpoint of the study was achieved with 22 of 57 (38.6%) evaluable pts displaying immunerelated PFS (irPFS) at 6 months. Preliminary analysis of secondary endpoints showed: median irPFS of 4.2 months (95% CI 2.8, 8.3), 1-year survival 75.0% (36 of 48 pts), on-going best objective response rate of 28.1% (16 of 57 pts), median time to response 2.8 months. Responses were observed in both injected and noninjected melanoma metastases. Here we report on a continuation study aimed at understanding the immune mediated effects of CVA21. Methods: To further elucidate the nature of the systemic antitumor responses, a CALM study extension cohort of 12 pts will receive up to 3 x 108 TCID50 CVA21 IT on study days 1,3,5 and 8 and then every three weeks for a further 6 injections. Sequential tumor biopsies of both injected and non-injected lesions will be monitored for levels of viral replication and evidence of viralinduced immune activation within the tumor micro-environment. Serial serum samples are being monitored for viral loads, antiCVA21 neutralizing antibody (nAb) and levels of immuneinflammatory cytokines. 20 Results: Active tumor-specific cytolytic viral replication is postulated to contribute to the generation of systemic antitumor responses. Normal kinetics of CVA21 decay would lead to complete viral clearance from the circulation around 24-30 hrs post-viral administration. Preliminary serum testing of CALM study pts for CVA21 load by viral-RNA RT-PCR revealed that at 48 hrs post-IT injection on treatment days 1 and 3, 40 % and 42 % of pts respectively possessed circulating CVA21, indicating possible tumor-specific cytolytic viral replication and detection of progeny virus. Detection of persistent serum CVA21 levels reduced to approx. 14% of pts at study day 8, a time where significant levels of anti-CVA21 nAb started to develop. Elevated levels of the inflammatory cytokines γ-IFN and IL-8 in the serum of some of these pts at similar time points provides further evidence for active viral infection and potential immune activation. Conclusions: Preliminary data indicate possible cytolytic-tumor specific CVA21 replication in a number of treated patients. Serial biopsies of CVA21-injected and non-injected lesions of advanced melanoma pts are being examined to confirm the nature of IT viral replication and its direct impact on immune activation in the tumor micro-environment and generation of potential systemic antitumor immune responses. Poster Section 25 Poster Board 15 CT215 CHRONOS-1: Open-label, uncontrolled phase II trial of intravenous phosphatidylinositol-3 kinase alpha/delta inhibitor copanlisib in patients with relapsed, indolent Non-Hodgkin’s lymphomas (iNHL). Martin Dreyling,1 Marius Giurescu,2 Julia Grunert,3 Felipe Fittipaldi,4 Lisa Cupit,5 Barrett H. Childs5. 1Klinikum der Universität MünchenGrosshadern, Munich, Germany; 2Bayer Pharma AG, Berlin, Germany; 3Bayer Pharma AG, Wuppertal, Germany; 4Bayer S.A., Sao Paulo, Brazil; 5Bayer HealthCare Pharmaceuticals, Whippany, NJ. Background: Copanlisib is a novel pan-Class I phosphatidylinositol-3-kinase (PI3K) inhibitor with potent preclinical inhibitory activity against both PI3K-δ and PI3K-α isoforms. Results from a phase II study of copanlisib in 67 patients with relapsed/refractory indolent or aggressive lymphoma have been reported, with a promising overall response rate for 53% seen for patients in the indolent lymphoma group (Dreyling et al., ASH 2014; Dreyling et al., ENA 2014). Enrollment in an expansion cohort of 120 patients with indolent lymphoma has been initiated. The objective of the study is to evaluate the efficacy and safety of copanlisib in patients with indolent B-cell NHL relapsed after or refractory to standard therapy. Methods: In this study (NCT01660451), patients meeting the following criteria will be eligible for enrollment: histologically confirmed diagnosis of indolent B-cell NHL, with follicular lymphoma (FL) grade 1-2-3a, marginal zone lymphoma (MZL; splenic, nodal, or extra-nodal), small lymphocytic lymphoma (SLL) with absolute lymphocyte count < 5 x 109/L at the time of diagnosis and at study entry, or lymphoplasmacytoid lymphoma/Waldenström macroglobulinemia (LPL/WM), and who have relapsed or are refractory after ≥ 2 prior lines of therapy (refractory defined as not responding to a standard regimen or progressing within 6 months of the last course of a standard regimen; patients must have received Rituximab and alkylating agents). Patients will receive 60 mg of copanlisib administered intravenously on days 1, 8 and 15 of a 28day cycle. Dose reductions due to toxicities to 45 mg and 30 mg will be allowed. Patients will be followed until disease progression or intolerable toxicity. Radiologic tumor assessment will be performed every 2 cycles. Adverse events will be collected and graded using NCI-CTCAE v4. The primary endpoint will be overall objective response rate (ORR), defined as a complete response (CR) or partial response (PR) up to 16 weeks after the last patient fully evaluable for the primary endpoint started treatment. Secondary objectives include duration of response (DOR), progression-free survival (PFS), overall survival (OS), and quality of Life questionnaire (FACT-Lym symptoms subscale and total score) Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 21 Clinical Trials Poster Session: Clinical Trials in Progress at week 16. Assuming a one-sided alpha of 0.025, 90% power and a true ORR of 75%, a total of 120 patients will be required. The trial is currently enrolling patients. Poster Section 25 Poster Board 16 CT216 Phase I/IIa non-randomized open-label trials with mouse renal adenocarcinoma (RENCA) cell containing agaroseagarose macrobeads in patients with treatment-resistant metastatic colorectal carcinoma. Allyson J. Ocean,1 Nathaniel Berman,2 Tapan Parikh,2 Zoe P. Andrada,2 Angelica Nazarian,2 Joanne Thomas,2 Eugene Akahoho,2 George Stoms,3 Alex Yaroshinsky,3 Thomas J. Fahey,4 David J. Wolf,2 Lawrence S. Gazda,5 Barry H. Smith2. 1Weill Cornell Medical College, New York, NY; 2The Rogosin Institute, New York, NY; 3Vital Systems, Rolling Meadows, IL; 4NewYork-Presbyterian Hospital, New York, NY; 5The Rogosin Institute-Xenia Division, Xenia, OH. Background: The purpose of phase I/IIa trials of RENCA tumor cells encapsulated in two concentric agarose layers is to evaluate safety and efficacy of agarose-agarose MB in various cancers, particularly advanced colorectal cancer (CRC) as reported in this abstract. MB release signals that inhibit tumor growth at primary or metastatic tumor locations. This novel approach has been substantiated in in vitro and in vivo models as reported in Cancer Research 71(3), 716-735, 2011. We report results of phase I/IIa studies in advanced metastatic CRC patients. Methods: CRC patients who have progressive disease after all available treatment modalities with ECOG PS of 0-2 were enrolled to the trial after informed consents were obtained. Patients underwent laparoscopic intraperitoneal implantations up to 4 times with 8 (43/46 patients- established dose) or 16 (3/46 patients) RENCA MB/kg. Serial clinical examinations, blood tests, and imaging were obtained before and 3 months (mo.) after implantations to assess safety and efficacy. Endpoints of safety as measured by description of adverse events, and efficacy as measured by tumor marker response, were noted. Results: 46 pts were implanted with RENCA MB (12 pts phase I; 34 pts phase II); of which 18 were males and 28 were females. Mean age was 58.2 years. 29/46 pts had a total of 1 implant (14 had a total of 2 implants; 1 had a total of 3 implants; 2 had a total of 4 implants). Response to MB is marked by a prominent initial rise in CRP, ESR, and IL-6, indicating a systemic inflammatory response (SIR) (100% of pts) and a parallel decrease in CEA and/or CA 19-9 in approximately 72%. SIR including its accompanying fatigue and anorexia lasts days to 3 wks. Overall, there was a significant difference in OS between the 72% of pts showing a decrease in tumor markers by at least 20% during the first 30 days post-implant (mean OS; 10.76 mo.) and those who did not (mean OS; 4.9 mo.). On PET-CT imaging, an important feature of response, most often seen in patients with the longest survival, was tumor necrosis. MB were well-tolerated. No Grade ≥3 adverse events were determined to be treatment-related. Conclusions: In reported phase I/IIa trials in advanced, metastatic, treatment-resistant CRC patients, response to RENCA MB after implantation was characterized by decrease in tumor markers in association with SIR. This response was correlated with a significant increase in OS. RENCA MB represent a possible new therapeutic option for advanced, metastatic CRC. Poster Section 25 Poster Board 17 CT217 Phase I/II study of dianhydrogalactitol in patients with recurrent malignant glioblastoma multiforme. Kent C. Shih,1 Manish R. Patel,2 Nicholas Butowski,3 Jeffrey A. Bacha,4 Dennis M. Brown,4 Anne Steino,4 Richard Schwartz,4 Sarath Kanekal,4 Lorena M. Lopez,4 Howard A. Burris1. 1Sarah Cannon Research Institute, Nashville, TN; 2Florida Cancer Specialists and Research Institute, Sarasota Cattlemen, FL; 3University of California, San Francisco, Department of Neurological Surgery, San Francisco, CA; 4DelMar pharmaceuticals Inc, Vancouver, British Columbia, Canada. Glioblastoma multiforme (GBM) is the most common and deadly form of human brain cancer. Median survival for patients with recurrent GBM is <6 months. Front-line systemic therapy is temozolomide, but resistance due to O6-methylguanine-DNAmethyltransferase (MGMT) activity is implicated in poor prognoses. Dianhydrogalactitol (VAL-083) is a structurally unique bi-functional DNA alkylating agent that crosses the blood-brain barrier and accumulates in brain tumor tissue. In recent in vitro studies, VAL083 overcame resistance to MGMT and demonstrated cytotoxic activity against GBM cell lines, as well as GBM cancer stem cells, and was shown to act as a radiosensitizer. Previous clinical trials suggest that VAL-083 has activity against a range of tumors, including GBM. In light of extensive safety data and promising efficacy in CNS tumors, we initiated a new phase I/II clinical study to establish the maximum tolerated dose (MTD) using an optimized dosing scheme. The goal of the current clinical trial is to determine an appropriate dose for advancement into registration trials as a potential new therapy for the treatment of refractory GBM. Historical NCI-sponsored studies in GBM achieved promising results with limited toxicity using a dosing regimen of 25mg/m2/day for five days every five weeks . The present dosing regimen utilizes a daily dose for three days every three weeks. Seven cohorts have completed the current trial with no drug-related serious adverse events: MTD was not yet reached at 40mg/m2/day; 50mg/m2/day is being studied and higher doses may be explored. Compared to historical trials, the present regimen delivers substantively more drug as measured by Cmax and dose density. A dose density of 25 mg/m2/week in combination with radiation was previously shown superior to radiation alone against GBM; a dose density of 50 mg/m2/week is being enrolled in the current trial. Pharmacokinetic analyses show dose-dependent linear systemic exposure with a short plasma 1-2h terminal half-life; Cmax at 40mg/m2 in the current trial ranged from 1130-739ng/mL (7.7-5.1µM). Calculated CNS tissue concentrations, based on the plasma concentrations, exceed concentrations known to be effective against glioma cell lines in vitro. Methods: Open-label, single-arm phase I/II doseescalation study in patients with histologically-confirmed initial diagnosis of primary WHO Grade IV malignant glioma (glioblastoma). Patients enrolled have previously been treated with surgery and/or radiation, if appropriate, and must have failed both bevacizumab and temozolomide, unless contraindicated. The study utilizes a 3+3 dose-escalation design. Patients receive VAL-083 IV on days 1, 2, and 3 of a 21-day cycle. Tumor response is assessed according to RANO criteria prior to every other 21-day treatment cycle, and patients exhibiting stable disease or tumor regression are allowed to remain on study drug. ClinicalTrials.gov Identifier NCT01478178. Poster Section 25 Poster Board 18 CT218 Results from the early cancer clinical trials for 4demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOCPEN). Roy S. Weiner,1 P Friedlander,2 T Mahmood,3 Adilia Hormigo,2 C Gordon,4 Y Saenger,2 ML Ware,5 VK Thirukonda,6 VM Patel,7 TJ Cosgriff,7 AH Rodgers,8 LR Morgan,8 G Bastian9. 1Tulane University, New Orleans, LA; 2Mount Sinai Medical School Tisch Cancer Institute, New York, NY; 3Detroit Clinical Research Center, Lansing, MI; 4Detroit Clinical Research Center, Farmington Hills, MI; 5Ochsner Medical Center, New Orleans, LA; 6Billings Clinic, Billings, MT; 7 Crescent City Research Consortium, Marrero, LA; 8DEKK-TEC, Inc, New Orleans, LA; 9Service de Pharmacologie, Paris, France. Purpose: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN), is a poly-chlorinated pyridine cholesteryl carbonate whose MOA is via alkylation of DNA @ N7 - guanine and N6 - cytosine and via oxidative stress. DM-CHOC-PEN underwent a phase I study in patients with advanced cancer +/- CNS involvement and is being evaluated in a phase II trial in patients with primary brain cancer and brain metastases from melanoma, breast, and lung cancers. The aims are to assess clinical responses when American Association for Cancer Research • AACR ANNUAL MEETING 2015 21 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 22 Clinical Trials Poster Session: Clinical Trials in Progress DM-CHOC-PEN is administered I.V, at maximum tolerated dose (MTD) and to monitor safety/toxicities, pharmacokinetics, and cardiac functions - IND 68,876. Patients and Methods: In phase I, DM-CHOC-PEN was administered as a 3-hr IV infusion once every 21-days to patients with advanced cancer - melanoma (n=3), colorectal CA (n=4), breast (n=3), lung (n=8) and glioblastoma multiforme (GBM) (n=9) the most common tumor treated. Cohorts were treated with escalating doses from 39 to 111 mg/m2. The phase II dose schedule is 2-tiered: 85.8 mg/m2 for patients with liver involvement and 98.7 mg/m2 for patients with normal livers. Results: Forty (40) patients have been treated to date - 27 in phase I and 13 in phase II. The drug was well tolerated; the most common adverse effects were fatigue (n=2), liver dysfunction elevated bilirubin (Gr-3, n=3; Gr-2, n=1), ALT/AST (Gr-2, n=3), alk phos (Gr-2, n=3), nausea (Gr-1/2, n=5) and an allergic reaction (Gr2, n=1). Three (3) patients with liver metastasis had hyperbilirubinemia (Gr-3 SLT) - two (2) at 98.7 mg/m2 and one (1) at 111 mg/m2 levels. No neuro/psychological, hematological, cardiac or renal toxicities were observed. PK studies revealed the following profile for DM-CHOC-PEN 98.7 mg/m2: AUC o-t = 1850 mg.h/L, CL - 3.0 L/h, T1/2 α - 3.3 h & Tβ - 79.1 h. DM-CHOC PEN and DM-PEN (metabolite) showed a rebound phenomenon at ~50 hours postinfusion with a T release of 26.7 h for plasma and rbcs. DM-CHOCPEN and DM-PEN were detected 3 and 15 days bound to RBCs (70 - 111 mg/m2); DM-CHOC-PEN was also detected in the urine (Cmax=17.5 µg/mL) until day 21. The AUC was linear for all doses. DM-CHOC-PEN was detected in spinal sarcoma and in lung cancer tissues (75 & 190 ng/g, resp.) surgically obtained from patients 21days post single injection of 39 & 98.7 mg/m2, resp. Patients receiving dexamethasone demonstrated lower blood levels of DMCHOC-PEN along with induction of steroid esterase activities. After multiple doses, DM-CHOC-PEN also induced steroid esterase levels, which reversed within 4 weeks. Steroid esterase assays may be a valuable companion assay. Conclusion: DM-CHOC-PEN is safe at the presented dose levels and shows a favorable PK profile. To date, 15 patients have had responses with significant PFS/OS, including 10 with CNS involvement. DM-CHOC-PEN is well tolerated with manageable toxicities. Complete patient responses/toxicities will be presented. Supported by NCI/SBIR grant - R43/44CA132257 Poster Section 25 Poster Board 19 CT219 Clinical and immunological analysis in a phase II trial of the glypican-3 peptide vaccine for patients with ovarian clear cell carcinoma. Shiro Suzuki,1 Kiyosumi Shibata,1 Fumitaka Kikkawa,1 Tetsuya Nakatsura2. 1Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan. Compared with other epithelial ovarian carcinoma subtypes, ovarian clear cell carcinoma (OCCC) is associated with a poorer prognosis and increased chemoresistance. Therefore, new treatment modalities are urgently required for patients with OCCC refractory to chemotherapy. Glypican-3 (GPC3) is useful not only as a novel tumor marker, but also as an oncofetal antigen for immunotherapy. It is specifically overexpressed in hepatocellular carcinoma (HCC). Previous studies demonstrated that GPC3 was also overexpressed in several malignant tumors, including OCCC. We previously reported the safety of and immunological and clinical responses to a GPC3-derived peptide vaccine in a phase I clinical trial of patients with advanced HCC. Although the efficacy of the GPC3-derived peptide vaccine against HCC patients was evaluated, other GPC3-positive cancer patients have not yet been investigated. Therefore, we conducted a phase II trial to evaluate the clinical outcome of OCCC patients treated with a GPC3-derived peptide vaccine (UMIN-CTR: 000003696). In this study, we describe the effect of vaccination with the HLAA24 or A2-restricted GPC3 peptide on patients with OCCC. The 22 patients were divided into three groups such as adjuvant therapy group, combined therapy group (combined to second-line chemotherapy) and recurrence or advanced group (single of vaccine treatment). The dose of GPC3 peptide injected was 3 mg per body. Patients received the intradermal injection of GPC3 peptide emulsified with incomplete Freund’s adjuvant. Vaccinations were carried out biweekly from the first until the 6th and repeated at 6-week intervals after the 7th. Immunological responses were analyzed by ex vivo IFN-γ enzyme-linked immunospot assay (ELISPOT). Seventy OCCC patients were entered into clinical trial until the end of October 2014. In adjuvant group, twenty-nine of thirty-five patients have no recurrence. Recurrence pattern of all 6 patients was peritoneal metastasis. Three of twenty-nine patients with refractory OCCC achieved a significant clinical response. Ex vivo IFN-γ ELISPOT analysis in most OCCC patients revealed vaccineinduced immune reactivity against the GPC3 peptide. Our current data provide preliminary evidence of clinically meaningful benefit for GPC3 peptide vaccines in OCCC and support further evaluation of this approach in these patient populations. Poster Section 25 Poster Board 20 CT220 A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic cancer (NCT02305186). Matthew H.G Katz,1 Todd W. Bauer,2 Gauri Rajani Varadhachary,1 Reid B. Adams,2 Amy R. Lankford,2 Gina Petroni,2 Timothy N. Bullock,2 Craig L. Slingluff,2 Osama E. Rahma2. 1MD Anderson Cancer Center, Houston, TX; 2University of Virginia, Charlottesville, VA. Background: Immunotherapy has recently emerged as a promising modality in cancer treatment, but little is known about the application of this modality in pancreatic cancer (PC). Tumorinfiltrating lymphocytes (TILs) play a major role in anti-tumor immune responses, and their presence is correlated with survival in a variety of tumors. These TILs do not reach the PC cells in significant numbers due to the presence of stroma and a suppressive microenvironment. One of the leading causes for immune suppression is elevated expression of PD-L1 either by the tumor cells or the surrounding regulatory cells, resulting in dysfunction of TILs. Neoadjuvant chemoradiation therapy (CRT) has been advocated as a potential way to improve outcomes of patients with resectable or borderline resectable PC. More importantly, there is recent evidence to suggest that CRT can increase the presence of TILs in the PC microenvironment (PCME), leading to production of interferon-γ (IFN-γ), which could increase the expression of PDL1 through a negative feedback loop. Accordingly, we hypothesize that blocking the PD-1 receptor will synergize with CRT to increase the density and activation of TILs in the PCME. Methods: This is a prospective multicenter randomized trial which will accrue subjects with resectable or borderline resectable pancreatic cancer who had not received prior treatment for PC. The primary objectives of the study are: (1) to determine the safety of neoadjuvant CRT in combination with Pembrolizumab. (2) To estimate the difference in the number of TILs in pancreatic cancer subjects receiving neoadjuvant CRT in combination with Pembrolizumab to the number of TILs in subjects receiving neoadjuvant CRT alone. This study will also investigate the effect of CRT+/-anti-PD-1 on the other effector and suppressive immune cells and immune checkpoints in PCME. Eligible subjects will be randomized 2:1 to the investigational treatment (Arm A) to receive Pembrolizumab administered IV every 3 weeks on days 1, 22, and 43 during concurrent CRT with capecitabine (825 mg/m2 orally twice daily, Monday through Friday, on days of radiation only) and radiation (50.4 Gy in 28 fractions over 28 days) or Arm B to receive only concurrent CRT with capecitabine. In all subjects, restaging CT scan or MRI will be performed at 4-6 weeks after completion of Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 23 Clinical Trials Poster Session: Clinical Trials in Progress neoadjuvant treatment to determine resectability. Patients without local or distant disease progression will be taken to the operative room for planned surgery (within 2 weeks of imaging). Postoperatively, resected patients will receive off study standard of care adjuvant gemcitabine (1000mg/kg IV weekly for 3 out of 4 weeks for 6 months). Post operatively resected patients will be followed for up for PFS and OS for up to 2 years. Poster Section 25 Poster Board 21 CT221 CHRONOS-3: A phase III, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of phosphatidylinositol-3 kinase (PI3K) alpha/delta inhibitor copanlisib in combination with rituximab in patients with relapsed indolent B-cell non-Hodgkin’s lymphoma (iNHL). Pier Luigi Zinzani,1 John F. Gerecitano,2 Marius Giurescu,3 Rodrigo Ito,4 Katharina Mueller,5 Barrett H. Childs4. 1Institute of Hematology “Seràgnoli” University of Bologna, Bologna, Italy; 2Memorial SloanKettering Cancer Center, New York, NY; 3Bayer Pharma AG, Berlin, Germany; 4Bayer HealthCare Pharmaceuticals, Whippany, NJ; 5 Bayer Pharma AG, Wuppertal, Germany. Background: Copanlisib is a novel pan-Class I PI3K inhibitor with potent preclinical inhibitory activity against both PI3K-δ and PI3K-α isoforms. In a phase II study of copanlisib monotherapy in patients with relapsed/refractory indolent or aggressive lymphoma, an overall response rate of 53% was seen in patients with iNHL (Dreyling et al., ENA 2014). Rituximab in combination with chemotherapy is standard first-line therapy for indolent iNHL and is a therapeutic option for relapsed patients who cannot tolerate chemotherapy or who had a long response following the last rituximab-based therapy. The objective of this study is to evaluate the efficacy and safety of copanlisib in combination with rituximab versus placebo plus rituximab in patients with iNHL who relapsed after one or more lines of therapy, including rituximab and alkylating agents, and who are either unfit for chemotherapy or had a treatment-free interval of at least 12 months following last rituximab-based therapy. Methods: Patients must meet the following criteria: histologically confirmed diagnosis of iNHL, with follicular lymphoma (FL) grade 1-2-3a, marginal zone lymphoma (splenic, nodal, or extra-nodal), small lymphocytic lymphoma, or lymphoplasmacytoid lymphoma/Waldenström macroglobulinemia, and who have previously received at least one line of therapy including rituximab and alkylating agents. Patients must be not refractory to rituximab during any prior line of therapy (response <6 months). Approximately 567 patients (435 FL patients and 132 other iNHL patients) will be randomized 2:1 and stratified according to the four factors: NHL histology (FL vs. other iNHL), treatment-free interval after rituximab treatment vs. contraindication for chemotherapy, bulky disease (yes vs. no), and previous treatment with PI3K inhibitors (yes vs. no). Patients will receive 60 mg of copanlisib or placebo administered intravenously on days 1, 8 and 15 of a 28-day cycle in combination with 375 mg/m2 of rituximab administered on days 1, 8, 15 and 22. Radiologic tumor assessment will be performed every 8, 12, or 24 weeks for years 1, 2, and 3, respectively. The primary endpoint will be progression-free survival (PFS) as assessed by central review. Secondary objectives include objective tumor response rate, duration of response, complete response rate, time to progression, overall survival, time to improvement and the time to deterioration in disease-related symptoms - physical (DRS-P) of at least 3 points as measured by the FLymSI-18 questionnaire. The study is planned to detect a 50% increase in median PFS in copanlisib plus rituximab arm versus the placebo plus rituximab arm (hazard ratio of 0.6667) within the FL patients, using a stratified log-rank test. Poster Section 25 Poster Board 22 CT222 Ferumoxytol enhanced MRI for lymph node staging in genitourinary cancers. Anna M. Brown,1 Sandeep Sankineni,1 Marcelino Bernardo,1 Dagane Daar,1 Juanita Weaver,1 Yolanda McKinney,1 Anna Couvillon,2 James L. Gulley,3 Bradford J. Wood,4 Peter A. Pinto,5 William L. Dahut,3 Ravi Amrit Madan,3 Peter L. Choyke,1 Baris Turkbey1. 1Molecular Imaging Program, National Cancer Institute, NIH, Bethesda, MD; 2Genitourinary Malignancies Branch, National Cancer Institute at the National Institutes of Health, Bethesda, MD; 3 National Cancer Institute at the National Institutes of Health, Bethesda, MD; 4Center for Interventional Oncology, National Cancer Institute, NIH, Bethesda, MD; 5Urologic Oncology Branch, National Cancer Institute at the National Institutes of Health, Bethesda, MD. Background: Conventional imaging has limited accuracy in genitourinary (GU) cancer staging. This study examines the utility of ferumoxytol enhanced MRI in lymph node (LN) staging of GU cancers. Methods: This ongoing IRB-approved phase II clinical trial enrolls patients with prostate cancer, renal cell carcinoma, or bladder cancer at high risk for LN metastases. Patients undergo baseline T2 and T2* weighted MRI scans followed by injection of 7.5mg/Kg ferumoxytol. Repeat scans are acquired at 24hr and 48hr postinjection. The criterion for positive LNs was preservation of hyperintense signal indicating failure to take up ferumoxytol. Validation was by histopathology when available or on clinical grounds, for which LNs that changed size on routine imaging were considered true positives. Results: To date, 13 patients have completed the study. Of 11 prostate cancer patients, one was studied pre-operatively while 10 had suspected therapy failure. Median age and PSA were 65yrs (36-75) and 5.6ng/mL (0.3-201). The other 2 patients had renal cell carcinoma and bladder cancer. Overall, 20 LNs were identified with mean size 1.9cm (0.7-3.8) long axis by 1.3cm (0.6-2.6) short axis. There were 14 true positive LNs, 1 false positive, 1 false negative, and 4 nodes pending validation. Validation was by histopathology for 7 LNs, with 2 nodes pending biopsy, and clinical grounds for 13 LNs, with 2 inconclusive nodes awaiting further validation. Ferumoxytol correctly identified LN status in 9 of 10 patients with validated nodes (Table 1; see next page). Conclusions: Ferumoxytol enhanced MRI shows promise in detecting malignant LNs >6mm in GU cancer patients. Since the method involves a conventional MRI unit with off-label use of an FDA-approved agent, it could be widely available. However, further validation is necessary before routine use. Poster Section 25 Poster Board 23 CT223 CCX872: Pharmacodynamic study of a potent and selective CCR2 antagonist in human volunteers and plans for phase Ib trial in patients with pancreatic cancer. Anne-Marie Duliege, Stefan Sleijfer, Ashley Bischof, Joanne Tan, Penglie Zhang, Lisa Seitz, Daniel Dairaghi, Pirow Bekker, Israel Charo, Thomas Schall. ChemoCentryx, Mountain View, CA. Recent clinical results have highlighted the importance of myeloid-derived suppressor cells in enhancing the ability of the host immune system to limit the growth of tumors. Chemokine receptors control the directed trafficking and persistence of peripheral blood mononuclear cells, including effector and suppressor cells, to the tumor microenvironment. Myeloid derived suppressor cells in particular express high levels of the chemokine receptor CCR2, and pre-clinical data suggests that CCR2 plays an important role in the recruitment of these suppressor cells to tumors. Here we report pharmacodynamic details of a potent and selective small molecule antagonist of CCR2, CCX872, and our plans for a phase Ib trial in patients with pancreatic cancer. CCX872 has been successfully evaluated in a phase I single and multiple ascending dose study (3 to 300 mg) in 40 healthy volunteers. All dose levels were well tolerated and safe. CCX872-B American Association for Cancer Research • AACR ANNUAL MEETING 2015 23 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 24 Clinical Trials Poster Session: Clinical Trials in Progress RP=retroperitoneal, TP=true positive, FN=false negative, FP=false positive, ext=external, int=internal showed a dose-linear PK profile. Ex vivo CCR2 occupancy and internalization assays revealed that blood monocytes from CCX872, but not placebo-treated subjects, were impaired in their ability to bind to or internalize exogenously added CCL2, indicating that CCX872 blocked CCR2 in the treated subjects. The 300 mg daily dose of CCX872 exhibited 104 ± 3% blockade at 2 hours and 93 ± 7% blockade at 24 hours. We calculated that 150 mg twice a day would provide over 90% CCR2 inhibition at all times. Analysis of data from the Tumor Cancer Genome Atlas revealed high levels of expression of CCL2 in pancreatic tumors, and coexpression of CCR2 with monocyte/macrophage markers such as CD68 and CSF1R. In pre-clinical studies, we found that a CCR2 24 antagonist reduced the growth of the pancreatic tumor cell line, PancO2, in rodent xenograft models. Based in part on these data we have initiated a phase Ib single arm, open label, multicenter study of CCX872 in patients with un-resectable pancreatic cancer. In Part A, subjects will receive a single dose of CCX872 for pharmacokinetic and pharmacodynamics analyses. In Part B, up to 50 subjects will be treated for at least 12 weeks with CCX872 in addition to FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin). Responders and those with stable disease after 12 weeks will continue treatment until disease progression, unacceptable toxicity or death. The primary endpoints are safety/tolerability and efficacy (progression-free rate at 6 months Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 25 Clinical Trials Poster Session: Clinical Trials in Progress based on RECIST criteria). Other endpoints include overall survival, and effect on the tumor microenvironment and immunological biomarkers in the blood. This trial will test the hypothesis that CCR2 plays an important role in enabling the myeloid suppressor cell response to pancreatic cancer, and will determine if addition of a CCR2 antagonist can enhance the standard of care for these patients Poster Section 25 Poster Board 24 CT224 A phase Ia clinical trial of a therapeutic prostate cancer vaccine containing PSA/IL-2/GM-CSF in PSA-recurrent prostate cancer patients. Jonathan F. Head,1 Gregory A. Daniels,2 Michelle McKinney,2 Weg Ongkeko,2 Jessica Wang-Rodriguez,2 Kyoko Sakamoto,2 Robert L. Elliott1. 1Oncbiomune, Baton Rouge, LA; 2VA San Diego Healthcare System/VA Medical Center, San Diego, CA. Immunotherapy for cancer has had two main approaches that have lead to clinical applications. The first is stimulating immune responses to tumor cells with cytokines or cellular immunotherapy and the second is blocking tumor immune evasion and the associated inhibition of T-cell activation with antibodies to the CTLA-4 receptor. We have taken a different approach and have developed therapeutic cancer vaccines that are a combination of tumor antigens (whole cells or proteins) with biological adjuvants (the cytokines IL-2 and GM-CSF). This study is a phase Ia/Ib clinical trial of a PSA/IL-2/GM-CSF vaccine in recurrent prostate cancer in hormone-naïve and hormone-independent patients. Major inclusion criteria include adenocarcinoma of the prostate, rising serum PSA and no measurable disease. phase Ia examines the rate of dose limiting adverse events (DLAEs) in an initial course of 6 vaccinations (“induction vaccination”). The phase Ib examines the rate of DLAEs with a continued coarse of an additional 6 vaccinations (“maintenance vaccine”). All patients will receive intradermal injections of the PSA/IL-2/GM-CSF vaccine at weeks 1, 2, 3, 7, 11, and 15. In an additional 28 patients the six maintenance vaccines will alternate IL-2 and the complete vaccine (PSA/IL-2/GM-CSF) at weeks 23, 27, 31, 35, 39 and 43. To date, twelve of twenty patients in the phase Ia portion of the trial have received at least one vaccine injection and ten patients have received all 6 vaccine. Seven of the nine patients that have received 3 vaccines had increased responses to PSA in a lymphocyte blastogenesis assay and five of the eight patients had an increase in their response after 6 vaccines. None of the patients vaccinated in the phase Ia portion have had a DLAE and enrollment continues in the phase Ia. Poster Section 25 Poster Board 25 CT225 Ado-trastuzumab emtansine for HER2 amplified or HER2 overexpressed cancers: A phase II “basket” trial. Bob T. Li, Marjorie Zauderer, Jamie Chaft, Alexander Drilon, Juliana Eng, Camelia Sima, Vicky Makker, Gopa Iyer, Yelena Janjigian, David Hyman, Maria Arcila, Jose Baselga, Mark G. Kris. Memorial Sloan Kettering Cancer Center, New York, NY. Background: The use of therapies targeting the human epidermal growth factor receptor 2 (HER2, ERBB2) has transformed care in breast and gastric cancers. HER2 amplification has emerged as a therapeutic target in 2-5% of lung cancers, 6% of bladder cancers, 5-12% of endometrial cancers, and 2-5% of ovarian and colorectal cancers. High level HER2 protein overexpression by immunohistochemistry correlates with HER2 amplification. Adotrastuzumab emtansine is an antibody drug conjugate linking the HER2 targeted monoclonal antibody trastuzumab, with the cytotoxic anti-microtubule drug emtansine. This agent improves response and survival in patients with HER2 amplified or HER2 overexpressed breast cancers. We hypothesize that adotrastuzumab emtansine will be effective in any tumor with HER2 amplification or overexpression, regardless of the primary site. Methods/Design: This phase II “basket” trial at Memorial Sloan Kettering (MSK) will evaluate ado-trastuzumab emtansine across 4 cohorts of patients with HER2 amplified or HER2 overexpressed advanced lung, bladder, endometrial, and other cancers. All patients will receive ado-trastuzumab emtansine at 3.6 mg/m2 IV every 21 days until disease progression or unacceptable toxicity. The primary endpoint is objective response rate (ORR). Patients will be molecularly selected primarily through the MSK-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), where all patients with advanced cancers can have tumor next generation sequencing (NGS) performed with a capacity to sequence 15,000 tumors each year. MSK-IMPACT uses the Illumina HiSeq platform to screen for potentially actionable genetic alterations, including single base substitutions, indels, copy number alterations and selected fusions across 341 cancer-related genes, including HER2 amplification. HER2 amplification assessment by our NGS assay correlates well with amplification by in-situ hybridization, is less operator-dependent and is performed concurrently with the mutation profile. In the first 6 months since introducing MSK-IMPACT into routine patient care, we have already identified HER2 amplification in 7 of 227 (3%) lung cancers sequenced, 4 of 67 (6%) bladder cancers sequenced, and 2 of 50 (4%) endometrial cancers sequenced. Using a Simon optimal twostage design, a one-sided Type I error rate α at 10% and power of 80%, a true ORR ≤ 10% will be considered unacceptable (null hypothesis) whereas a true ORR ≥ 30% will merit further study (alternative hypothesis). In each cohort, 7 patients will be accrued in the first stage; if there are no responses observed, the cohort will be closed. Otherwise, 11 additional patients will be accrued for second stage. A cohort will be deemed worthy of further investigation if ≥4 responses are observed in 18 patients. Exploratory analysis will examine the concordance among the HER2 biomarkers: gene amplification, protein overexpression and gene mutation. Poster Section 25 Poster Board 26 CT226 A pilot study of adoptive cell therapy with in vitro educated MART1 T cells in combination with ipilimumab for the treatment of metastatic melanoma. leila Khoja, Anthony M. Joshua, Lisa Wang, David Hogg, Linh Nguyen, Valentin Sotov, Vinicius Motta, Liz Scheid, Diana Gray, Nato Hirano, Marcus O. Butler. The Princess Margaret Cancer Centre, Toronto, Ontario, Canada. Background: The majority of ACT approaches use preconditioning lymphodepletion as a prerequisite for achieving clinical activity. For TIL therapy, high dose IL-2 is also required. As an alternative to the toxicity of this approach our group has developed a method for efficiently generating anti-tumor CD8+ T cells, using a novel clinical grade artificial (HLA-A*0201+, CD80+ and CD83+) antigen presenting cell (aAPC). A completed phase I clinical study (Butler 2011) demonstrated that this aAPC can in vitro-educate tumor antigen specific T cells enabling them to induce responses and establish anti-tumor memory without the use of lymphodepletion, vaccination, or cytokine injections. Detection of transferred aAPC educated MART1 T cells showed trafficking to tumor and an increased frequency of MART1 T cells with a central or effector memory phenotype. Durable responses were seen particularly in patients subsequently treated with ipilimumab. Trial design: A phase I trial of ACT with our novel in vitro educated CD8+T cells in combination with ipilimumab was designed to evaluate safety, feasibility and efficacy of this novel combination. Eligible patients must be HLA-A*0201+, ECOG 0/1, have evaluable disease which is MART1+ by IHC and have adequate organ function. Any number of previous therapies are allowed, including ipilimumab. Patients with stable brain metastases will also be eligible. Patients will undergo leukapheresis to harvest autologous PBMC and a CliniMACs will be used to isolate CD8 T cells for in vitro expansion and priming with MART1 peptide pulsed and irradiated aAPC. One infusion of CD8+ in vitro educated T cells will be given followed by ipilimumab for 4 cycles. Tumor biopsies will be performed at baseline, after 4 cycles of ipilimumab treatment, and American Association for Cancer Research • AACR ANNUAL MEETING 2015 25 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 26 Clinical Trials Poster Session: Clinical Trials in Progress at the time of progression. CT assessment scans will be performed at baseline, upon completion of treatment and every three months thereafter. Responses will be determined according to RECIST v1.1 and irRC. Immune correlates will be assessed by serial peripheral phlebotomy at defined time points during treatment; at baseline, prior to each treatment, at the end of all treatment, 3 monthly thereafter and at the time of progression. A target of ten patients will be recruited. As the primary aim is to assess feasibility and safety of this treatment no formal statistical power is required. We will however use Welch’s t test to assess for statistically significant changes (during treatment and follow up) in MART1 T cell frequency and phenotype in two-sample comparisons and the Wilcoxon signed-rank test for paired comparisons. Poster Section 25 Poster Board 27 CT227 MC1273: Phase II evaluation of aggressive dose deescalation for adjuvant chemoradiation in HPV associated oropharynx cancer. Daniel J. Ma, Katharine A. Price, Eric J. Moore, Joaquin J. Garcia, Scott H. Okuno, Daniel L. Price, Jeff A. Sloan, Nathan R. Foster, Robert L. Foote. Mayo Clinic, Rochester, MN. Background: Traditional adjuvant therapy for oropharyngeal squamous cell carcinoma (OPSCC) consists of 60-66 Gy of radiation therapy (XRT) given in 2 Gy daily fractions along with high dose cisplatin if the patient has high risk factors. Despite the excellent cure rates for HPV+ OPSCC, one in three patients treated with conventional treatment will develop grade >3 long-term sequelae from therapy. There is intense interest in de-intensifying adjuvant therapy for this patient population in order to maximize quality of life while maintaining excellent historical rates of disease control. Methods: MC1273 is a phase II non-randomized trial open at Mayo Clinic Rochester testing a novel course of aggressive therapy de-escalation following surgery for HPV+ OPSCC. The primary endpoint is local/regional control at 2 years while secondary endpoints include toxicity and quality of life (QOL). The eligibility criteria include all patients with p16-positive OPSCC with less than a ten pack-year smoking history who have had a complete surgical resection. Exclusion criteria include positive surgical margins, prior history of malignancy, and history of connective tissue disorders. Patients are divided into two prospective cohorts depending upon risk factors found at surgery. Patients with intermediate risk disease (≥T3, ≥N2, lymphovascular invasion, or perineural invasion) are enrolled in MC1273A while patients with extracapsular extension (ECE) are enrolled in MC1273B. Patients on MC1273A receive 30 Gy of radiation delivered in 1.5 Gy twice-daily fractions over the course of two weeks along with weekly docetaxel (15 mg/m2) given on day 1 and day 8. Patients on MC1273B receive a similar treatment regimen but also have the nodal level with positive ECE concurrently boosted to 36 Gy in 1.8 Gy twice-daily fractions. In addition to standard of care follow-up, patients receive a swallowing assessment with speech therapy immediately before XRT, one month post-XRT, and one year post-XRT. Patients also have QOL assessment consisting of the XeQOLS, Eq-5D, FACT H&N (Vers 4) and Dermatology Life Quality Index assessed at preXRT and 3, 12, and 24 months post-XRT. Results: Each cohort of MC1273 is powered to detect a 10% local/regional failure rate with 85% confidence. Each cohort will accrue 35 evaluable patients and 5 additional patients to account for ineligibilities and violations (40 patients total per cohort.) MC1273A began accrual in September 2013. The first five patients were monitored for grade ≥4 acute toxicities before proceeding to open accrual. MC1273B began accrual in May 2014 and has also proceeded to open accrual. Accrual will also begin in Mayo Clinic Scottsdale in the first quarter of 2015. Conclusions: MC1273 is meeting its accrual targets and should 26 finish accrual by 2016. We anticipate that preliminary results for toxicity will be available by 2017 and local/regional data will be available by 2018. Poster Section 25 Poster Board 28 CT228 Formulation switch and pharmacokinetics/pharmacodynamics of Debio 1347 (CH5183284), a novel FGFR inhibitor, in a first-in-human dose escalation trial in solid tumors patients. Valerie Nicolas-Metral,1 Anne Vaslin,1 Jeffrey G. Supko,2 Kiyohiko Nakai,3 Nobuya Ishii,3 Annick Menetrey,1 Marie-Claude RoubaudiFraschini,1 Judith Marfurt,1 Sebastien Chabaud,1 Andreas Layer,1 Daniela Purcea,1 Jerome Douchain,1 Claudio Zanna1. 1Debiopharm International SA, Lausanne, Switzerland; 2Clinical Pharmacology Laboratory, Massachusetts General Hospital, Boston, MA; 3Chugai Pharmaceutical Co., Ltd, Tokyo, Japan. Background: Deregulated fibroblast growth factor receptor (FGFR) signaling is associated with tumorigenesis. The oral selective FGFR 1, 2, 3 inhibitor Debio 1347, a Biopharmaceutical Classification System Class II drug, is currently investigated in a phase I trial in selected patients harboring FGFR genetic alterations (NCT01948297). A formulation switch from capsules to tablets was investigated in the course of the dose escalation part of the trial. Methods: Comparative in vitro dissolution tests and PK data after single dose (20 mg) in Cynomolgus monkeys were generated with capsules and tablets to evaluate the suitability of the new tablet formulation prior to human use. The first-in-human, phase I dose-escalation multiple tumor type “basket” study enrolled patients with advanced solid malignancies harboring defined activating alterations of FGFR 1, 2, or 3. Patients received Debio 1347 orally once daily and were assessed for dose-limiting toxicities (DLT) during the first 4 weeks. With a starting dose level of 10 mg the study followed a 3+3 algorithm with dose-escalation on a modified Fibonacci sequence. Capsules of 10- and 20-mg strength were administered in patients up to the 3rd dose level. At the 4th dose-level, the pharmacokinetic (PK) profile and intra-patient relative oral bioavailability of the new tablet formulation versus the original capsule formulation were determined in a two-period, 7-day washout, cross-over design after single dose in standardized food intake conditions. Debio 1347 plasma levels were measured using a validated LC-MS/MS assay. The pharmacodynamic (PD) profile of Debio 1347 was also assessed by measuring plasma levels of several biomarkers using standardized assays. A comparison of the PK and PD of the two dosage forms and their tolerability was performed prior to selection of the dosage form for pursuing the dose escalation. Results: Adequate in vitro dissolution profiles were observed for both capsules and tablets. In addition, oral bioavailability of 20mg tablets was comparable to that of 10- and 20-mg capsules in monkeys. In a majority of solid tumor patients administered with 40 mg of Debio 1347, intra-patient relative oral bioavailability of the tablet versus capsule was > 80%. In addition, PD and safety profiles after 4-week once daily dosing with tablets were comparable to that of capsules. Conclusion: A formulation switch from capsules (only 10 and 20 mg capsules available) to tablets (20, 30, 50, and 100 mg tablets) of the FGFR inhibitor Debio 1347 was successfully implemented in the course of the first-in-human trial to facilitate dose escalation and improve treatment compliance at high doses by reducing the number of units to be swallowed by solid tumor patients. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 27 Clinical Trials Poster Session: Clinical Trials in Progress Poster Section 25 Poster Board 29 CT229 Autologous dendritic cells loaded with allogeneic tumor cell lysate in patients with mesothelioma: A phase I study. Joachim G. Aerts, Robin Cornelissen, Cor van der Leest, Ferry Eskens, Koen bezemer, Margaretha Kaijen, Rudi Hendriks, Joost Hegmans, Henk C. Hoogsteden. Erasmus MC, rotterdam, Netherlands. Malignant Mesothelioma (MM) is an aggressive disease without curative treatment options. Treatment with chemotherapy and surgery is accompanied with high incidences of local recurrences. Novel therapeutic treatment options are urgently needed and immunotherapy may be a new player in the field of MM. We have previously shown the feasibility of dendritic cell (DC) immunotherapy in MM (Hegmans, et al. Am J Respir Crit Care Med 2010). In these studies DC were loaded with autologous tumor cell lysate. Apart from safety also radiographical responses were found and survival was promising. However autologous tumor cell lysate has major drawbacks both in availability, quality and logistics. Therefore a new concept was developed, based on DC loading with a tumor cell lysate derived from human MM cell lines. In a murine MM model allogenic loading of DC’s was equally safe, and effective as autologous (data submitted). We have developed a clinical grade allogenic batch of tumorcell lysate which is now used in a phase I study where patient diagnosed with MM are treated. This batch has been generated according to GMP and GLP regulations and a patent is pending. Orphan drug designation has been obtained from both FDA and EMA. For the trial, major inclusion criteria are chemonaïve patients or patients with a disease controle according to modified RECIST after standard chemotherapy. Patients with a need for high dosages of immunosuppressive therapy are excluded. In the study patients undergo a leukapheresis to collect monocytes. These monocytes are in vitro differentiated to DC’s and pulsed with tumor lysate according to the previously described protocol. DCs are re-injected intravenous and intradermally every 2 weeks to determine toxicity. A 3*3 design study is initiated where chosen dosages are 10, 25 and 50* million cells. Primary endpoint is safety. Secondary endpoints are radiological responses according to modified RECIST for mesothelioma, progression free survival and overall survival. At present the study is open for inclusion. At the meeting results of the first dose cohorts will be presented. This will be the first in human study in MM with allogenic tumor cell loaded DC. In case no safety issues are encountered this may open the field of combination treatments (e.g. immunecheckpoint inhibition combined with DC treatment) to increase the population of patients who benefit from immunotherapeutic treatment options. Poster Section 25 Poster Board 30 CT230 A phase I first in human dose escalation trial of MNK010 in subjects with advanced solid tumors. Louise S. Rochon, Krishna Devarakonda, Jose Martinez, Kelly Williams. Mallinckrodt Pharmaceuticals, Hazelwood, MO. Background: Taxanes possess broad activity and are widely used for a variety of tumor types. Currently available formulations are associated with dose limiting myelosuppression, neurotoxicity, fluid retention and other toxicities. MNK-010, a liposomal prodrug formulation of docetaxel, is designed to act as a drug depot with the slow conversion and release of docetaxel resulting in a relatively lower Cmax, and enhanced systemic exposure (AUC) over a prolonged period of time. It is anticipated that this unique PK profile would improve efficacy with a better safety profile compared to docetaxel. Design: This study is a dose escalation first in human (FIH) study in subjects with advanced solid malignancies who have failed conventional therapy. MNK-010 is administered IV every 21 days for four cycles. The primary objectives are to evaluate the safety and tolerability and determine the MTD and DLT of MNK-010. The secondary objectives are to characterize the PK profile of docetaxel, the liposomal components (DSPE-PEG[2000]) and docetaxel prodrug (MP-3528) and preliminary anti-tumor activity of MNK-010.Twelve dose levels are planned: 3, 6, 12, 24, 48, 80, 120, 160, 190, 225, 270, and 320 mg/m2 Dosing at 160 mg/m2 is underway. The recommended phase II dose will be administered to 20 patients with metastatic SCCHN to further evaluate the safety, PK profile, and preliminary antitumor activity of MNK-010. Results: 25 subjects have received at least one dose of MNK010 for a total of 106 cycles administered. Efficacy results include six stable diseases (SDs) in tumor types including thymic cancer, NSCLC, prostate, ovarian, cervical and gastroesophageal cancer. Safety data shows that MNK-010 is well tolerated at doses up to 120 mg/m2. The major drug related adverse events reported are nausea, vomiting and fatigue. The Clearance (CL), volume of distribution (Vss), half-life (t1/2), peak level (Cmax) and extent of exposure (AUC) values are comparable between DSPE(PEG-2000) and MP-3528. The CL (~ 0.026 L/h/m2) and Vss (~ 1.6 L/m2) of MNK-010 are very low, with the mean t1/2 being about 60h. Cmax and AUC demonstrate dose proportionality and linearity for MP-3528, DSPE(PEG-2000) and docetaxel. The dose normalized Cmax of docetaxel released from MNK-010 is about nine-fold lower relative to a comparable dose of Taxotere. Conclusion: MNK-010 is well tolerated up to doses of 120 mg/m2. Stable diseases have been observed in several tumor types. American Association for Cancer Research • AACR ANNUAL MEETING 2015 27 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 28 Clinical Trials Plenary Session: Clinical Trials of New Drugs in Breast Cancer Clinical Trials Plenary Session Monday, April 20, 2015 10:30 AM-12:20 PM Room 103, Pennsylvania Convention Center Clinical Trials of New Drugs in Breast Cancer Co-Chairpersons: Fabrice Andre, Institut Gustave Roussy, Villejuif, France; Matthew J. Ellis, Baylor College of Medicine Cancer Center, Houston, TX The complete text of the abstracts in this session will be posted to the online Proceedings after presentation. 10:30 AM CT231 A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader GDC0810 (ARN-810) in postmenopausal women with ER+ HER2-, advanced/metastatic breast cancer. Maura Dickler,1 Aditya Bardia,2 Ingrid Mayer,3 Eric Winer,4 Peter Rix,5 Jeff Hager,5 Meng Chen,6 Iris Chan,6 Edna Chow-Maneval,5 Carlos Arteaga,3 Jose Baselga1. 1 Memorial Sloan Kettering Cancer Center, New York, NY; 2 Massachusetts General Hospital Cancer Center, Boston, MA; 3Vanderbilt-Ingram Cancer Center, Nashville, TN; 4Dana-Farber Cancer Institute, Boston, MA; 5Seragon Pharmaceuticals, San Diego, CA; 6 Genentech, Inc, South San Francisco, CA. 10:50 AM Discussant C. Kent Osborne, Baylor College of Medicine Cancer Center, Houston, TX 11:00 AM CT232 SU2C Phase Ib study of the PI3K-alpha inhibitor BYL719 (alpelisib) with letrozole in ER+/HER2-metastatic breast cancer (MBC). Ingrid A. Mayer,1 Vandana Abramson,1 Justin Balko,1 Melinda Sanders,1 Dejan Juric,2 David Solit,3 Yisheng Li,4 Lewis Cantley,5 Eric Winer,6 Carlos Arteaga1. 1Vanderbilt University Medical Center, Nashville, TN; 2 Massachusetts General Hospital, Boston, MA; 3 Memorial Sloan Kettering Cancer Center, New York, NY; 4 MD Anderson Cancer Center, Houston, TX; 5Weill Cornell Medical College, New York, NY; 6Dana Farber Cancer Institute, Boston, MA. 28 11:20 AM CT233 A phase I study evaluating continuous and intermittent AZD2014 in combination with fulvestrant in patients with ER+ advanced metastatic breast cancer. Manish Patel,1 Erika Hamilton,2 Patricia M. LoRusso,3 W. Larry Gluck,4 Suzanne F. Jones,5 Muaiad Kittaneh,3 Sabina Cosulich,6 Elizabeth A. Harrington,6 Stephen Green,6 Wendy Burke,6 Donald K. Strickland,5 Elisabeth Oelmann,6 Howard A. Burris2. 1Sarah Cannon Research Institute/Florida Cancer Specialists, Sarasota, FL; 2Sarah Cannon Research Institute/Tennessee Oncology, PLLC, Nashville, TN; 3Karmanos Cancer Institute, Detroit, MI; 4 Greenville Health System Institute for Translational Oncology Research, Greenville, SC; 5Sarah Cannon Research Institute, Nashville, TN; 6AstraZeneca, Macclesfield, United Kingdom. 11:40 AM Discussant Matthew J. Ellis, Baylor College of Medicine Cancer Center, Houston, TX 11:50 AM CT234 A phase I study of MM-302, a HER2-targeted PEGylated liposomal doxorubicin, in patients with HER2+ metastatic breast cancer. Patricia LoRusso,1 Ian Krop,2 Kathy Miller,3 Cynthia Ma,4 Barry A. Siegel,4 Anthony F. Shields,5 Istvan Molnar,6 Thomas Wickham,6 Joseph Reynolds,6 Karen Campbell,6 Bart Hendriks,6 Ty McClure,6 Victor Moyo,6 Pamela Munster7. 1Yale, New Haven, CT; 2Dana Farber Cancer Institute, Boston, MA; 3Indiana University, Indianapolis, IN; 4Washington University in St. Louis, St. Louis, MO; 5 Karmanos, Detroit, MI; 6Merrimack Pharmaceuticals, Cambridge, MA; 7UCSF, San Francisco, CA. 12:10 PM Discussant Fabrice Andre, Institut Gustave Roussy, Villejuif, France Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 29 Clinical Trials Minisymposium: Clinical Trials of Novel Therapeutics Clinical Trials Minisymposium Monday, April 20, 2015 3:00 PM-5:00 PM Room 103, Pennsylvania Convention Center Clinical Trials of Novel Therapeutics Co-Chairpersons: Lillian L. Siu, University Health Network Princess Margaret Hospital, Toronto, Ontario, Canada; Paul Haluska, Mayo Clinic, Rochester, MN 3:00 PM Introduction 3:10 PM CT236 Advanced solid cancer therapy with a novel antibody-drug conjugate (ADC), sacituzumab govitecan (IMMU-132): Key preclinical and clinical results. Alexander N. Starodub,1 Allyson J. Ocean,2 Aditya Bardia,3 Michael J. Guarino,4 Wells Messersmith,5 Jordan Berlin,6 Vincent J. Picozzi,7 Sajeve S. Thomas,8 Gregory Masters,4 Linda T. Vahdat,2 Ingrid A. Mayer,6 Rebecca Moroose,8 Jennifer S. Diamond,5 Scott T. Tagawa,2 Manish A. Shah,2 Francois Wilhelm,9 William A. Wegener,9 Pius Maliakal,9 Robert M. Sharkey,9 David M. Goldenberg9. 1Indiana University Health Center for Cancer Care, Goshen, IN; 2Weill Cornell Medical College, New York, NY; 3Massachusetts General Hospital, Harvard Medical School, Boston, MA; 4Helen F. Graham Cancer Center, Newark, DE; 5University of Colorado Cancer Center, Aurora, CO; 6Vanderbilt-Ingram Cancer Center, Nashville, TN; 7Virginia Mason Cancer Center, Seattle, WA; 8University of Florida Health Cancer Center, Orlando, FL; 9Immunomedics, Inc., Morris Plains, NJ. Background: Sacituzumab govitecan (IMMU-132) is a new ADC comprising SN-38, the active metabolite of the topoisomerase inhibitor, camptothecin (irinotecan), conjugated to an anti-Trop-2 antibody. In vitro and in vivo preclinical data suggest that IMMU-132 is a unique ADC, being most efficacious at a high drug-antibody ratio (DAR) of 7.6, and capable of delivering up to 135fold more SN-38 than its parental drug, irinotecan, in a human cancer xenograft. In vitro studies also demonstrate specific double-stranded DNA breaks by the internalizing ADC. Methods: IMMU-132 is completing a phase I/II clinical trial with phase II expansion after MTD determination (ClinicalTrials.gov.NCT01631552) in patients with advanced cancers that typically express high levels of Trop-2, at doses of 8 and 10 mg/kg on days 1 and 8 of 21-day repeated cycles. Efficacy (N=91) and safety (N=130) results are provided. Results: The % of grades 3/4 AEs for both dose levels are neutropenia (16/4), febrile neutropenia (4/4), anemia (4/0), diarrhea (4/0), and fatigue (4/0) (Table 1). No patient discontinued therapy due to toxicity, and no patient showed immunogenicity despite repeated therapy. Patient dose reductions were 15-16%, and dose delays after first 2 cycles were 3-4%. Conclusions: IMMU-132 shows activity in patients with diverse cancers, even when they no longer responded to a topoisomerase inhibitor. It appears to have a manageable toxicity profile, with promising efficacy at a high therapeutic index in patients with heavily-pretreated metastatic cancers, especially TNBC, SCLC, and NSCLC. Based on these results, this ADC carrying a moderately-toxic drug that is the active metabolite of a currently-used camptothecin analogue represents a novel cancer therapeutic that challenges the current dogma of requiring ultratoxic drugs conjugated at low DARs for ADC therapy. 3:30 PM American Association for Cancer Research • AACR ANNUAL MEETING 2015 CT237 Preclinical characterization and first-inhuman study of MM-141, a dual antibody inhibitor of IGF-1R and ErbB3. Alexey A. Lugovskoy,1 Michel Curley,1 Jason Baum,1 Sharlene Adams,1 Sergio Iadevaia,1 Victoria Rimkunas,1 Adam Camblin,1 Lin Nie,1 Gege Tan,1 Bryan Johnson,1 Sara Mathews,1 Kerry Horgan,1 Chrystal U. Louis,1 Akos G. Czibere,1 Monica Arnedos,2 Jean-Charles Soria,2 Rastilav Bahleda,2 Anthony Shields,3 Patricia M. LoRusso,3 Mansoor Saleh,4 Steven J. Isakoff5. 1 Merrimack Pharmaceuticals Inc., Cambridge, MA; 2 Institut Gustav Roussy, Villejuif, France; 3Karmanos Cancer Institute, Detroit, MI; 4Georgia Cancer Specialists/Northside Hospital Cancer Institute, Atlanta, GA; 5Massachusetts General Hospital, Boston, MA. Background: MM-141 is a tetravalent bi-specific monoclonal antibody that binds IGF-1R and ErbB3, oncogenic receptors commonly co-expressed in solid tumors. In preclinical models, MM-141 blocks both ligand-dependent and -independent PI3K/AKT/mTOR signaling initiated through IGF-1R and ErbB3 complexes and potentiates the activity of gemcitabine, paclitaxel, nab-paclitaxel, docetaxel, irinotecan, tamoxifen, and everolimus. A multi-arm phase I study is ongoing, with continuing patient enrollment in Arm B (MM-141 in combination with everolimus). Monotherapy Arm A and combination Arm C (MM-141 with nab-paclitaxel and gemcitabine) are completed. Methods: Tumor expression of IGF-1R and ErbB3 was measured by immunohistochemistry. In vitro expression and degradation of IGF-1R and ErbB3 in pancreatic cell line models post-treatment were measured by immunoblotting and ubiquitination, respectively. The phase I dose-escalation study evaluated safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) properties of MM-141 as monotherapy (Arm A, n=15) and in combination with everolimus (Arm B) or with nab-paclitaxel and gemcitabine (Arm C, n=11). Pre- and post-treatment biopsies were acquired where mandated. Patients in the monotherapy Arm A received MM-141 at 6, 12, 20 mg/kg weekly or 40 mg/kg biweekly. Patients in the dose-escalation portion of Arm C received MM-141 at a weekly dose of 12 or 20 mg/kg in combination with weekly nab-paclitaxel (125 mg/m2) and gemcitabine (1000 mg/m2) on a schedule of 3 weeks on, 1 week off. Enrollment in Arm B (MM-141 in combination with everolimus) is ongoing. Patient serum free IGF-1 levels were detected using an in-house developed CLIA validated ELISA-based assay. Results: Here we report common co-expression of IGF-1R and ErbB3 in solid tumors. In stage IV metastatic 29 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 30 Clinical Trials Minisymposium: Clinical Trials of Novel Therapeutics pancreatic cancer, co-expression of IGF-1R and ErbB3 was associated with decreased patient survival. In preclinical models, increased expression of IGF-1R and ErbB3 desensitized tumors to gemcitabine and paclitaxel. However, co-treatment with MM-141 reversed this acquired resistance through blockade of growth factor binding and induction of IGF-1R and ErbB3 degradation. In the monotherapy arm of a phase I study, no dose-limiting toxicities were observed at any of the studied dose levels. The safety, tolerability, PK and PD profile of MM-141 support 2.8g bi-weekly MM141 phase II recommended dose. The analysis of preand post-treatment biopsies confirmed that levels of IGF-1R and ErbB3 were decreased following MM-141 administration. In Arm C, the observed safety profile of MM-141 in combination with nab-paclitaxel and gemcitabine was comparable to expected toxicities reported with the chemotherapy combination when used alone. Retrospective analysis of serum free IGF-1 levels in breast cancer patients (Arm B) demonstrated that patients with elevated levels of this potential biomarker remained on study longer and received a greater number of doses of MM-141. Conclusion: These data support continued development of MM-141 in biomarker-selected patient populations and the upcoming phase II study of MM141 in combination with nab-paclitaxel and gemcitabine in front-line metastatic pancreatic cancer patients with detectable free IGF-1 serum levels. 3:50 PM 30 CT238 Phase I safety and biodistribution study of 124I-PEG-AVP0458 diabody in patients with TAG-72 positive ovarian and prostate cancer. Andrew M. Scott,1 Timothy Akhurst,2 Fook-Thean Lee,1 Marika Ciprotti,1 Ian Davis,3 Andrew Weickhardt,1 Hui Gan,1 Pece Kocovski,1 Nancy Guo,1 Linda Mileshkin,2 Scott Williams,2 Declan Murphy,2 Rod Hicks,2 Kunthi Pathmaraj,4 Sze Ting Lee,4 Graeme O’Keefe,4 Sylvia Gong,4 Maggie Oh,5 Michael Wheatcroft,5 Peter J. Hudson5. 1Ludwig Institute for Cancer Research, Austin Hospital, Melbourne, Australia; 2Peter MacCallum Cancer Centre, Melbourne, Australia; 3Monash University Eastern Health Clinical School, Melbourne, Australia; 4Department of Nuclear Medicine and Centre for PET, Austin Hospital, Melbourne, Australia; 5Avipep Pty Ltd and Victorian Cancer Biologics Consortium, Melbourne, Australia. Background: The development of antibody therapeutics for imaging and payload delivery is complex, and intact IgG have long half-lives that impact on tumor:blood ratios and tumor penetrance. Smaller molecular weight antibody constructs (eg diabodies) have been developed for improved penetrance into tumor, faster blood clearance, and enhanced tumor: normal tissue uptake, however renal uptake may impact on imaging and therapeutic effects. Through a novel pegylation strategy to surface disulphides, a diabody to TAG-72 (AVP0458) has been generated, and produced under cGMP for a first-in-human clinical trial. Materials and Methods: We have conducted a phase I, open label, first-in-human trial of PEG-AVP0458. The primary study objective was the safety of single dose of I-124 PEG-AVP0458 in patients (pts) with TAG-72 +ve relapsed / metastatic prostate or ovarian cancer. Secondary study objectives were evaluation of the biodistribution, tumor targeting, pharmacokinetics (PK) and immunogenicity of I-124 PEG-AVP0458. Pts were infused with I-124 PEG-AVP0458 (3-5mCi) at one of two dose levels (1mg/m2 and 10mg/m2), and imaged sequentially over a one week period. Safety, PK, and immunogenicity was assessed up to 30 days post infusion. Results: Six pts (1F:5M; age range 62-85yrs; 1 ovarian cancer, 5 prostate cancer) were entered into the study, 3 at each dose level. I-124 PEG-AVP0458 was well tolerated, with no infusion-related adverse events, and no serious adverse events observed. There was consistent biodistribution on PET imaging of I-124 PEGAVP0458, with no normal tissue uptake. High tumor uptake was evident in metastatic disease in liver and lymph nodes, with lesion uptake seen within 1-2 days post injection. PK analysis showed a T½β of 46.8 ± 12.4 hrs. There was no impact of protein dose on biodistribution, tumor uptake or PK. No immunogenicity to PEG-AVP0458 was evident. Conclusions: I-124 PEG-AVP0458 is safe, and demonstrates excellent, rapid targeting of tumor in vivo, with no specific normal organ uptake, and high tumor: blood ratios. This data demonstrates the feasibility of using pegylated diabodies for imaging and for delivery of radioisotopes (RIT) or cytotoxic drug payloads (ADC) in cancer patients. 4:10 PM CT239 Clinical and preclinical evidence of an immune modulating role for the STAT3-targeting ASO AZD9150 and potential to enhance clinical responses to anti-PDL1 therapy. Patricia E. Mccoon,1 Rich Woessner,1 Shaun Grosskurth,1 Chris Womack,1 Mason Yamashita,2 Gene Hung,2 Robert MacLeod,2 Kirsten Bell,1 Mike Collins,1 Rachel DuPont,1 Vivian Jacobs,1 Michele Johnstone,1 Margaret Veldman-Jones,1 Paul Lyne1. 1AstraZeneca Pharmaceuticals, Waltham, MA; 2Isis Pharmaceuticals, Waltham, MA. AZD9150 is a therapeutic Generation 2.5 antisense oligonucleotide (ASO) targeting STAT3 that has completed two phase I clinical studies, in patients with HCC and DLBCL, with durable clinical responses seen in both trials. Biomarker studies using patient samples and related preclinical experiments were performed to investigate the mechanism of action of AZD9150. Patients were treated with three loading doses of AZD9150 in the first week followed by weekly dosing, at doses ranging from 1.0 to 3.0 mg/kg. In the DLBCL study, paired tumor biopsies were collected pretreatment and on-treatment to evaluate drug uptake and target knockdown by immunohistochemistry (IHC). In the HCC study, blood samples were collected at baseline and at multiple time points on-treatment to evaluate target knockdown and gene expression changes. IHC staining of DLBCL patients’ tumor biopsies (at 2 & 3 mg/kg) demonstrated that the drug distributes to the tumor, with strongest uptake in stromal cells, including endothelium, fibroblasts, and immune cells. Pronounced decreases (absence of staining on-treatment) in STAT3 were observed in the endothelium of several samples. More limited STAT3 modulation was observed in tumor cells. Flow cytometry analysis of HCC patients’ blood samples revealed an average decrease in STAT3 protein staining of 49% across all peripheral leukocyte populations in the 1 mg/kg cohort. Clinical pharmacodynamics and mechanism of action were explored further by conducting a gene expression study with the Nanostring nCounter Human Immunology Panel v2 to evaluate STAT3 RNA knockdown and 593 additional immune genes in Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 31 Clinical Trials Minisymposium: Clinical Trials of Novel Therapeutics evaluated as a single agent in expansion cohorts of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN). Methods: This was a phase I, multicenter, nonrandomized, open-label study in patients with advanced cancer (NCT01115790). Based on results from the dose-escalation phase, dose-expansion cohorts were comprised of patients with SCC given LY2606368 at the maximum tolerated dose (MTD) of 105 mg/m2 on day 1 of a 14-day schedule. Cohorts were defined by tumor location and by line of treatment. Patients were assessed for safety, tolerability, and preliminary efficacy. Pretreatment biopsies were obtained for pharmacogenomic analysis, including human papillomavirus (HPV) status. Aggregate results from patients with recurrent/metastatic SCCHN are presented. Results: Fifty-seven patients with recurrent/metastatic SCCHN were enrolled in the doseexpansion phase. Over 50% of patients had received ≥2 prior lines of treatment (median of 3 cycles; range: 1 to 5) in the recurrent/metastatic setting. The most frequently reported adverse event (AE) was a transient (typically <5 days) decrease in neutrophil/leukocyte count, which occurred in 91% of patients (grade 4 in 63% of patients). Ten patients (18%) experienced febrile neutropenia. Other study drug-related AEs occurring in >10% of patients included thrombocytopenia (44%), anemia (25%), fatigue (23%), and headache (14%). The majority of non-hematologic AEs were grade 1 or 2 (per Common Terminology Criteria) in severity. Three patients (5%) had a partial response and 25 patients (44%) had stable disease for at least 3 cycles. The duration of response ranged from 4.8-7.8+ months, and the median progression-free survival (PFS) was 1.6 months (90% confidence interval: 1.4, 2.8). Biopsy samples were evaluable from 34 patients. The median PFS by HPV status was 4.5 months in 15 patients who were HPV positive, and 1.4 months in 19 patients who were HPV negative (log-rank test, p=.0012). Conclusions: LY2606368 has an acceptable safety profile and demonstrates modest preliminary activity in a subset of patients with recurrent/metastatic SCCHN. The MTD of 105 mg/m2 is confirmed as the recommended dose for phase II testing. peripheral leukocytes collected from HCC patients. Statistically significant decreases of >30% in STAT3 expression were observed in 14/32 patients by the fourth week of treatment. These STAT3 changes are accompanied by +/- 40% changes in expression by additional genes associated with decreased myeloid trafficking and function, increased antigen presentation, and increased CD8 effector cell function. These data provide evidence that AZD9150 treatment may remove or reprogram immunosuppressive elements employed by tumors, leading to therapeutic benefit. Preclinical studies were carried out to investigate immune cell changes within tumors and the benefit of combining STAT3 ASO with PDL1 blockade. Monotherapy STAT3 ASO treatment resulted in CT26 tumor growth inhibition (80%) when tested in immune competent Balb/c but not immune-deficient NSG mice, and was associated with two-fold increases in CD45+ and CD8+ cell infiltrate into tumors. Mice treated with STAT3 ASO and anti-PD-L1 blocking antibody resulted in a 50% response rate for the combination treatment, vs. only 14% with anti-PD-L1 Ab alone. These data suggest that the effects of STAT3 ASO are mechanistically complementary to immune checkpoint inhibitors and that the combination with AZD9150 could broaden clinical responses to these important therapies. This hypothesis will be tested in upcoming clinical trials with AZD9150 and MEDI4736. 4:30 PM CT240 Checkpoint kinase (CHK) 1/2 inhibitor LY2606368 in a phase I dose-expansion study in patients with squamous cell carcinoma of the head and neck. Johanna Bendell,1 Stefan Grant,2 Filip Janku,3 Jeffrey Infante,1 William N. William,3 Todd M. Bauer,1 Sarina Piha-Paul,3 Ricardo Martinez,4 Sameera Wijayawardana,4 Ji Lin,4 Lisa Golden,4 Aimee Bence Lin,4 David Hong3. 1Sarah Cannon Research Institute, Nashville, TN; 2Wake Forest University, Winston-Salem, NC; 3The University of Texas MD Anderson Cancer Center, Houston, TX; 4Eli Lilly and Company, Indianapolis, IN. Background: LY2606368 is a CHK1/2 inhibitor. In addition to its role in DNA damage response, CHK1 also phosphorylates multiple downstream targets that regulate DNA replication, chromosome alignment, spindle checkpoints, and exit from cytokinesis. Since potent inhibition of CHK1 is predicted to generate DNA damage and mitotic catastrophe, LY2606368 was 4:50 PM American Association for Cancer Research • AACR ANNUAL MEETING 2015 Discussion 31 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 32 Clinical Trials Poster Session: Phase I Clinical Trials Poster Session Tuesday, April 21, 2015 8:00 AM-12:00 PM Poster Section 24 Halls B-E (Level 200), Pennsylvania Convention Center Phase I Clinical Trials Poster Section 24 Poster Board 1 CT301 Phase I study of SurVaxM in patients with survivinexpressing recurrent malignant gliomas. Michael J. Ciesielski,1 Laszlo Mechtler,1 Kathleen Mogensen,1 Jingxin Qiu,1 Manmeet Ahluwalia,2 Kelvin Lee,1 Alex Adjei,1 Robert Fenstermaker1. 1Roswell Park Cancer Inst., Buffalo, NY; 2Cleveland Clinic, Cleveland, OH. Background: Recent studies using SurVaxM, a survivin based multi-epitope cryptic peptide mimic, show specific CD8+ T cell responses and specific CD4+ T cell stimulation. Currently SurVaxM has completed a phase I clinical trial designed to study its safety, tolerability and immunological effects in patients with survivinpositive recurrent malignant gliomas. Survivin is an intracellular inhibitor of apoptosis protein (IAP) that mediates a number of anti-apoptotic and oncogenic effects and is highly expressed in malignant gliomas and other cancers. The vaccine is immunogenic in humans with HLA-A*02, HLA-A*03, HLAA*24 and other haplotypes and pre-clinical studies demonstrate potent and specific cytokine-supported antitumor CTL responses. Methods: Nine patients with survivin-positive, recurrent malignant gliomas and either HLA-A*02 or HLA-A*03 haplotypes received a series of 4 subcutaneous injections of SurVaxM at 2 week intervals. MRIs were performed at baseline, week 8, and at subsequent intervals. Results: SurVaxM was well tolerated with no SAE related to vaccine administration. Most AE were grade 1, including 6 of 9 patients with grade 1 localized erythema at the injection site. Three patients reported grades 1 or 2 fatigue, 2/9 experienced myalgia, possibly related to the study drug. Grade 1 lymphopenia was seen in 3/9 patients and grade 1 or 2 leukopenia was recorded in 3 patients. The only grade 3 AE, a seizure, was not related to the vaccine. The majority of patients developed specific cellular and humoral immune responses to survivin and 3 of 8 patients (all with recurrent glioblastoma), who are evaluable for clinical response, had stable disease after 17-26 months of ongoing follow-up. Five others have had progressive disease, although 4 of these maintained stable disease for 8-14 months. Conclusion: This study demonstrated the safety and tolerability of SurVaxM in patients with recurrent or progressive malignant glioma following failure of standard therapy. SurVaxM proved to be immunogenic in most patients. By activating multiple CD8+ CTL responses and CD4+ helper support, SurVaxM has a significant theoretical advantage as an active specific immunogen compared with survivin vaccines using a single class I-restricted peptide, or ones that incorporate generic helper peptides, which produce nonspecific helper support. Early indicators point to a strong rationale for continued study. A phase II clinical trial of SurVaxM is planned. Poster Section 24 Poster Board 2 CT302 Pharmacokinetics (PK) and safety of ARN-509 with abiraterone acetate (AA) and prednisone (P) in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). Edwin M. Posadas,1 Kim N. Chi,2 Ronald de Wit,3 Maja JA de Jonge,3 Gerhardt Attard,4 Terence Friedlander,5 Margaret Yu,6 Peter Hellemans,7 Caly Chien,8 Charlene Abrams,9 Martha Gonzalez,10 Géralyn C. Trudel,6 Vijay Chauhan,10 Fred Saad11. 1Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA; 2British Columbia Cancer Agency-Vancouver Cancer Agency, Vancouver, British Columbia, Canada; 3Erasmus MC- 32 Kanker Instituut, Rotterdam, Netherlands; 4The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom; 5Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA; 6Janssen Research & Development, Los Angeles, CA; 7Janssen Research & Development, Beerse, Belgium; 8Janssen Research & Development, Titusville, NJ; 9Janssen Research & Development, Spring House, PA; 10Janssen Research & Development, Raritan, NJ; 11University of Montréal, Montréal, Quebec, Canada. Background: ARN-509 and AA target the androgen axis via different mechanisms and may have complementary activity in mCRPC. ARN-509, a potent and selective androgen receptor (AR) antagonist, inhibits AR nuclear translocation and DNA binding without significant AR agonist properties (Clegg et al. Cancer Res. 2012). AA is the prodrug of abiraterone, which directly inhibits androgen biosynthesis. No overlapping toxicities are expected for the combination. This ongoing phase Ib study evaluates the potential PK drug-drug interaction and safety of ARN-509 in combination with AA + P. Methods: Pts had progressive mCRPC and ECOG score ≤ 2. Pts received AA (1000 mg/d) + P (5 mg BID) beginning on Cycle 1 Day 1 (C1D1) with the addition of ARN-509 (240 mg/d) on C1D8 in 28-day treatment cycles until disease progression or toxicity. Serial blood samples for PK analysis were collected on C1D7 and C2D8 for abiraterone analysis and on C2D8 for ARN-509 analysis. Primary objective: evaluate effect of ARN-509 on abiraterone PK. Secondary objective: evaluate safety of ARN-509 in combination with AA + P. Results: As of November 21, 2014, 28 pts have been enrolled. At baseline, the median age was 70 years (range: 49-83); median prostate-specific antigen was 56.8 µg/L (range: 4.1-2597.0 µg/L); bone, nodal, and visceral disease were present in 24 (86%), 17 (61%), and 8 (29%) pts; and 13 (46%) pts were pretreated with docetaxel, 11 (39%) with AA, and 12 (43%) with enzalutamide. 9 pts thus far completed 1 cycle, 6 completed 2 cycles, and 4 completed 3 cycles. 26 pts are continuing therapy. 10 pts were evaluable for PK assessment and 28 pts were evaluable for safety assessment. Most drug-related adverse events (AEs) were grade 1-2, and included fatigue (n = 5), diarrhea (n = 3), dysgeusia (n = 3), vomiting (n = 4), abdominal pain (n = 2), anorexia (n = 3), dyspepsia (n = 2), rash (n = 2) and nausea (n = 3). Grade 3 drug-related AEs included hyponatremia (n = 1), fatigue (n=1), and increased alanine aminotransferase (n = 1), and were managed by drug interruption and supportive measures. Interim data indicate a small reduction in abiraterone PK exposure when AA + P is coadministered with ARN509. PK of ARN-509 were consistent with historical data when ARN-509 was given as monotherapy. Conclusions: This ongoing phase Ib study (NCT02123758) indicates no clinically significant PK interaction between ARN-509 and AA + P. The combination is well tolerated in pts with mCRPC; interim AE data were consistent with those seen in the AA + P phase III trials (Fizazi et al. Lancet Oncol. 2012; Ryan et al. NEJM. 2013). These preliminary results justify further evaluation of the safety and efficacy of ARN-509 in combination with AA + P for mCRPC. Poster Section 24 Poster Board 3 CT303 A phase I pharmacokinetic and pharmacodynamic evaluation of the combination of everolimus and buparlisib for concurrent mTOR and PI3K pathway blockade in patients with advanced solid tumors. Taofeek Kunle Owonikoko, R.Donald Harvey, Colleen Lewis, Zhengjia Chen, John S. Kauh, Meredith Renfroe, Rijalda Deovic, Gabriel L. Sica, Bradley C. Carthon, Wayne Bernard Harris, Bassel F. El-Rayes, Suresh S. Ramalingam, Fadlo R. Khuri. Emory University, Atlanta, GA. Background: Preclinical work showed improved anticancer efficacy with the combination of an mTOR and a PI3K inhibitor over either agent alone. We conducted a phase I study to determine the Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 33 Clinical Trials Poster Session: Phase I Clinical Trials recommended phase II dose (RP2D) of the combination of everolimus (E), an mTOR inhibitor and buparlisib or BKM120 (B), a pan-PI3K inhibitor. Methods: Patients with advanced solid malignancies who have exhausted standard treatment options were enrolled. Main eligibility criteria include ECOG performance status 0-2, adequate end organ function and absence of glucose intolerance, uncontrolled hepatitis, anxiety or depression. Dose escalation was performed using a Bayesian Escalation with Overdose Control (EWOC) design to evaluate different doses of E (5mg or 10mg) and B (20, 40, 60 and 80 mg,) once daily continuously. Eligible patients were enrolled in cohorts of 3 patients. Pharmacokinetic (PK) assessment was conducted in cycle 1 on day 8 using peripheral blood samples collected at time 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours and prior to dosing on cycle 1 D day 15. Pharmacodynamic (PD) impact on mTOR/PI3K pathway modulation was evaluated in skin punch biopsies collected at baseline and at end of cycle 1. Results: We enrolled 35 patients: Median age 63yrs (range:4079), 21 females (58%); 2 Latinos (6%), 7 Blacks (20%) and 26 Caucasians (74%); with cancers of the lung (8), colorectal (7), sarcomas (3), salivary gland (3), breast (3), thyroid (3), thymic (2), bladder (2), ovarian (2), head and neck (1) and PNET (1). The safety of 6 different dose combinations of E and B (5/20; 10/20; 5/40; 5/60; 10/60; 5/80) was assessed. The most frequent toxicities were: hyperglycemia, diarrhea, nausea, fatigue and AST elevation. The dose limiting toxicities observed in 6 patients were: fatigue (3), hyperglycemia (1), mucositis (1), acute renal failure (1) and urinary tract infection (1). The 5/60 combination was defined as the RP2D. The median number of cycles completed was 2 (range: 0-20). Of the 25 patients evaluable for efficacy, 8 (32%) had disease progression while 17 (68%) achieved stable disease (SD). Median duration of SD was 18 weeks (range: 2-83) and 7 patients had SD lasting ≥6 months. Steady-state PK data for both agents in 24 evaluable patients showed no evidence of drug-drug interaction, with dose-normalized maximum concentrations (Cmax) and areaunder-the-curve (AUC0-∞) values for E and B in combination being comparable to single agent data. Preliminary signal of efficacy for the combination was observed in patients with thymic, breast and lung cancer. PD analysis is ongoing and will be presented at the meeting. Conclusion: The safety profile of the combination of everolimus plus buparlisib is well tolerated and the RP2D is 5mg/day and 60mg/day respectively on a continuous daily schedule. The efficacy data are encouraging and warrant further evaluation in phase II studies. Poster Section 24 Poster Board 4 CT304 Phase Ib trial of trastuzumab emtansine (TE) in combination with lapatinib (L) plus nab-paclitaxel (A) in metastatic HER2-neu overexpressed breast cancer patients: STELA trial results. Tejal Patel, Jaime Mejia, Angel Rodriguez, Jenny Chang. Houston Methodist Hospital, Houston, TX. Background: Multiple large randomized clinical trials have demonstrated that dual HER2 targeted therapies are synergistic and result in improved efficacy. We conducted a phase Ib trial of trastuzumab-emtansine (TE) and lapatinib (L), together with Nab paclitaxel (A) in patients with HER2 over-expressed stage IV breast cancer. Methods: Key inclusion criteria are stage IV HER2 positive breast cancer, LVEF ≥ 45%, and Peripheral neuropathy < grade 2. Primary phase Ib objective was to evaluate the maximum tolerated dose (MTD) of TE with L and A using a standard 3+3 dose deescalation design for up to 9 patients. Safety, tumor response and pharmacokinetics (PK) were also assessed. Dose limiting toxicities (DLTs) were defined as ≥ grade 3 non hematological toxicity attributed to the study drugs. Results: Nine patients, median age 47 (range 44-64) years were enrolled. All patients are currently off the study. The DLTs were grade 3 diarrhea (n=1) and grade 3 elevated transaminase (n = 1). Grade 3-4 hematological toxicities included neutropenia (N=6) and thrombocytopenia (N= 1). Other AEs included grade 1-2 mucositis (n = 2), diarrhea (N = 2), and liver function tests abnormality (N = 5). The 24 hour trough levels of each of the drug were within the range of the values reported in the literature. The MTD of TE, L and A was reached at: TE 3.0 mg/kg, Lapatinib 750mg and Abraxane (A) 80 mg/m2. Eight patients, with a median of 1 prior metastatic therapy (range 0-5) were evaluable for response. Five patients, 62.5% derived clinical benefit, including 1 patient with complete response and 4 patients with partial response. Conclusions: TE plus L and A therapy was well tolerated with antitumor activity observed. STELA Ib trial has been expanded to include twelve additional patients for safety and efficacy evaluation. Poster Section 24 Poster Board 5 CT305 Phase I studies of a selective cMet inhibitor AZD6094 (HMPL504/volitinib) in patients with advanced solid tumors. Ye Hua,1 Lin Shen,2 Hui Gan,3 Jason Lickliter,4 Michael Millward,5 Jianming Xu,6 Jian Wang,1 Yang Sai,1 Weiguo Su,1 Melanie M. Frigault,7 Chuan Qi1. 1Hutchison MedPharma, shanghai, China; 2 Beijing cancer hospital, China; 3Ludwig Institute for Cancer Research, Australia; 4Nucleus Network, Australia; 5University of Western Australia, Australia; 6The 307th hospital of chinese people’s liberation army, China; 7AstraZeneca, MA. Background: Volitinib is a selective oral small molecule inhibitor of cMet kinase, which has demonstrated potent in vivo inhibitory effects on a variety of human tumor xenografts. Methods: Two phase I dose-escalation studies have been conducted in Australia (AU) and China (CN) in parallel to determine the maximum tolerated dose (MTD) or phase II Recommended Dose (P2RD), to evaluate pharmacokinetics (PK) profile, and to assess antitumor activity of Volitinib. Treatment was given orally in 21-day cycles until disease progression or unacceptable toxicity. Results: By July 2014 both studies completed dose-escalation phase. A total of 61 patients were enrolled (40 in AU and 21 in CN). Patients were treated with daily (QD) volitinib from 100mg to 1000mg or twice daily (BID) from 300 mg to 600mg. Median age at baseline was 63 years, and 60% patients were male in the AU study; whereas median age was 53 years, and 57% patients were male in the CN study. In both studies, the most common treatment related adverse events included nausea, vomiting, fatigue, peripheral edema and decreased appetite, mostly of grade (G) 1/2. Four patients experienced 5 dose limiting toxicities (DLTs) in the AU study: 1 G3 abnormal liver function test at 600mg QD, 1 G3 fatigue at 800mg QD, and 2 G3 fatigues and 1 G3 headache at 1000mg QD. One DLT of G3 fatigue at 600mg BID was reported in the CN study. The MTD for the QD regimen was identified as 800mg whereas the MTD for the BID regimen had not been reached in either study. 500mg BID was determined to be the P2RD as monotherapy based on the favorable benefit/risk profile demonstrated in both studies. In the AU study, 2 patients in the 600mg QD cohort and 1 patient in 1000mg QD cohort achieved partial response (PR). All 3 responders were papillary renal cell carcinoma patients. Two of the 3 responders remain PR with volitinib treatment of approximately 10 and 18 months respectively by July 2014. One CRC patient at 600mg QD achieved a 29% tumor reduction. Tumor sample analysis showed that all responders had both MET gene copy number increase (Chr7 gains or Met gene amplification) and high MET protein expression. Volitinib was rapidly absorbed with Tmax around 2~4 hours and rapidly eliminated with half-life around 3~7 hours in both studies. Both Cmax and AUC were roughly dose-proportional up to 800 mg QD and 500 mg BID. No obvious accumulation was found after 21-day of continuous QD or BID dosing. Drug exposure did not show racial difference between Caucasian and Asian patients. Conclusions: Volitinib was well tolerated up to 800 mg QD and 600 BID with acceptable safety profile. 500mg BID was determined to be the P2RD as monotherapy. Preliminary efficacy data demonstrated promising anti-tumor activity in patients with Met gene copy number increase or high protein expression. Volitinib American Association for Cancer Research • AACR ANNUAL MEETING 2015 33 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 34 Clinical Trials Poster Session: Phase I Clinical Trials demonstrated linear PK profile without marked drug accumulation. Further clinical studies are warranted. Poster Section 24 Poster Board 6 CT306 Radiolabeled anti-PSMA antibody J591 immunotherapy is associated with favorable circulating tumor cell (CTC) count control in men with castration-resistant prostate cancer. Pravin Date, Beerinder S. Karir, Jaspreet S. Batra, Yuliya Jhanwar, Himisha Beltran, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Weill Cornell Medical College, New York, NY. Background: J591 is a monoclonal antibody that selectively binds to prostate-specific membrane antigen (PSMA). Prospective phase I and II trials with therapeutic radiolabeled 177Lu-J591 have shown favorable PSA decline rates. Additionally, our group has previously reported that non-invasive evaluation of PSMA expression by imaging with planar gamma camera imaging is associated with response and survival. Initial anecdotal experience showed declines in CTC counts which led to prospective study. Methods: Three clinical trials of 177Lu-J591 in men with metastatic CRPC have included prospective evaluation of CTC counts (CellSearch methodology) prior to and following therapy (single-dose 177Lu-J591, fractionated-dose 177Lu-J591, and fractionated-dose 177Lu-J591 + docetaxel). Based upon prior experience of J591-based therapy without an effector molecule as immunotherapy, and an anecdotal report of CTC count decline following a low (imaging) dose of J591 without an effector molecule, a cohort of patients who received J591 without 177Lu labeling was also retrospectively analyzed. Results: 44 pts unselected for PSMA expression received 177Lu-J591 therapy had prospectively measured CTC counts. 39 of 44 pts (88.6%) had imaging evidence of PSMA expression. 57% of pts had baseline unfavorable (≥ 5 cells/7.5 mL) CTC counts. Of overall evaluable pts, 72.4% had decline in CTC counts (7.5 to 100% decline), with 50% of those with unfavorable baseline counts converting to favorable counts at 4-6 weeks. 17.1% with undetectable CTC counts remained undetectable at follow up. 7 pts with mCRPC and elevated CTC counts who received low-dose (naked J591) prior to imaging were also identified, and 4 (57%) of these pts demonstrated CTC count decline. Conclusion: In addition to previously reported PSA declines, treatment with 177Lu-J591 is associated with favorable CTC count decline in men with metastatic CRPC. Infusion with unlabeled J591 may also lead to CTC count clearance. Based on this finding, we are planning a prospective clinical trial with unlabeled (naked) J591 in men with metastatic CRPC and unfavorable CTC counts. interval due to delayed cardiac repolarization increases the risk of developing a potentially fatal cardiac arrhythmia. Risk of trametinib inducing QT prolongation at supratherapeutic exposure was evaluated. (ClinicalTrials.gov NCT01658553). In this 2-wk study, pts with histologically/cytologically confirmed solid tumors received placebo on Day 1, followed by once-daily trametinib 2mg doses on Days 2-14. On Day 15 all pts received a single 3mg trametinib dose. The regimen was expected to achieve supratherapeutic dosing for QTc measurement on Day 15. ECG was monitored by 12-lead ambulatory continuous 24-hr Holter monitoring pre-study, and on Days 1 and 15. Pharmacokinetic (PK) and pharmacodynamic (PD) parameters were also measured. Thirty-two of 35 pts completed the study. There was no effect of trametinib compared to time-matched placebo on the change from baseline in QTcF (Table), QTcB or QTci. Mean AUC0-24 and Cmax following trametinib 2mg repeat doses were 364ng*hr/mL and 22.9ng/mL, respectively, consistent with those previously reported; the values for 3mg were 454ng*hr/mL and 29.2ng/mL. Median Tmax was approximately 2 hrs for both trametinib doses. There was no relationship between QTcF interval and trametinib plasma concentrations, as supported by statistical analysis and PK/PD modeling. Trametinib had no effect on ambulatory BP. 33/35 pts (94%) reported at least one AE; these were consistent with AEs previously reported. No ECG abnormalities were reported as AEs. This study showed no statistical difference between trametinib and placebo. Trametinib has no significant effect on QT prolongation at the supratherapeutic dose. Trametinib PK parameters were consistent with those previously reported. No new safety signals were seen. Poster Section 24 Poster Board 7 CT307 Phase I, placebo-controlled, patient (pt)-blinded study to evaluate the effect of repeat oral dosing of the MEK inhibitor trametinib on cardiac repolarization in pts with solid tumors. Drew Rasco,1 Amita Patnaik,1 Anthony W. Tolcher,1 Kyriakos P. Papadopoulos,1 Muralidhar Beeram,1 Glenda Chambers,1 Theresa L. Werner,2 John W. Bauman,3 Anita Scheuber,4 Donna Cox,4 YanYan Zhou,4 Mohammed Hamid,4 Daniel Schramek,4 Peggy Criscitiello,4 Sunil Sharma2. 1South Texas Accelerated Research Therapeutics (START), San Antonio, TX; 2Huntsman Cancer Institute – University of Utah, Salt Lake City, UT; 3GlaxoSmithKline, Research Triangle Park, NC; 4GlaxoSmithKline, Collegeville, PA. Trametinib is a reversible, selective inhibitor of the MAPKs, MEK1, and MEK2. Trametinib cardiotoxicity is an infrequent, but potentially life-threatening, adverse event (AE); elevated blood pressure (BP), and decreased left ventricular ejection fraction and heart rate have been seen with trametinib. Prolongation of the QT 34 Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 35 Clinical Trials Poster Session: Phase I Clinical Trials Poster Section 24 Poster Board 8 CT308 Pharmacokinetics and safety of vismodegib in patients with advanced solid malignancies and hepatic dysfunction. Ghassan K. Abou-Alfa,1 Lionel D. Lewis,2 Patricia LoRusso,3 Michael Maitland,4 Sravanthi Cheeti,5 Dawn Colburn,5 Sarah Williams,5 Brian Simmons,5 Richard A. Graham,5 Priya Chandra5. 1Memorial Sloan Kettering Cancer Center and Weill Cornell Medical College, New York, NY; 2Norris Cotton Cancer Center and The Geisel School of Medicine at Dartmouth, NH; 3Barbara Ann Karmanos Cancer Institute, MI; 4University of Chicago, IL; 5Genentech/Roche Inc., CA. Background: Vismodegib is an inhibitor of the hedgehog signaling pathway approved for treatment of advanced basal cell carcinoma. Pharmacokinetics (PK) and safety of vismodegib in patients (pts) with hepatic dysfunction are unknown. Methods: Pts with advanced solid malignancies and hepatic impairment (HI) were enrolled into 4 cohorts as defined by NCI Organ Dysfunction Working Group Criteria: normal (bilirubin [bili] < upper limit of normal [ULN]), mild (ULN<bili ≤1.5xULN), moderate (1.5xULN<bili ≤3xULN), and severe (3xULN<bili<10xULN) dysfunction. Pts received 150 mg of oral V/d for 8 days. Predose blood PK samples on Days 1, 3, 5, and 8 were collected; additionally on Day 8, serial blood and urine samples were collected up to 24 hours post-dose. vismodegib therapy was continued until disease progression, intolerable toxicity, or withdrawal of consent. Safety and tolerability were assessed throughout the study and up to 45 days after the last dose of vismodegib. Results: The average concentration of vismodegib in semen on Day 8 was 1.16 µM, approximately 7% of the average Css observed in plasma from these same male patients of the normal cohort (n=3) (Table 1; see next page). Best response among the 21 patients with HCC was 38% stable disease, 19% disease progression, and 43% not assessed. Conclusion: HI does not appear to impact vismodegib PK. However, the study is influenced by the high number of patients with HCC with advanced cirrhosis, rendering it difficult to draw any causality relationships between DLT and SAE, and vismodegib. This is confirmed by inability to deduce any correlation between Css values and AST or total bilirubin concentrations. Stable disease is reported in 38% of patients with HCC. Further study of vismodegib in pts with severe HI and HCC should be carefully considered within the context of the DLT and SAE events reported herein. Poster Section 24 Poster Board 9 CT309 Rapid evaluation of a novel small molecule cMet tyrosine kinase inhibitor in healthy subjects. A Dawn Millington,1 Sandra R. Chaplan,1 Zuleima Aguilar,1 Dennis M. Fisher,2 Jo Collier,3 Marielena Mata,4 Geert Mannens,5 Nico Goyvaerts,5 Vijay Peddareddigari,6 Chris Takimoto6. 1Janssen Pharmaceutical Research & Development, LLC, San Diego, CA; 2P Less Than, San Francisco, CA; 3Quotient Clinical Ltd., Nottingham, United Kingdom; 4Janssen Pharmaceutical Research & Development, LLC, Raritan, NJ; 5Janssen Pharmaceutical Research & Development, Beerse, Belgium; 6Janssen Pharmaceutical Research & Development, LLC, Springhouse, PA. Inhibiting cMet has potential therapeutic value in oncology. The Janssen WAVE Early Development unit, a team focused on efficient proof-of-concept evaluations, sought to rapidly assess the safety, pharmacokinetics, and pharmacodynamics of a novel, highly selective small molecule cMet TK inhibitor with a favorable preclinical safety profile. We conducted a placebo-controlled, randomized, double blind single ascending dose and multiple dose trial in 84 healthy males at Quotient Clinical LTD (UK). The trial design was adaptive in several respects: to expedite determination of an optimal formulation and to maintain sufficient systemic exposure to sustain target inhibition. In the single ascending dose phase, doses ranged from 6-350 mg, with early transition from a solution to capsule formulation at 18 mg. Two multiple dose cohorts received 60 mg twice daily for 7 days (inter-dose intervals of 4.5 and 12 hours). Target engagement biomarkers were plasma hepatocyte growth factor and soluble cMet. Evaluation of renal safety was of particular interest. Clinical evaluation, including both single ascending and multiple dose phases, was completed in only 6 months. All doses were safe and well tolerated. Pharmacokinetic analysis revealed doseproportional increases in exposure. Dose escalation was limited by concentrations of a major metabolite that approached the ceiling established in preclinical toxicology studies. Renal toxicity was not identified. Initial evaluation of this oncology drug in healthy subjects offered the advantages of rapid trial enrollment and completion, robust placebo comparison data, absence of concomitant illness or laboratory abnormalities, rapid dose escalation in a carefullymonitored, controlled setting, a thorough preliminary safety evaluation, and reduced costs. This approach, enabled by the acceptable preclinical safety profile of the compound, efficiently produced high quality clinical safety and pharmacokinetic data to support compound progression decisions. Poster Section 24 Poster Board 10 CT310 First-in-man (FIM) pharmacodynamic (PD) and pharmacokinetic (PK) phase I trial of PQR309 in advanced solid tumors. Andreas Wicki,1 Cristiana Sessa,2 Alexa Childs,3 Anastasios Stathis,2 Dagmar Hess,4 Markus Joerger,4 Roger von Moos,5 Jordi Rodon,6 Cinta Hierro,6 Natasa Cmiljanovic,7 Vincent Bize,8 Simona Berardi,8 Alexandros Xyrafas,8 Rebecca Kristeleit3. 1Univ. Hospital of Basel, Basel, Switzerland; 2Ospedale San Giovanni, Bellinzona, Switzerland; 3University College Hospital, London, United Kingdom; 4 Cantonal Hospital St. Gallen, St. Gallen, Switzerland; 5Cantonal Hospital Chur, Chur, Switzerland; 6Hospital Universitari Vall d’Hebron, Barcelona, Spain; 7Piqur Therapeutics, Basel, Switzerland; 8Swiss Group for Clinical Cancer Research, Bern, Switzerland. Background: PQR309 is a novel, oral, balanced pan-PI3K, mTORC1 and mTORC2 inhibitor. Preclinical experiments show promising anti-cancer activity. The primary objectives of this FIM trial of PQR309 were definition of maximal tolerated dose (MTD), and safety. Methods: The trial was designed as an accelerated 3+3 study. Patients with advanced solid tumors with no standard management options were eligible. Dose-limiting toxicity (DLT) was defined as either grade 4 neutropenia >7 days, febrile neutropenia, grade 4 thrombocytopenia, non-hematological toxicity of either grade 4, uncontrolled grade 3 >7 days or dose-limiting grade 2, or treatment delay >14 days. The DLT period was 21 days. Starting dose of PQR309 was 10mg once daily (OD) continuously, based on preclinical data and a No observed adverse effect level (NOAEL) of 4 mg/kg in non-rodent species. The predefined dose levels were 10, 20, 40, 80, 120mg adjusted by dose banding method to the patient weight. Additional flat doses at 80 and 100 mg were investigated. The highest dose reached with this schedule was 150mg OD. Toxicities were assessed throughout dose escalation. 21 patients were included in the trial. Results: No DLT has been observed to date. Drug-related adverse events (AE) included hyperglycemia, rash, loss of appetite, weight loss, nausea, diarrhea, and fatigue. The most common AE ≥ grade 3 was hyperglycemia in 5 patients. Blood sugar levels were manageable with oral antidiabetic drugs or insulin. Preliminary PK evaluation estimates a half-life of about 20 hours. Cmax and AUC show dose proportionality. Preliminary PD assessment of 16 phospho-proteins involved in PI3K and MAPK signaling in freshfrozen tumor biopsies, indicates downregulation of p-Akt, pS6 and p4EPB from 40mg OD. In addition, moderate inhibition of p-Erk was also observed. This correlates with preclinical data. Preliminary signs of clinical anti-cancer activity include 1 patient with clear cell cancer of the Bartholin’s gland experiencing stable disease for over 16 weeks. No DLT has been observed up to doses of 150mg OD. Based on these data, further dose escalation in this study will continue with intermittent dosing schedules. Trials of PQR309 in combination with other anticancer agents are planned. American Association for Cancer Research • AACR ANNUAL MEETING 2015 35 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 36 Clinical Trials Poster Session: Phase I Clinical Trials Poster Section 24 Poster Board 11 CT311 Results of a phase I/IIa dose-escalation study of the combination of decitabine and genistein against advanced solid tumors. Noël J. Raynal,1 Elie Kassouf,2 Luc Daigneault,3 Patrick Colin,4 Isabelle Plante,5 Guylaine Lassonde,5 Richard Momparler,4 Michel Charbonneau,5 Normand Blais2. 1Université de Montréal / Sainte- 36 Justine Research Center, Montréal, Quebec, Canada; 2Notre-Dame Hospital, Montréal, Quebec, Canada; 3Scimega, Montréal, Quebec, Canada; 4Université de Montréal, Montréal, Quebec, Canada; 5 INRS-IAF, Laval, Quebec, Canada. Background: Epigenetic aberrations, including DNA hypermethylation of tumor suppressor genes are a common feature in solid tumors. Thus, targeting epigenetic pathways is a promising Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 37 Clinical Trials Poster Session: Phase I Clinical Trials approach for cancer chemotherapy. However, only occasional proof-of-principle responses can be seen in solid tumors. The activity of hypomethylating drugs, such as decitabine could be improved by a treatment combination. Preclinical studies have suggested that adding genistein, a natural isofavone may synergize with demethylating drugs and improve treatment results. Methods: Here, we describe a phase I/IIa dose-escalation study of decitabine with a fixed dose of genistein. Primary objective of the phase I trial was to determine the Maximum Tolerated Dose (MTD) and to evaluate pharmacokinetic parameters in patients with advanced solid tumor. The objective of the phase IIa was to assess its efficacy in lung cancer. Decitabine was administered over 10hours at increasing doses (60, 120, 240 mg/m2) with continuous administration of genistein 300 mg/day orally. Treatment was repeated every 4 to 6 weeks. Results: For the phase I, ten patients with advanced solid tumors were recruited. The recommended dose of decitabine (for the phase IIa) was 120 mg/m2 with neutropenia as limiting side effect. Decitabine at 120 mg/m2 and genistein produced plasma levels of 0.62 ± 0.06 µM and 8.5 ± 5.6 µM, respectively. The drug combination was well tolerated and produced stable disease for more than 7 months (7-14 months) in 50% of the patients. One patient had a 50% reduction in tumor burden after 6 months of therapy. In the phase IIa trial against advanced lung cancer, patients progressed rapidly, within 2 months of treatment mainly because of the apparition of new lesions in the liver. Conclusion: The combination of decitabine and genistein is well-tolerated in advanced solid tumors and shows interesting anticancer effects. This study on decitabine and genistein warrants future investigations to address this issue of liver metastasis in advanced lung cancer. Poster Section 24 Poster Board 12 CT312 Preliminary results of a phase I clinical trial with the Delta24RGD oncolytic adenovirus administered by CED in patients with recurrent glioblastoma. Erik P. van Putten,1 David Noske,2 Anne Kleijn,1 Daphna Hoefnagel,1 Sander Idema,2 Arnold Vulto,1 Anna L. de Goede,1 Winald Gerritsen,2 David Curiel,3 Martin Schutten,1 Rene Vernhout,1 Victor W. van Beusechem,2 Martine LM Lamfers,1 Clemens MF Dirven1. 1 ErasmusMC, Rotterdam, Netherlands; 2VU Medical Center, Amsterdam, Netherlands; 3Washington University, St. Louis, MO. A phase I trial testing the oncolytic adenovirus Delta24-RGD in recurrent glioblastoma patients was initiated in 2010 and 20 patients have been treated with escalating doses of the virus. Delta24-RGD is a double mutated serotype 5 adenovirus. It harbors a deletion of 24 bp that restricts viral replication to cells with deregulated Rb pathway, and has an expanded tropism by insertion of an RGD peptide in the fiberknob, enabling viral entry via alpha v integrins. This virus was administered in patients with recurrent Glioblastoma after failure of standard and often second line treatments. The virus was delivered by Convection Enhanced Delivery (CED), which consisted of prolonged microinfusion through up to 4 catheters in and around the tumor over a period of 2 to 3 days. Doses of 1 x 10e7 to 1 x 10e11 were scheduled in 6 cohorts of 3 patients each. Data on toxicity, adverse events, and survival were collected. The MTD was reached at a dose of 1 x 10e10 viral particles. Dose limiting toxicities with respect to patient safety were not encountered. Viral titers in CSF support the occurrence of prolonged viral replication in the tumor. Poster Section 24 Poster Board 13 CT313 Phase Ib trial of dodecafluoropentane as a radiation sensitizer during chemoradiation for glioblastoma. Jason Lickliter,1 Jeremy Ruben,2 Olivia Longacre,3 David Wilson,3 Evan Unger3. 1Nucleus Networks, Melbourne, Australia; 2The Alfred, Melbourne, Australia; 3NuvOx Pharma, Tucson, AZ. Purpose: Using post-resected glioblastoma multiforme (GMB) patients to evaluate the pharmacokinetics, safety and potential survival benefit of Dodecafluoropentane emulsion (DDFPe) in combination with the current standard of care; radiation therapy (RT) and temozolomide (TMZ). Glioblastoma multiforme (GBM) is known to be a hypoxic tumor. Tumor hypoxia limits response to RT. DDFPe is an oxygen therapeutic (OT) which transports more than 100x more oxygen as other tested fluorocarbons OT’s. Studies in tumor xenografts show that DDFPe increases tumor pO2 by up to 400% and mitigates radiation resistance. Methods: Previously untreated GBM patients with postresected residual tumor visible on MRI post surgery were enrolled. Patients received standard RT (60 Gray in 30 fractions over 6 weeks) with concurrent and adjuvant TMZ. In addition, DDFPe was infused intravenously over 30 minutes immediately prior to each fraction of RT while patients simultaneously breathed carbogen (98%O2:2% CO2). DDFPe was dosed using an accelerated titration design with 1 patient per dose level. Blood samples were drawn to evaluate DDFP pharmacokinetics. Objective tumor responses were assessed with gadolinium-enhanced MRI scans and RANO criteria, and tissue oxygen level dependent (TOLD) MRI was employed as an exploratory biomarker of tumor oxygenation. Results: Two patients have been enrolled to date. The first patient received DDFPe at 0.05 cc/kg and completed chemoradiation and 2 cycles of adjuvant TMZ with dose reduction or delay. Therapy was well tolerated except for transient grade 3 ALT elevation during recovery from chemoradiation. A postradiation MRI scan showed stable disease. The second patient has completed a regimen of chemoradiation in combination with DDFPe at 0.1 cc/kg. Conclusions: DDFPe administration prior to each radiation fraction during standard chemoradiation of GBM is feasible. Further dose escalation od DDFPe is planned to establish a maximum tolerated dose and recommended dose for an envisioned Phase IIb multi-center GBM trial of DDFPe in combination with the accepted standard of care. Poster Section 24 Poster Board 14 CT314 Interim results from a phase I/II trial of TPI 287, a novel brain penetrable antimicrotubule agent, in combination with bevacizumab for the treatment of recurrent glioblastoma. Sandra L. Silberman,1 Samuel Goldlust,2 L. Burt Nabors,3 J Paul Duic,4 Tara Benkers,5 Nimish Mohile,6 Donald Picker,1 Samuel Singer,2 George Farmer1. 1Cortice Biosciences, Inc., New York, NY; 2 John Theurer Cancer Center, Hackensack University, Hackensack, NJ; 3University of Alabama at Birmingham, Birmingham, AL; 4 Neurological Surgery, P.C., Rockville Center, NY; 5Swedish Medical Center, Seattle, WA; 6University of Rochester, Rochester, NY. Background: TPI 287 is a novel anti-microtubule agent designed to evade inactivation and efflux by multiple drug resistance pathways, which enables meaningful penetration of the blood-brain barrier. Preclinical results demonstrate CNS accumulation at pharmacologically relevant concentrations and significant anti-neoplastic activity in a murine model of brain metastases (Fitzgerald, et al Mol Can Ther 2012 11:1959-67). Safety data from clinical trials enrolling over 200 cancer patients have shown TPI 287 to be well tolerated. Based on these data, a doseescalation phase I/II trial was designed to determine the maximum tolerated dose (MTD) and efficacy of TPI 287 for recurrent glioblastoma (GBM) in combination with the angiogenesis inhibitor bevacizumab. Interim results from the dose escalation portion of this trial are reported. Methods: GBM patients at first or second relapse after failure of standard chemoradiation and without prior exposure to angiogenesis inhibitors are eligible. Standard-of-care bevacizumab is administered at 10 mg/kg as an IV infusion once every 2 weeks. TPI 287 is administered as an IV infusion every 3 weeks. Employing a 3+3 design, dose escalation of TPI 287 is ongoing until determination of MTD, followed by transition to the randomized phase II stage of the trial. MRIs are obtained every six-weeks with American Association for Cancer Research • AACR ANNUAL MEETING 2015 37 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 38 Clinical Trials Poster Session: Phase I Clinical Trials response assessment via RANO criteria. Results: As of December 2014, 16 subjects have been enrolled in the first 5 TPI dose-escalation cohorts (140-180 mg/m2). No dose-limiting toxicities have been observed to date. Of 12 patients evaluable for safety, myelosuppression has been the only Grade 3 adverse event attributed to treatment (2 patients). Among the 13 patients evaluable for efficacy, there were 5 confirmed objective responses (1 CR; 4 PR) and 2 responses awaiting confirmation (1 CR; 1 PR). Durability of the confirmed responses has ranged from 5.5 to 8.2+ months. Conclusions: These interim results demonstrate that TPI 287 in combination with bevacizumab is well tolerated. Moreover, efficacy data is promising, with a high rate of durable responses achieved below the MTD. This study also highlights the power of translational studies for evaluating effective cancer therapy. Evolving safety and efficacy results will be presented. Poster Section 24 Poster Board 15 CT315 Phase I study of BPM 31510 (ubidecarenone) in patients with advanced solid tumors. Manish A. Shah,1 Peter Yu,2 Niven Narain,3 Rangaprasad Sarangarajan,3 Michael Kiebish,3 Vivek Vishnudas,3 Yezhou Sun,3 Leonardo Rodrigues,3 Viatcheslav R. Akmaev,3 Susan Brouwer,3 Janice Stevens,3 Ely Benaim,3 Ralph Zinner4. 1Weill Cornell Medical College, New York, NY; 2Palo Alto Medical Foundation, Sunnyvale, CA; 3Berg, Framingham, MA; 4The MD Anderson Cancer Center, Houston, TX. Background: BPM 31510 is a novel small molecule that targets the metabolic machinery of the cancer microenvironment to create a hallmark shift from lactate dependency towards mitochondrial oxidative phosphorylation, reversing the Warburg effect. Preclinical data indicates Ubidecarenone causes this shift resulting in tumor regression and enhances the antitumor activity in combination with chemotherapy agents in a priming schedule. This is the first clinical study to evaluate the BPM 31510 at a 4-days continuous infusion in four arms; as a single agent, and in combination with Gemcitabine, 5-FU or Docetaxel. Methods: Eligible patients (pts) (aged ≥18 y) had previously treated relapsed/refractory solid tumors. Pts in the monotherapy arm received IV BPM 31510 for 4 days in continuous infusion in 28d cycles. Patients in the combination arms were primed for 3 weeks with BPM 31510 and then started in a weekly dosing (either gemcitabine, 5-FU or docetaxel) after the BPM 31510 infusion in a 6-week cycle. Doses were escalated in a 3+3 schema. Phase I endpoints were safety, pharmacokinetics (PK) and Multi-Omics based pharmacodynamics (PD). Dose limiting toxicities (DLTs) are determined using Cycle 1 safety data. Tumor response is evaluated at week 2 and every 4 -6 weeks. Results: As of 01 Dec 2014, 56 pts with advanced solid tumors have been enrolled. Pts have been treated at 3 dose levels up to 137 mg/kg of BPM 31510. No DLTs or study treatment-related SAEs have been reported. The MTD has not yet been established. The most frequently reported related AEs in all 4 arms were grade 1-2 INR prolongation that was resolved after Vitamin K administration. Pre-load of pts with Vitamin K have resolved these events. No bleeding reported. Grade 1-2 thrombocytopenia has been seen in the Gemcitabine arm requiring dose modification. Preliminary PK data indicated linear distribution. Tmax and Cmax are associated with the end of the infusion. Twelve out of twenty five patients (48%) that are evaluable for efficacy after cycle 2 showed various responses including: tumor reductions, decrease FDG, arrested tumor progression, stable disease, decrease in tumor markers, clinical improvements reflected on QOL. Conclusions: Emerging data from this study suggest that BPM 31510 is well tolerated in monotherapy or in combination with chemotherapy agents. Early anti-tumor activity is seen. Doseescalation on a 6-day infusion schedule is ongoing to determine the recommended phase II dose. 38 Poster Section 24 Poster Board 16 CT316 A phase I trial of oral TRC102 (methoxyamine HCl) in combination with temozolomide (TMZ) in patients with relapsed solid tumors. Woondong Jeong,1 Khanh Do,1 Alice Chen,1 Jennifer Zlott,2 Lamin Juwara,3 Yvonne Horneffer,1 Robert Kinders,3 Lihua Wang,3 Priya Balasubramanian,3 Larry Anderson,1 Elad Sharon,1 Howard Streicher,1 Richard Piekarz,1 Barbara Conley,1 Jerry Collins,1 James H. Doroshow,1 Shivaani Kummar1. 1Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD; 2Center for Cancer Research, National Cancer Institute, Bethesda, MD; 3 Leidos-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD. Background: Base excision repair (BER), one of the pathways of DNA damage repair, has been implicated in chemoresistance. TRC102 acts through a novel mechanism to inhibit BER and cause DNA strand breaks, potentiating the antitumor activity of TMZ in preclinical models. We conducted a phase I trial of TRC102 in combination with TMZ to determine the safety, tolerability, and maximum tolerated dose of the combination; to evaluate the pharmacokinetics (PK) of each agent alone and in combination; and to assess DNA damage response (percent nuclear area of γH2AX foci) in circulating tumor cells (CTCs). Methods: Eligible pts were required to have refractory advanced solid tumors that had progressed following standard therapy; ≥ 18 yrs of age; ECOG PS 0-2; and adequate organ function. TRC102 and TMZ were administered orally once daily, D15 of q28d cycles; Starting dose level (DL 1) was TRC102 25 mg and TMZ 125mg/m2. Accrual to DL6 (TRC102 125 mg; TMZ 150 mg/m2) is ongoing. CTCs were obtained during C1 and on C2D1. Blood samples for PK analysis were obtained during C1. Results: Twenty pts have been enrolled to date; median age 59 yrs (range 45-78 yrs); median # of prior therapies: 3.5 (1-9); Dx: GI (6), H&N (4), breast (3), GYN (3), lung (2), soft tissue sarcoma (2). Fourteen pts are evaluable for response; 2 partial responses by RECIST have been observed to date (≥ 6 cycles; NSCLC and granulosa cell tumor of the ovary). Grade 3/4 toxicities (#pts): neutropenia (2), thrombocytopenia (1), lymphopenia (1), anemia (1), leucopenia (1), hypophosphatemia (1). PK in combination was similar to single agent PK reported for both drugs, with no evidence of a PK interaction. TRC102 levels required for preclinical activity (50ng/mL) were achieved at DL1. T1/2 of TRC102 was 26 hr. CTC analysis is ongoing. Conclusions: Combination of TRC102 with TMZ is well tolerated and clinical activity was observed with 2 partial responses to date. MTD has not been reached; accrual is ongoing. Paired tumor biopsies to assess for evidence of DNA damage response and apoptosis are planned at the MTD in the expansion phase. Poster Section 24 Poster Board 17 CT317 Quantitative ultrasound for personalized chemotherapy in locally advanced breast cancer: Clinical trial results. Gregory Jan Czarnota, Ali Sadeghi-Naini, Hadi Tadayyon, Lakshmanan Sannachi, Mehrdad Gangeh, William Tran, Frances Wright, Sonal Gandhi, Kathleen Pritchard, Sunil Verma, Maureen Trudeau. Univ. of Toronto Sunnybrook HSC, Toronto, Ontario, Canada. Many cancer therapies are intended to induce cell death within a target tumor. A substantial body of research using in vitro and in vivo models has demonstrated that cell death can be detected via quantitative ultrasound techniques. This study investigated the potential to quantify tumor responses to therapy in patients, using quantitative spectral and textural biomarkers extracted from lowfrequency ultrasound data (4-10 MHz). Results demonstrate for the first time in a large cohort of patients the ability to predict clinical responders from non-responders as early as one week after the start of chemotherapy with over 95% sensitivity and 95% specificity. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 39 Clinical Trials Poster Session: Phase I Clinical Trials A clinical study was undertaken investigating the efficacy of ultrasound to quantify cell death in tumor responses with cancer treatment. Patients (n=100) with locally advanced breast cancer received anthracyline and taxane-based chemotherapy treatments over four to six months. Data collection consisted of acquiring tumor images and radiofrequency data prior to treatment onset and at 4 times during neoadjuvant chemotherapy (weeks 0, 1, 4, 8 and pre-operatively). Data collection was carried out using an Ultrasonix-RP and an L15-5 6cm transducer pulsed at frequencies of ~5 and ~7 MHz, respectively. The majority of patients went on to have a modified radical mastectomy and correlative whole mount histopathology. Results obtained from both ~5 and ~7 MHz data indicated considerable increases in ultrasound spectral backscatter power in patients who clinically responded to treatment within one week of starting their chemotherapy. This was accompanied by significant increases in quantitative ultrasound spectral parameters such as mid-band-fit (up to 9.1 ± 1.2 dBr) and 0-MHz intercept (up to 10.8 ± 2.4 dBr). Patients categorized as poor responders clinically demonstrated significantly lower increases (1.9 ± 1.1 dBr and 1.4 ± 2.7 dBr for mid-band-fit and 0-MHz intercept, respectively). Textural biomarkers extracted from quantitative ultrasound spectral parametric maps also demonstrated considerable differences in trend between treatment responding and non-responding patients early after the treatment initiation. Statistically significant differences were found between treatment responding and non-responding patient populations using quantitative ultrasound spectral biomarkers 4 and 8 weeks after treatment initiation. Applying quantitative ultrasound textural biomarkers in order to incorporate response heterogeneities resulted in statistically significant differences between these two populations only one week after the start of chemotherapy. There were also associated survival differences between the two groups of patients. Patients with ultrasound-detected responses at week 1 had an over 90+/-5% 3 year survival whereas patients with no ultrasound-detected response at the same time had a 30+/10% survival (p<0.05). This study demonstrates the potential of ultrasound to quantify changes in tumors in response to cancer treatment administration in a clinical setting. The results indicate that such responses can be detected early during a course of chemotherapy. This can potentially permit ineffective treatments to be changed to more efficacious ones potentially leading to improved treatment outcomes within a framework of personalized medicine. Poster Section 24 Poster Board 18 CT318 Evaluation of salivary transcriptome markers for early detection of squamous cell cancer in a prospective blinded trial. Neil Gottehrer, Jack Martin. PeriRx, Havertown, PA. Oral squamous cell cancer (OSCC) is often diagnosed in late stages. Informative biomarkers could play a key role in early diagnosis. Prior case-control studies identified discriminatory salivary mRNA markers for OSCC. The National Cancer Institute (NCI) recommends prospective-specimen-collection, retrospectiveblinded-evaluation (PRoBE) design study for rigorous biomarker identification and validation. A PRoBE design study enrolled 170 patients with lesions suspicious for OSCC. Saliva was collected before performing oral biopsy. Six mRNAs (IL1β, IL8, OAZ1, SAT1, S100P, and DUSP1) were measured by quantitative polymerase chain reaction (PCR) without knowledge of tissue diagnosis. A pre-specified multi-marker panel from prior NCI - Early Detection Research Network (EDRN) studies was evaluated in this new PRoBE dataset. Individual marker cycle thresholds (Ct) from PCR were also compared in cancer versus control and new discriminatory models were generated. The EDRN model was validated based on pre-specified statistical analysis plan. Ct values of individual mRNAs reflect an approximately two to nearly fourfold increase in concentration in invasive OSCC (p<0.01 for all). A new model from this intended use population demonstrates a maximal sum of sensitivity and specificity of 153.5% with an area under the receiver operating characteristic (ROC) curve of 0.81. The validation of six pre-specified individual salivary transcriptome markers of OSCC and a pre-specified multi-marker model in a new prospective population, support the robustness of these markers and the multi-marker methodology. New models generated in this intended use population have the potential to further enhance the decision process for early biopsy. Trial ID: NCT01587573 Poster Section 24 Poster Board 19 CT319 Determination of FGF-19, 21, and 23 protein levels in a phase I clinical trial of ARQ 087, an oral pan-FGFR inhibitor. Terence Hall, Julia Kazakin, Brian Schwartz, Ronald Savage. ArQule, Woburn, MA. Background: Early biomarker development is vital in the current clinical landscape of targeted oncology therapies. Biomarkers that can be used to assess target engagement are increasingly being developed in parallel with novel agents. Fibroblast growth factors (FGFs) represent a potentially useful biomarker for use with pan-FGFR inhibitors such as ARQ 087. To evaluate this possibility, levels fibroblast growth factors 19, 21, and 23 were monitored using during the phase I trial of ARQ 087. Study Design: Plasma was collected from 61 pts (dose range 25-425mg QOD or QD). FGF levels were quantified using commercially available ELISA kits for FGF-19 and 21 (R&D Systems, Minneapolis, MN), and FGF-23 (EMD-Millipore, Billerica, MA). The kits were validated at ArQule for use with human clinical samples. Experiments were designed based on the FDA guidance for bioanalytical method development, and conducted using standard curves and quality control samples prepared in plasma. Performance parameters included selectivity, specificity, precision, accuracy, stability, and effect of sample collection tube additives on measured protein levels. Results: The kits were successfully validated for all three FGFs. Changes from baseline levels of all three FGFs were observed in patients. Full data tables and results from our evaluation, along with clinical and preclinical data will be presented. Conclusions: Although there are previous reports of altered FGF-23 levels in patients treated with an FGFR inhibitor, FGF-19 and 21 represent novel biomarkers, which have not previously been reported in the literature. These biomarkers may assist in identifying ARQ 087 activity in patients, and contribute to defining an optimal clinical development strategy. Poster Section 24 Poster Board 20 CT320 An open-label, multicenter, phase Ib study of daratumumab in combination with backbone regimens in patients with multiple myeloma. Raymond Comenzo,1 Philippe Moreau,2 Maria-Victoria Mateos,3 Joan Bladé,4 Lotfi Benboubker,5 Javier de la Rubia,6 Thierry Facon,7 Joseph Fay,8 Xiang Qin,9 Tara Masterson,9 Jordan Schecter,10 Tahamtan Ahmadi,9 Jesus San-Miguel11. 1Tufts Medical Center, Boston, MA; 2University Hospital of Nantes, Nantes, France; 3 University Hospital of Salamanca/IBSAL, Salamanca, Spain; 4 IDIBAPS, Hospital Clinic de Barcelona, Barcelona, Spain; 5CHU Tours Hopital Bretonneau, Tours, France; 6Doctor Peset and Universidad Católica “San Vicente Mártir”, Valencia, Spain; 7Centre Hospitalier Régional Universitaire de Lille, Lille, France; 8Baylor Institute for Immunology Research, Dallas, TX; 9Janssen Pharmaceutical Research & Development, Spring House, PA; 10 Janssen Pharmaceutical Research & Development, Raritan, NJ; 11 Clinica Universidad de Navarra, Pamplona, Spain. Daratumumab (DARA) is a human anti-CD38 IgG1κ MAb in development for multiple myeloma (MM). DARA has shown single agent efficacy and manageable safety in patients with MM relapsed from or refractory (RR) to ≥2 prior therapy lines as well as in American Association for Cancer Research • AACR ANNUAL MEETING 2015 39 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 40 Clinical Trials Poster Session: Phase I Clinical Trials combination with lenalidomide (LEN) and dexamethasone (D) in relapsed or RR MM. This ongoing open-label, 4-arm, multicenter, phase Ib study evaluated safety and tolerability of DARA combined with other MM backbone treatments: bortezomib-dexamethasone (VD), bortezomib- thalidomide-dexamethasone (VTD), bortezomibmelphalan-prednisone (VMP), pomalidomide-dexamethasone (POM-D). Newly diagnosed patients will be included in the VD (n = 6) and VTD (n = 12) arms (irrespective of transplant eligibility) and VMP arm (transplant ineligible; n = 12). Patients (up to 50) in the POM-D arm are RR to ≥2 lines of therapy including 2 consecutive cycles of LEN and V. Patients were treated with DARA 16 mg/kg and the approved label or standard of care of each backbone treatment: VD and VTD (DARA qw, 2 cycles; q3w, 16 cycles); VMP (DARA qw, 1 cycle; q3w, 8 cycles); POM-D (DARA qw, 2 cycles; q2w, 4 cycles; q4w remaining cycles). An independent data safety monitoring board evaluated safety and clinical responses. Data from 25 subjects with newly diagnosed (VD [n = 6], VTD [n = 6], and VMP [n = 6]) and RR MM (POM-D [n = 7 safety; n = 6 efficacy]) are presented. There was no additional toxicity when DARA was added to backbone therapy, other than DARA-specific infusion related reactions (IRR). There were 11/25 subjects with IRRs; nearly all occurred on Cycle 1 Day 1, were grade 1 or 2 in severity, and resolved with supportive treatment. Median (range) numbers of DARA infusions were: VD, 9 (8-11); VTD, 8 (7-11); VMP, 12.5 (10-14); POM-D, 11 (1-17). All other adverse events (AEs) were consistent with those associated with the backbone agents. There were 4 serious (S) AEs (pneumonia, soft tissue infection, pre-renal failure [not DARA-related], indirect Coombs test interference [possibly DARA-related]) in the VTD arm and 1 SAE (infectious pneumonia [possibly DARA-related]) in the POM-D arm. Most common AEs were hematologic toxicity, likely related to backbone therapy. The overall response rates were 100% in the VD, VTD and VMP arms and 50% in the POM-D arm. In the VD arm there were 3 very good partial responses (VGPR) and 3 partial responses (PR). In both the VMP and VTD arms there were 1 VGPR and 5 PRs. In the POMD arm there was 1 stringent complete response, 2 VGPRs, 2 minimal responses and 1 patient with progressive disease. Median (range) times to first responses were: VD, 23.5 (22-44) days; VTD, 22 (22-43) days; VMP, 32.5 (22-135) days; POM-D, 31 (29-57) days. In VTD/VD patients who discontinued for ASCT mean stem cell yield was 6.25 x 106 CD34+ cells/kg. DARA plus backbone MM therapies was well tolerated, with no significant additional toxicity and encouraging efficacy. The study is ongoing. Poster Section 24 Poster Board 21 CT321 First results of a 10-day regimen of SGI-110 (guadecitabine), a second generation hypomethylating agent (HMA) in previously untreated elderly AML who are not candidates for intensive chemotherapy. Gail Roboz,1 Hagop Kantarjian,2 Patricia Kropf,3 Ellen Richie,1 Nitin Jain,2 Elizabeth Griffiths,4 Nikola A. Podoltsev,5 Katherine Walsh,6 Casey O’Connell,7 Wendy Stock,8 David Rizzieri,9 Raoul Tibes,10 Todd Rosenblat,11 Woonbok Chung,12 Pietro Taverna,13 Xiang Yao Su,13 Sue Naim,13 Mohammad Azab,13 Jean-Pierre Issa12. 1Weill 40 Cornell Medical College, New York, NY; 2The University of Texas MD Anderson Cancer Center, Houston, TX; 3Fox Chase Cancer Center, Philadelphia, PA; 4Roswell Park Cancer Institute, Buffalo, NY; 5Yale University School of Medicine, New Haven, CT; 6The Ohio State University, Columbus, OH; 7University of Southern California, Keck School of Medicine, Los Angeles, CA; 8The University of Chicago Medical Center, Chicago, IL; 9Duke University Medical Center, Raleigh, NC; 10Mayo Clinic Arizona, Scottsdale, AZ; 11New YorkPresbyterian/Columbia University Medical Center, New York, NY; 12 Fels Institute, Temple University, Philadelphia, PA; 13Astex Pharmaceuticals, Inc., Pleasanton, CA. Background: SGI-110 (guadecitabine) is a novel HMA that prolongs in vivo exposure of the active moiety decitabine following subcutaneous (SC) administration. We previously reported guadecitabine clinical activity in previously untreated elderly AML with Overall Complete Response (CR+CRi+CRp) of 55% using a standard 5-day regimen (Yee et al, 2014). Here we report the first results of a more intensive 10-day regimen in the same patient population. Methods: Elderly AML patients (≥ 65y) with at least one of the following characteristics were enrolled: age ≥ 75y; poor Performance Status (PS) ≥ 2; significant comorbidities particularly cardiopulmonary dysfunction; poor risk cytogenetics; or secondary AML. Guadecitabine 60 mg/m2/d SC was administered for 10 days (Days 1-5; and 8-12) for the first 1-2 cycles followed by the 5-day regimen (Days 1-5) for subsequent cycles. Cycles were scheduled every 28 days. Primary endpoint was Overall Complete Response (Overall CR). Secondary endpoints included Overall Survival, Safety including all-cause 30-day and 60-day early mortality, and global maximum DNA demethylation from baseline by LINE-1 assay. Results: Fifty two patients were treated with median age 77 (range 66-92) including 40 (77%) ≥75y; 34 (65%) male; 21 (40%) PS ≥ 2; 19 (36%) poor risk cytogenetics; and 13 (25%) secondary AML. The median bone marrow blasts percentage was 49% (range 1698%). All patients had a minimum follow up of 3 months; 26 (50%) patients remain on treatment at the time of the analysis. Overall CR was observed in 24 patients (46%):14 CR (27%), 8 CRi (15%), and 2 CRp (4%). There was no significant difference between median DNA demethylation in responders (- 27%) and non-responders (28%). Early all-cause 30-day and 60-day mortality occurred in 2 (4%) and 10 (19%) patients respectively. The most common Grade ≥3 Adverse Events considered by investigators to be drug-related were febrile neutropenia (33%), thrombocytopenia (27%), neutropenia (21%), anemia (15%), bacteremia (10%), and pneumonia (6%). Conclusions: Administration of guadecitabine as a more intense 10-day regimen for the first 1-2 cycles was active with acceptable toxicity. However, and although it was not a randomized comparison, the 10-day regimen did not result in higher rate of Overall CR in previously untreated AML than what was previously reported for the 5-day regimen. A Phase III randomized clinical trial in previously untreated AML patients not considered candidates for intensive chemotherapy has been initiated with the 5-day regimen of guadecitabine. Reference: Yee K, Daver N, Kropf P, et al: European Hematology Association Meeting, June 12-15, 2104; Milan, Italy. Abstract S647. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 41 Clinical Trials Plenary Session: Clinical Trials Using PARP Inhibitors Clinical Trials Plenary Session Tuesday, April 21, 2015 10:30 AM-12:40 PM Room 120, Pennsylvania Convention Center Clinical Trials Using PARP Inhibitors Co-Chairpersons: Geoffrey I. Shapiro, Dana-Farber Cancer Institute, Boston, MA; Yves G. Pommier, National Cancer Institute Center for Cancer Research, Bethesda, MD The complete text of the abstracts in this session will be posted to the online Proceedings after presentation. 10:30 AM CT322 DNA repair defects and antitumor activity with PARP inhibition: TOPARP, a phase II trial of olaparib in metastatic castration resistant prostate cancer (mCRPC). Joaquin Mateo,1 Shahneen Sandhu,1 Susana Miranda,2 Suzanne Carreira,2 Suneil Jain,3 Christy Ralph,4 Andrew Protheroe,5 Syed Hussain,6 Robert Jones,7 Tony Elliot,8 Ursula McGovern,9 Alexa Gillman,10 Claire Paulding,10 Helen Mossop,10 Nuria Porta,10 Diletta Bianchini,1 Zafeiris Zafeiriou,1 Gunther Boysen,2 Daniel Nava Rodrigues,2 Penelope Flohr,2 George Seed,2 Jane Goodall,2 Ines Figueiredo,2 Raquel Perez-Lopez,1 Nina Tunariu,1 Aurelius Omlin,1 Roberta Ferraldeschi,1 Lakshmi P. Kunju,11 Rosalind Eeles,1 Gerhardt Attard,1 Dan Robinson,11 Arul Chinnaiyan,11 Emma Hall,10 Johann S. de Bono1. 1The Institute of Cancer Research & The Royal Marsden NHS Trust, London, United Kingdom; 2 The Institute of Cancer Research, London, United Kingdom; 3Queen’s University, Belfast, United Kingdom; 4 University of Leeds, Leeds, United Kingdom; 5Churchill Hospital, Oxford, United Kingdom; 6University of Liverpool, Liverpool, United Kingdom; 7Beatson West of Scotland Cancer Centre, Glasgow, United Kingdom; 8 Christie Hospital, Manchester, United Kingdom; 9 University College London Hospital, London, United Kingdom; 10Clinical Trials and Statistics Unit at The Institute of Cancer Research, London, United Kingdom; 11 The University of Michigan, Ann Arbor, MI. 10:50 AM Discussant to be announced 11:00 AM CT323 Accelerated phase I trial of two schedules of the combination of the PARP inhibitor Olaparib (Ola) and AKT inhibitor AZD5363 (AZD) using a novel intrapatient (intrapt) dose escalation design in advanced cancer pts. Vasiliki Michalarea,1 David Lorente,1 Juanita Lopez,1 Suzanne Carreira,2 Hasina Hassam,1 Mona Parmar,1 Nitharsan Sathiyayogan,1 Alison Turner,1 Emma Hall,2 Sonia Serrano Fandos,1 Satyanarayana Seeramreddi,1 Shaun Decordova,2 Karen Swales,2 Ruth Ruddle,2 Florence Raynaud,2 Nina Tunariu,1 Gerhardt Attard,1 L. Rhoda Molife,1 Udai Banerji,1 Ruth Plummer,3 Johann S. de Bono,1 Timothy A. Yap1. 1Royal Marsden Hospital and The Institute of Cancer Research, London, United Kingdom; 2The Institute of Cancer Research, London, United Kingdom; 3Northern Centre for Cancer Care, Newcastle upon Tyne, United Kingdom. 11:20 AM CT324 Phase I of oral BKM120 or BYL719 and olaparib for high-grade serous ovarian cancer or triple-negative breast cancer: Final results of the BKM120 plus olaparib cohort. Ursula A. Matulonis,1 Gerburg Wulf,2 William Barry,1 Michael Birrer,3 Shannon Westin,4 Tatum Spagnoletti,1 Katherine Bell-McGuinn,5 Elizabeth Obermayer,1 Christin Whalen,1 Carol Aghajanian,5 David Solit,5 Gordon Mills,4 Lewis Cantley,6 Eric Winer1. 1Dana-Farber Cancer Institute, Boston, MA; 2Beth Israel Deaconess Hospital, Boston, MA; 3Massachusetts General Hospital, Boston, MA; 4MD Anderson Cancer Center, Houston, TX; 5 Memorial Sloan Kettering Cancer Center, New York, NY; 6 Cornell Weill Medical College, New York, NY. 11:40 AM Discussant Geoffrey I. Shapiro, Dana-Farber Cancer Institute, Boston, MA 11:50 AM CT325 Combination of the PARP inhibitor veliparib (ABT888) with irinotecan (CPT-11) in patients with triple negative breast cancer: Preliminary activity and signature of response. Patricia M. LoRusso,1 Sara M. Tolaney,2 Shukmei Wong,3 Ralph E. Parchment,4 Robert J. Kinders,4 Lihua Wang,4 Jessica Aldrich,3 Alice Chen,5 Diane Durecki,1 Scott A. Boerner,1 Tina Guthrie,6 Adam Bowditch,6 Lance K. Heilbrun,6 Mary Jo Pilat,7 David Craig,3 Dongpo Cai,2 Tracy Bell,2 John Carpten,3 Geoffrey Shapiro2. 1Yale Cancer Center, New Haven, CT; 2Dana-Farber Cancer Institute, Boston, MA; 3Translational Genomics Research Institute, Phoenix, AZ; 4Frederick National Laboratory for Cancer Research, Frederick, MD; 5Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD; 6Karmanos Cancer Institute, Detroit, MI; 7 Wayne State University, Detroit, MI. 12:10 PM CT326 Pharmacokinetic/pharmacodynamic study of sequence specificity of the PARP inhibitor, olaparib and carboplatin in recurrent women’s cancers. Victoria L. Chiou,1 Christina Annunziata,1 Stanley Lipkowitz,1 Lori Minasian,1 Nicolas Gordon,1 Minshu Yu,1 Seth Steinberg,2 Nicole Houston,1 Elise Kohn,1 Jung-min Lee1. 1Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD; 2 Biostatistics and Data Management Section, Office of the Clinical Director, Center for Cancer Research, National Cancer Institute, Bethesda, MD. 12:30 AM Discussant Yves G. Pommier, National Cancer Institute Center for Cancer Research, Bethesda, MD American Association for Cancer Research • AACR ANNUAL MEETING 2015 41 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 42 Clinical Trials Minisymposium: Clinical Trials of Agents Targeting Breast and Prostate Cancers Clinical Trials Minisymposium Tuesday, April 21, 2015 3:00 PM-4:40 PM Room 103, Pennsylvania Convention Center Phospho/total AKT ratio was decreased by >50% in platelet-rich plasma in 21 of 23 pts across various doses. Similarly, phospho/total AKT ratio was decreased (median 35%) in post-treatment tumor biopsies compared to pre-treatment tumors in 4 of 5 pts treated at 400 mg dose. One pt (prostate) had a RECIST PR and 17 pts had SD, 8 of whom were on study for ≥6 months. Exploratory genomic analyses of tumor samples from 10 prostate cancer pts revealed a PIK3CB L1049R mutation in the tumor of one of the pts who remained on study for 33 weeks and an increased PIK3CB gene copy number in the tumor of a pt who achieved PR. Preclinical characterization of PIK3CB L1049R mutation is ongoing, as it is homologous to the oncogenic PIK3CA H1047R mutation. These data suggest potential clinical benefit with GSK2636771 in pts with tumors harboring genetic alterations in PIK3CB. Tumors from other histologies are being analyzed for alterations in key cancer-relevant genes, including PIK3CB. Conclusions: MTD and recommended phase II dose of GSK2636771 is 400 mg QD. Pathway inhibition was observed with limited anti-tumor activity in pts with PTEN deficient tumors. Pts with tumors harboring concomitant genetic alterations in PIK3CB may benefit from GSK2636771 treatment. Recruitment into phase II expansion is ongoing. Clinical Trials of Agents Targeting Breast and Prostate Cancers Co-Chairpersons: Nicholas C. Turner, Institute of Cancer Research, London, United Kingdom; Helen X. Chen, National Cancer Institute Cancer Therapy Evaluation Program, Rockville, MD 3:00 PM Introduction 3:10 AM CT328 Exploratory genetic analysis of tumors from a phase I/II dose escalation study of GSK2636771 in patients (pts) with PTEN deficient advanced tumors. Johann de Bono,1 Hendrik-Tobias Arkenau,2 Joaquin Mateo,1 Jeffrey R. Infante,3 Howard A. Burris,3 Yung-Jue Bang,4 Joseph Eder,5 Sunil Sharma,6 Hyun C. Chung,7 Shaun Decordova,8 Karen E. Swales,8 Michelle D. Garrett,8 Desamaparados Roda-Perez,1 Meichun Ding,9 Mark Russo,9 Li Yan,9 Ben Suttle,10 Jerry M. Tolson,10 Wendy S. Halsey,9 Ganji Gopi,9 Harjeet K. Van Der Keyl,9 Shanker Kalyana-Sundaram,9 Ganesh M. Sathe,9 Monica Motwani,9 Rakesh Kumar9. 1The Institute of Cancer Research & The Royal Marsden, Sutton, United Kingdom; 2Sarah Cannon Research Institute UK, London, United Kingdom; 3Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN; 4 Department of Internal Medicine and Cancer Research Institute, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea; 5Yale Cancer Center, New Haven, New Haven, CT; 6University of Utah Huntsman Cancer Institute, Salt Lake City, UT; 7Yonsei Cancer Center, Cancer Metastasis Research Center, Yonsei University College of Medicine, Shinchon-dong, Republic of Korea; 8The Institute of Cancer Research, Sutton, United Kingdom; 9 GlaxoSmithKline, Collegeville, PA; 10GlaxoSmithKline, RTP, NC. Background: GSK2636771 is a potent, orally bioavailable and selective inhibitor of PI3Kβ (apparent Ki = 0.89 nM), with >900-fold selectivity over PI3Kα/PI3Kγ and 10-fold over PI3Kδ. It inhibits AKT phosphorylation and downstream signaling measured as decrease of PRAS40-, GSK3β-, and RPS6-phosphorylation in PTEN deficient cell lines. Methods: An ongoing phase I/IIa FTIH, open label dose escalation study of GSK2636771, once daily (QD), is being conducted to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy in pts with advanced tumors deficient in PTEN. The study comprised 3 parts: starting-dose selection, 3+3 dose escalation, and phase II expansion. PD was assessed in tumor and platelet rich plasma samples. Archival tumor samples were analyzed for alterations in a custom panel of select oncogenes and tumor suppressors by nextgeneration sequencing and copy number analyses. Results: As of Nov 2014, 62 pts (38 m: 24 f, mean 60 yrs) were enrolled and dosed at 7 dose levels (25 500 mg). In total 5 DLTs (3x hypophosphatemia/ 2x hypocalcemia G3) were observed in 3 pts at 500 mg, defining MTD. AEs >20% (all grades) included diarrhoea, nausea, vomiting, fatigue, abdominal pain, anemia, decreased appetite and headache. Cmax was reached 4-6 h after single and repeat dosing, with a mean t1/2 of 17.1-38.6 h across cohorts. Increases in mean Cmax and AUC(0-24 h) were dose-proportional up to 350 mg, but less than proportional at 400 and 500 mg. 42 3:30 PM CT329 Phase I study of the PI3Kβ/δ inhibitor AZD8186 in patients with advanced castration resistant prostate cancer, triple negative breast cancer, squamous non-small cell lung cancer or PTEN deficient solid tumors: update from dosefinding. Lillian L. Siu,1 Johann De Bono,2 Kari B. Wisinski,3 Celestia S. Higano,4 Natalie Cook,1 Maria Jose De Miguel Luken,2 Rajiv Kumar,2 Joshua Lang,3 Gurkamal S. Chatta,5 Sara M. Tolaney,6 Stefan M. Symeonides,7 Gilmour Morrison,8 Patrick D. Mitchell,9 David G. Brooks,9 Geoffrey I. Shapiro6. 1Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 2The Royal Marsden Hospital, Surrey, United Kingdom; 3University of Wisconsin, Madison, WI; 4University of Washington/Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance, Seattle, WA; 5University of Washington, Seattle, WA; 6Dana Farber Cancer Institute, Boston, MA; 7 AstraZeneca and currently Edinburgh Cancer Centre, Macclesfield, United Kingdom; 8Covance, Alnwick, United Kingdom; 9AstraZeneca, Waltham, MA. Background: A frequent mechanism of dysregulation of the PI3K/AKT/mTOR pathway, commonly altered in cancer, is loss of function of the tumor suppressor PTEN, leading to increased PI3K signalling, particularly through the PI3Kβ isoform. AZD8186 is a potent and selective inhibitor of PI3Kβ/δ with significant activity in PTEN-deficient preclinical models. We report the dose-finding portion of the study, assessing the safety/tolerability of an intermittent dosing schedule of AZD8186. Trial design and eligibility criteria: AZD8186 tablets were administered twice daily 5 days on treatment, 2 days off (5/2 schedule) in 3 week cycles. Escalating doses of AZD8186 were evaluated in cohorts of 3-6 evaluable patients treated until confirmed disease progression, unacceptable toxicity, or withdrawal of consent. A safety review committee reviewed all available safety data and dose limiting toxicities (DLTs) prior to each dose modification. The dose/schedule finding, safety and activity will be updated at time of presentation. Adult patients were recruited with tumor types known to be Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 43 Clinical Trials Minisymposium: Clinical Trials of Agents Targeting Breast and Prostate Cancers PTEN deficient or to have prevalent PTEN loss, such as castration-resistant prostate cancer (CRPC), triple negative breast cancer, squamous non-small cell lung cancer or, that had relapsed and/or were refractory to suitable therapies. Results: As of 25 Oct, 32 patients have received treatment (30 mg n=7, 60 mg n=6, 120 mg n=5; 240 mg n=6, 360 mg n=6; 300 mg n=2). Pharmacokinetic parameters show that systemic exposures to parent drug and its major active metabolite increased in a dose proportional manner and exceeded preclinical exposures that have demonstrated robust anti-tumor activity in PTEN deficient xenograft models. DLTs of maculopapular rash (CTCAE Grade 3) were observed at 360 mg (in 2/6 patients) and at 300 mg (2/2 patients). Additional AEs occurring in >10% of patients included diarrhea, nausea, vomiting, fatigue, rash, decreased appetite and QTc prolongation. AEs of ≥ grade 3 included: rash, hypophosphatemia, hypokalemia, diarrhea elevated aspartate transaminase and 1st degree atrioventricular block; there were no grade 5 events. Overall, 11 patients remained on study for at least 60 days; one CRPC patient remained on study for more than 160 days with minor PSA response, symptomatic improvement and stable disease by CT and bone scan. Conclusions: AZD8186 is a potent oral inhibitor of PI3Kβ/δ, with potential for treatment of PTEN-deficient tumors. Investigation of the safety/tolerability of the 5/2 schedule is continuing. This agent may hold potential for treatment of PTEN deficient tumors. 3:50 PM CT330 Phase I study of PI3Kα inhibitor BYL719 + aromatase inhibitor (AI) in patients (pts) with hormone receptor-positive (HR+) metastatic breast cancer (MBC). Payal D. Shah, Mary E. Moynahan, Shanu Modi, Nicola Hamilton, Betty Ann Caravella, Stephen Zamora, Chau Dang, Theresa Gilewski, Tiffany Traina, Elizabeth Comen, Steven M. Sugarman, Gabriella D’Andrea, Diana Lake, Shari Goldfarb, Sujata Patil, Anne Covey, Michael Berger, Mario Lacouture, Larry Norton, Clifford A. Hudis, Jose Baselga, Sarat Chandarlapaty, Maura Dickler. Memorial Sloan Kettering Cancer Center, New York, NY. Background: Phosphatidylinositol 3-kinase (PI3K) hyperactivation contributes to endocrine therapy resistance. An α-selective PI3K inhibitor (BYL719) added to hormonal therapy may overcome this resistance. We conducted a phase I study to evaluate the safety and determine preliminary efficacy of BYL719 and an AI in pts with HR+ MBC. Methods: This 3+3 dose-escalation trial added oral BYL719 to letrozole (L) or exemestane (E) at standard doses using daily (Arm A: L; Arm B: E) and then intermittent dosing (Arm C: L + BYL719 every other week; Arm D: E + BYL719 on 5 of 7 days weekly). Pts with HR+ MBC, any or no PIK3CA mutation, and already on L/E were eligible. Endpoints were dose-limiting toxicity (DLT), tolerability (CTCAE 4.0), and efficacy. Paired tumor biopsies and serial plasma collection facilitated genomic, proteomic, and cell-free (cfDNA) correlatives. Results: 32 patients (Arm A/B: n=7/7; Arm C/D: n=9/9) received a mean of 99 days (range: 6-473d) of BYL719 + L or E. All were evaluable for toxicity; 25 (Arm A/B: 5/5; Arm C/D: 7/8) were evaluable for response. Median (M) age was 58.5 (30-83). M number of prior MBC therapies was 3 (1-12). PIK3CA status was mutant(MT)/wild-type(WT)/unknown in 8/5/1 pts on Arms A+B and 17/1/0 on Arms C+D. During dose escalation on Arms A+B, 14 pts received BYL719 doses up to 300mg/d, where 4 pts had 5 distinct DLTs: maculopapular rash (N=3), hyperglycemia (N=1), abdominal pain (N=1). 8 week (w) best response on continuous dosing arms (n=10 evaluable pts) was 1 PR (pt heavily pre-treated, including prior L, MBC to liver, Arm A, completed 10C); 7 SD (included -29.9%, -19%, 12%), and 2 POD. Due to toxicity, enrollment to these arms was halted prior to MTD determination. The protocol was amended to include 2 intermittent dosing arms. 3 DLTs were seen: Arm C, grade (G)3 rash, G1 fever and hypotension with >7d therapy hold; Arm D, G3 rash. Toxicities (Arms C+D, all cohorts, n=18) included G≥4: hyperglycemia (n=1, while pt on corticosteroids); G3: rash (n=4); G1/2: mucositis (n=9), hyperglycemia (n=8), and anorexia (n=5). MTD determinations are pending with evaluation of 350mg BYL719 5d on, 2d off ongoing on Arm D. On Arm C, of 7 evaluable pts, best response was SD in 3 pts (28w, 12w, 8+w in 1 pt each) and POD in 4 pts. On Arm D, of 8 evaluable pts, best response is SD in 6 pts (28+w, 16+w in 1 pt each; 20w, 8+w in 2 pts each), and POD in 2 pts. Using serial cfDNA analysis, PIK3CA mutant allele fraction declined with SD and PR and increased in pts with POD. Correlative studies including genomics and proteomics are ongoing. Conclusions: BYL719 with L or E is an active combination. Skin toxicity warranted evaluation of alternate schedules. A 5d on, 2d off schedule of BYL719 + exemestane appears tolerable and may allow for higher doses than daily administration, with MTD determination pending. Further safety, efficacy, and correlative data will be presented. 4:10 PM American Association for Cancer Research • AACR ANNUAL MEETING 2015 CT331 “BEECH”, a phase I/II study of the AKT inhibitor AZD5363 combined with paclitaxel in patients with advanced or metastatic breast cancer: results from the dose-finding study, including quantitative assessment of circulating tumor DNA as a surrogate for response/resistance. Nicholas C. Turner,1 Mafalda Oliveira,2 Anne Armstrong,3 Marie-Paule Sablin,4 José A. Perez-Fidalgo,5 Sarah Herebien,1 Isaac Garcia-Murillas,1 Stan Johnson,6 Andrew Foxley,6 Adnan Mahmood,6 Justin P. Lindemann6. 1The Royal Marsden Hospital, London, United Kingdom; 2Vall d’Hebron University Hospital, 43 02_AACR_2015_Clin_Trials_pp1_44_Layout 1 4/2/15 4:52 PM Page 44 Clinical Trials Minisymposium: Clinical Trials of Agents Targeting Breast and Prostate Cancers Barcelona, Spain; 3Christie Hospital, Manchester, United Kingdom; 4Institut Curie, Paris, France; 5Hospital Clinico Universitario, Valencia, Spain; 6AstraZeneca, Cambridge, United Kingdom. Background: The PI3K/AKT/MTOR pathway is frequently de-regulated in breast cancer. AZD5363 (AZD) is a potent selective inhibitor of AKT1-3. We report the dose-finding phase assessing the safety/tolerability of two intermittent weekly dosing schedules of AZD combined with paclitaxel (P) in HER2 negative breast cancer. The study incorporates prospective assessment of whether circulating tumor DNA (ctDNA) analysis can predict response/resistance earlier than conventional RECIST. Design: AZD capsules were dosed p.o. in one of two schedules: “4/3” (4 days on-treatment followed by 3 days off, starting 360 mg twice daily (bd)) or “2/5” (2 days on-5 days off, starting 560 mg bd). Both schedules comprised 3 weeks P 90 mg/m2 IV on Day 1 with AZD started on Day 2, and 1 week off-treatment. Patients who stopped P were able to continue AZD alone at investigators’ discretion. Escalating doses of AZD were evaluated in cohorts of 3-6 evaluable patients treated until disease progression, unacceptable toxicity, or withdrawal of consent. A safety review committee reviewed safety data and dose limiting toxicities (DLTs) prior to dose modification. Plasma samples for ctDNA extraction were collected at baseline, weekly in cycle 1, day 1 of all subsequent cycles, and were analysed by digital PCR. Female patients aged ≥18 years with HER2 negative MBC and up to two prior chemotherapy courses for advanced cancer were recruited. 44 Results: 37 patients have received AZD. DLTs in 2/6 patients at 480 mg 4/3 comprised one patient with maculo-papular rash, and another with immune allergic reaction (both CTCAE grade 3). No DLT was observed in the 400 mg 4/3 cohort, which was defined as the maximum tolerated dose (MTD) in combination with P. DLTs in 2/6 patients at 640 mg 2/5 comprised one patient with prolonged neutropaenia (grade 4), and another with diarrhoea (grade 3). No DLTs were observed in 11 patients on the 560 mg 2/5 cohort. In all patients median progression free survival was 8.2 months, despite pre-treatment with taxane chemotherapy in 62% patients and a median of 2 prior lines of chemotherapy. A patient with Cowden’s syndrome (PTEN germline mutation) had a partial response maintained for 18 months, including 12 months on AZD5363 alone. ctDNA assessment suggests that lack of a fall in ctDNA abundance may predict for early progression. Conclusions: AZD5363 400mg bd 4/3 in combination with paclitaxel was determined to be well tolerated in patients with MBC, with investigation of the 2/5 schedule continuing. Two international double-blind randomised studies have commenced to assess AZD5363 400 mg bd 4/3 schedule combined with paclitaxel versus paclitaxel plus placebo in ER positive HER2 negative MBC stratified by PIK3CA mutation status (BEECH Part B), and in triple negative breast cancer (PAKT). 4:30 PM Discussion Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 45 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 Late-Breaking Poster Session Sunday, April 19, 2015 1:00 PM-5:00 PM Poster Section 39 Halls B-E (Level 200), Pennsylvania Convention Center Late-Breaking Research: Experimental and Molecular Therapeutics 1 Poster Section 39 Poster Board 1 LB-001 Development and evaluation of a fluorescent antibodydrug conjugate for molecular imaging and targeted therapy of pancreatic cancer. Steve Knutson,1 Erum Raja,1 Ryan Bomgarden,1 Marie Nlend,1 Aoshuang Chen,2 Ramaswamy Kalyanasundaram,2 Surbhi Desai1. 1 Thermo Fisher Scientific, Rockford, IL; 2University of Illinois College of Medicine, Rockford, IL. Developing new strategies to effectively diagnose and treat various types of cancer is paramount to increasing patient survival rates. Although chemotherapeutic small molecules are effective for some cancer types, they often have harmful side effects, resulting in significant damage to healthy tissue. Targeted therapy, where anti-cancer drugs are more precisely delivered to specific cells, has the potential to revolutionize chemotherapy, through increased localized effective doses with minimized systemic toxicity. Antibody-drug conjugates (ADCs) are one such class of targeted therapy biopharmaceuticals, employing the inherent specificity of antibodies as a targeting mechanism to yield a potent drug delivery system. In addition to being used as ADCs, antibody-conjugates are also commonly used as a highly effective tool for cancer typing and longitudinal treatment monitoring by immunohistochemistry staining or diagnostic imaging. However, antibody-conjugates used for tumor diagnosis and treatment are different molecules. Fluorescent dye labeling of therapeutic antibodies has been previously demonstrated for cancer imaging. However, therapeutic antibodies dual-labeled with both chemotherapeutic small molecules and fluorescent dyes has not been reported. Here, we demonstrate the development of a directly-labeled, fluorescent antibody-drug conjugate for simultaneous targeted drug delivery and in vivo molecular imaging of cancer. Our novel biopharmaceutical entity is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to an average of one molecule of paclitaxel and two molecules of a nearinfrared fluorophore (DyLight 680-4xPEG). Preliminary data show that this fluorescent ADC selectively binds CEA positive cells and is cytotoxic using in vitro model systems. Ongoing studies using an in vivo mouse xenograft cancer model will demonstrate the utility of this fluorescent ADC as a new concurrent pancreatic cancer detection, monitoring and treatment technology. Poster Section 39 Poster Board 2 LB-002 A novel c-Met/EGFR bispecific targeting antibody drug conjugate for NSCLC. Gang Chen, Lingna Li, Pia Muyot, Edwige Gros, Yanliang Zhang, Yingqing Sun, Hong Zhang, Yanwen Fu, Alice Lee, Jian Cao, Gunnar Kaufmann, Zhenwei Miao. Concortis Biosystems, San Diego, CA. c-Met and EGFR are both widely and highly expressed, share and cross talk common signaling pathways in a variety of carcinomas including lung, breast, ovary, kidney, colon, thyroid, liver, and gastric carcinomas. Therapeutics like tyrosine kinase inhibitors (TKIs) and monoclonal antibodies, targeting EGFR and cMet are on the cutting-edge of cancer therapy. In preclinical studies and clinical trials, though these anti-c-Met and EGFR therapeutics showed promising anti-tumor activities but usually not sufficient for sustained treatment efficacy, their individual efficacies are limited due to the development of resistance. c-Met amplification has been considered to be a major escape route for EGFR-targeted therapies. We believe that antibody drug conjugates (ADCs) offer the promise and potential of delivering potent anti-tumor activity with the advantage of reduced side effects. We generated a novel bispecific antibody drug conjugate containing a proprietary human anti-c-Met antibody and anti-EGFR antibody, and a potent toxin by our novel C-lock conjugation method. The conjugate retained high binding affinity and targeting both c-Met and EGFR on tumor cell surface. The ADC showed potent cell killing in a lower nM range in a variety of c-Met and EGFR positive cell lines in vitro and strong in vivo anti-tumor efficacy in several c-Met and EGFR positive human NSCLC xenograft models without significant toxicity. The in vivo anti-tumor growth efficacy of bispecific c-Met/EGFR targeting antibody conjugates is superior to either single anti-c-Met or EGFR ADC. Poster Section 39 Poster Board 3 LB-003 High complete and partial response rate in a phase Ib pilot trial with cisplatin plus albumin-bound paclitaxel and gemcitabine in patients with advanced pancreatic cancer. Gayle S. Jameson,1 Erkut Borazanci,1 Elizabeth Poplin,2 Michael T. Barrett,3 John Crowley,4 Adam Rosenthal,4 Amy Stoll-D’Astice,4 Karen L. Ansaldo,1 Steven Boone,1 Leticia Lebron,1 Ramesh K. Ramanathan,1 Ronald L. Korn,1 Daniel D. Von Hoff5. 1Virginia G Piper Cancer Center at Scottsdale Healthcare, Scottsdale, AZ; 2 Rutgers-Cancer Institute of New Jersey, New Brunswick, NJ; 3 Mayo Clinic Arizona, Scottsdale, AZ; 4Cancer Research and Biostatistics, Seattle, WA; 5TGen/Virginia G Piper Cancer Center at Scottsdale Healthcare, Scottsdale, AZ. Background: The genomes of metastatic pancreatic cancers contain a myriad of intrachromosomal aberrations indicating a likely high prevalence of DNA repair deficiencies indicating sensitivity to DNA damaging agents such as the platinum’s. Because of this, the drug cisplatin was added to an albumin-bound paclitaxel + gemcitabine regimen, which has already been determined to improve survival over gemcitabine alone in a randomized phase III trial (NEJM 2013; 369:1691-1703). Objectives: To determine the efficacy and safety of albuminbound paclitaxel and gemcitabine plus cisplatin for patients with advanced pancreatic cancer Methods: Eligibility criteria included Stage IV pancreatic cancer, no prior chemotherapy for systemic disease, KPS ≥ 70; life expectancy ≥ 12 weeks and measurable disease. The doses were albumin-bound paclitaxel 125 mg/m2 undiluted, gemcitabine 1000 mg/m2 in 500 ml of normal saline (NS), each infused over 30 minutes on days 1 and 8 of a 21 day cycle, along with 3 different dose levels of cisplatin (25, 37.5 or 50 mg/m2) in 500 ml of NS infused over 60 minutes, after the nab-paclitaxel infusion. Pre and post cisplatin hydration was given. Results: To date, 10 patients have been entered on study with all patients being evaluable, (baseline and at least one follow up CT scans completed). There have been 2 complete responses (20%), 6 partial responses (PR), (60%), 1 stable disease (10%), and 1 patient with progressive disease (10%), by RECIST 1.1 criteria. An exponential decrease in CA19-9 correlating with the t1/2 of the marker was noted. Response was seen rapidly with PR observed at the first staging evaluation at 9 weeks in 7 of 10 patients. The 8th patient achieved a PR at 18 weeks. Serious adverse events occurred in 4 patients: non-neutropenic sepsis/pneumonia (n=1), and non-neutropenic bacteremia (n=1) in the cisplatin 25 mg/m2 cohort; clostridium difficile colitis (n=1) with cisplatin 37.5 mg/m2; and neutropenic fever/pneumonia (n=1) with cisplatin 50mg/m2. Discussion: The study has completed phase Ib and will be expanded at the phase II dose of cisplatin 25 mg/m2 for a total of 25 patients. If this favorable response rate is confirmed, this 3 drug regimen could be further developed both for patients with advanced disease as well as in neoadjuvant and adjuvant settings. Supported by grants from the Seena Magowitz Foundation and the SU2C Dream Team American Association for Cancer Research • AACR ANNUAL MEETING 2015 45 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 46 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 Poster Section 39 Poster Board 4 LB-004 Mouse PDX Trial Suggests Combination Efficacy of Raf and EGFR Inhibition in Colorectal Cancer with BRaf or KRas mutation. Yung-mae M. Yao, Gregory P. Donoho, Philip W. Iversen, Yue Wang Webster, Yong Gang Yue, James R. Henry, Gregory D. Plowman, Sheng-Bin Peng. Eli Lilly and Company, Indianapolis, IN. MAPK activation through KRas, NRas or BRaf mutation occurs in approximately 70% of colorectal cancer patients. Due to their epithelial origin, colorectal tumors generally have high levels of EGFR expression and activation. EGFR therapies such as cetuximab are effective for treatment of a subset of colorectal cancer, particularly patients with wild type (WT) KRas. EGFR signaling is also recently identified as a key resistance mechanism in BRaf mutant colorectal cancer to BRaf inhibitors. In this study, we have genetically characterized 78 patient-derived xenograft (PDX) models of colorectal tumors, and conducted an “n=1” (single mouse per treatment group) trial in these PDX models with cetuximab, LSN3074753, a pan-Raf and Raf dimer inhibitor, and their combination in collaboration with Oncotest GmbH and Champions Oncology. Among these 78 PDX models, 42 (53.8%) have a KRas mutation, 12 (15.4%) have BRaf V600E or an atypical BRaf mutation, and 26 (33.3%) are WT KRas and BRaf. Consistent with clinical results, cetuximab is primarily active in WT KRas and BRaf PDX models, with disease control rate (DCR) of 53.8% (14/26) in this subgroup. These results suggest that the mouse n=1 PDX trial paradigm could reliably predict clinical results. For pan-Raf and Raf dimer inhibitor LSN3074753, it is active in a subset of PDX models, particularly those with BRaf or KRas mutation(s), with DCR of 21.2% among models with a KRas or BRaf mutation. Importantly, a synergistic effect is observed when cetuximab and LSN3074753 are combined for treatment of these 78 PDX models. The overall DCR in the combination arm is 50% (39/78), while cetuximab or LSN3074753 alone has an overall DCR of 24 or 18%, respectively. Further statistical analyses reveal that BRaf mutations including V600E or other atypical mutations (G469E, G76E, G596V, G203V, etc) are the best predictor of combination synergy, and are significantly associated with synergistic effect with a p value of 0.004. In models with BRaf mutations, the combination arm has a DCR of 50% (6/12), whereas cetuximab or LSN3074753 alone has a DCR of 8.3 or 17%, respectively. BRaf or KRas mutations are also significantly associated with combination synergy with p value of 0.01. Among 42 KRas mutation models, LSN3074753 or cetuximab alone has a DCR of 21.4 or 16.7%, and the combination arm has a DCR of 43%. Overall, these results indicate that combination of EGFR and Raf inhibition by cetuximab and a pan-Raf inhibitor has the potential for treatment of colorectal cancer patients with BRaf or KRas mutation. Poster Section 39 Poster Board 5 LB-005 Monepantel a new first in class potent inhibitor of P70S6K potentiates the anti-tumor effects of gemcitabine and doxorubicin: In vitro and in vivo studies. Parvin A. Ataie-Kachoie,1 Javed Akhter,2 David L. Morris1. 1St George Hospital, Sydney, Australia; 2University of New South Wales, Sydney, Australia. Aims: Monepantel (MPL) is a new nematode-specific anthelmintic agent. We have recently communicated the preliminary results showing the anti-tumor activity of this agent in ovarian cancer through attenuation of mTOR/P70S6K pathway. As emerging data indicate that mTOR inhibitors are most effective when combined with other target agents, we evaluated whether MPL could favorably be combined with the clinically approved chemotherapeutic agents to improve therapeutic efficacy. Methods: The effects of MPL and/or chemotherapeutic agents gemcitabine (Gem)/ doxorubicin (Dox) on the growth of a panel of cancer cell lines were determined using SRB assay. In vitro drug 46 synergy was determined using combination index (CI) methods derived from Chou-Talalay equations using CalcuSyn software. For in vivo studies, we evaluated the effect of MPL, alone and in combination with Gem/ Dox on the growth of established human ovarian (OVCAR-3) tumors implanted subcutaneously in BALB/C nude mice. Toxicity was evaluated measuring animal weight. In vivo combination effects was determined using Fractional Tumor Volume (FTV) method. Results: In vitro, MPL in combination with Gem or Dox synergistically reduced survival rates of a panel of malignant cells from different origins while having no additive effect upon nonmalignant cell (HOSE) survival rates. In vivo, antitumor activity was observed in all treatment groups compared to the mock-treated animals after 4 weeks of treatment. However combination therapy with MPL and chemotherapeutics significantly attenuated tumor growth, compared to monotherapy without showing any toxicity. Combining MPL (50 mg/kg) increased the tumor inhibitory effect of low dose Gem (2 mg/kg), high dose Gem (5 mg/kg), low dose Dox (2 mg/kg) and high dose Dox (5 mg/kg) by 32.29, 35.1, 15.2 and 24.38% respectively. Moreover, MPL (25 mg/kg) enhanced the tumor inhibitory rate of Gem (5 mg/kg) by 36.26%. These resulted in complete tumor growth inhibition in Gem 5 mg/kg + MPL 25 or 50 mg/kg and Dox 2 or 5 mg/kg+ MPL 50 mg/kg treatment groups. Assessment of therapeutic synergy with FTV method revealed strong synergy in Gem 5 mg/kg + MPL 25 or 50 mg/kg, Gem 2 mg/kg + MPL 50 mg/kg and Dox 2 or 5 mg/kg+ MPL 50 mg/kg treatment groups with odds ratio of 2.48, 2.59, 1.48, 1.63 and 2.97 (>1 indicates synergy) respectively. Conclusion: These findings provide a rational to further investigate MPL in combination with standard chemotherapeutics as novel combination regimens which could hopefully provide strong anticancer synergy. Poster Section 39 Poster Board 6 LB-006 Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer. Fernanda Staquicini,1 Andrey Dobroff,1 Fortunato Ferrara,1 Sara D’Angelo,1 Andrew RM Bradbury,2 Wadih Arap,1 Renata Pasqualini1. 1 University of New Mexico Cancer Center, Albuquerque, NM; 2Los Alamos National Laboratory, Los Alamos, NM. The concept of targeted cancer therapy is predicated on the assumption that tumors have unique and sustained genetic abnormalities, and that direct targeting of such distinctive biological features can only be accomplished through the identification and validation of certain specific molecular markers. These principles are particularly relevant to lung cancer, the leading cause of cancerrelated death worldwide. For patients with early-stage disease, surgical resection or primary radiotherapy are generally the standard treatments, whereas combined-modality therapy (radiation plus chemotherapy, with or without surgery) is preferred for locally advanced disease. However, most patients with lung cancer develop metastatic tumors and resistance to therapy, and ultimately die of their disease. Here, we identify EphA5 as a molecular target of lung cancer, and as a novel regulator of IR-induced cell cycle checkpoint and DNA damage repair, with unexpected roles in the resistance of lung cancer to radiotherapy. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ataxia-telangiectasia mutated (ATM) at sites of DNA repair. In addition, we produced a new monoclonal antibody against EphA5 that sensitizes lung cancer cells to IR in vitro and improves the overall survival of mice bearing human lung cancer xenografts in combination with radiation therapy. These findings present a readily available and potentially effective strategy for the detection and treatment of lung cancer. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 47 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 Poster Section 39 Poster Board 7 LB-007 Synergistic inhibition of human gastric and colorectal cancers by Bromelain and N-acetylcysteine: An in vivo study. Afshin Amini, Samar Masoumi-Moghaddam, Anahid Ehteda, Winston Liauw, Javed Akhter, Krishna Pilai, David L. Morris. The University of New South Wales, Sydney, Australia. Mucin is well understood to be an adverse prognostic factor in some cancers. We previously reported that a combination of Bromelain (BR) and N-acetylcysteine (NAC) were synergistic in dissolving pseudomyxoma peritonei mucin, had direct in vitro cytotoxic effects on some gastrointestinal cancer cells and sensitized them to some chemotherapy agents. In the present study, we aimed to evaluate the growth-inhibitory effect of BR and NAC as single agent or combination therapy in nude mice models of gastrointestinal cancer. Nude mice received 2 million and 1 million cells of MKN45 (gastric) and LS174T (colorectal) by intraperitoneal injection. At day 14 and 7 post inoculation for MKN45 and LS174T cells, respectively, animals were intraperitoneally administrated BR (3, 6 mg/kg), NAC (300, 500 mg/kg) or their combination every other day over 12 days for MKN45 and 17 days for LS174T models. At the end of the study, the animals were euthanized, the number of peritoneal nodules and their weight were collected, and the peritoneal tumors were subjected to immunohistochemistry for evaluation of MUC2. No toxicity was observed during the experiment. At necropsy, highly significant reductions in the number of tumor nodules and tumor burden were observed, in particular in the combination group, where almost complete inhibition in LS174T group was found. There was more than 95% and 70% decreases in tumor burden and tumor nodules, respectively, in LS174T group after combination treatment. For MKN45 model, the reductions in tumor burden and tumor nodules in combination groups were more than 60% and 70%, respectively. Performing immunohistochemistry on tumor samples, MUC2 staining of the LS174T xenografts showed a greater than 60% reduction of cytoplasmic staining. To the best of our knowledge, this is the first report of the in vivo use of this combination with synergistic inhibition of human gastric and colorectal cancers. The combination of BR and NAC in mucin secreting gastrointestinal tumors is interesting and could be of potential value in peritoneal cancer. Poster Section 39 Poster Board 8 LB-008 Mesofluidic platform for high throughput screening for inhibitors of metastasis. Adrianne Shearer, Chris Spruell, Victoria Le, Mar Creixell, Seema Nandi, Aaron B. Baker. University of Texas at Austin, Austin, TX. Background: A fundamental limitation in the development of new therapies to prevent metastatic cancer is a lack of in vitro systems that can accurately recapitulate the steps of cancer cell metastasis. Currently, most assays for examining the steps of metastasis fail to incorporate the biophysical forces experienced by tumor cells due to blood flow, or are low throughput and therefore not amenable to drug screening and high throughput experimentation. Methods: We have developed a novel high throughput mesofluidic platform for assaying cell adhesion under flow in a 96well format. This device functions like a cone and plate viscometer in each well by inducing shear stress on cells cultured in a standard 96-well plate. We validated the fluid flow and alignment of the device and studied the adhesion of cultured leukocytic monocytes (THP-1 cells) and multiple cancer cell lines (HCT116, MDA-MB-231 and MCF-7 cells) to purified extracellular matrix molecules (ECM), endothelial cells and immobilized platelets. All assays were carried out under flow (0.5 dynes/cm2 of shear stress) and static conditions. Results: Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Treatment of breast cancer cells with a small library of integrin inhibitors demonstrated that these compounds had minimal effect on cancer cell adhesion to endothelial cells under static conditions, whereas under shear conditions many of these compounds reduced adhesion of cancer cells. In addition, a static adhesion assay of breast cancer cells to various types of ECM showed higher adhesion of the less aggressive MCF-7 cell line in comparison to the more aggressive MDA-MB-231 cell line. In contrast, flow incorporating assays showed increased adhesion of the MDA-MB-231 in comparison to the MCF-7 cell line. Finally, we performed a high throughput screening experiment using a kinase inhibitor library with 80 compounds and found that the shear based assay yielded notably different results from a similar screen under static conditions for cancer cell adhesion to endothelial cells, immune cell adhesion to endothelial cells and cancer cell adhesion to platelets. Conclusions: Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Thus, this high-throughput screening platform may enable the development of novel compounds to inhibit cancer metastasis and facilitate the study of the systems level behavior of cancer-endothelium adhesion. Poster Section 39 Poster Board 9 LB-009 Collagen regulation in postpartum mammary gland involution, a novel breast cancer prevention target. Qiuchen Guo, Pepper J. Schedin. Oregon Health and Science University, Portland, OR. Collagen is the most abundant extracellular protein in breast tissue and plays a critical role in breast cancer progression. Intravital imaging shows collagen fibers as “highways” for tumor cell invasion, and collagen fibers arranged perpendicular to the tumor border (radial alignment) independently predict breast cancer metastasis. To understand the role of collagen in breast cancer, we argue it is necessary to understand collagen biology in the normal breast. While collagen production has been well studied in the context of wound healing, little is known about its regulation in normal breast physiology. Here we utilize a murine, weaninginduced mammary gland involution model, which is characterized by robust collagen deposition and remodeling, to investigate collagen regulation in the normal mammary gland. During mammary gland involution, we find increased collagen-I, -III, and -V gene expression and high levels of lysyl-oxidase (LOX), a major collagen cross-linker. By SHG analysis, we find collagen fibers around mammary ducts in the involuting gland are more tightly organized than nulliparous gland, with “hot spots” of radial alignment. Collagen is predominantly produced by fibroblasts and we identified mammary fibroblasts as PDGFRα+. Using flow cytometry-based cell sorting and gene expression analysis, PDGFRα+ fibroblasts isolated from the involuting mammary gland have increased gene expression of collagen-I,-III, and LOX. Further, there is ~2 fold increase in number of PDGFRα+ cells/mg weight of tissue in the involuting gland, suggesting that fibroblasts may be recruited and activated during involution. Increased production of collagen by fibroblasts and radial alignment of collagen are consistent with a tumor promotional microenvironment during mammary gland involution. In fact, our lab has demonstrated protumorigenic attributes of involution in rodent models of postpartum breast cancer, which can be mitigated by non-steroidal antiinflammatory drug (NSAID) treatment. To investigate the impact of NSAID treatment on collagen during involution, we isolated fibroblasts from mammary glands of animals undergoing involution in the presence and absence of NSAID intervention. Mammary fibroblasts flow sorted from NSAID treated hosts display reduced collagen I, TGFβ1, and MMP-3 gene expression by 30~60% compared to involution-fibroblasts isolated from untreated mice. American Association for Cancer Research • AACR ANNUAL MEETING 2015 47 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 48 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 Taken together, these data demonstrate that fibroblasts are responsive to COX-2 inhibition and suggest a novel mechanism of NSAIDs action that may be mediated through suppression of collagen deposition. Collagen and breast cancer are inseparable topics, as collagen abundance, architecture and tension impact breast cancer risk and prognosis. Weaning induced mammary gland involution provides a unique physiological window to study collagen regulation. Studying the mechanism of how NSAIDs regulate collagen may provide insight into the use of NSAIDs as a preventative and therapeutic intervention in breast cancer. Poster Section 39 Poster Board 10 LB-010 Hijacking the E3 ubiquitin ligase cereblon to create efficient BRD4 degraders. Jing Lu,1 Yimin Qian,1 Martha Altieri,1 Hanqing Dong,1 Jing Wang,1 Kanak Raina,1 Jim Winkler,1 Andy Crew,1 Kevin Coleman,1 John Hines,2 Craig Crews2. 1Arvinas, Inc, New Haven, CT; 2Yale University, New Haven, CT. BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as an attractive target in multiple pathological settings. For example, recent studies have demonstrated its importance in driving the proliferation of several cancers including NUT midline carcinoma, acute myelogenous leukemia (AML), multiple myeloma, Burkitt’s Lymphoma (BL), and prostate cancer. Thus, BET bromodomain inhibitors have showed promising effects in certain preclinical settings, particularly in MYCdriven hematological malignancies, such as BL. Interestingly, we found that BRD4 inhibitors lead to a rapid and robust accumulation of BRD4 that, together with the reversible nature of binding to BRD4, may account for their moderate suppression of MYC expression and inhibition of cell proliferation. To circumvent these limitations, we designed a Proteolysis Targeting Chimera (PROTAC) compound, containing a BRD4 binding moiety and an E3 ubiquitin ligase cereblon ligand. By actively engaging the E3 ligaseproteasome degradation machinery, BRD4 PROTAC leads to fast and efficient degradation of BRD4. Consequently, it is more effective than small molecule BRD4 inhibitors in suppressing MYC levels and downstream signaling, which are associated with proliferation and survival of malignant cells. More importantly, we also demonstrated that BRD4 PROTAC is more effective in inhibiting BL cell proliferation and inducing apoptosis compared to BRD4 inhibitors. Our findings strongly demonstrate that a degrader of BRD4, in the form of a cereblon-based PROTAC, provides a better and more efficient strategy in targeting BRD4 than traditional small molecule inhibitors. Poster Section 39 Poster Board 11 LB-011 Arginine deprivation using ADI-PEG20 leads to regression of an ASS1-ve intracranial GBM tumor in mice and potentiates gamma irradiation of ASS1+ve GBM in vitro. Fernando Abaitua, Justyna Przystal, Amin Hajitou, Nelofer Syed. Imperial College London, London, United Kingdom. Patients with Gliobastoma Multiforme (GBM) have an extremely poor overall survival rate. Novel molecular targeted therapies have failed to provide further improvements in these rates and as such the standard of care for GBM patients remains unchanged, consisting of radical surgery followed by chemo and radiotherapy. In light of this, novel therapeutic approaches have to be tested in combination with current protocols for their efficacy. We have previously demonstrated the metabolic dependency of ~30% of de novo GBM to extracellular arginine. Using ADI-PEG20 to deplete arginine, we showed a marked reduction in the proliferation of primary GBM explants that exhibited methylation in their ASS1 gene, the rate-limiting enzyme in the arginine biosynthetic pathway. We have validated these findings in an animal model where we have demonstrated that ADI-PEG20 treatment induces a profound delay in the growth of ASS1-ve intracranial human GBM tumors in 48 mice. In cell culture models, we show that ADI-PEG20 has comparable cell growth inhibition efficiencies as conventional chemotherapies namely temozolomide, lomustine and chloroquine. Moreover, these agents exhibited inhibitory effects on proliferation in hypoxic conditions (1% O2) representing activity in a more physiological environment. Importantly, combination with ADIPEG20 did not reduce efficiency of these conventional drugs. Although no synergistic effects of ADI-PEG20 with these chemotherapeutic agents was observed, synergy was detected in combination with irradiation. Here, we showed the ADI-PEG20 radio-sensitized GBM cell lines to photon irradiation regardless of their ASS1 methylation status: In clonogenic assays, there was a 50% enhanced decrease in colony formation with pre-treatment of ADI-PEG20 and subsequent photon irradiation when compared to irradiation alone. These results suggest arginine deprivation therapy as a novel therapeutic strategy for ASS1-ve GBM. Moreover, we show that when combined with irradiation, ADI-PEG20 can be used to treat GBM irrespective of their ASS1 status. Poster Section 39 Poster Board 12 LB-012 Inhibition of PI3K induces paracrine factors, which promote growth and survival of human breast cancer cells. Christian D. Young, James P. Koch, Rebecca S. Cook, Carlos L. Arteaga. Vanderbilt University Medical Center, Nashville, TN. Phosphoinositide 3-kinase (PI3K) is aberrantly activated in many human cancers. To blunt the mitogenic action of this oncogenic pathway, PI3K inhibitors are currently in clinical development. However, inhibition of PI3K results in feedback activation of receptor tyrosine kinases (RTKs) and cap-independent translation of pro-survival proteins, thus diminishing the net antitumor effect of PI3K inhibitors. Therefore, characterization of pathways potentially activated by PI3K inhibitors is necessary in order to identify drug combinations which will better eradicate tumors. We demonstrate herein that inhibition of PI3K in breast cancer cells resulted in the increased expression of EGFR ligands and activation of EGFR/ERK signaling. FoxO transcription factors are repressed by PI3K and previous studies have shown that inhibition of PI3K results in nuclear localization of FoxO and activation of FoxO-mediated transcription of RTKs and IGF-I/II. However, RNAi-mediated knockdown of FoxO3A only partially attenuated the activation of EGFR induced by PI3K inhibition. Using a panel of transcription factor luciferase reporters, we identified 10 transcription factors (including FoxO) which are activated upon PI3K inhibition in three breast cancer cell lines from different intrinsic subtypes (MCF7, BT20 and SUM159). In addition, the serum-free media conditioned by MCF7 or BT20 cells in the presence of the pan-PI3K inhibitor BKM120 induced the survival and proliferation of recipient cancer cells which otherwise undergo apoptosis in serum-free conditions. Thus, we hypothesized that inhibition of PI3K results in activation of paracrine factors, including EGFR ligands, which may promote the survival of a heterogeneous tumor cell population. Indeed, evaluation of media conditioned by MCF7 and BT20 cells with cytokine/growth factor antibody arrays demonstrated over 100 proteins to be induced by PI3K inhibition, including ligands for receptors in the EGF, FGF, cytokine, chemokine and TGFβ families. We are currently performing SILAC-based mass spectrometry profiling of media conditioned by breast cancer cells ± PI3K inhibitors to determine modulation of secreted factors upon inhibition of PI3K. We are also determining whether inhibition of PI3K results in increased ADAM protease activity, inducing the shedding of EGFR ligands from the cell membrane. The results of these experiments will identify extracellular factors activated by the inhibition of PI3K and suggest which pathways need to be simultaneously inhibited to maximize the clinical activity of PI3K inhibitors. Further, these secreted proteins may serve as circulating pharmacodynamics biomarkers indicative of effective blockade of PI3K in patients. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 49 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 Poster Section 39 Poster Board 13 LB-013 The immunomodulatory effect of JAK inhibitors enhances metastasis by impairing antitumor immunity in preclinical models of breast cancer. Nancy E. Hynes,1 Alessia Bottos,1 Jason Gill,1 Thomas Radimerski,2 Alexander Tzankov,3 Aleksandra Wodnar-Filipowicz1. 1Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; 2 Novartis Institute for Biomedical Research, Basel, Switzerland; 3 University Hospital Basel, Basel, Switzerland. The JAK-STAT pathway is an attractive therapeutic target in breast cancer due to its frequent activation. Clinical trials testing JAK inhibitors in breast cancer are ongoing, making it important to understand the effect of this therapeutic approach on metastasis. While it is recognized that tumor growth in primary and metastatic sites is influenced by the local environment, little is known about the effect of targeted therapies on metastases or on host cells interacting with tumor cells at distant locations. Our goal was to determine the effect of JAK inhibitors on breast cancer in the bone environment, a common site of metastasis. Using patient biopsies and preclinical models of breast cancer metastasis, we demonstrate that the JAK-STAT pathway is active in bone metastasis, both in the cancer cells and in the tumor environment. To study the effect of JAK inhibitors, we established preclinical models of bone metastatic tumors, taking advantage of the bone tropism when injected via the intracardiac route, of the breast cancer cell lines, human MDA-MB231 scp1833 and mouse EO771. In vivo, in both models, STAT3 was active and treatment with the JAK1-JAK2 inhibitor ruxolitinib decreased pSTAT3 levels in primary tumors and in bone metastases. Unexpectedly, blocking the pathway with ruxolitinib, or with the JAK2 inhibitor BSK805, enhanced the metastatic burden in both models. To investigate the effect of JAK inhibition on tumor cell dissemination from the primary site, we employed the 4T1.2 model, which spontaneously metastasizes from the mammary tumor to the bone and the lungs. As seen with the other models, there was a significant increase in tumor cell numbers in the bone and in the lungs in response to JAK inhibitor treatment. To understand the mechanism underlying the increase in metastatic load, we considered the host immune system as a potential bystander target of the JAK inhibitors. Indeed, in response to JAK inhibitor treatment we observed a major reduction in the NK cell population. The effect of JAK inhibitors on NK cells was systemic since they were also reduced in the bone marrow and in the peripheral blood of tumor-bearing mice. To mechanistically explain the impact of JAK inhibitors on NK cells, we used the NK cell line, NK-92. Upon treatment of NK-92 cells with JAK inhibitors, activation of multiple STATs decreased and cell proliferation was strongly inhibited. In cytotoxic assays, treatment with JAK inhibitors significantly decreased the killing ability of NK-92 cells against carcinoma cells. To test the in vivo relevance of NK cells in metastatic growth we used NSG mice, which are devoid of NK cell activity. Remarkably, in contrast to the results obtained with 4T1.2 mammary tumors grown in immunocompetent hosts, no increase in bone or lung metastases was observed in tumor-bearing NSG hosts treated with ruxolitinib, providing strong evidence that JAK inhibition increases metastasis by interfering with NK cell mediated anti-tumor innate immunity. These results suggest that the immunomodulatory effect of JAK inhibitors in breast cancer patients undergoing clinical trials should be monitored. Moreover, our work highlights the importance of evaluating the effect of targeted therapy on cell populations in the tumor environment in order to predict and overcame bystander effects that might impact on therapy response. Poster Section 39 Poster Board 14 LB-014 Targeting p53 aggregation in ovarian cancer chemoresistant cells. Yang Yang-Hartwich, Carlos Cardenas, Mary Pitruzzello, Eydis Lima, Ayesha B. Alvero, Gil Mor. Yale University School of Medicine, New Haven, CT. Background: About half of patients diagnosed with ovarian cancer develop chemoresistance and succumb to the disease. The underlying mechanisms that lead to the development of chemoresistance are poorly understood. We previously demonstrated that p53 protein aggregation inhibited p53 proapoptotic activities consequently leading to platinum resistance in ovarian cancer cells with cancer stem cell properties. Since heat shock protein 90 (HSP90), a molecular chaperone, can sustain the accumulation of protein aggregates, the purpose of this study is to determine if HSP90 inhibitors can inhibit the accumulation of p53 aggregates, reactivate p53 pro-apoptotic function, and sensitize ovarian cancer stem cells (OCSCs) to carboplatin. Method: Chemoresistant CD44+/MyD88+ OCSCs were treated with the HSP90 inhibitor, 17-AAG, carboplatin, or the combination of 17-AAG and carboplatin. Cell viability was monitored by IncuCyte ZOOM live-cell imaging system. Caspase activation was determined by Caspase-Glo assay. p53 aggregation was detected by non-denaturing gel and western blot. The interaction between p53 and HSP90 proteins was determined by coimmunoprecipitation (co-IP). Chromatin immunoprecipitation (CHIP) of p53 and RT-QPCR of p53 targets (PUMA, BAX, et al.) were performed to demonstrate p53 transcriptional activation. Result: OCSCs were resistant to single treatment with Carboplatin. In line with results from our previous studies, carboplatin neither decreased cell viability nor induced caspase activation in these cells. Interestingly, carboplatin enhanced the levels of aggregated p53 and hence failed to upregulate the expression of p53-related pro-apoptotic genes. Co-IP results demonstrated that 17-AAG blocked the interaction between p53 and HSP90. More importantly, 17-AAG was able to sensitize OCSCs to carboplatin. The combination therapy effectively induced the transcriptional activity of p53, upregulated pro-apoptotic genes, and stimulated caspase activity and cell death in the carboplatinresistant OCSCs. Conclusion: The HSP90 inhibitor, 17-AAG can inhibit the formation of p53 aggregates by blocking the interaction between p53 and HSP90. By releasing p53 from the aggregates, 17-AAG reactivates the ability of p53 to bind to DNA and to upregulate the expression of target genes, which leads to the apoptosis of OCSCs. 17-AAG sensitizes chemoresistant OCSCs to carboplatin treatment. Using HSP90 inhibitors to target p53 aggregation and sensitize chemoresistant cells with protein aggregates may significantly improve the response of ovarian cancer to conventional chemotherapies. Poster Section 39 Poster Board 15 LB-015 VAMP2-NRG1 fusion gene is a novel oncogenic driver of non-small cell lung adenocarcinoma. Yeonjoo Jung,1 Seunghui Yong,1 Pora Kim,1 Hee-Young Lee,1 Yeonhwa Jung,1 Juhee Keum,1 Suyeon Kim,1 Sanghyuk Lee,1 Jhingook Kim,2 Jaesang Kim1. 1Ewha Womans University, Seoul, Republic of Korea; 2Samsung Medical Center, Seoul, Republic of Korea. Neuregulin 1 (NRG1) has been discovered as the tail moiety of fusion genes with several distinct partner head genes in lung cancers. These fusion genes activate ERBB2/ERBB3 receptormediated cell signaling and thereby function as oncogenic drivers. We have carried out whole-transcriptome sequencing of 100 nonsmall cell lung carcinoma (NSCLC) tumors and isolated a novel fusion gene consisting of Vesicle-Associated Membrane Protein 2 (VAMP2) and NRG1. RT-PCR and genomic DNA analysis were used to demonstrate inter-chromosomal translocation. Immunoblotting and soft agar assays were used to examine stimulating activity of the fusion gene through ERBB2/ERBB3 signaling pathway. The most highly expressed splice variant of VAMP2-NRG1 fusion gene was shown to be membrane-bound and display EGF-like domain of NRG1 extracellularly. VAMP2-NRG1 promotes anchorageindependent colony formation of H1568 lung adenocarcinoma cells. Ectopic expression of the fusion gene stimulates phosphorylation of American Association for Cancer Research • AACR ANNUAL MEETING 2015 49 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 50 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 ERBB2 and ERBB3 as well as down-stream targets, AKT and ERK, confirming activation of the signaling pathway. VAMP2-NRG1 is a novel oncogenic fusion gene representing a new addition to the list of NRG1 fusion genes which together may form an important diagnostic and clinical category of lung adenocarcinoma cases. Poster Section 39 Poster Board 16 LB-016 Estrogen receptor stability is modulated by calpain activity in breast cancer cells. Shannon T. Bailey, Thomas Westerling, Myles Brown. Dana-Farber Cancer Institute, Boston, MA. Breast cancer is a disease that affects thousands of women worldwide, and despite a number of treatment options, death results from metastasis and disease resistance. Two-thirds of all breast cancer cases involve dysregulation of estrogen receptor (ER) signaling; thus, a number of treatment strategies employ using compounds that affect its activity. Included in these drugs is the selective ER modulator (SERM) tamoxifen, which binds ER, leading to its association with DNA and the recruitment of corepressor proteins that inhibit ER signaling. While therapies involving tamoxifen are useful in controlling the disease, resistance inevitably occurs, rendering these treatments ineffective. Thus, other compounds are used such as selective ER down regulators (SERDs), which leads to ubiquitin-mediated ER degradation and complete loss of its signaling. Because resistance to these compounds also occurs, other methods for regulating ER signaling must be explored. In this study, we report loss in ER protein expression in response to treatment with doxorubicin and MG132. We found that treatment of the human breast cancer cell lines MCF7 and T47D with doxorubicin plus MG132 leads to ER protein expression loss as demonstrated by western blotting. In contrast, no ER protein expression loss was observed by treatment with either drug alone. RNA-Seq analysis of cells treated with this drug combination demonstrated loss in downstream ER targets, confirming suppressed ER signaling. As MG132 inhibits both the proteasome and calpain activity, we next sought to determine which of these protease activities was involved. Cells were treated in the presence of the proteasome inhibitors bortezomib and lactacystin, and no effect on ER expression was found with doxorubicin treatment, suggesting that the proteasome does not play a role in the ER loss. In contrast, treatment with doxorubicin plus the calpain-specific inhibitors PD 150606 and EST led to ER protein expression loss, confirming that calpain activity is involved in ER protein regulation. Next, to determine which calpains were specifically involved in the loss of ER protein, we knocked down several calpain proteins found to be expressed in MCF7 cells including calpains 1, 7, 8, 9, and 13 and the regulatory subunit CAPNS1. While knockdown of calpains 1, 7, 8, 9, and 13 had no effect on ER protein expression in the presence of doxorubicin, knockdown of CAPNS1 led to a loss in ER that was similar to treatment with calpain inhibitors in the presence of doxorubicin. In conclusion, this study identifies a new pathway by which ER stability is affected i.e., DNA damage induction plus calpain inhibition, suggesting a novel chemotherapeutic treatment paradigm that may overcome resistance in patients with breast cancer. Thus, future studies will focus on identifying the proteins and mechanisms directly responsible for ER loss as well as analyzing preclinical in vivo mouse models. Poster Section 39 Poster Board 17 LB-017 Minnelide reduces castration-resistant and enzalutamide-resistant prostate cancer via downregulation of androgen receptor-mediated signaling. Sumit Isharwal, Shrey Modi, Usman Barlass, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee, Badrinath Konety. University of Minnesota, Minneapolis, MN. Prostate cancer is the second leading cause of cancer death in men in western countries. Advanced prostate cancer is often 50 resistant to hormonal treatment and systemic chemotherapy has limited efficacy. Androgen receptor (AR), a ligand dependent transcription factor plays pivotal role in the development and progression of prostate cancer. While majority of prostate cancers are initially androgen dependent and respond to androgen ablation therapy, most patients eventually recur with more aggressive castration-resistant prostate cancer (CRPC) where AR signaling is reactivated even in the absence of androgen stimulation. Therefore developing novel chemotherapeutic agents for castrate resistant prostate cancer (CRPC) treatment is critical to improve survival in men with CRPC. Triptolide, a diterpene triepoxide isolated from a chinese herb, is extremely effective against several cancers like pancreatic cancer, colorectal cancer and liver cancer both in vivo and in vitro. The water-soluble pro-drug of triptolide, Minnelide, downregulates HSP70 via inhibition of the activity of transcription factor Sp1. Since both Sp1 and HSP70 have been reported to be critical in functionality of AR, we assessed therapeutic potential of Minnelide on androgen dependent, CRPC in vitro and in vivo. Triptolide treatment resulted in dose- and time-dependent cell death in an androgen dependent cell line LNCaP, CRPC cell line C42 and enzalutamide resistant CRPC tumor cell line 22RV1. Triptolide treatment decreased expression of AR full length, AR splice variants and its downstream targets (PSA, NKX3.1) at the mRNA and protein levels. Further, reporter assay with AR responsive elements showed that triptolide decreased transcriptional activity of AR. Expression levels of Sp1 and HSP70 were also reduced following treatment with triptolide these cell lines. To test the efficacy of Minnelide in vivo, male athymic nude mice were castrated 7 days prior to implantation of enzalutamide resistant CRPC (22RV1) cells subcutaneously. The animals received daily intraperitoneal injection of Minnelide and tumor volume was measured weekly until tumor size reached 2cm3.Mice receiving daily injection of Minnelide had significantly smaller tumors than controls as early as two week of treatment (p=0.008). Triptolide therapy inhibited enzalutamide resistant CRPC growth both in vitro and in vivo. Further, our studies for the first time showed that triptolide induces prostate tumor cell death by reducing expression of both full length AR and AR splice variants in a similar manner. Poster Section 39 Poster Board 18 LB-018 Defining a molecular subclass of treatment-resistant prostate cancer. Himisha Beltran,1 Davide Prandi,2 Juan Miguel Mosquera,1 Eugenia Giannopoulou,3 Loredana Puca,1 Clarisse Marotz,1 David M. Nanus,1 Scott T. Tagawa,1 Olivier Elemento,1 Eliezer Van Allen,4 Andrea Sboner,1 Levi Garraway,4 Mark A. Rubin,1 Francesca Demichelis2. 1Weill Cornell Medical College of Cornell University, New York, NY; 2University of Trento, Trento, Italy; 3Hospital for Special Surgery, New York, NY; 4The Broad Institute of MIT and Harvard, Boston, MA. Background: A subset of advanced prostate cancers can progress from an androgen driven state to androgen receptor (AR) independence, often associated with low or absent AR expression and extensive neuroendocrine differentiation. Once neuroendocrine prostate cancer (NEPC) develops, patients typically demonstrate an aggressive clinical course, resistance to AR therapies, and poor overall survival. Early diagnosis is important but remains challenging as the clinical and pathologic features associate with AR independence and NEPC are currently poorly defined. Methods: To address this gap in knowledge, we performed whole exome sequencing (WES) of 124 metastatic tumors from 81 patients including 35 with morphologic features of NEPC. Patients with serial or synchronous samples were included to characterize disease heterogeneity and the transition from adenocarcinoma to NEPC. Immunohistochemistry was performed for neuroendocrine markers and AR in all cases. Computational analysis of clonality and allele specific quantification of copy number were performed Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 51 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 1 using CLONET. Expression profiling (RNA-seq and/or quantitative assessment of a targeted panel of AR signaling genes by Nanostring) and DNA methylation were evaluated in the context of genomic changes. Results: The mutational landscape of NEPC and castration resistant prostate cancer (CRPC) did not differ significantly by rate of non-synonymous mutations or copy number burden (on average >40% of the genome was aberrant), and polyploidy was frequently detected together with common allelic imbalances. Comparative analysis at the DNA and mRNA level identified significant decrease in AR signaling in NEPC and a range of AR signaling in CRPC, enrichment of copy number losses (including RB1 and multiple genes on 16q) in NEPC, and focal high level AR amplification in CRPC in contrast to NEPCs (p-val=0.0007). DNA allele specific analysis of multi-sample cases including patient matched adenocarcinoma-NEPC tumors suggested diverse genomic state of key lesions including aberrations in MYCN and CDKN1B. Conclusions: This is largest study to date focused on the molecular landscape of the NEPC resistance phenotype. NEPC is characterized by a molecular profile defined by distinct genomic alterations and decreased AR signaling. A subgroup of CRPC demonstrates lower AR signaling and molecular overlap with NEPC. This study supports clonal evolution of prostate adenocarcinoma to NEPC, provides new insight into NEPC biology and disease heterogeneity, and may aid in the detection of AR independence and emergence of the NEPC subclass of treatment resistance. Poster Section 39 Poster Board 19 LB-019 BRCA1 protein expression and subcellular localization in primary breast cancer: automated digital microscopy analysis of tissue microarrays. Abeer Mostafa Mahmoud, Umaima Al-alem, Virgilia Macias, Ryan J. Deaton, Andre Balla, Peter Gann, Garth Rauscher. University of Illinois at Chicago, Chicago, IL. Mutations in BRCA1 are associated with familial as well as sporadic aggressive subtypes of breast cancer. However, it is not clear to what extent BRCA1 expression or its subcellular localization contributes to breast cancer progression. The goal of this analysis was to examine the differential expression and subcellular localization of BRCA1 among normal breast tissue and breast cancer cases, and whether it could serve as an additional prognostic marker in an ethnically diverse sample of 287 patients (86 Non-Hispanic White, 84 Hispanic and 116 African American). Tissue microarrays (TMAs) were constructed from invasive breast cancer samples obtained from the Breast Cancer Care in Chicago study in addition to 46 age and race-matched normal breast tissue samples. In these TMAs, BRCA1 was immunolabeled with Alexa647; epithelial cytoplasm with Alexa488; and nuclei with DAPI. Slides were visualized and quantified using “VECTRA Automated Multispectral Image Analysis System” and “InForm software”. BRCA1 expression was evaluated based on the percentage of positive cells and staining intensity using the H-score. The H score is a product of the percentage of cells (0-100%) in each intensity category (0, 1+, 2+ and 3+). The final score is on a continuous scale between 0 and 300. The average nuclear score for BRCA1 (Nuc) was higher than that of the cytoplasmic score (Cyto) in normal breast tissue (Nuc: 157.7 ±6.0; Cyto: 150.7±7.5, p=0.02) and breast cancer cells (Nuc: 140.7 ±3.3; Cyto: 118.0±3.4, p<0.0001). Normal breast tissue had higher levels of BRCA1 protein than breast cancer cells for both the nuclear (p<0.01) and the cytoplasmic (p<0.0001) fractions. However, the nuclear to cytoplasmic ratio of BRCA1 protein was significantly higher in breast cancer cells (1.5±0.1) than in normal mammary epithelial cells (1.1±0.1, p=0.01). This alteration in the nuclear to cytoplasmic ratio may suggest either a role of BRCA1 shuttling in the pathogenesis of breast cancer or the increased need for BRCA1 inside the nucleus to help repairing cancer-induced DNA damage. Cytoplasmic and nuclear BRCA1 expression was then classified as low or high using the mean of the H-score as a cutoff. BRCA1 cytoplasmic and nuclear scores correlated negatively with breast cancer nuclear grade (r2= -0.2, p=0.02); 64% of grade 1 breast cancer cases expressed high levels of BRCA1 while only 36% expressed low levels. In contrast, 60% of the cases in grade 2 and 54% of the cases in grade 3 had low levels of BRCA1. Similarly, an inverse correlation was found between high BRCA1 nuclear scores and stage of breast cancer; 53% in stage 1 and 42% of stage 2 (p=0.05). In addition, high BRCA1 expression significantly correlated with progesterone receptor positivity (P < 0.01), bcl2 positivity (P=0.01), high androgen receptor expression (p<0.0001) and low Ki67 index (P=0.04). In conclusion, in this multi-ethnic sample of breast cancer patients, we found that BRCA1 was associated with less aggressive, lower grade disease. Accordingly, evaluation of BRCA1 expression could provide additional clinically relevant information in routine classification of breast cancer. American Association for Cancer Research • AACR ANNUAL MEETING 2015 51 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 52 Late-Breaking Poster Session: Molecular and Cellular Biology 1 Late-Breaking Poster Session Sunday, April 19, 2015 1:00 PM-5:00 PM Poster Section 41 Late-Breaking Research: Molecular and Cellular Biology 1 Poster Section 41 Poster Board 1 LB-020 Characterization of a novel BRAF in-frame deletion reveals a distinct mutational activating mechanism for oncogenic kinases. Scott Foster, Aysegul Ozen, Dan Whalen, Kyung Song, Georgia Hatzivassiliou, Sarah Hymowitz, Nick Skelton, Shiva Malek. Genentech, South San Francisco, CA. Kinase domain mutations are frequent drivers of many different types of cancer. While extensive studies of kinases such as BRAF and EGFR have provided insight into the mechanistic basis for many oncogenic point mutations such as the commonly occurring BRAFV600E and EGFRL858R mutations, the activation mechanism of short, in-frame deletions (such as the common EGFR exon19 deletion in non-small cell lung cancer) remains to be fully understood. In this work, we have discovered the activation mechanism for novel in-frame deletions in BRAF that have been reported in a small subset of thyroid cancers (TCGA Thyroid data set). These BRAF deletions structurally align with the EGFR exon19 deletion (most common being ΔELREA) and a similar deletion recently identified in HER2-positive breast tumor samples (ΔLRENT). These deletions target the β3-αC loop within the N-lobe of the kinase domain, which provides essential flexibility to the αCHelix allowing the kinase to toggle between inactive and active conformations. Using molecular modeling studies, we demonstrate that all of these deletions genetically compromise this flexibility and constitutively “snap” the αC-Helix in the active conformation. Further characterization of the BRAF deletions shows that similar to BRAFV600E mutations, the β3-αC deletions are kinase-activating, CRAF-independent, and confer dimer-independent activity. Interestingly, cells with this deletion are sensitive to BRAF kinase inhibitors that bind to the active conformation (such as GDC-0879), but are innately resistant to the BRAF kinase inhibitor vemurafenib (which binds BRAF in the αC-Helix inactive, outward-shifted conformation). Similarly, modeling predicts all known β3-αC deletions would confer innate resistance to αC-Helix outwardshifting drugs such as vemurafenib or lapatinib. Further, we speculate that a β3-αC deletion could confer acquired resistance in vemurafenib-treated or lapatanib-treated patients. While many of the more common oncogenic point mutations have been suggested to function in part by destabilizing the inactive conformation and hence promoting the active conformation, it is becoming increasingly clear that oncogenic events that alter the αC-Helix region to directly promote the active conformation provide an alternative mechanism of kinase activation in cancer. Taken together our work underscores the importance of conformation specific kinase inhibitors to target mutationally activated kinases in cancer. Poster Section 41 Poster Board 3 LB-022 Aptamer-mediated inhibition of EGFRvIII mutant in glioblastoma cells. Simona Camorani,1 Elvira Crescenzi,1 David Colecchia,2 Andrea Carpentieri,3 Angela Amoresano,3 Mario Chiariello,2 Laura Cerchia1. 1 Istituto per l’Endocrinologia e l’Oncologia Sperimentale del CNR “G. Salvatore, Naples, Italy; 2Istituto Toscano Tumori-Core Research Laboratory, Siena, Italy; 3Dipartimento di Scienze Chimiche, Università degli Studi di Napoli “Federico II”, Naples, Italy. High-grade glioblastoma multiforme (GBM) is the most aggressive and common of gliomas in human populations, accounting for 55% of primary brain tumors. The prognosis of GBM is very poor and most patients die of tumor recurrence. The epidermal growth factor receptor (EGFR) and the platelet- 52 derived growth factor receptor β (PDGFRβ) are hallmarks in GBM since they influence multiple aspects of tumor biology including cell proliferation, migration, invasiveness and resistance to treatment. In approximately half of the tumors with amplified EGFR, the EGFRvIII truncated extracellular mutant is detected. EGFRvIII does not bind ligand, is highly oncogenic and appears to be relatively resistant to treatment with conventional anti-EGFR agents such as ligand blocking monoclonal antibodies or EGFR tyrosine kinase inhibitors (TKIs). Recently, it has been demonstrated that EGFRvIII-dependent cancers may escape targeted therapy by developing dependence on PDGFRβ signaling, thus providing a strong rationale for combination therapy aimed at blocking both EGFRvIII and PDGFRβ signaling. We have generated two nuclease resistant 2 F-Py RNA aptamers, CL4 and Gint4.T, as high affinity ligands and inhibitors of human wild-type EGFR (EGFRwt) and PDGFRβ, respectively. Thanks to their unique characteristics (low size, good target affinity, no immunogenicity, high stability) aptamers represent a new class of molecules with a great potential to rival monoclonal antibodies in both therapy and diagnosis. Herein, by different approaches we demonstrate that CL4 aptamer binds to the EGFRvIII mutant even though it lacks amino acids 6273 in the extracellular domain. As a consequence of binding, the aptamer inhibits EGFRvIII activation by hampering receptor homodimerization and downstream STAT3 pathway, thus confirming the critical role of EGFRvIII dimerization for signaling. Further, we show that targeting EGFRvIII by CL4, as well as by erlotinib and gefitinib, causes upregulation of PDGFRβ as a compensatory response to support cancer cell survival. Importantly, CL4 and EGFRTKIs cooperate with the anti-PDGFRβ aptamer in inhibiting survival and proliferation of EGFRvIII-overexpressing glioblastoma cells. Given the paucity of selective inhibitors for receptor tyrosine kinases, this study could have impact in the fields of targeted molecular cancer therapeutics and may result in progress against GBM. Poster Section 41 Poster Board 4 LB-023 Caspase-9b directly interacts with cIAP1 to drive agonist-independent NF-κB activation and tumorigenesis in non-small cell lung cancer. Ngoc T. Vu, Margaret A. Park, Michael D. Shultz, Amy C. Ladd, Charles E. Chalfant. Virginia Commonwealth University, Richmond, VA. Caspase-9 has two isoforms with opposing functions, proapoptotic caspase-9a (C9a) and anti-apoptotic caspase-9b (C9b). C9b expression has been reported to augment the anchoragedependent growth (AIG) and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. The mechanism of this biological observation was revealed in this study. Specifically, C9b was demonstrated to have a dual caspase-9a-independent function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway. In particular, C9b was shown to activate the canonical arm and inhibit of the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for C9b contributes to the enhanced survival and AIG of NSCLC cells conferred by C9b expression. The link between C9b expression and NF-κB activation was also validated in human NSCLC tumors. Further mechanistic studies revealed a direct association of C9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif (IBM). Through this interaction, C9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the viability, AIG and tumorigenicity of NSCLC cells. Hence, C9b/cIAP1 interaction is a new attractive molecular target for developing therapeutics to treat NSCLC. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 53 Late-Breaking Poster Session: Molecular and Cellular Biology 1 Poster Section 41 Poster Board 5 LB-024 Inhibition of bone morphogenetic protein (BMP) type I receptors in lung cancer cells activates the TGFβ signaling cascades which increases Id1 expression by TAK1. John E. Langenfeld, Elaine Langenfeld, Monica Castle. Rutgers Cancer Institute of New Jersey, New Brunswick, NJ. Bone morphogenetic protein (BMP) signaling increases Id1 expression while the transforming growth factor (TGF ) signaling typically decreases Id1 expression. BMP antagonists decrease growth of cancer through the inhibition of Id1. Both the BMP and TGF signaling pathways activate TGF activated kinase1 (TAK1). TAK1 has been shown to phosphorylate and activate the BMP transcription factor Smad1/5. The purpose of this study was to understand the cross regulation between the BMP and TGFβ signaling pathways in lung cancer cell lines that occurs following the inhibition of BMP signaling. Antagonists targeting the BMP (DMH2, DMH1), TGFβ (SB-505124), and TAK1 ((5Z)-7-Oxozeaenol) signaling cascades were used to examine the cross regulation between these pathways. Here, we show using siRNA and BMP antagonists targeting the type I receptors that upon inhibition of BMP signaling in lung cancer cells, the TGFβ signaling cascade is activated. SB-505124 alone increases Id1 expression in H1299 cells. However, when BMP signaling was inhibited, SB-505124 decreases Id1 expression, which is associated with a decrease expression in pTAK1. When BMP signaling is inhibited, the TGFβ constitutively active alk5 receptor activates TAK1 and increases Id1 expression, which is attenuated with (5Z)-7-Oxozeaenol. A BMP antagonist together with a TGFβ antagonist further enhanced growth suppression. This study reveals that TGFβ signaling can increase the expression of Id1 when BMP signaling is inhibited in lung cancer cells, which is mediated by TAK1. The data suggests that the inhibition of both the BMP and TGF signaling pathways enhances growth suppression of lung cancer cells that involves the downregulation of Id1. Poster Section 41 Poster Board 6 LB-025 Survivin is trafficked into cancer cell-derived exosomes via a microtubule-dependent mechanism. Bridget T. Kreger, Marc Antonyak, Richard Cerione. Cornell University, Ithaca, NY. Cell-cell communication plays important roles in promoting tumor growth and metastasis, and these events are frequently targeted by therapies. The generation of extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are beginning to be appreciated as one such form of communication that has important consequences in cancer. MVs are small vesicular structures (0.1-2 μm) that are derived from plasma membranes and are shed into the cell’s surroundings; exosomes (30-100 nm) are rerouted endosomes that are released by the fusion of multi-vesicular bodies with the plasma membrane. Both types of EVs influence the behavior of recipient cells through the transfer of their cargo, which includes oncogenic factors, signaling proteins, RNA transcripts, and cytoskeletal components. However, the mechanism through which cargo is selectively trafficked to EVs has not yet been elucidated. We have discovered that disrupting microtubule dynamics in the MDAMB231 breast cancer cell line using the microtubule inhibitor nocodazole has the intriguing effect of significantly increasing the amount of the anti-apoptotic factor survivin that is contained within the EVs shed by these cells. This finding has been replicated in several additional human cancer cell lines and also when using the chemotherapy agent Paclitaxel (Taxol), which also inhibits microtubule dynamics. When MVs were separated from exosomes using procedures that were developed to resolve these two classes of EVs from MDAMB231 cell conditioned medium, survivin was found to be a specific cargo of exosomes and was absent from MVs. Moreover, the exosomes containing survivin enhanced the survival of recipient cells exposed to serum-starvation. Overall, these results show how a specific protein (e.g. survivin) can be selectively packaged into exosomes, as well as shed light on a novel mechanism that underlies Paclitaxel resistance. Poster Section 41 Poster Board 7 LB-026 Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1. Yu-Ling Tai, Tang-Long Shen. National Taiwan Univ., Taipei, Taiwan. Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein in cooperation with numerous tyrosine kinases to regulate various cellular signaling and functions. Although the regulatory mechanisms on the activation of Grb7 have been documented, the molecular mechanism governing Grb7 stability and its functional consequence is still not clearly understood. Here, we observed a novel negative regulatory mechanism of Grb7 by peptidyl-prolyl cis/trans isomerases Pin1 at the post-translational level. The Grb7 phosphorylation on the Ser194-Pro motif facilitated its binding to the WW domain of Pin1 and subsequently ubiquitination on Grb7, which, in turn, enhanced the process of proteosome-dependent proteolysis. Moreover, the interaction between Grb7 and Pin1 is dependent on the phosphorylation that is mediated by c-Jun N-terminal kinases MAPK. Both the phosphorylated Ser/Thr-Pro motif binding module and the peptidylprolyl cis/trans isomerase activity of Pin1 is essentail for Pin1mediated Grb7 ubiquitin-proteasome proteolysis. By contrast, inhibition of Pin by lentiviral-mediated gene silencing resulted in accumulation of Grb7 protein as well as prolonged Grb7 protein stability. A stable Grb7S194A mutant that cannot be bound to and modulated by Pin1 utilize its potential to influence cell cycle progression. Our finding revealed that the Pin1/Grb7 complex formation enables negatively regulating Grb7-mediated cell proliferation due to the influence of Grb7 protein stability. Poster Section 41 Poster Board 8 LB-027 Inhibition of mammalian target of rapamycin by Torin2, an ATP-competitive inhibitor, induces growth inhibition in adult T cell leukemia. Tatsuro Watanabe,1 Naoko Aragane,2 Eisaburo Sueoka1. 1 Department of Laboratory Medicine, Saga University Hospital, Saga, Japan; 2Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan. Adult T cell leukemia (ATL) is one of the aggressive malignant lymphomas induced by the infection of human T-cell Lymphotropic virus-1. Advanced stages of ATL patients still have a poor prognosis and novel therapeutic approaches are needed. Since mammalian target of rapamycin (mTOR) is a key molecule in cell growth and survival in a number of hematological malignancies, we here focused on mTOR signaling pathway consisting from mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). It has been already reported that inhibition of mTOR with rapamycin, a classical mTOR inhibitor, or its derivatives induces growth inhibition in ATL cells, and we found that Torin2, a second generation ATPcompetitive mTOR inhibitor, has more beneficial effects than rapamycin does. Although Torin2 showed weaker growth inhibitory effect against ATL cell lines than rapamycin did at low concentrations (< 10 nM), its effect exceeded at higher concentrations (> 10nM) in CCK-8 assay and Trypan blue staining assay. To understand differences of mechanism of action between Torin2 and rapamycin, we first studied apoptosis and cell cycle arrest of the ATL cell line stained with annexin-FITC; Although both Torin2 and rapamycin caused G1 cell cycle arrest, a small number of ATL cells died in apoptotic manner at higher concentration of Torin2 but not of rapamycin. It is important to note that inhibition of mTOR pathway with rapamycin induced feedback activation of Akt (phosphorylation of Akt at Ser471), downstream of mTORC2, at high concentrations (> 50 nM) but Torin2 inhibited phosphorylation of Akt dose-dependently. Based on these results, we think Torin2 inhibits mTORC1 and mTORC2 followed by G1 cell cycle arrest, however rapamycin inhibits only mTORC1 and it induces feedback activation of Akt at high concentration resulting in resistance to apoptotic cell death. Thus, inhibition of mTOR pathway with Torin2 is new potent therapeutic approach in the treatment of ATL. American Association for Cancer Research • AACR ANNUAL MEETING 2015 53 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 54 Late-Breaking Poster Session: Molecular and Cellular Biology 1 Poster Section 41 Poster Board 9 LB-028 Cell density sensing alters TGF-beta signaling in a cell type-specific manner, independent from Hippo pathway activation. Flore Nallet-Staub,1 Xueqian Yin,2 Cristèle Gilbert,1 Véronique Marsaud,1 Saber Ben Mimoun,1 Delphine Javelaud,1 Edward B. Leof,2 Alain Mauviel1. 1INSTITUT CURIE, ORSAY, France; 2Mayo Clinic, Rochester, MN. Cell-cell contacts inhibit cell growth and proliferation in part by activating the Hippo pathway that drives the phosphorylation and nuclear exclusion of the transcriptional coactivators YAP and TAZ. Cell density and Hippo signaling have also been reported to block TGF-Β responses, based on the ability of phospho-YAP/TAZ to sequester TGF-Β-activated SMAD complexes in the cytoplasm. Herein, we provide evidence that epithelial cell polarization interferes with TGF-Β signaling well upstream and independent of cytoplasmic YAP/TAZ. Rather, polarized basolateral presentation of TGF-Β receptors I and II deprives apically delivered TGF-Β of access to its receptors. Basolateral ligand delivery nonetheless remains entirely effective to induce TGF-Β responses. These data demonstrate that cell type-specific inhibition of TGF-Β signaling by cell density is restricted to polarized epithelial cells and reflects the polarized distribution of TGF-Β receptors, which thus impacts SMAD activation irrespective of Hippo pathway activation. Poster Section 41 Poster Board 10 LB-029 Signal transduction in EGFR-amplified and NFKBIAdeleted human gliomas. Alok Mishra,1 Jun Kong,2 Daniel J. Brat3. 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA; 2Department of Biomedical Informatics, Emory University School of Medicine, Atlanta, GA; 3Department of Pathology and Laboratory Medicine, Department of Biomedical Informatics, Emory University School of Medicine, Atlanta, GA. Nuclear factor Kappa B (NF-κB)- a nuclear transcription factor that is retained in the cytoplasm by IKBα (encoded by NFKBIA)- is implicated as a mediator of aggressive behavior in all glioblastoma (GBM) subclasses. We are interested in elucidating molecular mechanisms that explain the relative mutual exclusivity of EGFR amplification and NFKBIA deletion in GBMs. Bioinformatics analysis of TCGA GBM data showed that NFKBIA hemizygous deletions were significantly depleted in samples with EGFR amplification (one-tailed p value of Fisher Exact test, P = 5.97e-4). Moreover, we showed by immunohistochemistry that IKBα was more highly expressed in EGFR-amplified than non-amplified GBMs. We use Ingenuity Pathway Analysis (IPA) to identify gene sets common to both EGFR amplification and NFKBIA hemizygous deletion to identity shared pathways that might explain mutual exclusivity and identified TNF, p53, PI3K, and NF-κB proliferation and apoptosis signaling pathways. We next genetically modeled glioma cell lines and GBM neurosphere cultures by transient transfection/stable viral transduction to recapitulate EGFR/NFKBIA combinations. qPCR of DNA and mRNA for EGFR and NFKBIA verified genotypes. Among the top hits predicted by our in silico studies, we identified sparcl1 and hgpx4 by RT-qPCR and immunoblots as concordantly upregulated in the EGFRamp/NFKBIA+/+ and EGFRnon-amp/NFKBIA+/cell lines/neurospheres. We also showed differential DNA binding of NF-κB (by electro-mobility shift assay); target gene expression (eg. bcl-2, Il-6); and expression of upstream regulators of NF-κB pathways (viz. pAKT (S436) and IKKβ (Y466)) in GBM neurospheres with distinct EGFR/NFKBIA status. Moreover in U87MG cells (NFKBIA+/-), transiently silencing and overexpressing NFKBIA in different EGFR backgrounds (non-amplified and amplified) led to activation of PARP-dependent but caspase-3-independent apoptotic pathways. Interestingly, in U87MG cells and HPV E6/E7 transformed astrocytes, Annexin V/PI based FACS experiments indicated that NFKBIA overexpression suppressed necrosis. These data demonstrate a complex interplay of EGFR and IKBα/NF-κB in GBMs and identify shared pathways that might explain the relative mutual exclusivity of EGFR amplification and NFKBIA deletion. 54 Poster Section 41 Poster Board 11 LB-030 Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs. Nathalie Wong, Mian He. Chinese Univ. of Hong Kong, Shatin, NT, Hong Kong. Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET proto-oncogene, S100 family members and the caveolins. Of interest, we found exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis. Poster Section 41 Poster Board 12 LB-031 Biochemical profiling of cancer-associated KRAS mutants: clues towards an understanding of differential clinical outcomes. John C. Hunter, Deepak Gurbani, Martin Carrasco, Anuj Manandhar, Sudershan Gondi, Kenneth Westover. UT Southwestern Medical Center, Dallas, TX. As one of the first identified and most commonly activated oncogenes, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) has been a focus of cancer research for many decades. Despite progress in our understanding of RAS biology, significant questions remain unanswered regarding observed differences in clinical outcomes between tumors harboring different KRAS mutations. In an attempt to understand these clinical differences we characterized the pertinent biochemical properties of the most commonly observed KRAS mutants including, nucleotide exchange rate, intrinsic and GTPase activating protein (GAP)-stimulated GTP hydrolysis rate and affinity for one of the enzyme’s primary downstream effectors, Raf kinase. We additionally solved high resolution crystal structures of four of these mutants and attempt to explain the observed biochemical differences between the mutants in the context of these structures. Based on the individual biochemical properties of each KRAS mutant, we propose a classification scheme to predict the propensity of the various mutants to activate Raf-kinase and respond to therapies targeting this downstream signaling pathway. Poster Section 41 Poster Board 13 LB-032 Soluble E-cadherin as a microenvironmental factor that enhances tumor progression. Pratima Patil,1 Robert W. Mason,2 Julia D’ Ambrosio,3 Ayyappan K. Rajasekaran4. 1University of Delaware, Wilmington, DE; 2Alfred I. du Pont Hospital, Wilmington, DE; 3Sci Strategy, Conshohocken, PA; 4 Therapy Architects, Wilmington, DE. During malignant transformation of epithelial cells, their wellorganized architecture is disrupted. A key event observed during epithelial cancer progression is down regulation of E-cadherin (Ecad), a central adherens junction protein that plays a crucial role in Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 55 Late-Breaking Poster Session: Molecular and Cellular Biology 1 directing cell polarity, epithelial architecture and cell-cell adhesion. One mechanism by which tumor cells downregulate E-cad is by the proteolytic cleavage of its extracellular domain by proteases such as the matrix metalloproteinases (MMPs). This proteolytic cleavage results in the shedding of the E-cad ectodomain as an 80 kDa fragment known as soluble E-cadherin (sE-cad). Elevated levels of sE-cad are found in the serum and urine of cancer patients. In tumor cells physiological consequences include enhanced tumor cell migration and invasion, induction of MMP secretion, and increased cell signaling; all of which ultimately result in tumor progression. sEcad also causes morphological changes in epithelial cells, including disruption of the adherens junction. However, the influence of elevated sE-cad levels on normal tissue architecture is not well understood. Specifically, the mechanism by which sE-cad in the tumor microenvironment interacts with cellular E-cad in normal epithelium is yet to be elucidated. We used a three-dimensional (3D) cell-culture system to determine the effects of sE-cad on normal epithelial cyst morphology. In this study, we show that recombinant, purified sEcad can induce lumen-filling in normal non-tumorigenic MadinDarby canine kidney (MDCK) epithelial cysts. The acinar luminal filling and multiple lumen formation induced by purified sE-cad represent changes in epithelial tissue structure that are characteristic of premalignant lesions reported in human epithelial glandular tumors. We further demonstrate that co-culturing of tumor cells with epithelial MDCK cysts results in disruption of 3D epithelial architecture and filling up of the hollow lumen. Our results also show that tumor cell-induced lumen filling is trigged by sE-cad shedding, with a concomitant increase in MMP-9. Using an inhibitor of MMP-9, we provide evidence that MMP-9 is crucial for lumen filling. Together, these novel findings indicate that invasive carcinoma cells can induce structural alterations in adjacent normal epithelium, and that elevated sE-cad levels disrupt epithelial architecture and activate signaling pathways in normal epithelial cells. Poster Section 41 Poster Board 14 LB-033 FAK promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3ββ. Chenxi Gao,1 Guangming Chen,1 Shih-Fan Kuan,2 Dennis H. Zhang,3 Jing Hu1. 1University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2University of Pittsburgh School of Medicine, Pittsburgh, PA; 3University of Pittsburgh Dietrich School of Arts and Sciences, Pittsburgh, PA. Aberrant activation of Wnt/β-catenin signaling plays an unequivocal role in colorectal cancer (CRC), but identification of effective Wnt inhibitors for use in cancer remains a tremendous challenge. New insights into the regulation of this pathway could reveal new therapeutic point of intervention, therefore are greatly needed. Glycogen synthase kinase 3β(GSK3β) is a major component of the Wnt/β-catenin pathway, how GSK3β is regulated in Wnt signaling and tumorigenesis remains largely unknown. This study investigated the function and regulation of GSK3β phosphorylation in Wnt/β-catenin signaling and Wnt-driven intestinal tumorigenesis. The function of GSK3β phosphorylation at tyrosine 216 (Y216) in Wnt/-catenin signaling and CRC cells was analyzed by a series of biochemistry experiments and anchorage independent growth assays. Phosphorylation of GSK3βY216 by focal adhesion kinas (FAK) was studied in in vitro reconstitution system and in cells. The role of FAK-mediated phosphorylation of GSK3βY216 was tested by examining adenoma formation and associated molecular changes in control and FAK inhibitor-treated APCmin/+ mice. Immunohistochemistry was used to evaluate the expression levels of FAK, p-GSK3βY216 and β-catenin in CRC patients. Our results showed that GSK3βY216 phosphorylation was required for the activation of the Wnt/β-catenin pathway. Pharmacological inhibition of FAK suppressed adenoma formation in APCmin/+ mice accompanied by reduced intestinal levels of pGSK3βY216 and β-catenin. FAK, p-GSK3βY216 and β-catenin were elevated and positively correlated in human CRC tissues. Together, our findings indicate that FAK/GSK3βY216 axis is critical for the activation of Wnt/β-catenin signaling in APC-driven intestinal tumorigenesis, thus providing a compelling mechanistic justification for clinical exploration of FAK inhibitors in colorectal cancer patients carrying APC mutations. Poster Section 41 Poster Board 15 LB-034 Identification of novel autophosphorylation structures in crystals of protein kinases. Qifang Xu, Kimberly Malecka, Jeffrey Peterson, Roland L. Dunbrack. Fox Chase Cancer Center, Philadelphia, PA. Cancer therapy depends heavily on the ability to effectively control the activity of oncogenic kinases. Autophosphorylation is a common regulatory mechanism of kinases in signaling pathways, and commonly elevated in cancer. Several autophosphorylation complexes have been identified from within crystals of protein kinases, with a known autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We have utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystal structures in the Protein Data Bank by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of the autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. With this approach, we have identified 15 autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described. Of greatest interest are five structures of activation loop autophosphorylation - PAK1 (T423), IRAK4 (T345), IGF1R (Y1165 and Y1166), and LCK (Y394), two of which we have identified for the first time (IGF1R-Y1166 and LCK). We show that 269 human kinases have potential S/T or Y phosphorylation sites at positions analogous to the PAK1, IGF1R-Y1165/LCK, and IGF1R-Y1166 structures, and that there are 182 such positions that are annotated as phosphorylation sites in Uniprot. To assess the functional importance of the LCK dimer, we performed mutational analysis of residues in the autophosphorylation complex interface of LCK and found that mutations disrupting the interface either severely impaired autophosphorylation (T445D and N446D) or increased it (P447L,A,G). The P447L mutation has been previously found in a Tcell leukemia cell line and associated with activation of LCK. Three structures of receptor tyrosine kinases contain autophosphorylation complexes of the juxtamembrane segment just N-terminal to the kinase domain, two of which are identified for the first time. One of these, CSF1R (Y561) is a homologous site to a known c-KIT (Y568) autophosphorylation structure. The other is in EPHA2 (Y594), which is homologous to Y570 in c-KIT, which is also an autophosphorylation site. Twenty receptor tyrosine kinases contain autophosphorylation sites at one or both of these positions. Phosphorylation at these sites is associated with interaction with SRC family kinases and other downstream effectors of receptor tyrosine kinase signaling. These structures provide critical information on domain-domain interactions and substrate specificity in autophosphorylation, as well as opportunities for understanding the role of certain cancer driver mutations and the development of non-ATP-competitive inhibitors that block dimerization shown in these structures. Poster Section 41 Poster Board 16 LB-035 Hepatitis C virus NS3/4A hijacks the host B-cell receptor signaling pathway. Bojie Dai, Ari Landon, Simone Houng, Ronald B. Gartenhaus. Univ. of Maryland School of Medicine, Baltimore, MD. B-cell receptor (BCR) signaling is critical for the development of normal B-cells and B-cell lymphoma. Increasing epidemiological evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral American Association for Cancer Research • AACR ANNUAL MEETING 2015 55 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 56 Late-Breaking Poster Session: Molecular and Cellular Biology 1 proteins in B-cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B-cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Strikingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and up-regulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, lending further insight into the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders. Poster Section 41 Poster Board 17 LB-036 Neuronal activity-regulated secretion of neuroligin-3 promotes glioma growth. Humsa Venkatesh, Tessa Johung, Viola Caretti, Parag Mallick, Michelle Monje. Stanford University, Stanford, CA. Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). We demonstrate that active neurons similarly promote HGG proliferation and growth in vivo using optogenetic control of cortical neuronal activity in a patientderived pediatric glioblastoma orthotopic xenograft model. Activityregulated mitogen(s) are secreted, as the conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures. The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen; soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feed-forward expression of NLGN3 in glioma cells, providing mechanistic insight into its surprising role as a mitogen. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth. Poster Section 41 Poster Board 18 LB-037 Development and preclinical assessment of a first-inclass small molecule inhibitor of the major cell death regulator protein FLIP. Joanna Majkut,1 Catherine Higgins,1 Adnan Malik,1 Zsusannah Nemeth,1 Peter Blurton,2 Ray Boffey,2 Trevor R. Perrior,2 Patrick G. Johnston,1 David Haigh,1 Timothy Harrison,1 Daniel B. Longley1. 1 Queen’s Univ. Belfast, Belfast, United Kingdom; 2Domainex Ltd, Cambridge, United Kingdom. Background: Evasion of cell death is a major cause of resistance to cancer therapy, making proteins that regulate cell death clinically-relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically-relevant stimuli, including: ligation of death receptors, such as TRAIL-R1 and R2; and cytotoxic chemotherapeutics. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We recently reported that there are important differences between FLIP and procaspase-8 in terms of both their binding affinities and preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. We now report our subsequent work leading to the development and pre-clinical characterisation of first-in-class inhibitors of FLIP. Methods: Molecular modelling of the FLIP-FADD complex; virtual small molecule library screening; cell-free screening assays; cell-based activity assays; biophysical binding assays; in vivo antitumor studies. Results: Molecular modelling of the FLIP-FADD complex identified a putative drug-binding pocket on FLIP against which a 56 virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimisation of these hits afforded lead and back-up series with nanomolar activity in cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). Lead compounds have been shown to block recruitment of FLIP to the TRAIL-R2 death-inducing signalling complex (DISC), confirming their on-target activity. Moreover, the pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL; and lead-molecules have been shown to potentiate the effects of standard-of-care chemotherapeutics and radiotherapy. To further confirm the mechanism of action, FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors. Lead molecules have been identified with ADME profiles suitable for in vivo evaluation. Using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models Conclusions: The novel, first-in-class inhibitors of FLIP developed in this study have the potential for broad application in a range of cancers, either as monotherapy or in combination with other agents. Acknowledgements: This work was supported by a grant from the Wellcome Trust’s Seeding Drug Discovery Initiative (reference: 099470). Reference 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Poster Section 41 Poster Board 19 LB-038 Caspase-mediated iASPP cleavage inhibits NF-kB. Ying Hu,1 Wenjie Ge,1 Xinwen Wang,1 Xin Lu2. 1Harbin Institute of Technology, Harbin, China; 2Ludwig Institute of Cancer Biology, Oxford, United Kingdom. Constitutive activation of the transcription factor nuclear factorkB (NF-κB) plays an important role in oncogenesis and drug resistance of several types of human cancers. Inhibiting NF-κB pathways is therefore emerging as a promising approach to treat cancer. iASPP was first reported as an NF-κB binding partner and inhibitor in a yeast-two-hybrid system. However, it remains unknown how this activity is regulated in cells. For the first time, we show that the activity of iASPP is regulated by caspase-dependent cleavage at cellular level. iASPP was cleaved by caspases at a very early stage of treatment. Interestingly, in contrast to full-length iASPP that is predominantly cytosolic, the iASPP fragment accumulates in the nucleus. Cleaved iASPP shows increased interaction with NF-kB, which correlates with reduced activity of κB reporter. Overexpression iASPP fragment results in increased apoptosis. It also sensitizes cancer cells’ response to paclitaxe. Thus, this study reveals a novel mechanism by which caspase cleavage relocates iASPP from cytoplasm to the nuclear compartment and to pro-apoptotic machinery by binding and subsequent inhibiting NF-κB. This study also provides insights into new strategies to conquer drug resistance of NF-kB constitutive activated malignancies. Poster Section 41 Poster Board 20 LB-039 1,25 dihydroxyvitamin D3 and cisplatin combination modulate p73 in bladder cancer. Brittany L. Bunch, Anna Woloszynska-Read, Donald L. Trump, Candace S. Johnson. Roswell Park Cancer Institute, Buffalo, NY. Cisplatin-based combination chemotherapy is the standard approach to therapy of advanced bladder cancer. While there is a substantial initial response rate and occasional complete response, long term control is not common. There is a need to develop new therapeutic approaches to bladder cancer. 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D, enhances the antitumor effects of cisplatin in preclinical bladder cancer models. We Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 57 Late-Breaking Poster Session: Molecular and Cellular Biology 1 evaluated the mechanism of 1,25D3 potentiation of cisplatin cytotoxicity through in vitro studies in bladder cancer cell lines. Our studies suggest that enhanced cytotoxicity is mediated through modulation of p73. The ratio of the pro-apoptotic TAp73 isoform to the anti-apoptotic ΔNp73 isoform is important in determining cellular response to cisplatin. Our studies demonstrate that pretreatment with 1,25D3 (0.5 uM) for 24 hrs followed by cisplatin (0.75 ug/ml) for 48 hrs increases the ratio of TA/ΔNp73 mRNA transcripts 2-fold in the T24 bladder cancer cell line, and increases TAp73’s transcriptional target, BAX approximately 3-fold compared to cisplatin alone. Protein levels of p73 and BAX, are also increased with 1,25D3 pretreatment followed by cisplatin in T24 cells as determined by western blot. Using a TransAm p53 binding assay after p53 immunodepletion, cisplatin treatment was found to decrease TAp73 functional ability to bind DNA by approximately half, although this has not yet been determined to be statistically significant. 1,25D3 pretreatment followed by cisplatin prevents the decrease in TAp73 DNA binding found after cisplatin alone. These data suggest an increase in TAp73 pro-apoptotic functional abilities resulting from 1,25D3 pretreatment followed by cisplatin in T24 cells. These findings suggest that 1,25D3 may have potential to be used in combination with cisplatin to increase the apoptotic response. Further studies are being performed to determine the requirement of TAp73 in 1,25D3 potentiation of cisplatin cytotoxicity by using T24 cells transfected with TAp73 shRNA in assays previously used to determine synergism, ie. MTT, clonogenic, and apoptosis assays. Supported by NCI grants CA067267 and CA016056. Poster Section 41 Poster Board 21 LB-040 mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction upon 4E-BP1 dephosphorylation. Kan He, Xingnan Zheng, Mei Li, Lin Zhang, Jian Yu. Univ. of Pittsburgh Cancer Inst., Pittsburgh, PA. The mammalian target of rapamycin (mTOR) is commonly activated in colon cancer. mTOR complex 1 (mTORC1) is a major downstream target of the PI3K/ATK pathway and activates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. Rapamycin analogs Everolimus and Temsirolimus are non-ATP-competitive mTORC1 inhibitors, and suppress proliferation and tumor angiogenesis and invasion. We now show that apoptosis plays a key role in their anti-tumor activities in colon cancer cells and xenografts through the DR5, FADD and caspase-8 axis, and is strongly enhanced by TRAIL and 5-fluorouracil. The induction of DR5 by rapalogs is mediated by the ER stress regulator and transcription factor CHOP, but not the tumor suppressor p53, upon rapid and sustained inhibition of 4EBP1 phosphorylation, and attenuated by eIF4E expression. ATPcompetitive mTOR/PI3K inhibitors also promote DR5 induction and FADD-dependent apoptosis in colon cancer cells. These results establish activation of ER stress and the death receptor pathway as a novel anticancer mechanism of mTOR inhibitors. Poster Section 41 Poster Board 23 LB-042 MCPIP1 suppresses breast tumor progression by targeting anti-apoptosis pathway. Jianguo Liu, Wenbao Lu, Huan Ning, Hui Peng, Qinghong Wang, Rong Hou. Saint Louis University, St. Louis, MO. The ability to evade apoptosis is a hallmark of cancer and also plays a significant role in cancer resistance to conventional therapy. While recent progress has broadened our understanding of the apoptosis-evasion mechanisms by cancer cells, how apoptosis resistance develops in cancer cells through posttranscriptional mechanisms, especially by the newly discovered RNA-binding protein monocyte chemotactic protein induced protein 1 (MCPIP1), remains unknown. Here, we report that MCPIP1 expression is impaired in breast tumors and breast tumor cell lines, with the severest impairment in highly metastatic tumors. Ectopic expression of MCPIP1 causes apoptosis of breast tumor cells through selectively suppression of anti-apoptotic gene transcripts, including Bcl2L1, Bcl2A1, RelB, Birc3 and Bcl3. This suppression is medicated through a physical interaction between the PIN domain of MCPIP1 and the 3’UTRs of anti-apoptotic transcripts, resulting in mRNA degradation and tumor cell apoptosis. In difference from the RNA-binding protein tristetraprolin which binds to the ARE in the 3’UTR of target genes for mRNA decay, MCPIP1 specially recognizes a stem-loop structure in the 3’UTR of anti-apoptotic genes for binding and mRNA decay. Furthermore, induction of MCPIP not only ameliorates breast tumor formation but also completely shrinks the established tumors within six days. Lung metastasis is also significantly reduced by MCPIP1 induction. The tumor suppressive effect of MCPIP1 acts through activation of apoptosis. Importantly, by analysis of the excised breast tumors of 251 patients, we found that low MCPIP1 levels in tumors are strongly associated with poor survival of patients over 13 years of follow up. Taken together, we demonstrate that MCPIP1 is a novel potent tumor suppressor which induces tumor apoptosis through tipping the balance between pro-apoptotic and anti-apoptotic genes via selectively targeting the mRNAs of anti-apoptotic genes for degradation. MCPIP1 can serve as a new therapeutic target for treatment of breast cancer and other cancers as well. Poster Section 41 Poster Board 24 LB-043 Highly specific and sensitive nanoimmunoassay of PARP1 as cell death measurement toolkit. Michael A. Pontikos,1 Amos Zimmermann,1 Camilo Moncada,2 Karin V. Abarca Heidemann,2 Jordan B. Miller,2 Jonathan Birabaharan,2 Michael Boyer,2 Robin M. Zuck,2 Philip L. Lorenzi,1 Carl Ascoli2. 1MD Anderson, Houston, TX; 2Rockland Immunochemicals, Pottstown, PA. We developed and validated a highly specific toolkit to analyze PARP1 in a panel of control and siRNA knockdown cell lysates by multiple immunoassays. We can specifically and quantitatively distinguish PARP1 from 17 other PARP family members using highly specific antibodies and optimized methods. Poly (ADP-ribose) polymerase-1 (PARP1) is a chromatin associated, ADP-ribosylating enzyme essential for multiple cellular functions, including cardiac remodeling, vasoconstriction, regulation of astrocyte and microglial function, long term memory, aging, transcription regulation, and DNA repair. More recently, it has been implicated in a new form of cell death termed parthanatos. PARP1 can also promote tissue survival by shifting the balance of cell death programs between autophagy and necrosis. Since PARP1 can promote tumorigenicity, it has gained traction as a therapeutic target in cancer. In that regard, clinical studies have shown vulnerability to PARP inhibitors in DNA repair defective cancers. One of the difficulties in analyzing PARP1 activity is the promiscuity of the reagents toward other PARP family members. Furthermore, the ability to examine multiple tissue samples in parallel has been limited. In this study we developed highly sensitive and specific antibodies against PARP1 fragments. Using siRNAs against 18 PARP family members, we validated antibody specificity by western blotting and nanoimmunoassay (NIA). NIA is a highly sensitive platform capable of screening up to 96 cancer samples at submicroliter volume. Moreover, this platform permits quantitative analysis of non-modified PARP1 as well as post-translational modifications such as PARylation and phosphorylation using a single antibody for the measurement of all species. We anticipate this workflow will be amenable to a wide range of protein targets, ushering in a new frontier in diagnostic analysis. American Association for Cancer Research • AACR ANNUAL MEETING 2015 57 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 58 Late-Breaking Poster Session: Molecular and Cellular Biology 1 Poster Section 41 Poster Board 25 LB-044 Germline mutations in patients with hereditary breast and ovarian cancer establish ERCC2 as a cancer susceptibility gene. Evelin Schrock,1 Anna Benet-Pagès,2 Steffen Schubert,3 Ram nas Janavicius,4 Karl Hackmann,1 Elitza Betcheva-Krajcir,1 Luisa Mackenroth,1 Janin Lehmann,3 AM Nissen,2 Janine Altmueller,5 Holger Thiele,5 Nataliya Di Donato,1 Barbara Klink,1 Jan D. Kuhlmann,6 Andreas Tzschach,1 Karin Kast,6 Pauline Wimberger,6 Elke Holinski-Feder,2 Alfons Meindl,7 Steffen Emmert,3 Andreas Rump1. 1Institute for Clinical Genetics, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany; 2Medizinisch Genetisches Zentrum, Munich, Germany; 3Clinic for Dermatology Venerology and Allergology, Goettingen, Germany; 4Vilnius University Hospital Santariskiu Clinics, Hematology, oncology and transfusion medicine center, Molecular medicine laboratory, Vilnius, Lithuania; 5Cologne Center for Genomics, Cologne, Germany; 6 Department of Gynecology and Obstetrics, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany; 7Zentrum Familiärer Brust- und Eierstockkrebs, Frauenklinik und Poliklinik der Technischen Universität München, Munich, Germany. Introduction: Breast and ovarian cancer (BC/OC) predisposition is associated with a number of high- and low-penetrance susceptibility genes. Despite comprehensive testing there is still a large portion of high risk cases without mutation in any of the known susceptibility loci. Therefore novel candidate genes need to be identified. Here we report on the results of testing 94 genes in 717 BC/OC patients from Germany and Lithuania. Method: Inclusion criteria for the patients in this study were defined by the German Consortium for Breast and Ovarian Cancer. Next generation sequencing (NGS) was performed on an Illumina MiSeq sequencer, with 150 bp paired end sequencing chemistry and an average base coverage of 300 fold. Target enrichment was performed with the Illumina TruSight cancer panel, which includes 94 genes associated with both common (e.g., breast, colorectal) and rare cancers. Results: In 19.7 % of the patients, BRCA1 or BRCA2 variations have been found. These were either clearly pathogenic loss-offunction mutations (43 %) or very rare, unclassified missense variations with high probability of a deleterious effect (57 %). In 17.9 % of the patients we found null-mutations and rare, unclassified missense variants in the acknowledged BC/OC susceptibility genes ATM, CDH1, CHEK2, NBN, PALB2, RAD51C/D and TP53. Analysis of the non-BC/OC genes on the NGS panel identified the “excision repair cross-complementing rodent repair deficiency, complementation group 2” gene (ERCC2 or XPD) as a promising BC/OC predisposition candidate: we found 3 frame-shift mutations and 1 splice-site mutation in four independent BC/OC families. Additionally we found 20 rare, unclassified sequence variations in ERCC2. These variants have a cumulative allele frequency of 2.9 % in our BC/OC cohort, which is 14.5-fold overrepresentation compared to the “exome aggregation consortium” (ExAC) cohort (61486 exomes). In all affected families tested so far, the ERCC2 mutations co-segregate with the occurrence of BC and/or OC. Functional assays testing the ERCC2 variants have been initiated. First results show that at least some of the missense variants (e.g. NM_000400.3:p.Val536Met) have lost their DNA repair ability. Conclusion: Deleterious mutations and probably pathogenic missense variations in ERCC2, which are significantly overrepresented in our BC/OC families and co-segregate with the affected individuals, define ERCC2 clearly as a susceptibility gene for BC/OC predisposition. Functional assays will be continued in order to identify missense variations that diminish the DNA-repair capacity of ERCC2. Affected individuals with excluded mutations in the known BC/OC predisposition genes should be tested for mutations in ERCC2. 58 Poster Section 41 Poster Board 26 LB-045 Detection of keratin fusions in oral squamous cell carcinoma. Jim Jinn-Chyuan Sheu. National Sun Yatsen University, Kaohsiung, Taiwan. Keratin cytoskeleton proteins form intermediate filaments in epithelial cells to regulate cell shape, mobility, membrane trafficking and cellular signaling. Although keratin-6 (K6) and -14 (K14) are highly expressed in certain squamous cell carcinomas and have been suggested as tumor markers, molecular mechanisms of how keratins contribute to cancer development still remain elusive. Here, we demonstrated novel K6-K14 chimeras in oral squamous cell carcinomas (OSCCs) by pair-ended transcriptome sequencing and subsequent validation by fluorescence in situ hybridization and junction site mapping. Two unique fusion types (type-1 and type-2) were identified with a total of 23 in-frame fusion variants verified in OSCCs. Clinical screening confirmed high detection rate of K6-K14 fusions in tumor samples: 33% for type-1 and 25% for type-2. Notably, K6-K14 fusions could be only detected in tumor lesions at late carcinoma stage, but not the ones at early stages, suggesting potential benefits of K6-K14 fusions in promoting aggressive tumors. Functional domain analyses revealed a potent role involved in EMT and metastasis. Poster Section 41 Poster Board 27 LB-046 Evaluation of known low-penetrance thyroid cancer risk alleles in a Hispanic population from South America. Ana Estrada-Flórez,1 Mabel E. Bohórquez-Lozano,1 Rodrigo PrietoSánchez,1 Gilbert F. Mateus,2 Alejandro Rios,3 Alejandro Vélez Hoyos,3 Carlos S. Duque,3 Mirko A. Ledda,4 Maria J. Erazo,5 Fernando Bolaños,6 Cesar Panqueba,6 María Magdalena Echeverry de Polanco,1 Luis G. Carvajal-Carmona4. 1Grupo de Citogenética, Filogenia y Evolución de Poblaciones, Facultades de Ciencias y Facultad de Ciencias de Salud, Universidad del Tolima, Ibagué, Colombia; 2Hospital Federico Lleras Acosta, Ibagué, Colombia; 3 Hospital Pablo Tobón Uribe, Medellín, Colombia; 4Genome Center, Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA; 5Patólogos Asociados, Pasto, Colombia; 6Hospital Hernando Moncaleano Perdomo, Neiva, Colombia. TC is one of the most common malignancy that shows familial inheritance with ~8 fold increase in the risk of developing TC in firstdegree relatives affected by this disease. Recent genome-wide association studies (GWAS) aimed at characterizing the risk loci for TC have identified five single nucleotide polymorphisms (rs966423, rs2439302, rs965513, rs116909374 and rs944289) associated with increased risk for TC. However, effect of these polymorphisms have bot been studied in any Hispanic population. Therefore, the aim of this study was to analyze the above-mentioned SNPs in 235 TC patients and 588 healthy controls from Central Colombia using KASP genotyping system. Genotype frequencies, Hardy Weinberg equilibrium (HWE) and association testing was carried out using Plink. The cumulative genetic risk was assessed using unweighted and weighted approaches. Significant associations between thyroid cancer risk and rs965513A (OR=1.503, 95% CI: 1.203-1.886, P=0.00038) and rs944289T (OR=1.372, 95% CI: 1.095-1.699, P=0.00488) was observed in our study. Consistent, yet not significant associations were observed between disease risk and rs2439302T (OR=1.177, 95% CI: 0.931-1.477, P=0.1666) and rs116909374A (OR=1.626, 95% CI: 0.799-3.766, P=0.2443). For rs966423G, significant departure from HWE was observed therefore this SNP was excluded from further analysis. The combined analyses of rs2439302, rs965513, rs116909374 and rs944289 showed consistent associations between the number of disease alleles and cancer risk. Having three risk alleles increased disease risk by two-fold (OR=2.09, 95% CI:1.46 - 3.02),while carrying five risk alleles was associated with nearly 4-fold increase in the disease risk (OR=3.84, 95% CI:1.47 - 10.06). To our knowledge, this was the first study that assessed TC risk associated with known Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 59 Late-Breaking Poster Session: Molecular and Cellular Biology 1 polymorphisms in the Hispanic population and suggest that these variants could be used in future risk prediction profiling studies in these populations. Poster Section 41 Poster Board 28 LB-047 Single-cell genomics reveals the genesis of cancer: copy number variation precedes single nucleotide variation. Sunney Xie,1 Lei Huang,2 Fei Ma,2 Jingran Wang,2 Shigang Ding,3 Fang Gu,3 Wenjing Wang,3 Jing Zhang3. 1Harvard University, Cambridge, MA; 2Peking University, Beijing, China; 3Peking University Third Hospital, Beijing, China. Recent advances in single cell whole genome amplification methods, such as MALBAC [1], have allowed accurate determination of copy number variation (CNV) as well as single nucleotide variation (SNV) of a single cancer cell [2]. In a bulk tissue sample at an early stage of cancer development, detection of abnormal CNV is often difficult, especially when the number of cells with abnormal CNV is small. Single cell genomic analysis is essential in evaluating the relation, if any, between CNV and SNV. We have carried out for the first time single cell genomic analyses of colonoscopy biopsy at different adenoma stages. Some single cells in the Stage II adenoma have CNVs in the tumor suppresser gene APC (the copy number loss reduced from two to one) as well as SNVs in numerous cancer related genes. Interestingly, we found that while some single cells exhibited CNV reduction in APC without SNVs, all single cells with SNVs in COSMIC genes showed the CNV reduction in APC. Moreover we did not see a single cell that has SNVs but not CNVs in an adenoma. These data indicates that the CNV of APC precedes the SNVs in colon cancer development. Furthermore, we found that the single cells from the same adenoma exhibited CNV patterns reproducible among all the cells, indicating that these cells were derived from the same stem cell, in which CNVs resulted from double strand breaks that could not be repaired perfectly. Thus, we have established for the first time a correlation between SNVs and CNVs and propose that SNVs are generated as a consequence of abnormal CNVs in the genome. Therefore, our results could have a significant implication on the genesis of cancer. References 1. Zong, et al. Science 338: 1622-1626 (2012). 2. Ni et al. PNAS 110, 21083 (2013). Poster Section 41 Poster Board 29 LB-048 Copy number and loss of heterozygosity (LOH) analysis in 52 breast cancer FFPE samples using molecular Inversion probe array: detailed analysis of reproducibility and performance compared to NGS platforms. Candice L. Horn,1 Fabio Nunes,1 John Calley,1 Steven Bray,1 Isabella Wulur,1 Mark Farmen,1 Robert Gallavan,2 Iris Halfpenny,3 Paul Medlow,3 Keith McGreeghan-Crosby,3 Gera Jellema3. 1Eli Lilly and Company, Indianapolis, IN; 2Inventiv Health, Burlington, MA; 3 Almac Diagnostics, Craigavon, United Kingdom. Introduction: Somatic mutations are routinely identified using NGS cancer panels but these panels lack genome-wide coverage for copy number (CN) and LOH analysis. To investigate mutation, CN, and LOH in late-stage breast cancer we tested >50 samples using the Oncoscan molecular inversion probe (MIP) array and evaluated its reproducibility and performance compared to NGS platforms. Methods: 52 breast cancer samples (stage IIIA - IV) were analyzed using MIP array (Oncoscan, Affymetrix). Four samples were tested in technical triplicates to determine assay reproducibility. In addition, 28 samples were sequenced by amplicon-based NGS and five of these samples were also tested using a capture-based NGS platform for mutation, CN, and LOH comparison. Results: MIP array provided highly reproducible results for CN and LOH, with >98% of calls showing CN range in the technical triplicates of < 0.5 copy for 891 cancer genes analyzed. Variability in CN seems to be proportional to absolute copy number at the tested locus, with CN range in technical triplicates of >2 copies seen in only two cases, once for ERBB2 (CN range 32 - 35 copies) and once for FLOT2 (CN range 22 - 25 copies). Gene level results were then categorized in five groups: homozygous deletion, single copy loss, diploid, low grade amplification (<6 copies), or high grade amplification (>6 copies). Using these predetermined cut points, we saw >99% concordance rate among the technical replicates in the MIP array. We found a 93% concordance rate between MIP array and CN/LOH calls by capture-based NGS. Discordant calls between NGS and MIP array were either LOH calls or single copy number change (diploid vs. single copy loss or gain). MIP array mutation analysis of 28 samples showed good sensitivity, correctly detecting the 17 PIK3CA mutations and one TP53 mutation identified by NGS in this cohort. There were seven false positive calls by MIP array, five of them occurring in two genotypes (2x NRAS G12S/C, and 3x EGFR L858R). The other two false positives occurred in PIK3CA, with one false positive (H1047L) occurring in association with a high-grade PIK3CA amplification (7 copies). Increasing CN at the mutation locus was associated with a higher mutation score provided by MIP array (p<0.0001), which may explain some false positive calls. Conclusions: MIP array platform provides a great alternative for assessing CN and LOH in FFPE samples at lower cost and using less input DNA than NGS (80ng vs. 250ng). There was good correlation between CN and LOH results from MIP array and capture-based NGS, with discordant results limited to small CN differences or LOH calls. Mutation analysis by MIP array showed no false negatives when compared to NGS, while false positives seem to occur either due to probe-specific issues or in association with amplifications at the genotyping locus. American Association for Cancer Research • AACR ANNUAL MEETING 2015 59 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 60 Late-Breaking Minisymposium Genome Project, Welcome Trust Sanger Institute, Hinxton, United Kingdom; 4Cancer Research UK London Research Institute, London, United Kingdom; 5Institute for Genomics & Systems Biology and in the Department of Human Genetics, The University of Chicago, Chicago, IL; 6Division of Surgery and Cancer Medicine, Department of Oncology, Oslo University Hospital Radiumhospitalet, Oslo, Norway; 7Department of Pathology, Oslo University Hospital Radiumhospitalet, Oslo, Norway; 8Single-cell Genomics Centre, Welcome Trust Sanger Institute, Hinxton, United Kingdom. Metastasis is the main cause of death amongst breast cancer patients. Our knowledge of the metastatic cascade and how to inhibit it is limited. Here we dissect the genetic profile of multiple single disseminated tumor cells (DTCs) taken at various time points after diagnosis, and compare them to their matched primary tumors and lymph node metastasis. We have previously published a method for studying CNAs in single DTCs by whole genome sequencing, where we compared two primary breast carcinomas to two corresponding DTCs. Copy number profiles from whole genome sequencing (WGS) from 40 DTCs were analyzed. The single cell whole genome amplified (WGA) DNA was used to generate WGS libraries, and the DTCs were subsequently sequenced on the Illumina HiSeq 2000. The WGS reads were trimmed for WGA adapters and aligned to GRCh37 human reference using Burrows-Wheeler Aligner (BWA). LogR values were calculated for genomic bins and corrected for % GC-bias and segmented using the piecewise constant fitting (PCF) algorithm (the penalty parameter, γ, was set to 25). Copy number was estimated per segment as 2logR × Ψ, where Ψ is the average ploidy. B allele frequency (BAF) was calculated for each known SNP position from dbSNP (dbSNP build 135) and somatic mutations read-outs generated. In this study we compared the mutation spectre and CNAs in six primary tumors, one with corresponding lymph node metastasis and single DTCs. In total, CN profile from 40 DTCs showed evidence of dissemination at both early and late stage of disease progression. At large, the copy number profile of the examined DTCs exhibited either a limited number of alterations, or a pattern similar to the primary tumor and lymph node metastasis suggesting continuous dissemination of single tumor cells throughout the tumor evolution. By demonstrating subclonality in the lymph node metastasis we provide novel insight into the metastatic process. Further, the correlation in aberration pattern between the lymph node metastasis and multiple DTCs, implies that cells found in the bone marrow may have originated from the lymph node metastasis. The DTCs exhibited common aberrations typically found in breast carcinomas, and several DTCs had deletion of 16q and17p, and gain of 1q, 8q. Certain DTCs exhibited CNAs not visible in the primary tumor or lymph node including gain of 9q, 14q, 19q and Xq, and loss of 2p, 6p, 8p, 18p and 19p. Two DTCs from time of diagnosis exhibited gain of the whole chromosome 5 that was not observed in the primary tumor or the lymph node. These results reveal the importance of assessing the sub-clonal genetic alterations in the primary tumor, as well as in the lymph node metastasis and DTCs, in order to evaluate patient treatment and prognosis. Late-Breaking Minisymposium Sunday, April 19, 2015 3:15 PM-5:15 PM Room 122, Pennsylvania Convention Center Minisymposium: Late-Breaking Research Co-Chairpersons: Judy Lieberman, Harvard Medical School, Boston, MA; William C. Hahn, Dana-Farber Cancer Institute, Boston, MA 3:15 PM Introduction 3:20 PM LB-050 Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy. Minan Wang,1 James G. Keck,1 Mingshan Cheng,1 Danying Cai,1 Leonard Shultz,2 Karolina Palucka,2 Jacques Banchereau,2 Carol Bult,2 Rick Huntress2. 1The Jackson Laboratory, Sacramento, CA; 2The Jackson Laboratory, Bar Harbor, ME. Mouse models are frequently used to test the therapeutic efficacy of anti-cancer drugs. However, the translation of murine experimental data to treatments for patients with cancer often fails due to significant differences between the species, including the differences in the immune system. Our goal is to bridge this gap and to establish an in vivo preclinical model of human tumor immunotherapy by engrafting immunodeficient mice expressing a partial human immune system with human tumor implants. Humanized NOD-scid IL2Rγ (null) (huNSG) mice were initially generated by transplanting NSG mice with human CD34+ hematopoietic stem and progenitor cells (HSPCs) which support human hematopoietic and immune system development. HuNSG mice develop functional human T cells and B cells with high levels of TCR excision circles, complex TCR repertoire diversity and antigen-specific T cell proliferative responses. Several types of patient-derived tumors (non small cell lung cancer, sarcoma, triple negative breast cancer and invasive bladder cancer) were successfully implanted into HLA mismatched hu-NSG mice. Tumor growth curves show a delay in tumor growth in hu-NSG compared to non-humanized NSG mice. In a colon cancer xenograft model, treatment with chemotherapy agent (5-FU) or with a therapeutic antibody directed against VEGF (Avastin) resulted in decreased tumor growth. In addition to PDX tumors we have also tested human cancer cell lines. Tumor growth was observed in all hu-NSG mice implanted with human ovarian tumor cell line SKOV3-Luc-D3 cells at different time points post HSPC engraftment, showing no evidence of tumor rejection. Thus, our model of humanized mice bearing tumor-derived xenografts provides opportunities to study both the safety and efficacy of current cancer therapies. 3:35 PM LB-051 Tumor heterogeneity and dissemination in breast cancer: Deep sequencing of single disseminated cells from bone marrow compared to primary tumor and lymph node metastases. Elen Møller,1 Parveen Kumar,2 Silje Nord,1 David Wedge,3 Peter van Loo,4 April Peterson,5 Randi R. Mathiesen,6 Renathe Fjelldal,7 Masoud Z. Esteki,2 Jason A. Grundstad,5 Elin Borgen,7 Lars O. Baumbusch,1 AnneLise Børresen-Dale,1 Kevin P. White,5 Thierry Voet,8 Bjørn Naume,6 Vessela N. Kristensen1. 1Institute for Cancer Research, OUS, Oslo, Norway; 2Centre for Human Genetics, University Hospital Leuven, Department of Human Genetics, KU Leuven, Leuven, Belgium; 3Cancer 60 3:50 PM LB-052 Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification. Jon Roffey,1 Andrew Turnbull,1 Christian Dillon,1 Susan Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 61 Late-Breaking Minisymposium Boyd,2 Philippe Riou,3 Mark Linch,3 Peter Parker,3 Sven Kjaer,4 Neil McDonald4. 1Cancer Research Technology Discovery Laboratories, London, United Kingdom; 2 CompChem Solutions Limited, Cambridge, United Kingdom; 3Protein Phosphorylation Laboratory, Cancer Research UK London Research Institute, London, United Kingdom; 4Structural Biology Laboratory, Cancer Research UK London Research Institute, London, United Kingdom. Protein kinases are attractive targets for pharmacological intervention through their frequent disease associated aberration of cellular signalling networks. In this context an understanding of the myriad of substrates on which a protein kinase operates can help to reveal potential pharmacodynamic and patient stratification biomarkers. Kinase Identification of Proximal Substrates (KIPS) utilises an innovative chemical genetics approach to identify novel proximal biomarkers of kinase target response. This approach relies on the generation of a desensitized mutant protein that retains its kinase function, but loses sensitivity to small-molecule mediated inhibition. Comparative phospho-proteomics is performed on immunocomplexes of tagged wild type and mutant protein kinases in the presence and absence of inhibitor to isolate specific phospho-proteomic changes in proximal substrates. To exemplify the KIPS approach we have focused on the identification of novel substrates of the atypical protein kinase C isoform, PKCι. Through structure based design we identified two acidic amino acid residues in the PKCι nucleotide binding site that are crucial for efficacy of a previously characterized PKCι inhibitor, CRT0066854. Upon mutation to nonacidic alanine residues the PKCι protein is rendered insensitive to compound-mediated inhibition, allowing a comparison of wild type and mutant responses to PKCι inhibition to be made. Using HCT116 cells, we identified Myosin X as a putative substrate for PKCι, and demonstrated its specificity to the PKCι signalling axis through alanine-scanning peptide arrays and generation of phospho-specific antibodies. We show that the key acidic amino acids utilised in KIPS are highly conserved across the AGC and CAMK kinase super families. Furthermore, extensive compound SAR, diverse kinome profiling and structural biology have identified a tool box of inhibitor compounds with a broad range of activities across these kinase families leading to broader applicability of KIPS to enable the identification of individual kinase specific substrates and response across approximately 20% of the human kinome. 4:05 PM Pathology, VUmc, Amsterdam, Netherlands; 8 Department of Pulmonary Diseases, Maastricht University Medical Center, Maastricht, Netherlands; 9 Piovtal, Madrid, Spain. This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2015 Official Press Program. It will be posted online following its presentation. 4:20 PM LB-054 Normal stem cell divisions, cancer incidence, and driver gene mutations. Cristian Tomasetti, Bert Vogelstein. Johns Hopkins University School of Medicine, Baltimore, MD. Cancer arises through the sequential accumulation of mutations in oncogenes and tumor suppressor genes. We show that the incidence of many cancers is strongly correlated (0.80; P < 1.8 × 10-6) with the lifetime number of divisions of the normal stem cells maintaining that tissue’s homeostasis. These results suggest that only ~35% of the variation in cancer incidence among tissues is attributable to environmental factors or inherited predispositions. Moreover, we derive an approach to assess the evidence for environmental and inherited factors that contribute to a given cancer type vs. other cancer types. The slope of the correlation noted above suggests that the number of driver gene mutations required to develop cancer must be smaller than what was previously estimated. We describe a novel approach to derive this estimate that combines conventional epidemiologic studies with genome-wide sequencing data. In two well-documented cancer types (lung and colon adenocarcinomas), we find that no more than three sequential mutations are needed. Finally, we explore the role of chance and aging in acquiring the driver gene mutations required to develop cancer. We provide novel experimental evidence, in both mice and humans, of an unexpected relationship between the dynamics of division rates in normal tissues and age-specific cancer incidence. These data and analyses provide a firm basis for understanding the role of random, replicative mutations during stem cell division in cancer incidence. 4:35 PM LB-055 Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations. Leanne G. Ahronian,1 Erin M. Sennott,1 Eliezer M. Van Allen,2 Nikhil Wagle,2 Eunice L. Kwak,1 Jason E. Faris,1 Jason T. Godfrey,1 Koki Nishimura,1 Kerry D. Lynch,3 Craig H. Mermel,1 Elizabeth L. Lockerman,1 Anuj Kalsy,1 Joseph M. Gurski,1 Samira Bahl,4 Kristin Anderka,4 Lisa M. Green,4 Niall J. Lennon,4 Tiffany G. Huynh,3 Mari Mino-Kenudson,3 Gad Getz,1 Dora Dias-Santagata,3 A. John Iafrate,3 Jeffrey A. Engelman,1 Levi A. Garraway,2 Ryan B. Corcoran1. 1Massachusetts General Hospital Cancer Center, Boston, MA; 2Dana Farber Cancer Institute, Boston, MA; 3Massachusetts General Hospital Department of Pathology, Boston, MA; 4Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA. BRAF V600E mutations occur in ~10% of colorectal cancer (CRC), and are associated with poor prognosis. RAF inhibition alone has not been an effective treatment in BRAF-mutant (BRAFm) CRC patients, with response rates of only 5%, due to persistence of MAPK signaling. Combined RAF/EGFR, RAF/MEK, or RAF/MEK/EGFR inhibitors have produced improved efficacy in BRAFm LB-053 Monitoring rearrangement of EML4-ALK in blood platelets predicts outcome to crizotinib treatment in non-small-cell lung cancer patients. Jonas A. Nilsson,1 Niki Karachaliou,2 Pepijn Schellen,3 Ana Gimenez-Capitan,4 Jordi Berenguer,3 Cristina Teixido,4 Justine L. Kuiper,5 Esther Drees,3 Magda Grabowska,3 Marte van Keulen,6 Jihane M. Tannous,6 Danielle A.M. Heideman,7 Erik Thunnissen,7 Anne-Marie C. Dingemans,8 Santiago Viteri,2 Bakhos A. Tannous,6 Ana Drozdowskyj,9 Rafael Rosell,2 Egbert F. Smit,5 Thomas Wurdinger3. 1Radiation Sciences, Oncology, Umeå, Sweden; 2Translational Research Unit, Dr Rosell Oncology Institute, Quiron Dexeus University Hospital, Barcelona, Spain; 3Department of Neurosurgery, VUmc, Amsterdam, Netherlands; 4Pangaea Biotech SL, Barcelona, Spain; 5Department of Pulmonary Diseases, VUmc, Amsterdam, Netherlands; 6Department of Neurology, MGH/HMS, Boston, MA; 7Department of American Association for Cancer Research • AACR ANNUAL MEETING 2015 61 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 62 Late-Breaking Minisymposium CRC patients, yet ultimately resistance develops after an initial treatment response. Understanding the mechanisms of clinical acquired resistance that arise to RAF inhibitor combinations in BRAFm CRC patients may lead to valuable opportunities to overcome resistance and prolong clinical response. We sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations by obtaining tumor biopsies from BRAFm CRC patients upon disease progression, after initial response to RAF/EGFR or RAF/MEK inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA-seq. In one BRAFm CRC patient with prolonged stable disease on a RAF/EGFR combination, WES identified KRAS amplification in a progressing lesion. FISH confirmed the presence of KRAS amplification in the post-progression biopsy, and RNA-seq revealed KRAS transcript overexpression. Interestingly, in resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitors, KRAS exon 2 mutations were identified. Either KRAS amplification or KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. In a second patient with a minor response to a RAF/EGFR inhibitor combination, BRAF amplification was identified in a progressing lesion, which was confirmed by FISH and was not present in a pretreatment biopsy of the same lesion. BRAF amplification led to cross-resistance to the BRAF/MEK inhibitor combination. In a third patient with a minor response to a RAF/MEK inhibitor combination, WES identified the presence of an ARAF Q489L mutation and a MEK1 F53L mutation in a single progressing lesion, suggesting possible intra-lesional heterogeneity of acquired resistance mechanisms. However, utilizing a cell line derived from the patient’s post-progression biopsy, we found that 30 out of 30 single-cell clones harbored both the ARAF and MEK1 mutations, and that MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant cells. MEK1 F53L expression markedly abrogated the ability of RAF/MEK and RAF/EGFR inhibitor combinations to suppress MAPK signaling. Despite developing resistance to upstream MAPK pathway inhibitors, we found that each of the acquired resistance mechanisms we detected remained sensitive to ERK inhibition, which could effectively suppress MAPK signaling. Our findings demonstrate the central importance of MAPK pathway activity in BRAFm CRC, and highlight the critical need for MAPK pathway inhibition in the prevention of disease progression. Additionally, our work indicates ERK inhibitors may be valuable additions to future therapeutic combinations for BRAFm CRC patients. Further efforts to understand acquired resistance mechanisms will be vital to developing novel therapeutic strategies to overcome resistance and extend clinical benefit in this lethal CRC subtype. 62 4:50 PM LB-056 TP53 and RB1 alterations promote reprogramming and antiandrogen resistance in advanced prostate cancer. Ping Mu, Zhen Cao, Elizabeth Hoover, John Wongvipat, Chun-Hao Huang, Wouter Karthaus, Wassim Abida, Elisa De Stanchina, Charles Sawyers. Memorial Sloan Kettering Cancer Center, New York, NY. Castration-resistant prostate cancer (CRPC) is one of the most difficult cancers to treat with conventional methods and is responsible for nearly all prostate cancer deaths in the US. The Sawyers laboratory first showed that the primary mechanism of resistance to antiandrogen therapy is elevated androgen receptor (AR) expression. Research based on this finding has led to the development of next-generation antiandrogen: enzalutamide. Despite the exciting clinical success of enzalutamide, about 60% of patients exhibit various degrees of resistance to this agent. Highly variable responses to enzalutamide limit the clinical benefit of this novel antiandrogen, underscoring the importance of understanding the mechanisms of enzalutamide resistance. Most recently, an unbiased SU2C-Prostate Cancer Dream Team metastatic CRPC sequencing project led by Dr. Sawyers and Dr. Chinnaiyan revealed that mutations in the TP53 locus are the most significantly enriched alteration in CRPC tumors when compared to primary prostate cancers. Moreover, deletions and decreased expressions of the TP53 and RB1 loci (co-occurrence and individual occurrence) are more commonly associated with CRPC than with primary tumors. These results established that alteration of the TP53 and RB1 pathways are associated with the development of antiandrogen resistance. By knockdowning TP53 or/and RB1 in the castration resistant LNCaP/AR model, we demonstrate that the disruption of either TP53 or RB1 alone confers significant resistance to enzalutamide both in vitro and in vivo. Strikingly, the co-inactivation of these pathways confers the most dramatic resistance. Since upregulation of either AR or AR target genes is not observed in the resistant tumors, loss of TP53 and RB1 function confers enzalutamide resistance likely through an AR independent mechanism. In the clinic, resistance to enzalutamide is increasingly being associated with a transition to a poorly differentiated or neuroendocrinelike histology. Interestingly, we observed significant upregulations of the basal cell marker Ck5 and the neuroendocrine-like cell marker Synaptophysin in the TP53 and RB1 inactivated cells, as well as downregulation of the luminal cell marker Ck8. The differences between these markers became even greater after enzalutamide treatment. By using the p53stabilizing drug Nutlin, level of p53 is rescued and consequently the the decrease of AR protein caused by RB1 and TP53 knockdown is reversed. These results strongly suggest that interference of TP53 and RB1 pathways confers antiandrogen resistance by “priming” prostate cancer cells to reprogramming or transdifferentiation, likely neuroendocrine-like differentiation, in response to treatment. Futher experiments will be performed to assess the molecular mechanism of TP53/RB1 alterations in mediating cell programming and conferring antiandrogen resistance. 5:05 PM Discussion Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 63 Late-Breaking Poster Session: Molecular and Cellular Biology 2 Late-Breaking Poster Session Monday, April 20, 2015 8:00 AM-12:00 PM Poster Section 39 Late-Breaking Research: Molecular and Cellular Biology 2 Poster Section 39 Poster Board 1 LB-058 Novel interactions of the RAS oncoprotein. Sunita Shankar,1 Rohit Malik,1 Vishal Kothari,1 Yasuyuki Hosono,1 Sethuramasundaram Pitchiaya,1 Shanker Kalyana-Sundaram,1 Anastasia Yocum,1 June Escara-Wilke,1 Harika Gundlapalli,1 Krishnapriya Chinnaswamy,1 Matthew Shuler,1 Anton Poliakov,1 Xiaoju Wang,1 Vishalakshi Krishnan,1 Yasmine White,1 Ari Firestone,2 Xuhong Cao,1 Saravana M. Dhanasekaran,1 Jeanne Stuckey,1 Gideon Bollag,1 Kevin Shannon,2 Nils G. Walter,1 Chandan KumarSinha,1 Arul Chinnaiyan1. 1University of Michigan, Ann Arbor, MI; 2 University of California, San Francisco, CA. Approximately 30% of all cancers harbor activating mutations in the RAS family of small GTPase proteins, making it one of the most common oncogenic aberrations in humans. Normal RAS proteins (H, K or N-RAS) localize to the inner cell membrane and transduce extracellular growth signals by cycling between an “active” GTPbound state and “inactive” GDP-bound state, through interactions with various “GTPase activating proteins” (GAPs) that promote RAS mediated GTP hydrolysis. Oncogenic mutants of RAS lose their catalytic activity or association with the GAP proteins, resulting in constitutively active GTP-bound state that signals through direct interactions with effector kinases like RAF and PI3K and activate the MEK/ERK and/or Akt, leading to activation of hallmark cancer pathways including growth factor independence, uncontrolled cell proliferation, evasion of apoptosis and immune responses, increased metabolism as well as metastases. Although RAS is the most frequently mutated gene driving multifarious pathways of oncogenesis, our knowledge of protein interactions involving RAS proteins have been largely limited to RAS binding domains in RAF/PI3K/RalGDS. Targeting mutant RAS proteins or its direct effectors, or pathways activated by RAS effectors remains a challenging endeavor for treating RAS driven cancers. Towards the goal of a thorough understanding of RAS biology through a comprehensive identification of its interactors, we performed IP-Mass Spectrometric analysis of pan-RAS immunoprecipitates from multiple cell lines. Interestingly in our experiments, apart from the well-known interactor RAF, we found evidence of several novel RAS interacting proteins, including many with DNA and RNA binding motifs. Our study validates these findings through cell-free protein interaction analyses and explores the possible biological effects of these novel RAS interactions in mutant KRAS driven cellular transformation. Poster Section 39 Poster Board 2 LB-059 Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer. Priya S. Mathur,1 Jessica J. Gierut,2 Rosa M. Xicola,3 Xavier Llor,3 Angela L. Tyner1. 1University of Illinois Medical Center, Chicago, IL; 2 Beth Israel Deaconess Medical Center, Cambridge, MA; 3Yale Medical School, New Haven, CT. Background: Protein Tyrosine Kinase 6 (PTK6, also called BRK) is a non-receptor tyrosine kinase expressed in differentiated epithelial cells of the skin, gastrointestinal tract, and prostate. Disruption of the Ptk6 gene led to impaired intestinal differentiation and increased intestinal proliferation in mice. However, PTK6 also has a tumor-promoting role in the colon, as disruption of the Ptk6 gene impaired carcinogen-induced STAT3 activation and tumorigenesis in mice. PTK6 also promotes survival of human colon cancer cell lines following DNA damaging treatments including γirradiation and chemotherapeutic drugs via STAT3 activation. Results: We examined and manipulated colon cancer cell lines to understand these two seemingly contradictory roles for PTK6 in the colon. The human colon adenocarcinoma cell lines SW480 and SW620 represent a primary colon tumor and a metastasis from the same patient, respectively. PTK6 is highly expressed in SW480 cells and significantly reduced in SW620 cells. Active PTK6, phosphorylated on tyrosine residue 342, is not detectable in either cell line. Stable knockdown of PTK6 in SW480 cells results in an epithelial-to-mesenchymal transition (EMT), evidenced by decreased E-cadherin expression and increased ZEB-1 and Vimentin; as well as increased migration, wound-healing, and anchorage-independent growth of cells in culture. Additionally, knockdown of PTK6 led to increased in vivo tumorigenesis of xenograft cells in nude mice. Ectopic expression of wild-type PTK6 in SW620 cells drives the reverse process (MET), demonstrating a rescue of the EMT phenotype. When a constitutively active PTK6 construct is expressed in SW620 cells, it promotes activation of cytoplasmic oncogenic targets and downstream effectors, STAT3, FAK, BCAR1 and ERK5. Analysis of patient tissues demonstrates a downregulation of mRNA and protein levels, as well as a change in protein localization in tumor versus normal tissues; while PTK6 is detectable in the nuclei of differentiating normal cells it is excluded from nuclei in colon tumors. Conclusions: Our studies indicate that PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner and a kinase switch activates PTK6 to promote oncogenic signaling. PTK6 protein localization may function to regulate its specific role. Further examination of the complex role of this kinase could allow for a more nuanced understanding of colon cancer initiation and progression for the development of novel treatments. Poster Section 39 Poster Board 3 LB-060 A phosphoproteomic comparison of BRAF(V600E) and MKK1/2 inhibition in melanoma. Scott Stuart, Natalie Ahn, Stephane Houel, William Old. University of Colorado, Boulder, Boulder, CO. Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with BRAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the BRAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4,722 proteins. This included 1,317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. While the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation. American Association for Cancer Research • AACR ANNUAL MEETING 2015 63 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 64 Late-Breaking Poster Session: Molecular and Cellular Biology 2 Poster Section 39 Poster Board 4 LB-061 Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. Heather Schofield,1 Filip Bednar,1 Meredith Collins,1 Wei Yan,2 Yaqing Zhang,1 Nikhil Shyam,1 Jaime Eberle,3 Kenneth Olive,3 Nabeel Bardeesy,4 Daisuke Nakada,5 Diane Simeone,1 Sean Morrison,6 Marina Pasca di Magliano1. 1University of Michigan, Ann Arbor, MI; 2Fourth Military Medical University, Xi’an, China; 3 Columbia University, New York, NY; 4Massachusetts General Hospital, Boston, MA; 5Baylor College of Medicine, Houston, TX; 6 University of Texas Southwestern Medical Center, Dallas, TX. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Many characteristic mutations in PDAC are known, but this information has so far failed to produce the development of effective treatments, highlighting the need for deeper understanding of the processes that drive tumorigenesis. B-cell specific Moloney murine leukemia virus insertion site 1 (Bmi1), a Polycomb repressive group protein, is upregulated in PDAC and associated with poor prognosis. Our lab has shown that despite an oncogenic K-ras mutation, mice with pancreas specific loss of Bmi1 do not develop precancerous lesions, termed PanINs (Pancreatic Intraepithelial Neoplasia). This lack of PanIN development was also seen in the presence of pancreatitis, which is usually known to synergize with oncogenic K-ras to speed PanIN development. In other cancer types, Bmi1’s effect on tumorigenesis was mechanistically linked to its regulation of the Ink4a/ARF genetic locus. However, we found that in PDAC, PanIN initiation was independent of Bmi1 control this locus. Further, impairment in the regulation of ROS generation was seen in vitro in pancreatic cancer cell lines lacking Bmi1. Regulating ROS generation is a vital step in the neoplastic process and has been shown in other systems to be controlled by Bmi1. Overall, in this work we have shown that expression of the Polycomb group protein Bmi1 is necessary for the initiation of pancreatic precancerous lesions, and that the mechanism of Bmi1 requirement is independent of its repression of the Ink4a/ARF genetic locus. Given the recent pre-clinical development of a Bmi1 inhibitor, this work could provide rationale for future treatment of pancreatic cancer, a truly devastating disease. Poster Section 39 Poster Board 5 LB-062 XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas. Rossella Marullo,1 ShaoNing Yang,1 Tami Rashal,2 Yosef Landesman,2 Robert Carlson,2 Sharon Shacham,2 Leandro C. Cerchietti1. 1Weill Cornell Medical College of Cornell University, New York, NY; 2Karyopharm Therapeutics, Newton, MA. Mutation and constitutive expression of MYC and BCL2 and/or BCL6 (a.k.a. double and triple-hit lymphomas) defines subsets of diffuse large B-cell lymphoma (DLBCL) pts with particularly poor outcome. Almost 60% of pts with BCL2 and MYC translocations die within six months of diagnosis due to chemorefractory disease, a prognosis that cannot be overcome with intense chemotherapy. A further hindrance to patient survival is that these double and triplehit lymphomas are frequently found in the elderly who have limited tolerability to aggressive chemotherapeutic regimens. To identify chemoresistant double and triple-hit DLBCL, we screened 38 DLBCL cell lines for mutations in BCL2, MYC and BCL6 by FISH and response to chemotherapy administration in vitro. We found 3 triple-hit and 2 double-hit DLBCL cell lines. Previous work in our laboratory showed that the nuclear export protein, XPO1, is a critical regulator of MYC, BCL2 and BCL6 mRNA transport in DLBCL. We therefore analyzed XPO1 amplification (by PCR) and expression (by immunoblot) in these cell lines and found that all 5 cells expressed XPO1 at higher levels than centroblasts and that, in at least 2 of them, this was result of gene amplification. Inhibition of XPO1 with selinexor, a Selective Inhibitor or Nuclear Export (SINE) compound, increased nuclear localization 64 of MYC, BCL2 and BCL6 transcripts and consequently decreased their protein expression. Moreover, selinexor administration induced chemosensitization in doxorubicin-resistant DLBCL cells through decreasing DNA damage repair mechanisms as demonstrated by comet assays. To determine the potential extent of DLBCL patients that could benefit from such a treatment, we first analyzed 110 DLBCL patient samples by immunohistochemistry and found that XPO1 expression was increased in 85 cases compared to normal tonsils. Within the cohort analyzed, 6 patients had double or triple hit lymphoma (by FISH) and all overexpressed XPO1. Since this population has higher incidence of chemorefractory disease, we decided to develop a pre-clinical model of this disease using primary patient samples. We developed two patient-derived xenograft (PDX) models representing a double and a triple-hit lymphoma. In both cases XPO1 was also amplified. We compared the molecular and pathological characteristics of 12 generations of PDXs with the original pt sample, and found that protein expression and mutations of these genes were stable. We therefore administered selinexor alone, CHOP alone (cyclophosphamide, vincristine, doxorubicin and dexamethasone) and the combination of selinexor with CHOP to these PDXs. We found that selinexor significantly decreased tumor growth, compared to vehicle or CHOP alone. Moreover, combinatorial treatment indicated that selinexor was able to revert the chemorefractoriness of these DLBCLs, without inducing toxicity by clinical chemistry or pathologic examination. Analysis of selinexor vs. vehicle treated PDXs indicated a decrease in protein levels of MYC, BCL6, BCL2 as well as DNA damage and checkpoint regulators such as CHK1. In summary, selinexor has potent anti-proliferative effects in double/triple-hit DLBCL and can be safely combined with CHOP to enhance cancer cell death. These data present a new therapeutic approach for pts with double/triple hit lymphomas, and provide rational support for the study of selinexor/CHOP combination in clinical trials. Poster Section 39 Poster Board 6 LB-063 PTEN function is controlled by recruitment to cytoplasmic vesicles. Adam Naguib,1 Gylua Bencze,1 Hyejin Cho,1 Wu Zheng,1 Ante Tocilj,1 Elad Elkayam,1 Christopher R Faehnle,1 Nadia Jaber,2 Christopher Pratt,1 Muhan Chen,1 Wei-Xing Zong,2 Michael S Marks,3 Leemor Joshua-Tor,1 Darryl J Pappin,1 Lloyd C. Trotman1. 1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 2Stony Brook University, Stony Brook, NY; 3University of Pennsylvania, Philadelphia, PA. PTEN is thought to function at the plasma membrane where receptor tyrosine kinases activate PI 3-Kinases. Yet the majority of PTEN is located throughout the cytoplasm so that only a fraction of PTEN could be actively suppressing PI 3-K signaling at any time. Here we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via interaction with phosphatidylinositol 3- phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the C2 domain of PTEN specifically binds PI(3)P via the CBR3-loop. Mutations that render the CBR3-loop incapable of PI(3)P binding abrogate PTEN function in cells but not in vitro. The loss-of-function in cells is rescued by fusion of the canonical PI(3)P vesicle targeting domain, FYVE, to CBR3-loop mutant PTEN, demonstrating the functional relevance of PTEN activity on endosomal membranes. These findings introduce an entirely unexpected site of action of the PTEN tumor suppressor. Furthermore, they introduce the concept of PI 3-K signal activation over the vast surface of the plasma membrane that is contrasted by PTEN-mediated signal termination on the discretely sized and much smaller surfaces of endocytic vesicles. Implications of these results for cancer signaling and growth control will be discussed. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 65 Late-Breaking Poster Session: Molecular and Cellular Biology 2 Poster Section 39 Poster Board 7 LB-064 Characterization of the tumor suppressor function of the lysine-specific methyltransferase KMT2D in follicular lymphoma. Ana Ortega-Molina,1 Isaac Boss,2 Heng Pan,2 Yanwen Jiang,2 Deqing Hu,3 Xin Gao,3 Rita Shaknovich,2 Ali Shilatifard,3 Ari M. Melnick,2 Hans-Guido Wendel1. 1Memorial Sloan Kettering Cancer Center, New York, NY; 2Weill Cornell Medical College, New York, NY; 3 Northwestern University, Chicago, IL. Follicular lymphoma (FL) is a common and incurable form indolent B-cell lymphoma. Genomic studies have now catalogued many recurrent mutations in FL. Epigenetic regulators have emerged as the most common targets of somatic point mutations. For example, the histone methyltransferase KMT2D (MLL4/MLL2) is the most frequently mutated gene in FL with reported mutational frequencies of up to 84%. However, the transcriptional and biological consequences of KMT2D loss of function are currently unknown. Using a well-described murine model of FL, we showed that deficiency of KMT2D promotes the initiation of indolent FL in vivo. To explore the mechanism of KMT2D-mediated tumor suppression we used cross species comparisons of gene expression, histone H3K4 methylation marks and KMT2D genomic occupancy. These analyses converge on a relatively small group of conserved target genes. Strikingly, they include bona fide tumor suppressors and regulators of B-cell proliferation (e.g. TNFAIP3/A20, SOCS3, ARID1A, and TNFRSF14). Our results indicate that KMT2D restrains FL development through simultaneous effects on multiple key regulators of B-cell behavior. Poster Section 39 Poster Board 8 LB-065 Mutational and functional analysis of the tumor suppressor PTPRD in human melanoma. Vijay Walia,1 Todd Prickett,1 Jung-Sik Kim,2 Jared J. Gartner,1 Jimmy C. Lin,1 Steven A. Rosenberg,3 Randolph C. Elble,4 David A. Solomon,2 Todd Waldman,2 Yardena Samuels5. 1National Human Genome Research Institute, NIH, Bethesda, MD; 2Lombardi Cancer Center, Georgetown University School of Medicine, Washington, DC; 3Surgery Branch, National Cancer Institute, Bethesda, MD; 4 Southern Illinois University, Springfield, IL; 5The Weizmann Institute of Science, Department of Molecular Cell Biology, Rehovot, Israel. Protein tyrosine phosphatases (PTPs) tightly regulate tyrosine phosphorylation essential for cell growth, adhesion, migration, and survival. We performed a mutational analysis of the PTP gene family in cutaneous metastatic melanoma and identified 23 phosphatase genes harboring somatic mutations. Among these, receptor-type tyrosine-protein phosphatase delta (PTPRD) is one of the most highly mutated genes, harboring 17 somatic mutations in 79 samples, a prevalence of 21.5%. Functional evaluation of six PTPRD mutations show enhanced anchorage-dependent and anchorage-independent growth. Interestingly, melanoma cells expressing mutant PTPRD are significantly more migratory than cells expressing wild-type PTPRD or vector alone, indicating a novel gain-of-function associated with mutant PTPRD. To understand the molecular mechanism of PTPRD, we searched for its binding partners by converting the active PTPRD enzyme into a “substrate trap” form. Using mass spectrometry and coimmunoprecipitation, we report desmoplakin, a desmosomal protein that is implicated in cell-cell adhesion, as a novel PTPRD substrate. Further analysis showed reduced phosphatase activity of mutant PTPRD against desmoplakin. Thus our study has established a novel link between PTPRD and desmoplakin and identified a phosphatase implicated in desmosome formation, which is disrupted in melanoma. As both Met and EGFR signaling have been shown to be involved in desmoplakin phosphorylation and their respective inhibitors, SU11274 and Gefinitib reduce its phosphorylation; melanoma cells harboring PTPRD mutations may be selectively sensitive to these two inhibitors. Moreover, because PTPRD is also mutated in glioblastomas and adenocarcinoma of the colon and lung, our data might be applicable to a number of human cancers. Poster Section 39 Poster Board 9 LB-066 Adenomatous polyposis coli regulates epithelial morphogenesis and migration through FAK/Src signaling. Alyssa Lesko,1 Carolyn Ahlers,1 Jenifer R. Prosperi2. 1University of Notre Dame, Notre Dame, IN; 2IUSM-SB, South Bend, IN. Adenomatous Polyposis Coli (APC) is a multi-functional protein that is lost or mutated in many epithelial cancers including breast, colorectal, and pancreatic cancer. Although APC is well known as a negative regulator of the Wnt/β-catenin signaling pathway, it also binds to the cytoskeleton, microtubules and polarity proteins, such as Dlg and Scribble, suggesting functions in regulation of epithelial polarity and cell migration. Our lab has previously determined that the mammary glands of ApcMin/+ mice demonstrate mis-regulation of epithelial polarity, exhibit early neoplastic changes, and develop more aggressive mammary tumors when crossed to the MMTVPyMT model of breast cancer. Cells isolated from these tumors displayed activated FAK/Src signaling. Our lab has also shown that APC knockdown in the Madin-Darby Canine Kidney (MDCK) model altered epithelial morphogenesis, resulted in inverted polarity in 3D culture, and up-regulated gene expression of epithelial membrane protein 2 (EMP2). While restoration of the middle β-catenin binding domain was unable to rescue the phenotype, introduction of either full-length or a c-terminal fragment of APC partially restored these phenotypes. The current studies investigate the Wnt-independent mechanisms by which APC regulates these processes using the MDCK model and primary mammary epithelial cells (MECs) isolated from Apc mutant mice. We hypothesize that the c-terminal fragment of APC mediates FAK/Src signaling to regulate 3D morphogenesis, polarity, and migration. Treatment of APC knockdown MDCK cells with PP2, a Src kinase inhibitor, or AIIB2, a β1-integrin inhibitor, eliminated the drastic cyst size changes produced by APC knockdown. Furthermore, inhibition of Src partially restored the polarity phenotype in cysts with APC loss. In addition, shAPCMDCK cells and MECs isolated from ApcMin/+ mice exhibited increased cell migration compared to control cells indicating a role for APC in cell motility. Preliminary data demonstrates that treatment of shAPC-MDCK cells with PP2, AIIB2, and a FAK inhibitor decreases migration suggesting FAK/Src signaling as a possible mechanism by which APC mediates cell migration. Interestingly, EMP2 has been shown to bind integrin to activate FAK signaling suggesting an interaction in these pathways, and preliminary studies show EMP2 expression is increased in shAPCMDCK cells during migration. Future studies will aim to dissect the role of the c-terminal fragment and further devise the mechanism by which FAK/Src signaling and EMP2 play a role in APC regulating gene expression, cell migration, and polarity and 3D morphogenesis in MDCK cells and MECs isolated from Apc mutant mice. Investigating the interactions of APC with several targets such as those in the FAK/Src signaling pathway and EMP2 will help identify key players in the role of APC in Wnt-independent tumor development. Poster Section 39 Poster Board 10 LB-067 Critical role of Mieap, a p53-inducible protein, in intestinal tumor suppression. Yasuyuki Nakamura,1 Masayuki Tsuneki,1 Takashi Kinjyo,2 Noriaki Kitamura,1 Hirofumi Arakawa1. 1Division of Cancer Biology, National Cancer Center Research Institute, Tokyo, Japan; 2University of The Ryukyus, School of Medicine, Okinawa, Japan. Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria via MALM (Mieap-induced Accumulation of Lysosome-like organelles within Mitochondria) or MIV (Mieap-Induced Vacuole), respectively (1,2). Two mitochondrial outer membrane proteins, BNIP3 and NIX are essential mediators of MALM (3). 14-3-3gamma mediates the degradation of oxidized mitochondrial proteins in the MALM American Association for Cancer Research • AACR ANNUAL MEETING 2015 65 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 66 Late-Breaking Poster Session: Molecular and Cellular Biology 2 function (4). The Mieap-regulated mitochondrial quality control pathway was inactivated in almost 80% of colorectal cancer patients (5). Here, we report that Mieap plays a critical role in mouse intestinal tumor suppression. To investigate a role of Mieap in intestinal tumorigenesis, we generated the Mieap-deficient ApcMIN/+ mice. The ApcMIN/+ mice with the Mieap+/- and Mieap-/- genetic background revealed the remarkable shortening of the lifetime, compared to ApcMIN/+ mice. Mieap deficiency caused the increased number and size of the intestinal tumors in ApcMIN/+ mice. In addition, the tumors in the Mieap-deficient ApcMIN/+ mice showed more advanced grade of malignancy and often became cancerous. The mitochondria in the intestine and tumor of the Mieap-deficient ApcMIN/+ mice were morphologically unhealthy and generated high level of reactive oxygen species. These results suggest that Mieap plays a critical role in intestinal tumor suppression through mitochondrial quality control and prevention of mitochondrial oxidative stress. The Mieap-regulated mitochondrial quality control is a critical function for p53 tumor suppressor. 1. Miyamoto Y, Kitamura N, Nakamura Y, Futamura M, Miyamoto T, Yoshida M, Ono M, Ichinose S, Arakawa H. Possible existence of lysosome-like organella within mitochondria and its role in mitochondrial quality control. PLoS ONE 6: e16054, 2011. 2. Kitamura N, Nakamura Y, Miyamoto Y, Miyamoto T, Kabu K, Yoshida M, Futamura M, Ichinose S, Arakawa H. Mieap, a p53indicuble protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria. PLoS ONE 6: e16060, 2011. 3. Nakamura Y, Kitamura N, Shinogi D, Yoshida M, Goda O, Murai R, Kamino H, Arakawa H. BNIP3 and NIX mediate Mieap-induced accumulation of lysosomal proteins within mitochondria. PLoS ONE 7: e30767, 2012. 4. Miyamoto T, Kitamura N, Ono M, Nakamura Y, Yoshida M, Kamino H, Murai R, Yamada T, Arakawa H. Identification of 14-3-3γ as a Mieap-interacting protein and its role in mitochondrial quality control. Scientific Reports 2: 379, 2012. 5. Kamino H, Nakamura Y, Kitamura N, Futamura M, Yoshida M, Murai R, Saito Y, Sano H, Kanai Y, Moriya Y, Arakawa H. Frequent inactivation of the Mieap-regulated mitochondrial quality control in colorectal cancer. AACR annual meeting 2013 (Washington D.C.): abstract number 1687. Poster Section 39 Poster Board 11 LB-068 Inhibitors of Skp2 E3 ligase-mediated degradation of p27kip1 as a novel therapeutic approach to malignant pleural mesothelioma. Julien Daubriac, Jonathan Melamed, Unnati Pandya, Harvey I. Pass, Leslie I. Gold. New York University School of Medicine, New York, NY. Malignant pleural mesothelioma (MPM) is a highly aggressive cancer related to asbestos exposure for which there is no cure. Even with multimodal therapies median survival is only 24 months. Therefore, new therapeutic approaches are desperately needed for this cancer. MPMs are epithelioid, sarcomatoid, or composed of both patterns (biphasic); those tumors that are sarcomatous are most aggressive. Nuclear p27kip1 (p27) is a tumor suppressor critical to inhibiting cell proliferation by blocking Cdk2 activity to enforce cell cycle arrest. Cell cycle dysregulation has been shown to be involved in the pathogenesis of MPM. Accordingly, among the prognostic markers described for MPM, nuclear expression of p27 has been associated with increased progression-free survival. The levels of nuclear p27 are regulated post-translationally by the ubiquitin E3 ligase complex SCF-Skp2/Cks1, which causes its destruction. Accordingly, in many human cancers, Skp/Cks1 levels are high and associated with poor survival. Recently, we have shown that inhibitors of Skp2 E3 ligase activity block both, estrogen-induced degradation of nuclear p27 and inhibit proliferation of primary endometrial carcinoma cells in vitro as well as increase nuclear p27 and inhibit endometrial hyperplasia in vivo in estrogen-primed mice. Whereas nuclear p27 appears to be a 66 good prognostic biomarker for MPM, the mechanistic relationship to high levels of Skp2 has not been shown. Hence, the aim of our study is to determine whether MPM is a candidate cancer for targeted Skp2 inhibitor therapy to regain cell cycle control. By gene expression profiling of 53 surgically resected MPM tumors and matched control tissue, Skp2 gene expression was elevated by 3fold. In addition, by immunoblot analysis, we show that high levels of Skp2 protein are inversely related to p27 in four cell lines derived from MPM tumors of different tumor types. Interestingly, Skp2 expression was higher and associated with a higher proliferative rate in H2452 and HP-1 cells derived from biphasic tumors compared to those from epithelioid (H2591) or sarcomatoid (H2373) tumors and LP9 cells derived from normal pleural tissue expressed the lowest level of Skp2 and a lower proliferative rate. Similarly, by IHC of MPM tissue, Skp2 levels were inversely correlated with p27. These results suggest that Skp2 inhibitor therapy to stabilize nuclear p27 and regain growth control is a promising new approach for treatment of MPM. Poster Section 39 Poster Board 12 LB-069 Identification and characterization of the intercellular adhesion molecule-2 gene as a novel p53 family target. Kousuke Takeda,1 Yasushi Sasaki,1 Takafumi Nakagaki,1 Miyuki Tamura,1 Tomoko Ohashi,1 Kazuhiro Ogi,2 Masashi Idogawa,1 Hiroyoshi Hiratsuka,2 Takashi Tokino1. 1Department of Medical Genome Sciences, Sapporo Medical University, Sapporo, Japan; 2 Department of Oral Surgery, Sapporo Medical University, Sapporo, Japan. Background: p53 is one of the most important tumor suppressors, and is mutated in about half of human cancers. In response to DNA damage or other cellular stresses, activated p53 transactivates a number of target genes, which mediate various p53 functions, such as cell cycle arrest, apoptosis, DNA repair and senescence. Recent papers suggest that in addition to these functions, p53 contributes to the regulation of migration and invasion. We found that intercellular adhesion molecule-2 (ICAM2) is a target gene of p53 family, that is, p53, TAp73, and TAp63. Study Aims: The purpose of this study is to characterize the functional relevance of p53-mediated expression of ICAM2 in human cancer. Methods and Materials: The effect of p53 family on transcription of ICAM2 was investigated using real-time PCR, immunoblot analysis, and chromatin immunoprecipitation (ChIP) and reporter assays. Cancer cell migration and invasion were examined by wound healing assay and Matrigel invasion assay, respectively. The relationship between ICAM2 expression and p53 mutational status in tumor tissues was examined using the Oncomine utility. To examine whether ICAM2 expression affects prognosis in cancer patients, the PrognoScan databases were interrogated. Results: ICAM2 expression was upregulated by DNA damageinduced p53 activity, as well as by overexpression of p53 family genes. We found a specific binding site for p53 family proteins in the first intron of the ICAM2 gene by a ChIP assay. A heterologous reporter assay revealed that this binding site is a functional response element, suggesting that ICAM2 is a transcriptional target of the p53 family member genes. We also found that ectopic expression of ICAM2 inhibited cancer cell migration and invasion. Conversely, inactivation of ICAM2 stimulated cancer cell migration and invasion. Importantly, The inhibitory activity of ICAM2 on cancer cell migration and invasion was abrogated by the addition of neutralizing ICAM2 antibodies. We also demonstrated that silencing endogenous ICAM2 in cancer cells caused a marked increase in extracellular signal-regulated kinase-1/2 (ERK) phosphorylation levels, suggesting that ICAM2 inhibits migration and invasion of cancer cells by suppressing ERK signaling. We found six published cancer microarray data sets, in which the p53 status and ICAM2 expression have been reported. In all these six datasets, patients with mutant p53 show lower expression of ICAM2 mRNA than Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 67 Late-Breaking Poster Session: Molecular and Cellular Biology 2 patients with wild-type p53, although this did not reach statistical significance. Moreover, we have found that low expression of ICAM2 may serve as a poor prognostic factor for certain cancer patients. Conclusions: We identified ICAM2 as a direct transcriptional target of the p53 family member genes. Our finding suggests that ICAM2 is one of the effectors downstream of p53 to negatively regulate tumor metastasis. Poster Section 39 Poster Board 13 LB-070 The studies of tumor-associated gene C1orf35 in pathogenesis of human multiple myeloma. Wei-Xin Hu, Sai-Qun Luo, Yan Zhong, Xiu-Feng Bu, Yang Zhou. Xiangya School of Medicine, Central South University, Changsha, China. Multiple myeloma (MM) is a disease characterized by the uncontrolled proliferation and accumulation of malignant plasma cells in the bone marrow. Cancer cells are characterized by a profound genetic instability resulting in a complex set of numerical and structural chromosomal abnormalities. We previously cloned a tumor-associated gene C1orf35 from MM cell line ARH-77, which located the chromosome 1q42.13. The results showed that C1orf35 could promote the transformation, cell growth and proliferation of NIH3T3 cells, as well as cell transition from phase G1 to S. C1orf35 also possessed the potentials of promoting tumor formation in animal experiments. Based on this finding, we further explored the molecular mechanism of C1orf35 gene up-regulation and its effects on pathogenesis of human MM. The expression level of C1orf35 gene in MM patients was detected by RT-PCR. The gene expression of C1orf35 in mononuclear cells of MM patients was upregulated (68.9%), and was not detectable in normal controls. The lentivirus vector GV248/C1orf35-RNAi for interference experiments was constructed and the NIH3T3/C1orf35-RNAi cell line which stable expressed C1orf35-RNAi was set up. The expression level of C1orf35 in NIH3T3/C1orf35-RNAi cells was detected by real-time PCR and Western blotting. The results showed that the expression levels of both mRNA and protein were down-regulated and the cell proliferation was markedly decreased after interference. By detecting the c-myc expression level of NIH3T3/Clorf35-RNAi cells, we found that expression level of c-myc gene and protein all decreased. These results indicated that down-regulation of C1orf35 was followed by down-regulation of c-myc, which implied C1orf35 might play an important role in c-myc-dependent pathway. To investigate the effects of C1orf35 gene on proliferation of MM cells, we interfered and over-expressed C1orf35 gene. The results showed that the cell proliferation was obviously decreased after interference, and the cell proliferation was significantly accelerated after over-expression of C1orf35 gene. The bioinformatics analysis showed that C1orf35 protein was located in nucleus and might play an important role for transcription regulation, which may participate in the activation of some oncogenes. We detected the binding of C1orf35 protein and promoter region of proto-oncogene c-myc with chromatin immunoprecipitation assay (ChIP) and the results showed that C1orf35 protein binds promoter region of c-myc in 226 to -10 bp. This work may contribute to reveal the role of C1orf35 in MM pathogenesis. Poster Section 39 Poster Board 14 LB-071 Kub5-Hera/RPRD1B controls CDK1 regulation and synthetic lethality to PARP1 inhibition. Edward A. Motea,1 Farjana Fattah,1 Ling Xiao,1 Luc Girard,1 Amy Rommel,1 Julio Morales,1 Praveen Patidar,1 Andrew Porter,2 John Minna,1 David Boothman1. 1UT Southwestern Medical Center, Dallas, TX; 2Imperial College Faculty of Medicine, London, United Kingdom. Aberrant CDK1 regulation induces impulsive proliferation as well as genomic and chromosomal instability in cancer cells. However, the mechanism of how CDK1 is regulated at the transcriptional level, especially under pathological conditions (e.g. cancer), remains elusive. Here, we show that Kub5-Hera (K-H), a known transcription termination factor, plays a novel role in promoting CDK1 transcription to mediate DNA repair and cellular response to PARP1 inhibition in various cancer cell lines. At the molecular level, we demonstrate via luciferase reporter assays and mutagenesis studies that K-H binds the phospho-Serine 2-containing C-terminal domain (CTD) of RNA Pol II, which is then recruited to the cell-cycle homology region (CHR) of the CDK1 promoter to efficiently stimulate CDK1 transcription. Mechanistically, the loss of K-H concomitantly reduces CDK1 mRNA/protein expression levels, which consequently compromises homologous recombination (HR)mediated DNA repair due to the lack of BRCA1 phosphorylation for efficient function. Intriguingly, loss of K-H stimulates PARP1 activity needed for cancer cell survival. Genetic and pharmacological ablation of PARP1 activity in BRCA-proficient cells with aberrant KH reduction leads to synthetic lethality, which is reversed by reexpression of wild-type K-H but not the mutant with attenuated RNAPII binding. The mechanistic insights gained from this study should lay foundation toward promising new tumor biology-based targets (i.e., K-H as a novel therapeutic target) and innovative strategies to reduce normal tissue toxicity, increase tumor control, and expand the use of PARP1 inhibitors (e.g., Rucaparib) against BRCA-proficient cancers with aberrant K-H loss. Poster Section 39 Poster Board 15 LB-072 The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype. Etienne Dardenne, Kaitlyn Gayvert, Adeline Berger, Andrea Sboner, Brian Robinson, Kenneth Hennrick, Juan Miguel Mosquera, Cynthia Cheung, Martin Eilers, Himisha Beltran, Mark A. Rubin, Olivier Elemento, David S. Rickman. Weill Cornell Medical College, New York, NY. Although neuroendocrine prostate cancer (NEPC) rarely arises de novo, up to 30% of patients with prostate adenocarcinoma (PCa) develop NEPC features in later stages of their disease as a mechanism of treatment resistance to hormonal therapies including abiraterone and enzalutamide. NEPC is clinically more aggressive than PCa, commonly metastases to visceral organs such as liver and brain, and can be suspected in patients with progressive disease and a disproportionately low serum PSA. There is currently no effective therapy for NEPC, and most patients with NEPC survive less than one year. The development of novel treatment strategies for patients with NEPC represents a significant clinical unmet need. We have previously discovered significant overexpression and gene amplification of AURKA (encoding Aurora-A) and MYCN (encoding N-Myc) in NEPC as compared to prostate adenocarcinoma. As in neuroblastoma, N-Myc interacts with Aurora-A in NEPC which leads to a co-stabilization of both proteins that is targetable with allosteric inhibitors of Aurora A. We have also shown that ectopic expression of N-Myc induces neuroendocrine transformation of prostate adenocarcinoma cells. However, the molecular mechanisms that underlie N-Myc driven NEPC phenotype have yet to be characterized. To address this, we performed RNA-sequencing (RNAseq) and ChIP-sequencing from multiple stable prostate adenocarcinoma cells with and without NMyc over-expression. We have identified a signature of N-Myc deregulated genes that are both biologically and clinically relevant. Based on RNAseq data from 128 clinical samples (17 NEPC, 10 castrate resistant prostate cancer 68 prostate adenocarcinoma patient tumors and 33 matched benign prostate samples) we found that majority (70%) of the N-Myc deregulated genes identified in our in vitro models distinguish the NEPC from PCa tumor samples. Based on GSEA and pathway analysis we found that N-Myc induces a profile enriched in pro-metastatic, dedifferentiation and Polycomb Repressive Complex deregulated genes and dramatically reduces AR signaling. Furthermore we show that N-Myc is recruited to AR-bound enhancers of AR target genes. To further determine the role of N-Myc in driving the NEPC phenotype we have generated transgenic mice that carry an integrated MYCN gene American Association for Cancer Research • AACR ANNUAL MEETING 2015 67 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 68 Late-Breaking Poster Session: Molecular and Cellular Biology 2 behind a lox-stop-lox (LSL) cassette at the ROSA26 locus, a floxed Pten locus and a tamoxifen-activated Cre recombinase driven by Tmprss2. N-Myc over-expression in the context of Ptenfl/+ leads to focal high levels of activated AKT pathway that accompany mouse high grade prostatic intraepithelial neoplasia (mHGPIN) at 3 months post-induction and other clinically relevant molecular changes. Littermates that harbor Ptenfl/+ alone do not display mHGPIN or AKT activation. In the context of Pten homozygous loss (Ptenfl/fl), N-Myc is associated with diffuse mHGPIN in the ventral and dorsolateral prostate lobes and irregular gland borders, nuclear atypia and high levels of AKT activity at 3 months (MYCN homozygous) or 6 months (MYCN heterozygous) post-induction. In conclusion, our findings support the role of N-Myc as one of the drivers of the NEPC phenotype and have the potential to ultimately lead to the identification of a new class of disease specific biomarkers and therapeutic alternatives for this aggressive subgroup of prostate cancer. Poster Section 39 Poster Board 17 LB-074 The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc. Xiao-Xin Sun,1 Xia He,2 Li Yin,2 Rosalie Sears,1 Mushui Dai1. 1Oregon Health & Science University, Portland, OR; 2Jiangsu Cancer Hospital, Nangjing, China. c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCFFbw7 and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) USP28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ, but not the nucleoplasmic Fbw7α. Yet, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of cMyc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc’s nucleolar degradation pathway. Poster Section 39 Poster Board 18 LB-075 The histone demethylase JMJD1A is essential to prostate cancer cells through regulation of c-Myc expression. Lingling Fan,1 Guihong Peng,1 Natasha Sahgal,2 Ladan Fazli,3 Martin Gleave,3 Yuji Zhang,1 Arif Hussain,1 Jianfei Qi1. 1University of Maryland, Baltimore, MD; 2University of London, London, United Kingdom; 3University of British Columbia, Vancouver, British Columbia, Canada. Histone demethylase JMJD1A controls gene expression by epigenetic regulation of H3K9 methylation marks. To investigate role of JMJD1A in the development and progression of prostate cancer, we knock down JMJD1A in prostate cancer cells and examine its effect on gene expression and cancer cell biology. We find that silencing of JMJD1A results in the lethality of prostate cancer cells concomitant with inhibition of cell proliferation, induction of apoptosis and cell cycle arrest. Profiling array analyses reveal cMyc as one of the top transcriptional networks altered upon the JMJD1A inhibition. The gene ontology pathway analyses show that the primary functions of c-Myc targets regulated by JMJD1A are related to cell cycle, cell proliferation and cell survival. Mechanistically, JMJD1A promotes the recruitment of AR and induces demethylation at the c-Myc gene enhancer, thereby 68 increasing the AR-dependent transcription of c-Myc mRNA. In a parallel pathway, JMJD1A binds to E3 ubiquitin ligase HUWE1, attenuates the HUWE1-induced degradation of c-Myc, and thus increases the c-Myc protein level. Together, JMJD1A controls the expression of c-Myc at transcriptional and post-translational levels. Re-expression of c-Myc in the JMJD1A-knockdown cells partly recues the growth of prostate cancer cells in vitro and in vivo. The protein level of c-Myc is positively correlated with that of JMJD1A in a subset of human prostate cancer specimens. Collectively, our findings identify a critical role of JMJD1A for the viability of prostate cancer cells through regulation of c-Myc expression. Poster Section 39 Poster Board 19 LB-076 Spatial regulation of β-actin monomer synthesis controls epithelial-mesenchymal tissue fate specification: consequences for cancer and metastasis progression. Pavan Vedula, Lissette A. Cruz, Alexis J. Rodriguez. Rutgers University Newark, Newark, NJ. The purpose of this study is to investigate the extent to which spatially regulating β-actin gene expression controls epithelialmesenchymal fate specification in healthy and cancer tissue culture models. We utilize mRNA zipcode antisense oligonucleotide masking of the β-actin transcript and translation site imaging to assess the consequences of perturbing the location of monomer synthesis during cancer and healthy epithelial tissue establishment and maintenance. Additionally, we developed a novel method to quantify adherens junction assembly based on fluorescence covariance between E-cadherin and F-actin during Ca2+ switch experiments to assess the consequences of β-actin monomer synthesis mislocalization in epithelial tissue culture model systems. Using these methods we demonstrate that perturbing the location of β-actin monomer synthesis by mRNA zipcode antisense oligonucleotide masking in healthy 2D and 3D epithelial tissue culture models perturbs adherens junction assembly and causes epithelial-mesenchymal transition. Moreover, perturbing E-cadherin expression or function causes β-actin monomer synthesis mislocalization, perturbs adherens junction assembly, and causes epithelial-mesenchymal transition. By contrast, β-actin mRNA zipcode antisense nucleotide masking in 3D colon cancer tissue culture models causes no additional structural defects since adherens junctions are already disorganized. Importantly, western blot analysis of whole tissue lysates from these colon cancer models reveals altered expression of key components of the pathway used to regulate the spatial location of β-actin monomer synthesis such as E-cadherin or Zipcode Binding Protein-1. Together, these data support a model where E-cadherin regulates the location of β-actin monomer synthesis by modulating Zipcode Binding Protein-1/mRNA zipcode interactions to control adherens junction homeostasis and consequently epithelial-mesenchymal cell fate specification. Using β-actin antisense zipcode oligonucleotide masking during Ca2+ switch experiments we demonstrate 3D colon cancer tissue culture models have already lost function of the βactin spatial translation pathway and therefore are not further perturbed during this assay. Consequently, we hypothesize that spatially regulating β-actin monomer synthesis is a key aspect of controlling epithelial-mesenchymal cell fates specification and loss of this pathway is expected to predispose epithelial tissues to cancer and metastasis progression. This is an attractive hypothesis explaining the molecular and cellular mechanism of how E-cadherin and Zipcode Binding Protein-1 function as potent metastasis inhibitors and highlights the importance of spatially regulating βactin gene expression as an approach to establish and maintain healthy epithelial tissues. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 69 Late-Breaking Poster Session: Molecular and Cellular Biology 2 Poster Section 39 Poster Board 20 LB-077 Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. Daniel V. LaBarbera, Qiong Zhou, Adedoyin D. Abraham, Linfeng Li, Wells A. Messersmith. University of Colorado, Aurora, CO. The Wnt signaling pathway, which controls TCF/Lef/β-catenin (TCF) transcription, is well accepted as a dominate player in CRC tumor initiation and progression. More recently, this pathway has been implicated as a major driving force inducing epithelialmesenchymal transition (EMT), acquiring a drug resistant tumor initiating cell (TIC) phenotype that may promote metastasis. As a result, this pathway has been seriously pursued for novel drug development that has yielded no real translational success due to the lack of “druggable” targets. Thus, the major challenge moving forward is to identify a druggable target within the Wnt/TCF pathway that may result in effective clinical translation. Using a chemical biology approach targeting EMT we have identified topoisomerase IIα (TopoIIα) as a master regulator EMT by participating as a required component of TCF-transcription in CRC. Specifically, we show that inhibiting TopoIIα using small molecule ATP-competitive inhibitors as well as shRNA results in the blockade of TCF-transcription using the TOP/FOP flash reporter system. Inhibition of TopoIIα-dependent TCF-transcription results in significant reversion of EMT characterized by Western blot analysis in a panel of CRC cell lines. Time course studies reveal that inhibiting TopoIIα results in downregulation of TCF-transcription followed by the induction of apoptosis using 3D multicellular tumor spheroid models. We show that TopoIIα participates in proteinprotein complexation with β-catenin using co-immunoprecipitation studies, which is not prevented by small molecule inhibitors. However, chromatin immunoprecipitation (ChIP) studies reveal that ATP-competitive TopoIIα inhibitors can significantly block TCFtranscription by preventing TCF-complex-DNA binding. In addition, inhibiting TopoIIα-dependent TCF-transcription correlates with the inhibition of TIC function characterized by loss of colony formation using the clonogenic assay. Likewise, inhibiting TopoIIα-dependent TCF-transcription inhibits the invasive potential in a panel of CRC tumor cell lines. Interestingly, the clinically used TopoIIα drug, etoposide, had no effect on TCF-transcription, EMT, or invasion. In conclusion, we hypothesize that TopoIIα participates as a component of the TCF-complex via the C-terminus and is required for binding chromatin DNA and initiating gene transcription. TopoIIα participation in TCF-transcription may convey drug resistance due to altered poison binding sites. However, our data indicates that TopoIIα-dependent TCF-transcription requires ATP. Hence, central to our hypothesis is that N-terminal ATP sites are conserved providing an Achilles heel to drug resistance. Therefore, N-terminal ATP binding sites provide optimal druggable target sites that may be utilized for translational drug development preventing TCFtranscription, EMT and potentially metastasis in CRC patients. Poster Section 39 Poster Board 21 LB-078 RNAi and CRISPR/Cas9-based in vivo models for drug discovery. Prem K. Premsrirut, Christof Fellmann. Mirimus Inc., Woodbury, NY. Genetically engineered mouse models (GEMMs) are a powerful platform that enable the study of disease initiation and maintenance, the microenvironment and the responsiveness of disease to known or novel therapeutics; however, the long lead times and high costs required to develop, intercross and maintain models with various disease predisposing gene combinations have limited their practical utility in the drug discovery process. RNA interference (RNAi), a mechanism that controls gene expression, is a rapid and cost-effective alternative to gene deletion that can be exploited experimentally to reversibly silence nearly any gene target not only in vitro but also in live mice. By using our “Sensor assay” to biologically identify short hairpin RNAs (shRNAs) that induce potent gene suppression in combination with a new miRNA scaffold, miR-E, we have engineered a reliable system for in vivo gene suppression. Furthermore, by utilizing tetracycline-regulated miR-E based shRNAs with high efficiency ES cell targeting, we have developed a fast, scalable pipeline for the production of shRNA transgenic mice with reversible gene silencing. Recently, with the advent of new genome editing techniques, such as CRISPR/Cas9 technology, we are able to introduce additional sensitizing lesions to induce disease pathogenesis. In synergy with RNAi technology, complex multi-allelic ESC based GEMMs can be generated without extensive intercrossing. Using this combination of CRISPR/Cas9 and RNAi technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in mice, giving us unprecedented capabilities to perform preclinical studies in vivo. Here, we demonstrate that RNAi in combination with CRISPR/Cas9 genome editing enables us to recapitulate the phenotypes of knockout mice and further explore potential therapeutic approaches within the same model. Using our robust system, we have created a a cost-effective and scalable platform for the production of complex GEMMs with RNAi silencing of nearly any gene–mice with enormous predictive power that will shape our development of better tolerated therapies. Poster Section 39 Poster Board 22 LB-079 Specific epigenetic reader role for cyclin D1. Gabriele Di Sante,1 Mathew Craig Casimiro,1 Chenguang Wang,1 Zuoren Yu,1 Marco Crosariol,1 Ratna K. Vadlamudi,2 Monica Mann,2 Peter Tompa,3 Agnes Tantos,3 Richard G. Pestell1. 1Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA; 2School of Medicine, UTHSCSA, San Antonio, TX; 3Institute of Enzymology of the Hungarian Academy of Sciences, Budapest, Hungary. Amplification of the CCND1 gene occurs in approximately 30% of human breast cancer and is sufficient when overexpressed for the induction of mammary tumorigenesis. A DNA-associated form of cyclin D1 governs gene expression and chromosomal instability. Herein, the mechanisms governing gene activation by cyclin D1 were defined. Cyclin D1 was recruited to activate gene regulatory regions associated with enrichment of p300, HP1α and reduced HDAC3 and SUV39H1. The recruitment of cyclin D1 into local chromatin was dissociable from its kinase function and required an intrinsically disordered carboxyl-terminus flanked by a glutaminerich motif. The cyclin D1 epigenetic interaction motif bound acetylated or methylated H3 on specific residues (H3K36me2, H3K9me3). Cyclin D1 bound to the Top2A gene promoter induced Top2A transcription, abundance and sensitivity to Top2A inhibitors. The direct recognition of an epigenetic code by a motif within cyclin D1 and the induction of Top2A may facilitate communication of genome wide expression changes during cell-cycle progression and tumorigenesis. Poster Section 39 Poster Board 23 LB-080 Reactivating RBL2/p130 oncosuppressive function as a new, possible antitumoral strategy. Francesca Pentimalli,1 Luca Esposito,1 Iris Maria Forte,1 Carmelina Antonella Iannuzzi,2 Flavio Rizzolio,3 Tiziano Tuccinardi,4 Paola Indovina,2 Silvia Boffo,5 Antonio Giordano6. 1Oncology Research Center of Mercogliano (CROM); Istituto Nazionale Per Lo Studio E La Cura Dei Tumori “Fondazione Giovanni Pascale”, Mercogliano, Avellino, Italy; 2Department of Medicine, Surgery and Neuroscience, University of Siena and Istituto Toscana Tumori (ITT), Siena, Italy; 3 Experimental and Clinical Pharmacology, Centro di Riferimento Oncologico- National Cancer Institute, Aviano, Italy; 4Department of Pharmacy, University of Pisa, Pisa, Italy; 5Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA; 6Sbarro Health Research Organization, Philadelphia, PA. Deregulation of cell cycle control is the leading cause of cancer. The retinoblastoma (Rb) family members, including RB1/p105, RBL1/p107 and RBL2/p130, are crucial to restrain cell cycle progression and their inactivation, either direct or indirect, is a hallmark of most human tumors. In particular, RBL2/p130 emerging American Association for Cancer Research • AACR ANNUAL MEETING 2015 69 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 70 Late-Breaking Poster Session: Molecular and Cellular Biology 2 role in senescence and apoptosis, seems to contribute importantly to its tumor suppressor function. Furthermore, many studies largely contributed to establish RBL2/p130 as an important cancer target, which is inactivated by cell cycle kinases and whose deregulation underlies various cancer types. In this regard, we set out to restore RBL2/p130 function in tumors and exploit its tumor suppressive potential for cancer therapy. In particular, we have identified, through computational chemistry and molecular modeling studies, a small molecule able to act as a specific inhibitor of the CDK2-CycA complex and to reactivate the tumor suppressive function of RBL2/p130 in cancer. We analyzed by MTS assay the cytotoxic effect of our compound on a panel of different tumor cell lines and determined its IC50 values. We evaluated its effects on cell cycle and apoptosis by FACS and dissected its molecular mechanism of action by Western blot and RT-PCR. We found that our compound effectively inhibited proliferation of lung cancer and mesothelioma cell lines by specifically inhibiting CDK2-CycA activity towards RBL2/p130. The decrease in RBL2/p130 phosphorylation led to stabilization of its complex with the E2F4 transcriptional factor and consequent repression of their targets necessary for cell cycle progression, including CDK2 itself. Consistently, our compound arrested cell cycle and triggered apoptosis. Interestingly, apoptosis seemed to be mediated by RBL2/p130 itself because it was repressed in RBL2/p130-silenced cells. Furthermore, our compound proved to be active in an A549 xenograft model of lung cancer. Overall our findings indicate that the pharmacological reactivation of RBL2/p130 induces its cell-cycle restraining and proapoptotic functions, the latter being still largely unexplored, and identify a new possible therapeutic strategy for cancer treatment. Poster Section 39 Poster Board 24 LB-081 Elucidating the function of MCM3 ubiquitination by KEAP1: crosstalk between redox-sensing and cell cycle progression. Kathleen M. Mulvaney, Jacob Matson, Feng Yan, Dennis Goldfarb, Jeannette Cook, Michael Benjamin Major. UNC Chapel Hill, Chapel Hill, NC. While the KEAP1-NRF2 axis is essential for maintaining redox homeostasis, whether KEAP1 has alternative functions and how this pathway crosstalks with other important cellular processes remains unknown. KEAP1 targets the NRF2 transcription factor for proteasomal degradation in a redox-sensitive manner. Thus, this pathway serves as the cell’s primary response to elevated reactive oxygen species. Importantly, KEAP1-NRF2 are frequently mutated in cancer, most strikingly in non-small cell lung cancer, where KEAP1 or NRF2 are mutated in 20-30% of patient tumors. Though regulation of NRF2 has long been considered the only physiologically important role for the E3 ligase KEAP1, we have determined that KEAP1 binds the master cell cycle regulator, MCM3, a subunit of the hexameric DNA replication licensing complex, MCM2-7. Excitingly, our ubiquitination assay data establish MCM3 as a new substrate for KEAP1; however, KEAP1 intriguingly does not regulate total cellular levels of MCM3. Consistent with this, we determined that only a small pool of cellular MCM3 is bound to KEAP1, suggesting that KEAP1 may bind and ubiquitinate a highly specified pool of MCM3. To determine the function of KEAP1-dependent ubiquitination of MCM3, we recently applied a new proteomics technique to map the ubiquitinated residues within MCM3 and identify these lysines within the larger MCM2-7 helicase. This mapping and protein modeling has provided new insight into the structure-function relationship of ubiquitinated MCM3. As MCM2-7 chromatin loading is a highly coordinated, cell cycle-dependent process, we tested whether KEAP1 loaded concurrently onto chromatin. Strikingly, we found that KEAP1 indeed loads onto chromatin during G1 and unloads in late S phase in a similar fashion as the MCM complex, further suggesting KEAP1 70 regulates the function of this essential cell cycle regulator on chromatin. Given the role of MCM3 in cell cycle progression, we tested whether KEAP1 was required for normal G1 to S phase progression and saw that loss of KEAP1 retards S phase DNA synthesis, which is an MCM-dependent process. Intriguingly, primary, untransformed KEAP1 knockout fibroblasts show decreased growth and aberrant cell cycle patterns consistent with a defect in the G1 to S transition. Overall, these data suggest a novel function for KEAP1 in regulating the MCM complex and cell cycle progression. We postulate that KEAP1 promotes cell cycle progression in a redox-sensitive manner through its association with MCM3 and that this presents a novel mechanism by which cells may halt cell cycle to protect DNA from damage by reactive oxygen species. Poster Section 39 Poster Board 25 LB-082 FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones. Pin Ouyang. Chang Gung University, Taoyuan, Taiwan. Purpose of the Study: Identify and characterize a novel D boxcontaining protein, FLJ25439 for tetraploid induction and maintenance of genomic stability. Pertinent Experimental Procedures: Immunofluorescent microscopy and flow cytometry for cell cycle determination. Proteomics and bioinformatics approaches for protein category profiling. Summary of the Data: Investigation of the mechanisms leading to aneuploidy and polyploidy is critical to cancer research. Previous studies have provided strong evidence of the importance of tetraploidization as an early step in tumorigenesis. In cancer cells, tetraploid cells may contribute to abnormal mitotic progression, which may be associated with cytokinesis failure. Tetraploidy leads to genomic instability due to centrosome and chromosome overreplication. Until now, the mechanism by which cells maintain tetraploid status has been unknown. Here, we identified a novel D box-containing protein, FLJ25439, which displays a dynamic expression profile during mitosis/cytokinesis with the midbody as the most prominent associated structure. To understand the function of FLJ25439, we established stable cell lines overexpressing FLJ25439. FLJ25439-overexpression cells grew slower and displayed a tetraploid DNA content in comparison with diploid parental cells. They also showed aberrant mitosis and dysregulated expression of p53, pRb and p21, suggesting a defect in cell cycle progression. To explore the molecular mechanisms responsible for FLJ25439-induced tetraploidization, we conducted a comparative analysis of the global protein expression patterns of wild type and overexpressors using proteomics and bioinformatics approaches. Protein category profiling indicated that FLJ25439 is involved in pathways related to anti-apoptosis, protein folding, the cell cycle, and cytoskeleton regulation. Specifically, genotoxicstress- and ER stress-related chaperone proteins greatly contributed to the FLJ25439 overexpression phenotypes. Conclusion: The results of this study pave the way to our further understanding of the role of FLJ25439, a novel cytokinesisrelated protein in protecting cells from environmental stress and tetraploid formation. Poster Section 39 Poster Board 26 LB-083 Regulation of the mitotic centrosome by heat shock protein 70. Chieh-Ting Fang, Hsiao-Hui Kuo, Ling-Huei Yih. Academia Sinica Inst. of Cell. & Organismic Bio., Taipei, Taiwan. A functional centrosome is essential for the assembly of a bipolar mitotic spindle and accurate chromosome segregation. Loss of centrosome integrity frequently occurs in transformed cells or in tumor tissues. Pathways maintaining the quality control of proteins are known to regulate the biogenesis and duplication of the Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 71 Late-Breaking Poster Session: Molecular and Cellular Biology 2 centrosome. In this study, we explored the role of heat shock protein 70 (HSP70), an essential molecular chaperone for protein quality control, on maintaining the centrosome integrity during mitosis by treating cells with a HSP70 inhibitor or transducing cells with HSP70-specific shRNA. The effects of these treatments on mitosis progression, mitotic spindle assembly, and cell viability were investigated. Our results showed that inhibition or depletion of HSP70 disrupted microtubule nucleation and polymerization, induced abnormal mitotic spindles, and interfered with mitosis progression. In addition, HSP70 accumulated at the spindle pole and co-localized with γ-tubulin and pericentrin during mitosis. Loss of centrosomal HSP70 impeded the recruitment of pericentriolar components essential for centrosome maturation. These results indicate that HSP70 is required for the maintenance of a functional mitotic centrosome to support the assembly a bipolar mitotic spindle. Poster Section 39 Poster Board 27 LB-084 A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. Med Univ Vienna, Vienna, Austria. LINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeatcontaining RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells. Poster Section 39 Poster Board 28 LB-085 Protein phosphatase 1 and the RNA-binding protein CSTL2 function to constrain daughter centriole number. Jyoti Iyer,1 Neena Peel,2 Sean O’Rourke,3 Bruce Bowerman,3 Kevin O’Connell1. 1NIDDK, NIH, Bethesda, MD; 2The College of New Jersey, Ewing, NJ; 3University of Oregon, Eugene, OR. Centrioles are cylindrical microtubule-based structures that are required for the formation of cilia, flagella and the mitotic spindle. A centrosome is comprised of two centrioles (one mother and one daughter) that are organized in an orthogonal orientation. Centrioles are duplicated only once during each cell cycle and this involves the formation of a single daughter centriole next to each mother. Dysregulation of this process yields an abnormal centriole number and this can result in aneuploidy, a hallmark of cancer cells. Therefore, it is critical that centriole duplication (CD) is tightly regulated. The nematode C. elegans is an excellent model system to study the process of CD because the core components of the CD pathway in C. elegans are conserved in humans. The main purpose of this study is to identify novel regulators of CD using C. elegans as a model system. The kinase ZYG-1 is a functional ortholog of human PLK4 and is absolutely essential for CD. Using a zyg-1 suppression assay, we have identified Protein Phosphatase 1 (PP1) as a critical inhibitor of CD. Using super-resolution microscopy, we found that deactivating PP1 results in the formation of multiple daughter centrioles adjacent to a single mother. Western blot analysis indicated that the mechanism by which PP1 inhibits CD is by decreasing ZYG-1 levels. Our study has also identified an RNA-binding protein with homology to human cleavage stimulation factor subunit 2 tau variant (CSTL-2) as a potential PP1 substrate and a novel component of the C. elegans CD pathway. Utilizing a fluorescently tagged CSTL-2 transgenic worm line, we determined that CSTL-2 localizes to the centrosomes during mitosis. An immunoprecipitation assay followed by mass spectrometry has identified CSTL-2 as an interacting partner of PP1- thereby implicating it as a PP1 substrate. To evaluate the effect of CSTL-2 on ZYG-1-mediated CD, cstl-2-null; zyg-1-hypomorphic double mutants were constructed. At the restrictive temperature, the zyg-1hypomorphic mutant worms show 100% embryonic lethality due to a failure of CD. However, in the cstl-2-null; zyg-1-hypomorphic double mutants, approximately 20% of the embryos survive indicating a role for CSTL-2 as an inhibitor of CD. We performed cytological analyses on cstl-2-null; zyg-1-hypomorphic double mutant embryos and confirmed that CSTL-2, like PP1, also functions as an inhibitor of CD. Therefore, based upon our data, we conclude that PP1 is an important negative regulator of CD which functions to ensure that only one daughter centriole forms adjacent to a mother centriole and we propose that CSTL-2 is a substrate through which PP1 regulates CD. In summary, this study highlights a novel pathway that controls centriole number in C. elegans, which may be dysregulated in cancer. American Association for Cancer Research • AACR ANNUAL MEETING 2015 71 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 72 Late-Breaking Poster Session: Carcinogenesis Late-Breaking Poster Session Monday, April 20, 2015 8:00 AM-12:00 PM Poster Section 40 Late-Breaking Research: Carcinogenesis Poster Section 40 Poster Board 1 LB-086 Genes targeted by drugs and curcumin in a breast carcinogenesis model. Gloria M. Calaf,1 Debasish Roy,2 Richard Ponce-Cusi1. 1Universidad De Tarapaca, Arica, Chile; 2Department of Natural Sciences, Hostos College of the City University, Bronx, NY. Breast carcinogenesis is a multistage process that involves mutations and cellular phenotypic alterations attributed to exposure to exogenous environmental substances as well as endogenous agents as female hormones. Pamidronate (Pam), one of the nitrogen-containing bisphosphonates, is used in the treatment of bone metastases of breast cancer. It has recently been reported that it is also related to cell proliferation and apoptosis. 5Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of a variety of solid cancers that arrest cell cycle and induce apoptosis in cancer cells. Curcumin (Cur) is an antioxidant known as a dietary natural yellow pigment derived from the rhizome of the herb Curcuma longa. The aim of this study was to evaluate genes that could be targeted by these drugs and curcumin in a breast carcinogenesis in vitro model induced by radiation and estrogen. Such model was developed with a normal immortalized breast epithelial cell line, MCF-10F that was exposed to low doses of high LET (linear energy transfer) alpha particles (150 keV/μm) of radiation, and cultured in presence of 17β-estradiol. This model consisted of the following cell lines: i) MCF-10F, ii) Alpha3, a malignant non-tumorigenic, iii) Alpha5, a tumorigenic one and iv) Tumor2, derived from Alpha5 injected into the nude mice. Previous results showed that Alpha5 and Tumor2 increased cell proliferation, presented anchorage independency, invasive capabilities and tumor formation in nude mice, as well as microsatellite instability and loss of heterozygosity in chromosomes 6, 8, 11 and 17 and mutations of c-Ha-ras and Rho-A among others. Pam, 5-FU and Cur were analyzed with MCF-10F by MTT indicating that the mean LD50 was 10, 2 and 30 μM after 48 hrs, respectively. Such substances inhibited migration and invasion in both Alpha5 and Tumor2 cell lines compared to the control MCF-10F and their counterparts, and also decreased c-Ha-ras, Rho-A, p53, Serpin-1 and Cav-1 gene expression in Alpha5 and significantly in Tumor2 cell lines by RTqPCR. A significant apoptotic activity was observed by flow cytometry in Alpha5 and Tumor2 cell lines in comparison to control MCF-10F and their counterparts. These compounds had a direct antitumor and apoptotic effect on Bcl-xL and Bax by downregulation of a transcription factor as NF B gene expression in Alpha5 and Tumor2 cell lines. It can be concluded that c-Ha-ras, RhoA, p53, Serpin-1 and Cav-1 genes were targeted by drugs and curcumin in a model transformed by low doses of alpha particles and estrogen. Such genes are involved in critical steps in breast carcinogenesis. Supported by FONDECYT #1120006 (GMC) and Ministry of Education (MINEDUC), Universidad de Tarapacá, Arica, Chile Poster Section 40 Poster Board 2 LB-087 A facilitative role for caspase 3 in promoting genetic instability and carcinogenesis. XINJIAN LIU, Fang Li, Chuan-yuan Li. Duke university, Durham, NC. Apoptosis and apoptotic caspases are generally considered tumor-suppressive since there are involved in the elimination of unwanted or damaged cells. However, the relationship between caspases and carcinogenesis has not been thoroughly examined. In this study, we sought to determine the role of caspase 3 in chemical- and radiation-induced genetic instability and carcinogenesis. By use of a non-invasive caspase 3 reporter, we found that that a significant fraction of mammalian cells treated with 72 ionizing radiation could survive, despite caspase 3 activation. Moreover, this sublethal activation of caspase 3 promoted persistent DNA damage, chromosome aberrations and oncogenic transformation. In addition, chemically-induced skin carcinogenesis was significantly reduced in mice genetically deficient in caspase 3. We provided strong evidence that activated caspase 3 can indeed promote oncogenic transformation in human cells and in mice by inducing persistent genetic instability. Since a wide array of environmental and endogenous stressors can trigger caspase 3 activation, our findings suggest that rather than acting as a broad inhibitor of carcinogenesis, caspase 3 activation may contribute to genome instability and play a pivotal role in tumor formation following damage. Poster Section 40 Poster Board 3 LB-088 Ptch1 heterozygosity predisposes mice to developing IR-induced BCCs. Grace Y. Wang, Eileen Libove, Danielle Tucker, Ervin Epstein. Children’s Hospital Oakland Research Institute, Oakland, CA. Basal cells nevus syndrome (BCNS, Gorlin syndrome) patients carry heterozygous germline mutations in PATCHED1 (PTCH1) gene, which encodes a receptor of hedgehog (HH) ligands and represses HH signaling in the absence of ligands. Mutated PTCH1 leads to aberrant activation of the HH signaling pathway, which is the pivotal driver underlying BCC carcinogenesis in both sporadic and BCNS BCCs. BCNS (PTCH1+/-) individuals are very susceptible to developing more BCCs at an earlier age but the mechanism for this genetic predisposition to BCCs remains elusive. In this study, we assessed how heterozygosity of the murine Ptch1 gene contributes to IR-induced BCC carcinogenesis. Specifically, we treated Ptch1fl/+ K14CreER2 mice with tamoxifen either at age 4 weeks (group A) or at 9 weeks (group B) to delete one copy of Ptch1 in K14-expressing keratinocytes. We irradiated both groups of mice with IR at mouse age 8 weeks so that at the time of IR, keratinocytes in group A mice, like those in BCNS patients, were Ptch1+/-, and mice in group B remained Ptch1+/+ but subsequent to recovery from acute damage all mice had Ptch1+/- keratinocytes. We found that mice in both groups developed similar amounts of microscopic BCCs at age either 7- or 9-months. However, only mice of group A (3 out of 13 mice) developed visible BCCs; none of the mice of group B (n=30) developed visible BCCs by age 18-months, the longest time point monitored based on our studies of Ptch1+/- mice. The difference is statistically significant (p=0.03). Histologically, these visible BCCs closely resemble human BCCs. This finding indicates (i) that heterozygosity of the Ptch1 gene limited to keratinocytes (without gene deletion in stromal cells) is sufficient to produce susceptibility to IR-induced visible BCC development and (ii) surprisingly that heterozygosity of the Ptch1 gene during acute mutagenic damage dramatically increases eventual conversion of microscopic to visible BCCs, probably by affecting acute repair of this damage. To gain further insights into this possible mechanism, we are culturing keratinocytes from these mice and performing cell cycle and apoptosis analysis upon IR treatment in vitro. Poster Section 40 Poster Board 4 LB-089 Defining windows of susceptibility for low-dose exposure to endocrine disruptors in rat mammary development by microRNA profiling. Vasily N. Aushev,1 Maya Kappil,1 Kalpana Gopalakrishnan,1 Qian Li,1 Yula Ma,1 Luca Lambertini,1 Fabiana Manservisi,2 Laura Falcioni,2 Luciano Bua,2 Fiorella Belpoggi,2 Susan L. Teitelbaum,1 Jia Chen1. 1 Mount Sinai School of Medicine, New York, NY; 2Ramazzini Institute, Bologna, Italy. Background: Endocrine disruptors (EDs) constitute a class of chemicals that can interfere with the endocrine system, possibly influencing breast cancer risk. EDs exposure has been associated with changes in the epigenome such as methylation; their ability to modify other epigenetic processes, such as microRNA, has not Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 73 Late-Breaking Poster Session: Carcinogenesis been thoroughly investigated, especially in the context of mammary development. Methods: We investigated the influence on microRNA of three EDs widely used in personal care products: diethyl phthalate (DEP), methyl paraben (MPB) and triclosan (TCS). Sprague-Dawley rats were treated with these EDs at six windows of susceptibility (prenatal, neonatal, prepubertal and pubertal, parous, and nulliparous) from in utero to young adulthood. Oral treatment doses were selected to produce urinary metabolite levels comparable to those found in the US population. MicroRNAs were profiled using the NanoString platform. Results: Of 420 microRNAs analyzed, 90 were stably detected in >90% samples across all windows, with let-7 family members showing the highest expression. In the meantime, 132 microRNA species were expressed below the detection level in >90% of the samples; other miRNAs were expressed at different levels in some but not all samples. Principal component analysis revealed that different developmental windows displayed markedly different microRNA profiles. Importantly, we found that ED exposure, TCS in particular, resulted in measurable difference in rat mammary microRNAs, and the changes were window-specific. More specifically, the TCS-related changes were mainly present in prenatal, neonatal and pubertal windows; among these the neonatal period appeared to be the most susceptible window to EDs with the largest number of differentially expressed microRNAs (7 microRNA species with adjusted p<0.05). Conclusions: Low-dose ED exposure, even at levels comparable to human exposure, was able to modify microRNA expression in rat mammary tissues in a window-specific fashion. The study also demonstrates dynamic changes of microRNA profiles in developing mammary glands and highlights the importance of taking the normal developmental gene signal into account when searching for environment-induced gene signatures. Poster Section 40 Poster Board 5 LB-090 Estrogen metabolism within the human lung: impact of tumorigenesis, tobacco smoke, gender and race. Jing Peng,1 Xia Xu,2 William E. Smith,3 Sibele I. Meireles,4 Stacy L. Mosier,5 Guo Zhang,6 Shumenghui Zhai,6 Xiang Ma,6 Michael J. Slifker,1 Karthik Devarajan,1 Mindy S. Kurzer,3 Grace X. Ma,6 Margie L. Clapper1. 1Fox Chase Cancer Center, Philadelphia, PA; 2Leidos Biomedical Research, Inc, Frederick, MD; 3University of Minnesota, St. Paul, MN; 4Hospital Sirio Libanes, Sao Paulo, Brazil; 5Johns Hopkins University, Baltimore, MD; 6Temple University, Philadelphia, PA. Previous data from this group demonstrate that the murine lung can metabolize estrogen. Production of 4-hydroxyestrogens (4OHEs), putative carcinogens, is elevated within the lungs of females vs. males and accelerated by tobacco smoke exposure. The goal of this study was to assess the ability of the human lung to metabolize estrogen and determine if metabolism is altered either during lung tumorigenesis or by tobacco smoke, gender or race. Urine and lung tissue, tumor (T) and adjacent normal (N), were obtained from surgical non-small cell lung cancer (NSCLC) patients (men and women). Urine was also collected from healthy postmenopausal Caucasian and Chinese American women. Estrogen synthesis (CYP17A1, CYP21, HSD17B3 and HSD17B7) and metabolism (CYP1B1, GSTA4, GSTT1, NQO1 and COMT) genes were expressed in 50-100% of the lung specimens (T and N), as determined by qRT-PCR. Transcripts for CYP19 and HSD3B1 were detected only in tumors, while levels of HSD17B3 (produces testosterone) were lower in tumor vs. normal tissue (P=0.02). To determine the functional significance of the detected expression, the level of estrogen and its metabolites (EMs) were measured in human lung tissue by LC-MS2. Analyses of paired specimens (T and N) from NSCLC patients (18 women, 15 men) revealed 3 estrogens (E1, E2, E3) and 6 estrogen metabolites (2OHE1, 2-OHE2, 4-OHE1, 4-OHE2, 2-OMeE1, 2-OMeE2). Levels of each EM were higher in tumors than in adjacent normal tissue and in tissue (T and N) from women as compared to men (P<0.05). 4- OHEs were a larger proportion of the total EMs in lung tumors (from men and women) than in matching normal lung tissue (P=0.03), suggesting a shift towards the carcinogenic 4-hydroxylation pathway during tumorigenesis. In addition, 4-hydroxylation was increased in normal lung tissue from female current (n=10) vs. never (n=9) smokers (P=0.004), with similar trends observed in urine (P=0.04). P values were based on Wilcoxon tests. Based on the elevated risk of lung cancer among nonsmoking Chinese American women, the urinary EM profile of healthy, neversmoking Chinese vs. Caucasian American women (age 55 - 65) matched for age and body mass index (20/group) was compared. The level of 4-OHEs (4-OHEs/total EMs) in the urine of postmenopausal Chinese women was 1.8-fold higher than that of Caucasian women (P=0.015), suggesting estrogen metabolism may contribute to racial differences in lung cancer susceptibility. In summary, these data demonstrate that the human lung can metabolize estrogen, in particular to produce 4-OHEs. A shift towards 4-hydroxylation during lung tumorigenesis may contribute to the risk conferred by smoking, gender or race. Future studies will confirm these results in a larger population and evaluate the utility of urinary EM profiles as noninvasive biomarkers for the early detection of lung cancer. (Supported by the Estate of Jane Villon, the Kitty Jackson Fund, and Aurora and Timothy Hughes.) Poster Section 40 Poster Board 6 LB-091 Characterization of molecular changes occurring during long-term treatment of human bronchial epithelial cells with cigarette smoke total particulate matter. Marco van der Toorn, Niklas Kuehn, Stefan Frentzel, Diego Marescotti, Emmanuel Guedj, Nikolai Ivanov, Patrice Leroy, Manuel C. Peitsch, Julia Hoeng, Karsta Luettich. Philip Morris Products S.A., Neuchatel, Switzerland. Objectives: Cigarette smoking is the leading cause of lung cancer worldwide. Carcinogens in cigarette smoke are responsible for airway epithelial changes, although our knowledge about the molecular events underlying lung tumorigenesis are still not very detailed. Our aim was first of all to establish an in vitro model which mimics chronic exposure conditions found in the airways of smokers and to more comprehensively characterize the chronological changes that occur in bronchial epithelial cells under those conditions. Materials and Methods: Total particulate matter (TPM) was generated from the 3R4F reference cigarette according to the ISO smoking regimen. Human bronchial epithelial cells (BEAS-2B; ATCC) were repeatedly exposed to TPM for 4 weeks. Gene regulation and expression were investigated using Affymetrix GeneChip® twice weekly during exposure, and data were analyzed using R scripts and Ingenuity Pathway Analysis® (IPA®). Phenotypic alterations due to exposure including, for example, cell cycle changes and epithelial-to-mesenchymal transition (EMT) were studied on a weekly basis and at the end of the exposure period, respectively, using high content imaging. The imaging data were subjected to statistical analysis using linear models (SAS 9.2). Results: Treatment of BEAS-2B cells with TPM resulted in increased DNA damage after 1 week, continuously rising until the end of the 4-week exposure, although this trend was not statistically significant. We also observed a shift of the cell cycle to a G2/M-phase arrest, with a growing number of cells accumulating in this cell cycle phase over the treatment period. In addition, gradual activation of canonical pathways (e.g. PI3K/AKT, p53 and IL6 signaling) and enrichment in biological functions related to tumorigenesis over time was evidenced by overexpression of tumor promoting genes such as TP63, COX-2, GDF15, CCND1 and tumor suppressive genes like SERPINB5, THBS1, FAS and CDKN1A from as early as days 3-7. Furthermore, overexpression of miR-200 and miR-205 was identified first following 17 days of treatment, remaining significantly higher than controls until day 28. However, no visible morphological changes could be detected at the time when gene and miRNA expression changes first became significant. In contrast, those events typically occurred later. American Association for Cancer Research • AACR ANNUAL MEETING 2015 73 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 74 Late-Breaking Poster Session: Carcinogenesis Conclusion: Our experiments indicate that repeated exposure of bronchial epithelial cells to TPM induces ongoing alterations in gene expression as well as phenotypic changes related to tumorigenesis. The data here may lead to a better mechanistic understanding of the stepwise transformation of normal airway epithelial cells to full malignancy. Poster Section 40 Poster Board 7 LB-092 Programmed death-ligand 1 is overexpressed in bronchial preneoplastic lesions: can it be a risk indicator. Hee Sun Park,1 Bo Mi Park,1 Dong Il Park,1 Chung Jae Uk,1 Jae Young Moon,1 Jung Sung Soo,1 Choong Sik Lee,1 Ju Ock Kim,1 Sun Young Kim,1 Jaseok Peter Koo2. 1Chungnam National University College of Medicine, Daejeon, Republic of Korea; 2Yale School of Medicine, New Haven, CT. Compared to the vigorous development of targeted drugs and some of them are already become one of the best treatment of choices in lung adenocarcinoma, squamous lung carcinoma has no definite targets and treatment outcome is not yet superior to lung adenocarcinoma. Demonstration of initial carcinogenesis is benefitial in two aspects: cancer prevention and development of its targets. Studying preneoplastic lesions of bronchus can answer those questions. Preneoplastic lesion, which is considered to have malignant potential, may develop into invasive cancer by escaping from host immune response. CD274 (programmed death-ligand 1, PD-L1) interacts with PD-1, is known to inhibit CD8+ cytotoxic T lymphocyte and induce apoptosis and, also to promote the differentiation of CD4+ T cells into regulatory T cells, so finally evade immune surveillance. We hypothesized that PD-L1 may work as an immune evader in preneoplastic lesion during its carcinogenesis. We performed white light and/or autofluorescence bronchoscopy in patients who have risk factor(s) of lung cancer or are suspected to have a lung cancer. Interestingly, PD-L1 was also overexpressed in preneoplastic lesions especially in severe dysplasia of the bronchus. This finding implies overexpression of PD-L1 can involve at early step of carcinogenesis. Further studies are needed to demonstrate the role of PD-L1 in preneoplastic bronchial lesion potential to develop invasive cancer. Poster Section 40 Poster Board 8 LB-093 Activation of the FGFR1 signaling pathway by the epstein-barr virus-encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells. Kwok Fung Lo,1 Christopher W Dawson,2 Lawrence S Young,3 Kwok Wai Lo1. 1The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; 2University of Birmingham, Birmingham, United Kingdom; 3 University of Warwick, Coventry, United Kingdom. Undifferentiated nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) latent infection. The EBVencoded latent membrane protein 1 (LMP1) oncogene is believed to be important in NPC pathogenesis by virtue of its ability to activate multiple cell signaling pathways to induce proliferation, transformation, angiogenesis and invasiveness as well as 74 modulation of energy metabolism. In this study, we report that LMP1 increases cell uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentration. LMP1 also increases the phosphorylation of PKM2, LDHA and FGFR1 as well as expression of PDHK1, FGFR1, c-Myc and HIF-1α regardless of oxygen availability. This suggests that LMP1 promotes aerobic glycolysis. In addition to FGFR1, LMP1 also upregulates FGF2, the FGFR1 ligand, setting up a constitutive activation loop of FGFR1 signaling to promote aerobic glycolysis. FGFR1 inhibitors also abolish LMP1mediated cellular transformation (proliferation and anchorageindependent growth) as well as cell migration and invasion in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a high level of phosphorylated FGFR1 is common in primary NPC specimens, and that this correlated with the expression of LMP1. Inhibition of FGFR1 activity in NPC cells results in the suppression of cell proliferation and anchorageindependent growth. Our current findings demonstrate that LMP1mediated FGFR1 activation contributes to growth and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signaling activation in the EBV-driven pathogenesis of NPC. Poster Section 40 Poster Board 9 LB-094 Regorafenib and sildenafil interact to kill tumor cells. Mehrad Tavallai, Laurence Booth, Jane L. Roberts, Andrew Poklepovic, Paul Dent. Virginia Commonwealth University, Richmond, VA. The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as sildenafil (Viagra) to kill tumor cells. PDE5 and PDGFR (alpha and beta) were overexpressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death. Knock down of PDE5 or of PDGFR (alpha and beta) recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. RNS stimulated the activation of the death receptor CD95 as well as the formation of autophagosomes and autolysosomes. Inhibition of CD95 / FADD / caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK-1, Beclin1 or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas reexpression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib / regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. PDE5 inhibitors and sorafenib / regorafenib are FDA approved agents, and our data is now being translated into the clinic for further determination as to whether this drug combination is a useful anti-tumor therapy for solid tumor patients. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 75 Late-Breaking Poster Session: Cancer Chemistry Late-Breaking Poster Session Monday, April 20, 2015 8:00 AM-12:00 PM Poster Section 41 Late-Breaking Research: Cancer Chemistry Poster Section 41 Poster Board 1 LB-095 Distribution and clearance of single-walled carbon nanotubes in mouse tissues: in situ detection, imaging and analysis. Afsar Barlas, Ke Xu, Yevgeniy Romin, Simone Alidori, Dmitry Yarilin, Ning Fan, Mesruh Turkekul, Sho Fujisawa, David A. Scheinberg, Michael R. McDevitt, Katia Manova-Todorova. Memorial Sloan Kettering Cancer Center, New York, NY. Single-walled carbon nanotubes (SWCNT) are the subject of expanding research in the fields of targeted drug delivery and biosensors for disease treatment and monitoring. Functionalization of the SWCNT has proven to enhance the efficiency of distribution in the organism. The goal of this project was to establish and standardize methods of detection and characterization of the local tissue distribution of functionalized SWCNT in mouse models. We investigated the distribution and clearance of SWCNT in several tissues at various times after injection, up to one month. We also try to standardize methods of tracking SWCNT in both live and fixed tissues and image analysis protocols to quantify the distribution of the SWCNT in tissues. Another aim is to evaluate local immune response to SWCNT. We have analyzed liver, kidney, spleen, lung, brain, ovary, colon and small intestine from mice sacrificed 24 hours, 3 days, 7 days and 30 days following the injection of SWCNT. Our evaluation revealed that some organs, like kidney and small intestine, retain SWCNTs for at least a month. Slow rates of SWCNT removal from these organs do not appear to affect the overall health of the animals. We have made multiple attempts to image anesthetized live animals and detect SWCNTs in situ, however, the fluorescent signal emitted from SWCNT is too low to be reliably detected. The tissues must be fixed and signals amplified through immunodetection. Detection of immune markers, especially in immunologically active tissues such as spleen is a challenge for histological experiments, but is extremely important and such experiments is underway. Our findings of SWCNT persistence in certain organs 30 days post-injection is surprising and should be studied further. This observation opens further questions about the effect of long-term presence of SWCNT in some tissues and shows that careful investigation into the advantages and disadvantages of SWCNT retention is necessary. Poster Section 41 Poster Board 2 LB-096 Blood-brain barrier-penetrating terpolymer nanoparticles deliver docetaxel and trastuzumab to brain metastases of breast cancer. Chanson(Chunsheng) He,1 Ping Cai,1 Jason Li,1 Jeffrey T. Henderson,1 Andrew M. Rauth,2 Xiao Yu Wu1. 1University of Toronto, Toronto, Ontario, Canada; 2Ontario Cancer Institute, Toronto, Ontario, Canada. Background: Brain metastases occur in up to one third of all metastatic breast cancer patients, with high prevalence and earlier development in triple-negative and HER2-positive breast cancers. Treatment options for brain metastases are severely limited due to the inability of many therapeutic agents, including docetaxel (DTX), a small molecule hydrophobic drug, and trastuzumab (TRA), a macromolecular antibody, to cross the blood-brain barrier (BBB) at adequate levels. Here we developed multifunctional BBBpenetrating terpolymer-lipid nanoparticles (TPN) for delivering DTX and TRA to brain metastases of breast cancer, and evaluated their tumor accumulation and efficacy in multiple lesion brain metastases mouse models. Methods: Fluorescence-labeled TPN were prepared by microemulsion using poly(methacrylic acid) and polysorbate 80grafted starch loaded with either DTX (DTX-TPN) or TRA (TRA-TPN). In vitro cytotoxicity of the formulations was evaluated by the MTT assay. Brain metastases of triple negative MDA-MB-231-lucD3H2LN or HER2 positive BT474 human breast cancer were established in SCID mice by stereotactic intracranial inoculation of the cells into the cortex. The biodistribution and tumor accumulation of the NPs were examined by whole body fluorescence imaging at various times after tail vein injection. The NP distribution within the brain tumor microenvironment was detected ex vivo using laser scanning confocal microscopy. The efficacy of DTX-TPN against brain metastases was assessed by measuring bioluminescence intensity weekly following administration of 2×20 mg/kg DTX-TPN or Taxotere. Results: In vitro DTX-TPN exhibited greater cytotoxicity against MDA-MB-231 cells compared to free DTX (IC50=40 vs. 63 nM), while TRA-TPN decreased the IC50 by 4.5-fold (IC50=0.6 vs. 2.7 μg/mL) in the inhibition of BT474 cells compared to free TRA. In vivo TPN was able to transport DTX and TRA across the BBB to brain metastasis lesions. The TPNs were observed to extravasate the brain microvessels and accumulate within the perivascular tissue throughout the tumor core and periphery. DTX-TPN increased the median mouse survival time by 2-fold (Saline group: 20 ± 3 days; free Taxotore: 18 ± 4 days; DTX-TPN: 36 ± 3 days) and delayed tumor growth by 9.8-fold (58% vs. 5.9%) as compared to clinically used Taxotere in mice with brain metastasis of triple negative breast cancer. No change in tissue morphology was observed in the liver, lungs, heart and kidneys of mice treated with DTX-TPN compared to the saline control group. Conclusions: These results demonstrate that TPNs are a promising BBB-penetrating carrier for the delivery of small molecule therapeutic agents or antibodies to the brain and for the treatment of brain metastases of breast cancer. Poster Section 41 Poster Board 3 LB-097 Targeted degradation of the androgen receptor in prostate cancer. Meizhong Jin,1 James D. Winkler,1 Kevin Coleman,1 Andrew P. Crew,1 AnnMarie K. Rossi,1 Ryan R. Willard,1 Hanqing Dong,1 Kam Siu,1 Jing Wang,1 Deborah A. Gordon,1 Xin Chen,1 Caterina Ferraro,1 Craig M. Crews,2 Taavi K. Neklesa1. 1Arvinas, Inc., New Haven, CT; 2 Yale University, New Haven, CT. Progression of prostate cancer in patients treated with antiandrogen therapy usually involves several mechanisms of enhanced Androgen Receptor (AR) signaling, including increased intratumoral androgen synthesis, increased AR expression and AR mutations. We have developed a protein degradation technology called PROTACs (PROteolysis TArgeting Chimera), which uses bifunctional molecules that simultaneously bind a target of choice and an E3 ligase. PROTACs, via induced proximity, cause ubiquitination and degradation of the targeted, pathological protein. As opposed to traditional target inhibition, which is a competitive process, degradation is a progressive process. As such, it is less susceptible to increases in endogenous ligand, target expression, or mutations in the target. Thus this technology seems ideal for addressing the mechanisms of AR resistance in patients with prostate cancer. AR PROTACs were shown to degrade AR in LNCaP and VCaP cells, with low nM to pM potency, and had a >85% reduction in AR concentration (Dmax). Degradation was rapid, with 50% of AR lost within 15 minutes and maximal degradation observed by 4 hours. The degradation process in cells was specific, as the PROTAC activity can be competed with excess E3 ligand and PROTACs with an inactive epimer for E3 ligase binding did not degrade AR. AR PROTACs induced rapid apoptosis and cell death in VCaP cells. In LNCap and VCaP cell systems, AR PROTACs were anti-proliferative under conditions in which enzalutamide was inactive, such as increasing concentrations of the AR agonist R1881 and cells containing the ARF876L mutation. AR PROTACs typically exhibited good pharmacokinetic properties, with t1/2 values of several hours and bioavailability of >50% after ip or sc injection. In mice, AR PROTACs demonstrate in vivo activity, including reduction of AR protein levels, prostate involution and tumor growth inhibition. American Association for Cancer Research • AACR ANNUAL MEETING 2015 75 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 76 Late-Breaking Poster Session: Cancer Chemistry In summary, PROTACs designed to degrade AR are potent, specific, active in vitro and in vivo, and have cellular efficacy superior to enzalutamide. Targeted degradation of AR may provide a novel mechanism for providing efficacious therapy for patients with prostate cancer for whom current therapies have failed. Poster Section 41 Poster Board 4 LB-098 Antitumor studies: Design, synthesis, antitumor activity and molecular docking study of novel 2-deoxo-2substituted-5-deazaalloxazines. Sawsan A. Mahmoud,1 Mosaad S. Mohamed,2 Nageh A. Abou Taleb,3 Tomohisa Nagamatsu,4 Hamed I. Ali5. 1Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA; 2 Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Helwan University, Cairo, Egypt; 3Department of Pharmaceutical Chemistry, College of Pharmacy, Helwan University, Cairo, Egypt; 4Department of Medical Technology, Faculty of Health Science , Kumamoto Health Science University, Kumamoto, Japan; 5 Irma Lerma Rangel College of Pharmacy, Texas A&M UniversityHealth Science Center, Kingsville, TX. Cancer is the major threat to the public health worldwide, thereby we have a considerable interest to get potential antitumor agents via computational design, synthesis, functional elucidation, and biological evaluation of different deazaalloxazine analogs. Many of these compounds revealed higher selectivities against different tumor cell lines. In the study the structure activity relationships (SAR) of the proposed derivatives was investigated, by applying structure based drug design (SBDD) using most advanced molecular modeling tool programs, namely: AutoDock 4.2 and Accelrys Discovery studio 2.0. These computational approaches aim to increase the speed and efficiency in the drug discovery process. The reasonable drug candidates were subjected to the chemical synthesis and biological in vitro test against different tumor cell lines. The docking study of the synthesized and the rationally designed derivatives was carried out using PTKs as target enzymes which was early reported as a proposed pathway for inhibition of cancer. The main outcome of this study is the synthesis of novel 2-methylthio, 2-(substituted alkyl amino), 2-(heterocyclic substituted), 2-amino, 2,4-dioxo and 2deoxo-5-deazaalloxazine derivatives. Their antitumor activities against human T-cell acute lymphoblastoid leukemia cell line (CCRF-HSB-2), human oral epidermoid carcinoma cell line (KB), human breast cancer cell line (MCF-7) and human cervical cancer cell line (Hela) have been investigated in vitro. Many compounds showed promising antitumor activities. Furthermore, AutoDock study has been done by binding of the 5-deazaalloxazine analogs into c-kit PTK (PDB code: 1t46), where a good correlation between their IC50 and AutoDock binding free energy was exhibited. Poster Section 41 Poster Board 5 LB-099 A high-throughput screen of approved drugs uncovers a synergistic interaction targeting prostate cancer. Marco P. Licciardello,1 Patrick Markt,1 Freya Klepsch,1 CharlesHugues Lardeau,1 Gerhard Dürnberger,1 Vladimir Ivanov,2 Jacques Colinge,1 Stefan Kubicek1. 1CeMM, Vienna, Austria; 2Enamine, Ltd, Kiev, Ukraine. Libraries of approved drugs contain some of the best studied small molecules and represent invaluable resources for drug repurposing and chemical biology studies. Access to comprehensive libraries of clinical compounds is limited and these collections are otherwise difficult to arrange. Using cheminformatics approaches we have assembled a non-redundant set of representative FDA-approved small molecules that we call the CeMM Library of Unique Drugs (CLOUD). The CLOUD covers the chemical and biological space of approved drugs, contains active forms of prodrugs, and can be screened at clinically relevant concentrations. In a combinatorial viability screen of the CLOUD we uncovered a synergistic interaction between two approved drugs. 76 We show that the concomitant administration of these two small molecules induces apoptosis in the androgen receptor (AR)dependent prostate cancer cell line LNCaP. Mechanistically, the combination affects AR signaling and the stability of the receptor itself. We are currently dissecting further aspects of the mechanism of action and performing in vivo experiments. Altogether, our data suggest that the combination of these two drugs can be repurposed for the treatment of prostate cancer. Poster Section 41 Poster Board 6 LB-100 Development of novel selective tumor-associated carbonic anhydrase inhibitors as promising anticancer agents. Ahmed Mahmoud Alafeefy,1 Sabry Atia,2 Sheikh Ahmad,2 Khairy Zoheir,2 Abdelkader Ashour,2 Ashok Kumar2. 1Salman Bin Abdulaziz Univ., Alkharj, Saudi Arabia; 2King Saud Univ., Riyadh, Saudi Arabia. The molecular complexity of cancers and therapy-related side effects often limit efficacy of numerous anti-tumor therapies, and warrant development of new drugs that are specific for certain molecular targets while minimizing the off-target effects. We previously have synthesized a series of benzene-sulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues), and investigated such new compounds as inhibitors of the metalloenzyme carbonic anhydrase (CA). Specifically, we determined the inhibitory activity of such novel compounds against the cytosolic, house-keeping human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII. Four compounds, namely CS2, CS6, CS8 and CS13, were very potent CA IX/XII inhibitors whereas they were much less effective as inhibitors of CA I and II. To determine whether these selective tumor-associated CA inhibitors exert antitumor activity, we examined their antiproliferative activity against the human medulloblastoma Daoy cell line, human hepatoma HepG2 cell line and the human epithelial cervical cancer Hela cell line. Compounds CS6 and CS13 showed significant antiproliferative activity against the three cancer cell lines. Daoy was the most sensitive cell line, and compound CS6 was the most potent one. Remarkably, compound CS6 was more potent against Daoy cells than the multitargeted kinase inhibitor of BCR-ABL and SRC family kinases, dasatinib, since the former exerted smaller IC50 than that exerted by the latter (4.14 versus 7.28 µg/mL). In addition, flow cytometric Annexin-V/propidium iodide assay results showed that cell death induced by compounds CS6 is mediated, at least in part, by apoptosis. Moreover, CS6 significantly inhibited tyrosine kinase activity in Daoy cells, compared to DMSO-treated (control) cells. Taken together, these data indicate that compound CS6 is a novel multiple cancer pathways inhibitor, and warrants further investigation of its antitumor activity in medulloblastoma and other brain tumors. Currently, compounds CS6 and CS13 are being tested for their inhibitory activity against dihydrofolate reductase and thymidylate synthase. Poster Section 41 Poster Board 7 LB-101 Lycopene suppresses the NF-κB signaling pathway through inhibition of IκB kinase in human prostate cancer cells. Emelia A. Assar, Mridula Chopra, Sassan Hafizi. Univ. of Portsmouth, Portsmouth, United Kingdom. Prostate cancer is the fourth most common cancer worldwide and the second most common amongst American men. Alongside age and genetic factors, lifestyle and diet have also been implicated as significant factors involved in the pathology of cancer and prostate cancer risk. The tomato-derived antioxidant lycopene has been highlighted as a key protective nutrient amongst various dietary components, with several in vitro studies having reported anti-cancer properties. However, the mechanism of action of lycopene is not fully characterized. We present here a comprehensive investigation of the influence of lycopene on the cell signaling pathway regulating nuclear factor kappa B (NF-κB). NF-κB is a redox-sensitive transcription factor that is believed to play a Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 77 Late-Breaking Poster Session: Cancer Chemistry critical role in the development of the cancer phenotype in cells and has been linked to the early onset and progression of prostate cancer. Phosphorylation of IκB releases NF-κB from sequestration in the cytoplasm, allowing its translocation into the nucleus and consequent tumorigenic gene expression. We studied the effect of lycopene in vitro on multiple points along the NF-κB signaling pathway in two highly malignant, androgen-independent prostate cancer cell lines, DU145 and PC3. Lycopene (0.5-5 μM) was incubated with the cells for 48h, and significantly inhibited prostate cancer cell growth in a formazan cell growth assay at physiologically relevant concentrations of ≥1.25 μM. Similar concentrations of lycopene also caused 30-40% reduction in levels of both basal and TNF-stimulated IκB phosphorylation in cultured DU145 and PC3 cells after 20h, as determined by western blotting. Furthermore, the same degree of inhibition by lycopene was observed for NF-κB transcriptional activity in both cell lines, as determined by a reporter gene assay. NF-κB transcriptional activity in DU145 and PC3 cells was suppressed by 20-40% (p<0.05) and 20-50% (p<0.001) respectively. Further probing of lycopene’s effects on upstream elements of the NF-κB pathway showed a significant inhibition of both activity of recombinant IKKβ kinase in a cell-free in vitro assay by approximately 25% (p<0.01), as well as activity of IKKβ immunoprecipitated from cells treated with lycopene. In conclusion, our findings show that the anti-cancer properties of lycopene may occur through its inhibition of the NF-κB signaling pathway, beginning at the early stage of IKK kinase activity. Furthermore, these effects in prostate cancer cells were observed at concentrations of lycopene that are physiologically relevant and achievable in man. Ongoing work is exploring the mechanisms behind the redox regulation of IKK activity by lycopene, as well as the downstream genes regulated by its control of NF-κB signaling. Unraveling the mechanisms by which lycopene acts as an anticancer nutritional agent has implications for the control of prostate cancer development through diet. Poster Section 41 Poster Board 8 LB-102 Layer-by-layer engineering of upconversion nanoparticle based siRNA and miRNA delivery system for cancer therapy. Lin Min,1 Yan Gao,1 Francis J. Hornicek,1 Mansoor M. Amiji,2 Zhenfeng Duan1. 1Massachusetts General Hospital, Boston, MA; 2 Northeastern University, Boston, MA. Purpose: Nanoparticle based drug delivery systems have shown promising applications in cancer treatments. Among various nanoparticles, upconversion nanoparticles (UCNPs) allow for the conversion of near infrared (NIR) excitation to localized UV/visible emission providing unprecedented control over UCNP based delivery platforms for deep tissue therapeutic payload release, both spatially and temporally. This study reports the layer-by-layer engineering of UCNP based siRNA and miRNA delivery systems for application in cancer therapy. Experimental Design: To obtain the nanoparticle core, NaYF4:Yb,Er UCNPs were synthesized via thermal decomposition method. The morphology and size of the UCNPs were assessed by transmission electron microscopic (TEM). The upconversion fluorescence property was studied by fluorescence spectroscopy using a 980 nm laser as the excitation source. To enable gene delivery, the naked UCNPs were then surface functionalized with biocompatible polymers using polyacrylic acid (PAA) and poly allylamine hydrochloride (PAH). Surface functionalization was accomplished through layer-by-layer assembly of polyelectrolyte multilayers. Successful surface functionalization was demonstrated by measuring zeta potential after assembly of each layer. Biocompatibility of the developed delivery system was tested by MTT assay. Gel electrophoresis was employed to determine the loading capacity of the delivery system. We then tested the siRNA, miRNA, and EGFP expression vector transfection efficiency in different cancer cell lines. Results: The diameter of the synthesized UCNPs was ~40 nm. Upon 980 nm excitation, green emission and red emission were identified from the fluorescence spectrum. Zeta potential of the delivery system was ~+38 mV, indicating its capability to absorb negatively charged siRNA, miRNA, or EGFP vector by electrostatic interaction. The MTT assay demonstrated good biocompatibility. Significant retardation of siRNA, miRNA, or EGFP plasmid in gel electrophoresis assay was observed when mixed with PAA and PAH coated UCNPs. In contrast, UCNP-PAA without PAH coating did not retard siRNA, miRNA, or EGFP plasmid. Confocal laser scanning microscopy results provided direct evidence of siRNA, miRNA, or EGFP plasmid transfection. Conclusions: In this proof-of-concept study, a layer-by-layer engineered UCNP based siRNA and miRNA delivery system was successfully developed. Our novel delivery system opens up preparation of advanced UCNP based photoresponsive delivery systems that allow remote, precise, and trackable control over therapeutic payload (e.g., drug, gene) release for better cancer theranostics. Poster Section 41 Poster Board 9 LB-103 Structural characterization of SPRY2-Cbl interactions. Na Zhang,1 Xuan Zhang,1 Laurie Washington,1 Asokan Anbanandam,2 Kevin Battaile,3 Philip Gao,2 Aaron Smalter Hall,2 Scott Lovell,2 Anuradha Roy,2 Robert Hanzlik,2 Raymond Perez1. 1 University of Kansas Cancer Center, Fairway, KS; 2University of Kansas, Lawrence, KS; 3IMCA-CAT Hauptman-Woodward Research Institute, Argonne, IL. Sprouty 2 (SPRY2) is a feedback modulator of receptor tyrosine kinase (RTK)-ERK signaling, and a potential tumor suppressor. Cbl, an E3 ubiquitin ligase and scaffold protein, binds SPRY2 with high affinity, and sequesters or targets it for degradation. Cbl also binds several growth factor receptors. Hence SPRY2, Cbl and receptors are in a dynamic equilibrium. Truncation and site-mutagenesis studies previously mapped SPRY2-Cbl interactions to two discrete domains, TKB and RING. A pY binding pocket in the TKB SH2 region is critical for binding, but the role(s) of other TKB regions and/or RING are undefined. Interactions of three Cbl recombinant proteins (P47-G351 (TKBD), and two phosphomimetic RING activeconformation constructs, P47-D435 Y371D/E) with three SPRY2 peptides (P1: Q36-N53, a putative RING binder; P2: Q36-T60 (pY55), TKB binder; and P3: 54-60(pY55), a truncated TKB binder) were characterized by computational simulation, surface plasmon resonance spectroscopy (SPR), fluorescence polarization (FP), and X-ray crystallography. In silico modeling suggested possible lowaffinity binding of P1 in the groove between TKBD SH2 and 4-helix (4H) regions and near the RING, but this was not confirmed by FP, SPR, or crystallography. Apo and P2 ligand-bound structures agreed with prior published data; three previously unknown 4H interaction sites were also identified. Data on the effects of sitespecific 4H mutants on P2 binding affinities are pending. Binding affinities for P2 with the TKBD, Y371E, and Y371D constructs were 160±5, 92±9, and 45±3 nM, respectively, by SPR; FP results were similar. Direct interactions with the RING were not confirmed, but the higher P2 binding affinities observed with RING constructs suggest that this domain indirectly facilitates SPRY2 binding. Binding of P2 induced 16.9° rotation of the SH2 region and formation of new H-bonds to 4H, compressing the TKBD. Importantly, P4 induced identical conformational changes as P2, but had no direct contact with 4H. These observations are consistent with a refined model of SPRY2-Cbl interaction, where ligand docking at the pY SH2 site is sufficient to globally change TKB architecture, facilitating SPRY2 binding at previously unrecognized sites in the 4H region (Support: KU COBRE-PSF (NIH/RR017708, NIH/GM103420), KU Cancer Center, Kansas Bioscience Authority, and the George & Floriene Lieberman Endowment). American Association for Cancer Research • AACR ANNUAL MEETING 2015 77 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 78 Late-Breaking Poster Session: Cancer Chemistry Poster Section 41 Poster Board 10 LB-104 Excited electrons in melanin induce cyclobutane dimers in the dark. Sanjay Premi,1 Silvia Wallisch,1 Camila Mano,1 Adam Weiner,1 Antonella Bacchiocchi,1 Kazumasa Wakamatsu,2 Etelvino Bechara,3 Ruth Halaban,1 Thierry Douki,4 Douglas E. Brash1. 1Yale School of Medicine, New Haven, CT; 2Fujita Health Univ., Toyoake, Japan; 3 Univ. São Paulo, São Paulo, Brazil; 4Commissariat a` l’Energie Atomique, Grenoble, France. Sunlight-induced melanomas contain UV-signature mutations, which are caused by cyclobutane pyrimidine dimers (CPD). These photoproducts are typically created picoseconds after a UV photon is absorbed at adjacent thymines or cytosines. However, using immunohistochemistry, mass spectrometry, and RNAi, we find that melanocytes generate CPD for >3 hours after exposure to UVA or UVB, wavelengths found in sunlight and in tanning beds; these “dark CPD” constitute the majority of CPD induced. Using pharmacologic inhibitors, single-photon counting, and specific energy acceptors, we elucidated the mechanism. The process begins when UV-induced superoxide and nitric oxide combine to form peroxynitrite, one of the few biological molecules capable of exciting an electron. Excitation creates a quantum triplet state in the skin pigment melanin that has the energy of a UV photon but induces CPD by transferring its energy to DNA in a radiationindependent manner. Melanin is evidently carcinogenic as well as protective. These findings may underlie the dependence of UVinduced and spontaneous skin cancers on melanin type. The results also validate the long-standing suggestion that chemical generation of excited electronic states - the source of bioluminescence in lower organisms - is important in mammalian biology. Poster Section 41 Poster Board 11 LB-105 Methanol extract of Cochrorus olitorus protects against potassium dichromate toxicity in albino rats. Olabode O. Osifeso,1 Kazeem A. Akinwumi,2 Ayobami W. Adedoja3. 1 Moshood Abiola Polytechnic, Abeokuta, Nigeria; 2Bells University of Technology, Ota, Nigeria; 3City University of New York, New York, NY. Hexavalent chromate compounds are human carcinogens that pose great health hazard in several parts of the world especially in developing countries. In addition, an effective cure eludes the world. The study therefore evaluates the usefulness of the leafy vegetable and herb; Cochrorus olitorus (CO) against potassium chromate K2Cr2O7. Negative control animals were fed distilled water, while the positive control rats received K2Cr2O7 once per week. Test rats were exposed to 25, 50 and 100 mg/Kg body weight of CO alone for 42 days and / or 12 mg/kg body wt of K2Cr2O7 once per week before the animals were sacrificed. The frequency of micronucleated polychromatic erythrocytes (mPCEs) 78 was monitored in bone marrow cells while aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine levels were assessed in the serum. Haematological parameters were also monitored in test and control animals. The phytochemical analysis of CO was also carried out. K2Cr2O7 significantly (P<0.05) increased the levels of mPCE, AST, ALT, creatine, total white blood cells and lymphocytes as compared with the control. While percentage pack cell volume and neutrophils were reduced. In contrast, treatment with the different doses of CO restored the markers towards the levels of the negative control. Methanolic extract of CO is rich in flavonoids, saponins, anthraquinnones, terpernoid and phenols which may be responsible for the protection observed in this study. Our results suggest that methanol extract of CO has potentials in the treatment/management of chromate toxicity. Poster Section 41 Poster Board 12 LB-106 Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo. Angela Alexander-Bryant,1 Anca Dumitriu,2 Christopher Attaway,2 Hong Yu,2 Andrew Jakymiw1. 1Clemson University & Medical University of South Carolina, Charleston, SC; 2Medical University of South Carolina, Charleston, SC. Intracellular delivery and endosomal escape of functional small interfering RNAs (siRNAs) remain major barriers limiting the clinical translation of RNA interference (RNAi)-based therapeutics. Recently, we demonstrated that a endosome-disruptive peptide we synthesized termed, 599, could enhance the intracellular delivery and bioavailability of siRNAs designed to target the CIP2A oncoprotein (siCIP2A) into oral cancer cells and consequently inhibit oral cancer cell invasiveness and anchorage-independent growth in vitro. Thus, to further assess the therapeutic potential of the 599 peptide in mediating RNAi-based therapeutics for oral cancer and its prospective applicability in clinical settings, the objective of the current study was to determine whether intratumoral dosing of the 599+siCIP2A complex could induce silencing of CIP2A and consequently impair tumor growth using a xenograft oral cancer mouse model. Our results demonstrated that the 599 peptide was able to protect siRNAs from degradation by serum and ribonucleases in vitro, confirming the stability of the 599+siRNA complex and its potential for in vivo utility. Moreover, 599 peptide-mediated delivery of siCIP2A to tumor tissue induced CIP2A silencing without any associated toxicity, consequently resulting in reduction of the mitotic index and significant inhibition of tumor growth. Together, these data suggest that the 599 peptide carrier could be a clinically effective mediator of RNAi-based cancer therapeutics. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 79 Late-Breaking Poster Session: Clinical Research / Endocrinology Late-Breaking Poster Session Monday, April 20, 2015 1:00 PM-5:00 PM Poster Section 40 Late-Breaking Research: Clinical Research / Endocrinology Poster Section 40 Poster Board 1 LB-107 Targeting ATR using a novel ATR inhibitor AZD6738 in human gastric cancer cells. Ahrum Min, Seock-Ah Im, Hyemin Jang, Seongyeong Kim, Miso Lee, Jungeun Kim, Kyung-Hun Lee, Sae-Won Han, Tae-Young Kim, Do-Youn Oh, Tae-You Kim, Woo-Ho Kim, Yung-Jue Bang. Seoul National University, Seoul, Republic of Korea. Introduction: The DNA repair system is critical for maintaining genomic integrity. In particular, the homologous recombination (HR) repair pathway, on that repairs DNA double-strand breaks (DSBs), is directly associated with cancer development. Many cancer cells could contain mutations in genes involved in the HR pathway including BRCA1/2, ATM, ATR, and RAD51. ATR can be activated by various types of DNA damage and it initiates DNA damageinduces signaling cascade. As a result, ATR pathway components are considered promising therapeutic targets because ATR inhibition is likely to have greater deleterious effect on cancer cells. Materials and Methods: The antiproliferative activity of AZD6738 was examined in vitro using a cytotoxic assay, cell cycle analysis, and western blotting. To figure out the action mechanism of AZD6738 in gastric cancer cells, the expression of proteins which participate in DNA repair were investigated. The accumulation of DNA damage was also assessed with a DNA comet assay. These in vitro data were validated in vivo using a human gastric cancer xenograft model. Results: Human gastric cancer cell lines showed heterogeneous response to AZD6738. In AZD6738 sensitive cells, ATR inhibition leads to the accumulation of unrepaired DSBs due to dysfunctional RAD51 foci formation along with increased caspase 3-dependent cell death and S phase cell cycle arrest. Compared with sensitive cells, the activation of ATM-chk2 signaling pathway under the ATR inhibition was observed in the AZD6738 insensitive cells. It suggests that the activation of ATM-chk2 signal leads to attenuation of AZD6738 sensitivity. Furthermore, AZD6738 significantly suppressed tumor growth with increased apoptosis in vivo. Conclusion: We evaluated the anti-tumor activity of AZD6738 in gastric cancer in vitro and in vivo model. This is the first study to show that AZD6738 interferes with RAD51-mediated homologous recombination, as well as promotes cell death by inducing S cell cycle arrest and apoptosis in gastric cancer cells. Our findings can help to promote novel treatment strategies using AZD6738 in gastric cancer. Poster Section 40 Poster Board 2 LB-108 Automated immunohistochemistry-based identification of molecular subtypes in colorectal cancer. Anne Trinh,1 Filipe De Sousa E Melo,2 Xin Wang,1 Joan H de Jong,2 Evelyn Fessler,2 Marnix Jansen,2 Gerrit KJ Hooijer,2 Jan Paul Medema,2 Florian Markowetz,1 Louis Vermeulen2. 1CRUK Cambridge Institute, Cambridge, United Kingdom; 2Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands. Background: Recent molecular profiling of colorectal cancer has identified three distinct subtypes: two good-prognosis subtypes characterised by chromosomal instability (Subtype 1) and microsatellite instability (Subtype 2), and a poor prognosis subtype defined by epithelial-mesenchymal transition and stemness (Subtype 3). A key challenge lies in the subsequent implementation of this classification system in the clinic, where the requirement for sufficient bulk tumor and high cost hampers the widespread use of genomic profiling. In this proof-of-concept study, we have adapted our molecular signatures to an automated immunohistochemistry (IHC)-based classifier which can be used a rapid screening tool, and validated the feasibility of this approach in a multi-centre study. Patients and Methods: A total of 1080 patients from four different centres were used in this study. Tumor microarrays (TMAs) from each patient were stained using immunohistochemistry for a panel of five stains in conjunction with microsatellite instability status (MSS). An automated image analysis pipeline was developed to quantitate and normalize all staining. A classifier to distinguish colorectal cancer subtypes was trained on 74 patients from a single centre and applied to the three remaining cohorts. Results: We have developed an automated IHC-based classification system which demonstrated 84% correlation with our genomic profiling system. The prognostic value of colorectal cancer subtyping was validated in three independent cohorts, taking into account age, sex and stage (Hazards Ratios with 95% Confidence Interval: 1.8 (1.2 - 2.6), 1.4 (1.1 - 1.7) and 1.2 (1.0 - 1.6)). We evaluated the predictive value of the subtyping system in a retrospective analysis of a late-stage clinical trial and demonstrated the benefit of adjuvant cetuximab in KRAS/BRAF wild type Subtype 1 patients (Hazards Ratio 0.6 (0.4-0.9)) but not in Subtype 3 patients. Conclusion: In this proof-of-concept study we have demonstrated the effectiveness of an automated IHC-TMA classifier as a surrogate for genomic profiling. Using this approach, we have validated the prognostic value of colorectal subtyping in a multicentre study, and shown the predictive value of subtyping for adjuvant cetuximab therapy. This approach has demonstrated high clinical potential as a rapid screening tool, in particular in retrospective examination of patient cohorts where only formalinfixed paraffin-embedded (FFPE) tissue is available. Poster Section 40 Poster Board 3 LB-109 Exome sequencing identifies common somatic mutations in an adult patient with a concurrent germ cell tumor (GCT) and acute myeloid leukemia (AML) suggesting a single clonal origin. Charles Lu,1 Peter Riedell,2 Peter Westervelt,2 Christopher Miller,1 Ian S. Hagemann,3 Eric J. Duncavage,3 Elaine R. Mardis,4 Richard K. Wilson,4 Bradley A. Ozenberger,4 Lukas D. Wartman5. 1 Washington University, St. Louis, MO; 2Department of Internal Medicine, Washington University, St. Louis, MO; 3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; 4The Genome Institute, Washington University School of Medicine, St. Louis, MO; 5Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO. The Genomics Tumor Board at Washington University was established to increase knowledge, competence, and performance in the application of genomic testing in cancer care. Here we report the findings from a Tumor Board case of concurrent germ cell tumor and acute myeloid leukemia. A 33-year old male presented with generalized weakness, weight loss, and dyspnea on exertion. Initial workup was notable for a platelet count of 5,000 platelets/µL, hemoglobin of 13.1g/dl, and a white count of 9,200 cells/µL with a normal differential. His AFP was 237 (ULN 8.1 ng/ml), LDH was 6760 U/L (ULN 250 U/L) and β-hCG <5 (normal <5 IU/L). Chest CT scan revealed an anterior mediastinal mass. A bone marrow biopsy and aspirate showed a cellularity of 70%, with the core biopsy showing a fibrotic marrow with a population of larger mononuclear cells. The hemodilute aspirate showed 15% large blasts, and a subset of the larger cells expressed CD61 and weak CD117. These findings were consistent with a diagnosis of acute megakaryoblastic leukemia (AML M7). The patient underwent incisional biopsy of the mediastinal mass with pathology showing necrotic fragments of tissue with scattered foci of moderately to poorly differentiated adenocarcinoma. Immunostaining was consistent with a nonseminomatous germ cell tumor. Multiple prior studies have described associations between hematological malignancies, including AML M7 and nonseminomatous germ cell tumors, and a recent study identified a patient with a concurrent American Association for Cancer Research • AACR ANNUAL MEETING 2015 79 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 80 Late-Breaking Poster Session: Clinical Research / Endocrinology AML and GCT that shared several mutations including PTEN, TP53, and chromosome 12 abnormalities, suggesting that a common founding clone initiated both cancers (Oshrine, B. R., et al. Cancer Genet 2014). To investigate the clonal relationship in our samples, we studied the GCT (whole genome amplification was performed on 2 ng of DNA isolated by laser capture microdissection of viable cells from the FFPE tumor specimen) and the M7 AML (cryopreserved cells from the diagnostic bone marrow biopsy were flow-sorted using the above markers) by whole exome sequencing. We found both samples contained somatic mutations in PTEN (C136R missense) and TP53 (R213 frameshift). Both the mutations in PTEN and TP53 were present at ~100% variant allele frequency (VAF) in both tumors. A copy number comparison between the 2 samples revealed similar amplifications of chromosome 12p. In addition, we detected a heterozygous germline variant in FANCA (R858D), which is known to be associated with Fanconi anemia and is of uncertain significance here. In conclusion, the data not only support a common clonal ancestor for these cancers but also suggest that a specific set of distinct genomic alterations drives the rare association between GCT and AML, and likely underlies the poor outcome of these patients. Poster Section 40 Poster Board 4 LB-110 Expression of multiple androgen receptor splice variants in late stage prostate cancer. Zara Ghazoui,1 Emma Jones,1 Margaret Veldman-Jones,1 Vivien N. Jacobs,1 Neil R. Smith,1 Michele Johnstone,2 J. Carl Barrett,2 Elizabeth A. Harrington,3 Paul Elvin1. 1AstraZeneca, Macclesfield, United Kingdom; 2AstraZeneca, Boston, MA; 3AstraZeneca, Cambridge, United Kingdom. The androgen receptor (AR) remains a key driver of disease progression even with the advent of new therapeutic agents such as abiraterone (A) and enzalutamide (E). Relapse after treatment with A or E has been associated with the emergence of tumor cells expressing AR splice variants, and at least one mutation in AR has been associated with resistance to E. We have investigated the expression of AR splice variants in late stage prostate cancers using Nanostring (nCounter®) to quantify the levels of AR transcripts and by IHC using antibodies targeting the N- and Cterminal AR domains. We also examined the expression of putative AR regulated genes found to be expressed in prostate cancer reported in publically available expression data. Formalin fixed paraffin embedded tissue from 38 commercially sourced (TriStar Technology Group; Asterand; Avaden Biosciences) late stage prostate cancers including 10 castrate resistant prostate cancers (CRPC), were confirmed by pathology review. Total mRNA was isolated from a single 5µm section and gene expression from nCounter® analysis was expressed as normalised Log2 values. Immunohistochemical analysis of AR expression was determined by a pathologist using a H score. Our analysis of AR splice variant expression, relative to a Nanostring detection threshold based on housekeeping genes, has shown the presence of more than one constitutively active AR splice variant in individual tumors. ARV7 was detected in 4/10 CRPC and always found together with ARV1, AR45, and the constitutively active ARV12. All of the CRPC that were negative for ARV7 expressed AR45 and ARV12. Unsupervised clustering of 507 putative AR regulated genes revealed three molecular subgroups among the 38 prostate tumors. One of these subgroups was characterised by the presence of 12/14 AR splice variants including ARV7. Of the other two subgroups, one contained AR45, the other ARV12. Pathway analysis of genes that were expressed to a higher level (>1.5 Log2 fold) in the ARV7 positive compared to ARV7 negative tumors were predominantly associated with cell cycle and proliferation functions. Our data suggests complexity in the molecular landscape of prostate cancer associated with AR splice variants, and that multiple AR splice variants may be of importance to identify patients likely to relapse on treatment with emerging therapy. 80 Poster Section 40 Poster Board 5 LB-111 Comprehensive proteomic analysis of salivary gland cancer subtypes. Seema Mukherjee, Yoshitsugu Mitani, Robert Cardnell, You Hong Fan, Lixia Diao, Jing Wang, Adel K. El-Naggar, Lauren Averette Byers. U.T M.D Anderson Cancer Center, Houston, TX. Background: Salivary gland cancer (SGC) is a highly heterogenous disease with distinct histological and pathological features. Although subtypes of salivary gland cancer have been extensively characterised based on histological features, very little is known at the proteomic level, as to how and what key proteins and signaling pathways are differentially activated. Currently, there are no approved targeted therapies for SGCs, although there appears to be benefit of androgen receptor (AR) and Her2 blockade in a small subset of molecularly defined patients (<5% overall). To discover other potential therapeutic targets, we performed an RPPA analysis using paired normal and tumor samples. Method: Reverse phase protein array (RPPA) analysis was performed to measure 195 total and/or phosphorylated proteins in matched normal and tumor samples from 75 SGC patients, including 16 salivary duct carcinomas (SDC); 14 mucoepidermoid carcinomas (MEC); 30 acinic cell carcinomas (ACC) and 15 adenocystic carcinomas (ADCC). Differences in protein expression between normal and tumor samples was assessed by paired t test. Results: RPPA analysis showed distinct protein expression and pathway activation between and within subtypes. Using a conservative cutoff to account for multiple testing (FDR <1%, corresponding to p≤0.005), several proteins were found to be differentially altered between normal and tumor tissue. In SDC, proteins regulating the glucose metabolic pathway were found to be upregulated. For example, PKM2 (a key player in the Warburg effect) and other glycolytic proteins (e.g. ACC1, malic enzyme and IDH1) were elevated in SDC tumors; indicating that these tumors may have increased dependency on glycolysis. Increased expression of other potentially druggable targets such as p-MEK, Akt, vascular endothelial growth factor (VEGFR), and AR were also observed. Consistent with other reports, AR was found at higher levels in tumors (vs. normal tissue) in 62.5% of SDC tumors (fold change=2.33, p>0.001). Interestingly, AR overexpressing tumors also showed increased activation of EGFR (pY1173) compared to those with low AR expression. ACC tumor tissue expressed higher levels of proteins involved in cell cycle, DNA repair and metabolism. ADCC had higher expression of oncoproteins (c-kit and bcl-2); as well as IGF-binding protein 2 (IGFBP2) and its downstream effector proteins. MEC showed increased expression of EGFR. Conclusion: This study provides an insight into the molecular heterogeneity and enrichment of distinct signaling pathways in the different SGC subtypes. The observed changes in protein expression of EGFR, IGFR and PIK3CA could be driven by genetic aberrations, indeed copy number variations and mutations in several genes including EGFR, IGFR and PIK3CA have been observed in SDC. This study has identified proteins and signaling pathways that could potentially be targeted for the treatment of salivary gland cancer patients. Poster Section 40 Poster Board 6 LB-112 A retrospective study to assess the potential for the TheraLink® HER family assay, a reverse-phase protein microarray assay, to predict treatment benefit in breast cancer. Corinne Ramos,1 Nicholas Hoke,1 George J. Snipes,2 Pinar Yurt,1 Cody Thomas,2 Tuan Tran2. 1Theranostics Health, Inc., Rockville, MD; 2Baylor University Medical Center, Dallas, TX. Background: A significant percentage of patients with HER2+ breast cancer exhibit de novo resistance or develop an acquired resistance to anti-HER therapy. HER2 overexpression is currently used to guide anti-HER2 therapy by either IHC to measure the total amount of HER2 protein present, or FISH to measure the number of copies of the HER2 gene. Several potential mechanisms for this resistance have been proposed, ranging from mechanisms intrinsic Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 81 Late-Breaking Poster Session: Clinical Research / Endocrinology to the target itself to the involvement of compensatory signaling pathways. As current therapy-guiding assays provide a measure of the level of HER2 protein, a better predictor of response may be possible through an assessment of not only HER2 activation (phosphorylation status), but also the protein levels of the other HER family members, their activation, and the activation of downstream resistance signaling pathways. To provide evidence that HER receptors are actively signaling, and to identify alternative therapies, a measure of the activation status of key signal transduction pathways downstream of these receptors was performed. The availability of molecular diagnostic assays that can evaluate phosphoproteins is an appealing approach to predicting treatment-sensitivity and to select more effective therapies. Methods: De-identified breast tissue samples, including a subset of samples having their HER2 status identified by IHC and FISH were received for phosphoproteomic analysis using the TheraLink® HER Family Assay. The assay utilizes reverse-phase protein microarray (RPMA) to measure the total protein level and activation status of multiple proteins directly from a FFPE-biopsy lysate. The TheraLink® Assay measures the protein levels of EGFR, HER2, and HER3, their phosphorylation status, and the activation status of proteins in three downstream signaling pathways: AKT/mTOR; Mek/Erk; and Jak/STAT. Results: The TheraLink® Assay’s panel identified the cohort of tumors with known HER2 status (100% concordance between HER2 level measured by RPMA platform and IHC/FISH). Moreover, unsupervised clustering of this subset of samples identified three patterns of unique signaling. The first group showed high phosphorylation levels of HER2 and EGFR protein, suggesting the potential use of a dual kinase inhibitor therapy. The PI3 kinase and MAPK pathways were also elevated in this cohort. A second group that might not benefit from mono-therapy was observed. This group showed high levels of HER3 protein, a well-known dimerization partner for HER2. Therefore, this group might benefit from an antidimerization therapy, like pertuzumab. The PI3 Kinase and Jak/Stat pathways were also highly activated in this cohort. A third unique group exhibiting only activation of HER2 and HER3 was observed, with no downstream activity. Although a pan-HER kinase inhibitor has therapeutic potential, further downstream pathways, as well as other tyrosine kinase receptors, should be further examined. Conclusion: The TheraLink® Assay has the potential to identify therapeutic strategies that might provide benefit to patients that are HER2 positive but failing on anti-HER2 therapy. Alternative therapeutics can be more precisely identified using the TheraLinkTM HER Family Assay’s panel, based upon the molecular uniqueness of the groups described. Poster Section 40 Poster Board 7 LB-113 Recombinant HER2/Neu expressing Listeria combined with radiation safely delays tumor progression and prolongs overall survival in a phase I clinical study in canine osteosarcoma. Nicola Mason,1 Josephine Gnanandarajah,1 Danielle Laughlin,1 Julie Engiles,1 Anu Wallecha,2 Yvonne Paterson1. 1University of Pennsylvania, Philadelphia, PA; 2Advaxis Inc., Princeton, PA. The purpose of this study is to determine the safety and efficacy of a highly attenuated recombinant Listeria monocytogenes expressing a chimeric human HER2/neu (Lm-LLO-HER2/neu) in combination with palliative radiation (RT) to induce HER2 specific immunity and delay tumor progression in dogs with spontaneous osteosarcoma (OSA). Dogs develop OSA that recapitulates many aspects of human OSA, and are recognized as a clinically relevant, large animal model in which to evaluate novel therapies. Previously we have shown that Lm-LLO-HER2/neu administration to dogs with HER2+ OSA following amputation and chemotherapy is safe, breaks tolerance to HER2 and prolongs overall survival. Ten systemically healthy dogs with histopathologically confirmed, treatment naïve, HER2+ appendicular OSA, and no evidence of cardiac or metastatic disease were enrolled. All dogs received 2 x 8Gy fractions of RT on consecutive days, followed by the first of 8 intravenous doses of Lm-LLO-HER2/neu given once every 3 weeks. Dogs were monitored at each treatment and every 2 months after for systemic and cardiac toxicity, lameness and quality of life (QOL) (using a validated owner questionnaire). Radiographs were performed at baseline, week 10, week 22 and every 2 months thereafter to determine the effect on the primary tumor and development of pulmonary metastases. PBMCs were collected every 3 weeks to evaluate HER2/neu specific T cell responses. The primary endpoint was time to progression; secondary end points were safety and overall survival. Results are compared to historical canine controls that received 2 x 8Gy RT alone. Repeat Lm-LLO-HER2/neu administration was well tolerated with no systemic or cardiac toxicity. Lameness and QOL showed near uniform improvement over the study period. To date, limb radiographs of 7 dogs have been taken 10 weeks post enrollment and showed no evidence of tumor progression. 4 dogs have currently completed the scheduled 8 vaccinations, and limb radiographs of these dogs taken at 22 weeks, showed no progression of the primary lesion. At the time of writing, 4/10 dogs have died; 2 dogs were euthanized due to pathological fracture, and 2 dogs were euthanized due to metastatic disease. One dog, still alive, has developed pulmonary metastatic disease. At the time of writing, median time to progression is 243 days and median survival time (MST) is 285 days. Historically, MST of dogs treated with RT alone is 136 days. Results of IFN-γ ELISpot assays are pending. To conclude, our preliminary results show repeat Lm-LLOHER2/neu administration is safe and well tolerated with no cardiac toxicity. Our results also suggest that combination RT and Lm-LLOHER2/neu immunotherapy improves QOL, delays primary tumor progression and prolongs overall survival in canine OSA. These findings may have important translational relevance for human patients with OSA and other HER2/neu+ cancers. Poster Section 40 Poster Board 8 LB-114 The INFORM personalized medicine study for high-risk pediatric cancer patients. Barbara C. Worst,1 Cornelis M. van Tilburg,2 Gnana P. Balasubramanian,1 Petra Fiesel,1 David Capper,1 Miream Boudalil,1 Stephan Wolf,1 Sabine Schmidt,1 Melanie Bewerunge-Hudler,1 Matthias Schick,1 Angelika Freitag,2 Ruth Witt,2 Lenka Taylor,3 Andreas von Deimling,1 Matthias Schlesner,1 Angelika Eggert,4 Peter Lichter,1 David TW Jones,1 Olaf Witt,1 Stefan M. Pfister1. 1German Cancer Research Center, Heidelberg, Germany; 2NCT Clinical Trial Center, Heidelberg, Germany; 3University Hospital, Heidelberg, Germany; 4Charité - University Hospital, Berlin, Germany. Background: Despite substantial progress in treating primary childhood malignancies, relapses from high-risk entities remain a major clinical challenge. The German INFORM study (INdividualized therapy FOr Relapsed Malignancies) is attempting to address this problem using an integrated next-generation sequencing analysis to rapidly generate personalized tumor profiles and identify therapeutic targets. Methods: The INFORM pilot phase assessed the feasibility of integrating rapid molecular profiling into the clinical management of progressive or relapsed high-risk pediatric cancer patients. Wholeexome and low-coverage whole-genome sequencing of tumor and normal DNA was complemented with tumor RNA sequencing (Illumina HiSeq2500, ‘rapid’ mode). This allowed reliable detection of copy-number changes, point mutations, InDels, fusion genes and deregulated gene expression. Identified alterations were prioritized according to tumor biological relevance and potential as an actionable drug target, with results discussed in a weekly molecular tumor board. Results: From Oct 2013 to Jan 2015, 57 patients (average age 13 years) were enrolled from 20 centers throughout Germany. Entities included: high-grade glioma (n=12), Ewing’s sarcoma (n=11), rhabdomyosarcoma/DSRCT (n=7), medulloblastoma (n=5), American Association for Cancer Research • AACR ANNUAL MEETING 2015 81 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 82 Late-Breaking Poster Session: Clinical Research / Endocrinology ependymoma (n=5), osteosarcoma (n=4), neuroblastoma (n=4), and others (n=9). Tumor tissue was sufficient for analysis of 52 cases. Average turnaround time from tissue arrival to molecular results was 25 days. Actionable targets with at least ‘borderline’ evidence (according to a prioritization score harmonized with other pediatric precision oncology programs across Europe) were identified in 28 patients (49%). The most commonly affected targets included: tyrosine kinases (ALK, n=5; FGFR, n=4; MET, n=4; others, n=4), the PI3K/mTOR-pathway (PIK3CA, n=4; PIK3R1, n=1; TSC2, n=1), the MAPK pathway (BRAF, n=1; KRAS, n=1), the SHH-pathway (PTCH1, n=3) and cell-cycle control (CCND1/2, n=4; CDK4, n=4; CDKN2A/B, n=3). Based on these findings, targeted therapeutics were incorporated into the therapy regime of several patients (as single experimental treatments or in clinical trials), with anecdotal reports of marked responses. For example, one patient with a previously inoperable myofibroblastic sarcoma is now in complete remission more than 12 months after entering an ALK-inhibitor trial on the basis of our identification of an ALK fusion. Conclusion: A nationwide individualized diagnostic and treatment approach for pediatric cancer patients based on rapid next-generation sequencing is feasible. Our pilot phase results show that actionable targets can be identified in roughly half of the patients. The INFORM registry study has now opened (www.dkfz.de/en/inform/), to collect molecular and clinical data and establish the infrastructure for prospective clinical trials on personalized pediatric oncology. Poster Section 40 Poster Board 9 LB-115 Baseline neutrophil to lymphocyte ratio as a selection criterion for ipilimumab treatment in metastatic melanoma patients. Pier Francesco Ferrucci,1 Sara Gandini,1 Angelo Battaglia,1 Salvatore Alfieri,1 Laura Pala,1 Annamaria Di Giacomo,2 Giovanni Amato,2 Gian Carlo Antonini Cappellini,3 Diana Giannarelli,4 Emilia Cocorocchio,1 Chiara Martinoli1. 1European Inst. of Oncology, Milan, Italy; 2University Hospital of Siena, Siena, Italy; 3Istituto Dermopatico dell’Immacolata, Roma, Italy; 4National Cancer Institute Regina Elena, Rome, Italy. Background: Ipilimumab, a recently approved immunomodulatory drug, improves the survival of metastatic melanoma patients. Despite documented, durable objective responses, a significant number of patients fails to obtain clinical benefit from treatment. The aim of this study was to identify a criterion to select patients best suited to receive this drug. Methods: Sixty-nine metastatic melanoma patients treated at the European Institute of Oncology with 3 mg/kg ipilimumab, were evaluated. Neutrophil to lymphocyte ratio (NLR) was calculated from pre-therapy full blood counts. Progression free survival (PFS) and overall survival (OS) were assessed using the Kaplan-Meier method, and multivariate Cox models were applied. Findings were independently validated on a cohort of 27 patients treated with 10 mg/kg ipilimumab at the University Hospital of Siena and on a cohort of 88 treated in Rome. Results: Best overall response and disease control rates were 9% and 27%, respectively. Median PFS and OS were 3 and 5 months, respectively. Pre-therapy NLR was identified as the strongest and independent marker for treatment benefit in univariate and multivariate analyses. Patients with baseline NLR<5 had a significantly improved PFS (HR=0.38; 95% CI: 0.22-0.66; P=0.0006) and OS (HR=0.24; 95% CI: 0.13-0.46; P<0.0001) compared with those with a NLR≥5. Conclusions: Our findings show that baseline NLR is strongly and independently associated with outcome of patients treated with ipilimumab, and may serve as a selection criterion for this therapy. 82 Poster Section 40 Poster Board 10 LB-116 Strong MAGE-A3-specific humoral and cellular immune responses in multiple myeloma patients receiving MAGE-A3 protein immunotherapy and peripheral blood lymphocyte reconstitution. Sacha Gnjatic,1 Sarah Nataraj,1 Naoko Imai,1 Achim A. Jungbluth,2 Linda Pan,3 Ralph Venhaus,3 Rafik Fellague-Chebra,4 Olivier Gruselle,4 Adam Cohen,5 Nikoletta Lendvai,2 Hearn J. Cho1. 1Mt. Sinai Icahn School of Medicine, New York, NY; 2Memorial SloanKettering Cancer Center, New York, NY; 3Ludwig Institute for Cancer Research, New York, NY; 4GSK Vaccines, Rixensart, Belgium; 5 University of Pennsylvania, Philadelphia, PA. MAGE-A3 is an immunogenic tumor-associated antigen expressed in multiple myeloma (MM) patients and conferring poor prognosis, making it a rational target for immunotherapy. Recombinant MAGE-A3 protein was administered in AS15 immunostimulant (containing MPL, QS-21, and CpG 7909) to 13 MM patients pre- and post-autologous stem cell transplant (ASCT) coupled with infusion of vaccine-primed autologous peripheral blood lymphocytes (PBL) early post-ASCT (NCT01380145). All patients had MAGE-A+ myeloma cells at baseline and had an acceptable safety profile during immunotherapy. Robust antibody responses against MAGE-A3 (assessed by ELISA) were induced in all 13 subjects, with high antibody titers (1:10^410^6) that persisted to at least 1-year post-ASCT. Subclass analysis demonstrated a prevalence of IgG1 and IgG3. Epitope mapping identified 7 distinct epitopes clustering in hydrophilic regions of MAGE-A3. Peripheral blood T cell responses were evaluated in 8 subjects by IFNγ-ELISpot after in vitro re-stimulation with MAGE-A3 overlapping peptide pools. All patients quickly developed strong MAGE-A3-specific CD4 responses post-vaccination and ASCT, persisting 1-year post-ASCT. Intracellular cytokine staining confirmed polyfunctional, Th1-biased CD4 T cell responses. One patient developed CD8 responses against MAGE-A3 that recognized naturally processed antigen. To date, 6 patients relapsed and 1 died of progressive MM, with no notable difference in cytogenetics or antibody titers compared to non-progressors. MAGE-A expression was assessed by immunohistochemistry in relapse bone marrow biopsies, and interestingly, 4/6 were negative. MAGE-A3 protein-based immunotherapy and PBL reconstitution is generally well tolerated, feasible, and induces antibody and Th1-biased CD4 T cell responses, but only rare CD8 responses, in the setting of ASCT for MM. Cellular immune assessments are ongoing. The magnitudes of antibody and CD4 responses appear greater than those seen historically with older immunostimulant formulations of MAGE-A3 in other cancers, despite significant immune compromise after ASCT, suggesting a benefit from the new AS15 immunostimulant formulation, or from immunization and autologous PBL transfer in the peri-ASCT setting, or both. The frequent loss of MAGE-A3 expression in relapsing patients implies antigen-specific immune selective pressure, and suggests that combination strategies aimed at limiting immune escape should be investigated. Funding Sources: GlaxoSmithKline Biologicals SA, Ludwig Institute for Cancer Research, Cancer Research Institute. Poster Section 40 Poster Board 11 LB-117 Prognostic significance of minimal residual disease detection in bone marrow and peripheral blood samples of infants with MLL-rearranged acute lymphoblastic leukemia treated by MLL-baby protocol. Grigory Tsaur,1 Tatyana Riger,1 Alexander Popov,1 Alexander Solodovnikov,2 Anatoly Kustanovich,3 Tatyana Nasedkina,4 Egor Shorikov,1 Leonid Saveliev,5 Larisa Fechina1. 1Regional Children’s Hospital, Research Institute of Medical Cell Technologies, Yekaterinburg, Russian Federation; 2Research Institute of Medical Cell Technologies, Yekaterinburg, Russian Federation; 3Belarusian Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 83 Late-Breaking Poster Session: Clinical Research / Endocrinology Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus; 4Engelgardt Institute of Molecular Biology Russian Academy of Science, Moscow, Russian Federation; 5Ural State Medical University, Yekaterinburg, Russian Federation. Background: Minimal residual disease (MRD) is powerful tool for prediction of treatment outcome in acute lymphoblastic leukemia (ALL). Objective: To estimate prognostic significance of MRD in peripheral blood (PB) and bone marrow (BM) by detection of different MLL fusion gene transcripts in infant ALL enrolled into multicenter MLL-Baby protocol. Methods: Fifty three infants (20 boys and 33 girls) with median age of 5.3 months (range 0.03-11.80) and defined MLL rearrangements were included in the current study. Among them there were 25 patients (47.2%) carrying MLL-AFF1 fusion gene transcripts, 10 (18.9%) MLL-MLLT3-positive, 9 (17.0%) MLL-MLLT1-positive, 5 (9.4%) MLL-MLLT10-positive and 4 (7.5%) MLL-EPS15-positive ones. MRD evaluation was performed by detection of MLL fusion gene transcripts in BM and PB samples using real-time PCR and nested RT-PCR with sensitivity non-less than 1E-04. MRD-negativity was defined as absence of fusion gene transcripts in both assays. Median of follow-up period in the observed group was 5.2 years. Time points (TP) for MRD assessment were as follows: day 15 of remission induction (TP1), at the end of remission induction (TP2), after each course of ATRA administration (TP3-TP7). Results: We estimated 142 paired BM/PB samples. 77 samples were double positive, 43 were double negative Thus concordance between MRD results in BM and PB samples achieved 84.5%. Concordance varied between different TPs of MLL-Baby protocol from 79.0% to 100%. The highest concordance rate was at TP4 and TP7 (92.3% and 100%, respectively). Interestingly, all discrepant results (22 samples 15.5%) were BM-positive/PBnegative. Median level of ABL gene, used for normalization, was similar in BM and PB samples (p=0.760). Evaluation of prognostic significance of MRD in BM in TP1-TP7 revealed that TP4 was the earliest TP when discriminative data between MRD-positive and MRD-negative patients were obtained. MRD-positivity at TP4 in BM led to unfavorable outcome. EFS was significantly lower in MRDpositive group (n=22) in comparison to MRD-negative one (n=31) (0.06±0.06 vs 0.70±0.09 p=0.0001), while cumulative incidence of relapse in MRD-positive patients was remarkably higher (0.92±0.01 vs 0.29±0.08, p<0.0001). MRD-positivity at this TP in BM was the only significant factor in the diagnostic model where initial risk factors (age at diagnosis, initial WBC count, immunophenotype, CNS disease, presence of MLL-AFF1) were combined to response criteria (number of blast cells at day 8 of dexamethasone prophase and MRD in BM at TP4). MRD data obtained from PB data did not bring extra advantages as compared to TP4 in BM. Conclusions: Despite high qualitative concordance rate between MRD detection in BM and PB samples we could not show prognostic value of MRD monitoring in PB by fusion gene transcripts. Univariate and multivariate analysis revealed that MRDpositivity at TP4 in BM was the only independent prognostic factor of unfavorable outcome in the observed group of patients. Poster Section 40 Poster Board 12 LB-118 High APOBEC3B mRNA levels in estrogen receptorpositive primary tumors predict short time to progression for hormone-naive breast cancer patients treated with 1st line tamoxifen. Anieta M. Sieuwerts,1 Marion E. Meijer-van Gelder,1 Fred C.G.J. Sweep,2 Paul N. Span,2 John A. Foekens,1 John W.M. Martens1. 1 Erasmus MC Cancer Institute, Erasmus University Medical Center and Cancer Genomics Netherlands, Rotterdam, Netherlands; 2 Radboud University Medical Center, Nijmegen, Netherlands. Introduction: Recent evidence has implicated APOBEC3B as a source of mutations in cervical, bladder, lung, head and neck, and breast cancers. The innate immune enzyme APOBEC3B contributes to mutagenesis and is a biomarker for poor prognosis in estrogen receptor positive (ER+) breast cancer1. APOBEC enzymes normally function in the innate immune system and in the protection against viral pathogens, but these enzymes can also generate C to T and DNA rearrangements in the host genome2-4. The role of this enzyme as a predictive biomarker for the response to first-line tamoxifen, a common therapy for ER+ breast cancer patients, is unknown. Here, we evaluated the predictive potential of APOBEC3B mRNA expression measured in the primary tumor using 2 independent Dutch retrospective ER+ cohorts of a total of 285 hormone-naive recurrent breast cancer patients treated with first-line tamoxifen. Materials and Methods: APOBEC3B mRNA levels were measured by reverse transcriptase quantitative PCR (RT-qPCR) as described1 and the length of progression-free survival (PFS) was used as the primary endpoint. Cox univariate and multivariate regression analysis for PFS were used to assess the predictive potential of APOBEC3B mRNA expression. Results: In both cohorts individually, high levels of APOBEC3B were associated with an unfavorable response to 1st line tamoxifen (Dutch cohort-1, 225 patients: HR=1.58, P=0.0009; Dutch cohort-2, 60 patients: HR=2.12, P=0.009). Stratified for study cohort, high APOBEC3B mRNA levels were also significantly associated with an unfavorable PFS in multivariate analysis that included the traditional predictive factors: age, dominant relapse site, disease-free interval, ER and progesterone receptor (PGR), and adjuvant chemotherapy (HR=1.67, P=0.0001). Conclusion: Altogether, our data show that high APOBEC3B mRNA expression is an independent and validated predictor of not only poor prognosis but also of poor PFS after 1st line tamoxifen in recurrent breast cancer, which supports the notion that APOBEC3B is a promising therapeutic target to prevent metastasis and tamoxifen failure in ER+ disease. 1. Sieuwerts, A. M. et al. Elevated APOBEC3B correlates with poor outcomes for estrogen-receptor-positive breast cancers. Hormones & cancer 5, 405-413, doi:10.1007/s12672-014-0196-8 (2014). 2. Stephens, P. J. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462, 10051010, doi:10.1038/nature08645 (2009). 3. Burns, M. B. et al. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366-370, doi:10.1038/nature11881 (2013). 4. Nik-Zainal, S. et al. Association of a germline copy number polymorphism of APOBEC3A and APOBEC3B with burden of putative APOBEC-dependent mutations in breast cancer. Nature genetics 46, 487-491, doi:10.1038/ng.2955 (2014). Poster Section 40 Poster Board 13 LB-119 MGMT immunohistochemistry (IHC) improves patients’ selection for temozolomide (TMZ) treatment in advanced chemorefractory MGMT-methylated colorectal cancer. Filippo De Braud, Filippo Pietrantonio, Maria Di Bartolomeo, Katia Fiorella Dotti, Claudia Maggi, Roberto Iacovelli, Marta Caporale, Rosa Berenato, Federica Perrone, Elena Tamborini, Alessio Pellegrinelli, Stefano Federici, Fabrizio Festinese, Giuseppe Pelosi, Ilaria Bossi, Paola Valentina Consonni, Susanna Maggi, Massimo Milione. Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. Background: We showed that standard dose TMZ (150 mg/mq/day day1-5q28) is tolerable and active in 32 heavily pretreated patients with advanced CRC and MGMT promoter methylation (Pietrantonio et al., Ann Oncol 2014). In a subsequent study, we included 32 pts treated with dose-dense TMZ (75 mg/mq/day day 1-21q28, for up to 6 cycles or until progression/unacceptable toxicity). Additional predictive markers are needed for improved selection of patients for TMZ therapy in metastatic CRC. Retained MGMT expression by IHC was proposed as marker for negative selection of pts with brain tumors. We conducted a retrospective analysis to assess MGMT expression in our 2 studies and to correlate this with the outcomes. Methods: All 64 patients included in the 2 studies were defined American Association for Cancer Research • AACR ANNUAL MEETING 2015 83 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 84 Late-Breaking Poster Session: Clinical Research / Endocrinology as MGMT methylation-positive according to methylation-specific PCR. A total of 40 patients had tissue available for IHC. Expression of nuclear MGMT protein was defined scored semiquantitatively according to extension and intensity. Using ROC analysis, IHC score <4 was considered as MGMT-low expression vs. IHC score 412 as MGMT-high. Extended RAS-BRAF mutations were assessed by Sanger sequencing. Results: Herein, we report for the first time the results of the dose-dense TMZ study. We obtained 4 confirmed partial responses and 2 stable disease, accounting for a response rate of 12% and a clinical benefit of 18%. Median PFS was 1.8 months. OS data are not mature. Thus, the results are comparable to those obtained in the previous study. MGMT-low expression was found in 15 (38%) samples and MGMT-high in 25 (62%). RAS-BRAF mutations were found in 28 (70%) pts and were not correlated with MGMT expression (p=1). Response rate was significantly higher in patients with MGMT-low (53%) vs. those with MGMT-high (p<0.0001). Progression-free survival was significantly longer in MGMT-low vs. MGMT-high(5 vs. 2.3 months; p=0.001). Further analyses are ongoing to validate MGMT IHC as a biomarker and to correlate it with quantitative gene methylation analysis. Conclusions: In this translational study on MGMT methylated CRC, absence of MGMT expression was significantly correlated with tumor response and benefit from TMZ treatment. Poster Section 40 Poster Board 14 LB-120 HER2 as a potential predictive marker and target for therapy in cervical cancer. Mari K. Halle,1 Akinyemi I. Ojesina,2 Ingvild L. Tangen,1 Frederik Holst,1 Hilde R. Engerud,1 Bjørn I. Bertelsen,3 Camilla Krakstad,1 Helga B. Salvesen1. 1Department of Clinical Science, Centre for Cancer Biomarkers, University of Bergen, Bergen, Norway; 2Broad Institute of MIT and Harvard, Cambridge, MA; 3Department of Pathology, Haukeland University Hospital, Bergen, Norway. Cervical cancer is the third leading cause of cancer in the female population worldwide, causing the death of more than 240,000 patients annually in developing countries. Increased molecular knowledge is crucial to identify robust prognostic and predictive biomarkers that can better guide treatment. The tumor response to trastuzumab is well established and strongly links to HER2 expression status evaluated by the Hercep Test, hence, it is essential to define the level of expression of this receptor using the FDA-approved Hercep Test to stratify cervical cancer patients with potential benefits from trastuzumab treatment. Comprehensive molecular characterization has been conducted on 88 paired normal and tumor cases identifying ERBB2 to be frequently altered in cervical cancers. We here explore the protein expression of HER2 by immunohistochemical staining in a larger validation series (n=220) and relate HER2 expression to the ERBB2 gene alterations, patients molecular profile and clinicopathological features. We find a highly significant correlation between Hercep Test score and mRNA ERBB2 expression (p<0.001). The level of ERBB2 mRNA was also significantly associated with copy number status (p=0.007). Further clinocopathological parameters like high FIGO stage, high grade, adenocarcinomas and normal p53 status was significantly linked to high HER2 protein expression. Kaplan Meier survival analysis revealed that within the squamous cell carcinomas, high protein levels of HER2 was linked to poorer disease specific survival. Our results show a link between ERBB2 amplification, high mRNA expression and protein levels for HER2 in aggressive cervical cancers. Further studies of HER2 as a potential predictive marker for response to trastuzumab treatment in cervical cancer are needed. Poster Section 40 Poster Board 15 LB-121 Identification of Carboxylesterase-2 as a determinant of response to irinotecan and neoadjuvant FOLFIRINOX therapy 84 in pancreatic ductal adenocarcinoma. Michela Capello,1 Minhee Lee,2 Hong Wang,1 Ingrid Babel,2 Matthew H. Katz,1 Jason B. Fleming,1 Anirban Maitra,1 Huamin Wang,1 Weihua Tian,1 Ayumu Taguchi,1 Samir M. Hanash1. 1UT MD Anderson Cancer Center, Houston, TX; 2Fred Hutchinson Cancer Research Center, Seattle, WA. Background: Serine hydrolases (SHs) are among the largest classes of enzymes in human and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the pro-drug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. Methods: CES2 expression in PDAC and paired non-tumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. Results: Significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared to paired nontumor tissue (P < .0001), with 48/118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in PDAC cell lines was inversely correlated with irinotecan IC50 values (P = .021), while no molecule involved in irinotecan metabolism yielded a significant correlation between its expression and sensitivity to the drug. Remarkably, we recently found that high CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (P = .024). Conclusion: Our findings suggest that CES2 expression and activity, by mediating the intra-tumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy. Poster Section 40 Poster Board 16 LB-122 molecular imaging of early colorectal dysplasia using fluorescently-labeled lectins. Andre Neves, Joe Kuo, Ashraf Ibrahim, Kevin Brindle. University of Cambridge, Cambridge, United Kingdom. Objective: Screening for colorectal cancer (CRC) is currently performed using bright-field colonoscopy, which has low specificity and sensitivity for the detection of early dysplasia in the colon. Fluorescently labeled lectins have been applied topically for detecting glycosylation changes in early dysplastic lesions in the esophagus, providing a route for improved endoscopic detection using fluorescence endoscopy [1]. We have identified here fluorescently labeled lectins that can detect changes in glycosylated biomarkers on the surface epithelium of early dysplasia in CRC. These probes may enable fluorescence-guided resection of dysplastic lesions at colonoscopy and subsequent automated assessment of colon histopathology. Study Design: Ninety-nine colon lesions in 48 patients (34 males, 14 females; mean patient age 68.8 ± 8.3 yr, range 53-95 yr) were identified during routine colonoscopy at Addenbrooke’s Hospital, Cambridge, UK. Lesions suspected of being dysplastic or neoplastic, were detected using magnification colonoscopy or surgery, and removal performed at biopsy (11.7%) and/or polypectomy (62.1%), or partial colon resection (11.7%). Colon histological sections were stained with fluorescently labeled lectins, and the lectin binding to surface epithelium, which would be observable in colonoscopy, was compared with conventional histopathology for the presence of disease and disease stage. Results: Normal colonic epithelium (NE) occupied 40.1% of the area of all sections, hyperplastic polyps (HP) occupied 10.2%, low(LGD) dysplasia 25.6%, high-grade (HGD) dysplasia 13.9%, and Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 85 Late-Breaking Poster Session: Clinical Research / Endocrinology adenocarcinoma (C) 10.2%, as assessed by conventional histopathology based on H&E staining. Lesions were located in the ascending (10.4%), transverse (15.6%), descending (14.3%), sigmoid (42.9%) colon and rectum (7.8%), with the mean lesion size being 16.0 ± 14.2 mm (range 2-60 mm). The lectin wheat germ agglutinin (WGA), when fluorescently-labeled, was capable of distinguishing epithelial regions containing NE from regions containing LGD, HGD and cancer, with >81% sensitivity, >79% specificity and >93% positive predictive value. Conclusions: Automated analysis of fluorescently-labeled WGA binding to the surface epithelium of excised colon sections may be used for improving the assessment of early dysplasia in CRC. The same fluorescent lectin may also be useful as a topical imaging agent for improving detection of early dysplasia using fluorescence colonoscopy, increasing the early diagnosis of CRC and improving patient survival. Reference 1. Bird-Lieberman, E.L., Neves, A.A. et al. (2012). Nat Med. 18(2): p. 315-21. Poster Section 40 Poster Board 17 LB-123 Analysis of cell-free plasma DNA (cfDNA) identifies 3 molecular subtypes of acquired resistance to AZD9291, a novel EGFR tyrosine kinase inhibitor (TKI), in patients (pts) with advanced lung cancer. Geoffrey R. Oxnard,1 Kenneth S. Thress,2 Cloud P. Paweletz,1 Enriqueta Felip,3 Byoung Chul Cho,4 Daniel Stetson,2 Brian Dougherty,2 Zhongwu Lai,2 Aleksandra Morkovets,2 Ana Vivancos,3 Yanan Kuang,1 Dalia Ercan,1 Mireille Cantarini,5 J Carl Barrett,2 Pasi A. Janne1. 1Dana-Farber Cancer Institute, Boston, MA; 2 AstraZeneca, Waltham, MA; 3Vall d-Hebron Institute of Oncology, Barceloma, Spain; 4Yonsei Cancer Center, Seoul, Republic of Korea; 5 AstraZeneca, Macclesfield, United Kingdom. Introduction: EGFR T790M is the most common mechanism of acquired resistance to EGFR TKIs in pts with EGFR-mutant lung cancer. AZD9291 is an irreversible, mutant-selective EGFR TKI developed to have potency against both sensiziting EGFR mutations and T790M. In the ongoing phase I study of AZD9291 (AURA, NCT01802632), the response rate in pts with T790Mpositive lung cancer was >60%. The molecular mechanism underlying acquired resistance to AZD9291 is not known. Methods and Results: To explore for mechanisms of resistance to AZD9291, we studied cfDNA extracted from pretreatment and post-progression plasma collected on AURA.Next-generation sequencing (NGS) of cfDNA was first performed on an exploratory cohort of 7 pts. All exons of a 20 gene panel (including EGFR) underwent PCR amplification and NGS using an Illumina HiSeq. In 1 pt, NGS of progression plasma identified a new EGFR C797S mutation in exon 20, not present in pretreatment plasma. Stable expression of C797S in Ba/F3 cells induced a >100-fold increase in IC50 to AZD9291 compared to EGFR activating and T790M mutations alone. To validate the plasma NGS, digital droplet PCR (ddPCR) assays were developed to detect key EGFR mutations including C797S. 15 T790M-positive cases were identified with progression plasma available for analysis. Serial plasma ddPCR showed that both the EGFR activating and T790M mutation levels decreased with AZD9291 treament and increased at progression, with 3 molecular subtypes of resistance apparent. In 6 pts (40%), C797S was detected in addition to T790M; NGS of resistance biopsies from 2 of these pts confirmed presence of acquired C797S. In 5 pts (33%), T790M was detected without evidence of C797S. Intriguingly, in 4 pts (27%), the T790M levels became undetectable with treatment despite high levels of the EGFR activating mutation at progression, suggesting overgrowth of a competing non-T790M resistance mechanism. Further NGS of progression plasma revealed additional evidence of the genomic heterogeneity of resistance. Individual sequencing reads indicate that C797S and T790M can occur either in cis or in trans (i.e. on competing resistant alleles). In the 2 pts with tumor NGS demonstrating C797S, plasma NGS identified both the DNA alteration seen in tumor as well as a second DNA alteration encoding for C797S. Conclusion: Using complementary assays for genomic analyses of cfDNA, we identified 3 molecular subtypes of acquired resistance to AZD9291, including an EGFR C797S mutation never before reported in pts. Due to the key role of the C797 residue in drug binding, C797S is expected to induce resistance to all irreversible EGFR TKIs currently in clinical development. Plasma NGS revealed substantial genomic heterogeneity and highlights the need for combination therapies to effectively prevent or treat drug resistance in cancer. Poster Section 40 Poster Board 18 LB-124 Tumor-educated platelets allow for multiclass liquid biopsy-based diagnosis of cancer. Myron Best,1 Nik Sol,1 Irsan Kooi,1 Jonas Nilsson,2 Bart Westerman,1 Bauke Ylstra,1 Josephine Dorsman,1 Egbert Smit,1 Henk Verheul,1 Jaap Reijneveld,1 Bakhos Tannous,3 Pieter Wesseling,1 Thomas Würdinger1. 1VU Medical Center, Amsterdam, Netherlands; 2Umeå University, Umeå, Sweden; 3Massachusetts General Hospital, Boston, MA. Background: Cancer diagnosis is frequently hampered by limited access to adequate tissue of the primary tumor or of metastatic lesions. To overcome such limitations, the use of bloodbased liquid biopsies has been suggested. Blood represents a biosource of tumor-educated platelets (TEPs) that sequester biomolecules during tumor growth, thereby altering the platelet mRNA profile. Methods: Blood platelet samples of 175 patients with cancer covering five tumor types (40 non-small cell lung cancer, 39 glioblastoma, 37 colorectal cancer, 35 pancreatic cancer, and 24 breast cancer) and of 52 healthy donors were isolated from whole blood by differential centrifugation. Total RNA was isolated, subjected to SMARTer mRNA amplification and submitted for whole transcriptome mRNA sequencing on the Illumina platform. Healthy donors, pan-cancer, and individual cancer classes were distinguished by a self-learning support vector machine (SVM) algorithm, using transcripts with moderate to high expression. Results: The 227 blood platelet samples were successfully sequenced and demonstrated a good intersample correlation of the detected mRNAs. Based on mRNA profiles, all tumor samples were clearly distinguished from healthy individuals: the pan-cancer SVMsupported classification test reached a sensitivity of 97% and a specificity of 92% to distinguish cancer patients from healthy controls. Also, all patients without overt metastases were correctly predicted as cancer patients. Moreover, a multiclass cancer diagnostics TEP-test, to distinguish multiple tumor subclasses and healthy controls provided an overall accuracy of 70%, far exceeding random classification. In addition, we distinguished HER2-positive, and mutant KRAS and EGFR tumors from their wild-type counterparts. Also, patients with metastatic tumors in lung, brain, and liver were accurately diagnosed according to the tumor in the tissue of origin. Conclusion: Molecular interrogation of TEP-based liquid biopsies may leverage cancer diagnostics. TEPs provide a circulating biosource for pan-cancer, multiclass, and molecular cancer classification. Of interest, this tool might also allow for blood-based highly sensitive early-stage cancer screening. American Association for Cancer Research • AACR ANNUAL MEETING 2015 85 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 86 Late-Breaking Poster Session: Clinical Research / Endocrinology Poster Section 40 Poster Board 19 LB-125 The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer. jon roffey,1 Aurore Lejeune,1 Michelle Barnard,1 Julie Stock,1 Ai Ching Wong,1 Jenny McKelvie,1 Kelly Holmes,2 Jason Carroll,2 Stephen Myatt1. 1Cancer Research Technology Discovery Laboratories, London, United Kingdom; 2Cancer Research UK Cambridge Institute, Cambridge, United Kingdom. Estrogen receptor alpha (ERα) is expressed in ~75% of breast cancers where it binds and regulates specific target genes which drive cancer cell proliferation. Hormone-based treatments such as selective estrogen receptor modulators (SERMs) and aromatase inhibitors prevent estrogen signalling through the ERα, but in many cases resistance to these therapies develops. FOXA1 is a pioneer factor for ERα that is required to facilitate ERα binding to its target genes and its expression is considered to be a defining gene that characterizes ERα positive luminal breast cancers. Here we show that FOXA1 is an essential gene in ERα positive, tamoxifen resistant and aromatase-resistant breast cancer cell lines suggesting inhibitors that target the FOXA1/ERα pathway could have broad utility in these settings. To this end we performed a cell-based screen to identify novel small molecule regulators of FOXA1/ERα pathway activity. Our novel cell-based assay allowed us to screen over 180, 000 discrete compounds in a high through-put manner. Counter screens and phenotypic profiling in FOXA1 positive and negative breast cancer cell lines were used to remove non-specific compounds and direct ERα modulators. Second batches of confirmed actives were profiled through the screening cascade and by using ChIP assays we have observed a reduction in FOXA1 binding at the chromatin that is commensurate with a similar loss of ERα binding, demonstrating for the first time pharmacological modulation of FOXA1/ERα occupancy at the chromatin level. Poster Section 40 Poster Board 20 LB-126 Declining T levels provide increased opportunity for mutagenesis in prostate stem cells. Ye Zhou, Jeremy Jones. City of Hope, Duarte, CA. Because both normal prostate development and the growth of prostate cancers depend on androgen-mediated activation of androgen receptor (AR) signaling, it is reasonable to hypothesize that androgens and AR signaling play a role in prostate tumor initiation as well; however, little is known about androgens and AR in this process. We hypothesize that the decline in androgen production that occurs naturally with age contributes directly to tumorigenesis. Previously, we showed that low serum testosterone (T) causes changes in the expression of genes in the androgen/AR signaling axis which allows the prostate tissue to sustain functional androgen levels in rodents. These changes may contribute to prostate tumorigenesis. There is ongoing debate concerning the cell of origin for prostate cancer, but most evidence would suggest that it can arise from stem cells or cells with stem-like features. Here, we used a murine model to manipulate serum androgen levels to assess the changes in prostate stem-like cells in response to low serum T and to investigate the potential mechanisms by which androgens and AR contribute to prostate tumorigenesis. Using flow cytometry, we observed that chronic exposure to low T causes a consistent expansion of prostate stem-like cell populations, including Lin-/CD133+/CD117+ cells (from 0.23% to 0.33%), Lin-/CD49fHigh/Trop2+ cells (from 6.47% to 8.42%) and intermediate CK5+/CK8+ cells (from 0.43% to 1.26%). Using an in vitro sphere forming assay, we found that stem-like cells isolated from low T treated animals had an increased ability to form spheres, suggesting that low T increases self-renewal capacity. We are currently testing the ability of low T to increase self-renewal capacity in rodent models. Using qPCR, we found that low T causes changes in the expression of genes in the androgen/AR signaling pathway as well as in the oxidative stress response in stem-like cells. We are currently using RNA-seq to understand on a 86 global scale the changes that chronic low T exposure causes in these stem-like cells. Our studies suggest that chronic exposure to low T, as is the case in older men, could provide a greater opportunity for damage to cells that can become cancer initiating cells. Poster Section 40 Poster Board 21 LB-127 Enrichment of chromosome 1 and 6p21 genes associated with worse progression-free and overall survival in endometrial cancer patients. Rusheeswar Challa,1 GVR Chandramouli,2 Alyssa Fedorko,1 Thomas P. Conrads,3 John I. Risinger1. 1Michigan State University, Grand Rapids, MI; 2GenePria Consulting Inc, Columbia, MD; 3Womens Health Integrated Research Center at Inova Health System, Annandale, VA. Endometrial cancers are generally considered to have favorable outcomes. However, recurrent, and/or non-endometrioid types and some high-grade endometrioid lesions often have very poor survival with limited treatment options. The genes specifically associated with these poor outcomes are not well described. The integrated genomic characterization of endometrial cancers was published in 2013 and the associated RNA-Seq data of these uterine corpus cancers are publicly available at The Cancer Genome Atlas [Nature 497 (7447):67-73]. However, studies on the association of gene expression with the overall survival and recurrence of these endometrial cancers are not yet reported. In this work, we identify those genes associated with survival and recurrence endpoints. RSEM Normalized RNA-Seq (UNC Illumina HiSeq RNASeq V2, level 3) data was downloaded from the TCGA website and survival data extracted from this resource. Cox regression analysis was performed using the survival package in R-environment. The cohort of the patients used in this study cover a large proportion of endometrioid type, while the remaining are serous and mixed histologic types. The proportion of grades 1 to 3 is 84:101:168 and stages I & II (low) to III & IV (high) is 263:87. The ratio of microsatellite unstable to stable cases is about 1:2 approximately both in living and diseased. The tissues were not micro-dissected leaving tumor infiltrates, stroma and other debris. This analysis identified 160 genes significant at p ≤ 0.001 for either overall survival and recurrence free survival or both. In the overall survival analysis, 82 genes were at hazard ratio (HR) ≥ 1 and 48 were at HR ≤ 1. We noted that chromosome 1 was over-represented among gene locations from these gene transcripts. In addition to enrichment of chromosome 1 genes we noted a cluster of genes located on 6p21 with elevated hazard ratios associated with poor clinical outcome. Ontology analysis of the survival associated genes was assessed by EASE software which pointed out the immune response genes have hazard ratios under 1 suggesting that it is a predominant mechanism associated with positive clinical outcome. The finding of immune response genes association with positive clinical outcome (HR ≤ 1) is provocative that prompts further investigation in the assessment and therapy of endometrial cancer. The enrichment of genes on chromosomes 1 and 6 offer further clues to the mechanisms underlying those endometrial cancers with poor clinical outcome. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 87 Late-Breaking Poster Session: Tumor Biology 1 Late-Breaking Poster Session Monday, April 20, 2015 1:00 PM-5:00 PM Poster Section 41 Late-Breaking Research: Tumor Biology 1 Poster Section 41 Poster Board 1 LB-129 Identifying tumor subpopulations and the functional consequences of intratumor heterogeneity using single-cell profiling of breast cancer patient-derived xenografts. Paul Savage,1 Sadiq M. Saleh,1 Ernesto Iacucci,1 Timothe Revil,1 Yu-Chang Wang,1 Nicholas Bertos,1 Anie Monast,1 Hong Zhao,1 Margarita Souleimanova,1 Keith Szulwach,2 Chandana Batchu,2 Atilla Omeroglu,1 Morag Park,1 Ioannis Ragoussis1. 1McGill University, Montreal, Quebec, Canada; 2Fluidigm Corporation, South San Francisco, CA. Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patientderived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. Functional studies distinguishing these subpopulations are ongoing. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells showed high and homogeneous target enrichment with >75% of reads mapping uniquely on target. Variant calling using GATK and Samtools revealed founder mutations in key genes as BRCA1 and TP53, as well as subclonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 cancer driver genes. These findings may be important in understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drugresistance. Poster Section 41 Poster Board 2 LB-130 Reporter genes for a rapid in vivo screen of PDA therapeutics are required for energy homeostasis in pancreatic cancer-associated malnutrition. Ozhan Ocal, Yalda Zolghadri, Galvin H. Swift, Rolf A. Brekken, Thomas M. Wilkie. UT Southwestern Medical Center, Dallas, TX. Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths. Limited progress in developing effective therapy for PDA is partially due to the lack of a robust in vivo screen for effective drug combinations. Kras mutations (e.g. KrasG12D) are found in over 90% of human PDA and occur early in tumor progression. G Protein Coupled Receptor (GPCR) and protein kinase signaling can initiate Ras activation. Regulators of G-protein Signaling (RGS) proteins are coincidence detectors of Ras activation that feedback regulate, by virtue of their GTPase Activating Protein (GAP) activity, the intensity and duration of Giand Gq-coupled GPCR signaling. RGS-resistant mutations in Gq have been associated with PDA. We show a Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of neoplasia in the LSL-KrasG12D; Cdkn2af/f; p48Cre (KIC) mice. GFP is proportional to and coincident with tumor burden. Although KrasG12D is expressed in embryonic pancreas progenitor cells and in all mature acinar cells, Rgs16::GFP expression in tumors first emerges in ductal PanINs as early as 12 days post birth. The receptor tyrosine kinase Axl is highly expressed in PDA progenitor cells. The Gas6 ligand evokes Axl signaling in epithelial progenitor cells and contributes to activation of KrasG12D, PDA initiation and progression. In a proof-of-principle for drug screens, we determined that warfarin, which blocks maturation of Gas6, an Axl agonist, combined with the standard of care Gemcitabine and Abraxane (GA), significantly reduced PDA progression. In humans, partial pancreatic deficiency often precedes pancreatic cancer. Pancreatic insufficiency develops by 5 weeks in KC (LSL-KrasG12D;p48Cre) mice that express KrasG12D in all pancreas cells. KrasG12D, in the context of wild type Cdkn2a, causes dedifferentiation of acinar cells and a drastic reduction in digestive enzymes secreted by the pancreas. KC mice become malnourished but can survive over one year before succumbing to PDA. We find Intraductal Papillary Mucinous Neoplasm (IPMN) in KC mice express Rgs16::GFP by 2 weeks of age. We crossed the Rgs8-16 double knockout into KC (KC-R) mice to test if Rgs8-16 are tumor suppressor genes. Most KC-R mice die before 4 months of age because they can not maintain energy homeostasis - Rgs816 are required in liver to conserve energy utilization in malnourished mice. The effects of Rgs8-16 deficiency on exocrine pancreas function, acinar-to-ductal metaplasia (ADM), apoptosis and tumor progression in KC-R mice are under investigation. As a reporter gene, Rgs16::GFP faithfully tracks PDA progression and sensitivity to new drug regimens that inhibit KrasG12D mediated oncogenesis. Supported by NCI CA161624. Poster Section 41 Poster Board 3 LB-131 In vivo chromosomal engineering with the CRISPR/Cas9 model: a new frontier for mouse modeling. Danilo Maddalo. Memorial Sloan Kettering Cancer Center, New York, NY. Introduction: Chromosomal rearrangements are often detected in several types of cancer and result in expression of aberrant genes with oncogenic functions. In spite of their importance in tumor formation, modeling such rearrangements has been proven challenging and requires heavy and long germline manipulation. Purpose of the Study: In this study we propose to induce chromosomal rearrangements in vivo by delivering the CRISPR/Cas9 system making use of viral vectors. Experimental Procedures: For in vitro studies we transfected NIH/3T3 cells with plasmids expressing the Cas9 protein and the specific sgRNA. For in vivo delivery of the CRISPR/Cas9 system to the lung of adult mice we made use of Adenoviral vectors containing the Cas9 and two sgRNAs expressed under the control of two independent U6 promoters. Imaging of the lungs was performed with µCT scan. Results: Two sgRNAs to direct the endonuclease Cas9 to the intron 19 of Alk and the intron 14 of Eml4 to induce the Eml4-Alk inversion were designed to induce Eml4-Alk inversion, detected in a subset of patients with non-small cell lung cancer (NSCLC). Codelivery of the sgRNAs and the Cas9 resulted in the expected rearrangements, indicating that two sgRNAs and the endonuclease are sufficient to induce chromosomal rearrangements in vitro. For in vivo delivery to the lung of adult mice, we generated an adenoviral system for the concomitant delivery of the Cas9 and the two sgRNAs (Ad-EA) or the Cas9 alone (Ad-Cas9). Within 8 weeks postinfection with Ad-EA, mice developed lung adenomas while mice infected with Ad-Cas9 were tumor free. All the tumors carried the genomic inversion Eml4-Alk and were negative for p63 and positive for TTF-1 resembling the histological feature of NSCLC. Finally we tested the sensitivity of the tumors to the ALK/MET inhibitor crizotinib, used in therapy for the treatment of ALK+ NSCLC. Radiological and histological analysis showed in all cases tumor regression, indicating that our CRISPR-induced mouse model closely resembles human NSCLC. American Association for Cancer Research • AACR ANNUAL MEETING 2015 87 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 88 Late-Breaking Poster Session: Tumor Biology 1 Conclusion: In conclusion the strategy here presented expands significantly our possibility of modeling cancers driven by chromosomal rearrangements and represents an opportunity to test novel targeted therapies and thee mechanisms leading to drug resistance. Poster Section 41 Poster Board 4 LB-132 Adult lineage-restricted CNS progenitors specify distinct glioblastoma subtypes. Sheila R. Alcantara Llaguno, Zilai Wang, Daochun Sun, Jian Chen, Jing Xu, Euiseok Kim, Kimmo Hatanpaa, Jack Raisanen, Dennis Burns, Jane Johnson, Luis F. Parada. UT Southwestern Medical Ctr., Dallas, TX. A central question in glioblastoma multiforme (GBM) research is the hierarchy of tumor-initiating cells, and its contribution to the malignant phenotype and genomic make-up of GBM. We examine the potential of adult lineage restricted central nervous system (CNS) progenitors to form malignant gliomas. We show that targeting of Nf1,p53 and Pten mutations in adult CNS progenitors but not stem cells gives rise to fully penetrant GBM. We identify two phenotypically and molecularly distinct GBM subtypes that arise from different adult progenitor lineages. Using multiple inducible cre transgenic lines, we demonstrate that murine GBMs are molecularly separable based on the cell of origin. The oligodendrocyte progenitor cell-derived murine tumors have parallels with a subset of human high-grade gliomas with oligodendrocytic component, tumors that have been ascribed better prognosis compared to classic malignant astrocytomas. These studies indicate that two independent sources of GBM-initiating adult progenitors are susceptible to identical mutations and point to the cell of origin as a major determinant of GBM subtype. Poster Section 41 Poster Board 5 LB-133 Excessive activation of the Src-kinase HCK in the tumor stroma promotes progression of solid tumors. Robert O’Donoghue,1 Ashleigh Poh,2 Adele Preaudet,2 Matthias Ernst1. 1Olivia Newton-John Cancer Research Institute, Heidelberg, Australia; 2Walter and Eliza Hall Institute, Parkville, Australia. Hemopoietic cell kinase, HCK, is a Src-family kinase expressed in myeloid and B-lymphoid cells, regulates innate immune cell functions, and is aberrantly activated and/or expressed (incl HCK gene amplification) in solid tumors of the colon, stomach, lung and other organs. Here we exploit the Hck(CA) mouse model, which carries a constitutively activating germline mutation and spontaneously develops pulmonary inflammation, airspace enlargement and cellular consolidation highly reminiscent of patient with chronic obstructive pulmonary disease, COPD [1]. To establish whether aberrant activation of endogenous Hck promotes tumorigenesis, we tested three established cancer models. First, we found lung adenocarcinoma burden in Hck(CA)x Kras(LSL-G12D) mice compared to Kras(LSL-G12D) mice, where the oncogenic KrasG12D transgene becomes activated in response to transnasal administration of adenovirus-encoded Cre recombinase. Second, using gp130(F/+) mice as a strain predisposed to the development of intestinal-type gastric cancer [2], we established that compound Hck(CA)x gp130(F/+), developed tumor burden similar to that of gp130(F/F) animals. Furthermore, tumors from Hck(CA)x gp130(F/+) mice, but not from gp130(F/F) mice, showed sub-mucosal invasion. Third, we modeled sporadic colorectal cancer by weekly administration of the alkylating agent azoxymethane for 6 consecutive weeks [3]. Compared to wild-type mice, Hck(CA) mice exhibited increased tumor frequency and multiplicity, and colonic tumors in Hck(CA) mice showed mucosal invasion, which was absent from tumors in wild-type mice. In all models the Hck(CA) allele increased tumor cell proliferation and associated Stat3, Erk, Akt and S6 cytoplasmic signaling, although the total number of tumor-associated macrophages remained unchanged by the presence of the Hck(CA) allele. However gene expression profiling of the latter cells revealed excessive alternative 88 macrophage activation from Hck(CA) mice with prominent expression of Il4, Il10, Il13, Arg1 and Ym1. Accordingly, adoptive transfer of Hck(CA) bone-marrow was sufficient to confer increase tumor burden and promote alternative macrophage polarisation, while reconstitution with triple Hck(KO)x Fgr(KO)x Lyn(KO) bone marrow reduced tumor burden. Collectively, our data suggest that mutations affecting the stroma, and macrophage polarization specifically, confer tumor progression and that HCK may provide a novel target for rational cancer drug development. References 1. J. Exp. Med. 196:589-604 (2002) 2. Nat Med 11:845 (2005) 3. Cancer Cell 24:25 (2013) Poster Section 41 Poster Board 6 LB-134 A novel model to investigate Kras mutation specificity in the skin. Peter MK Westcott, Minh D. To, Reyno Delrosario, Kyle D. Halliwill, Peter Vuong, Melissa Q. McCreery, Allan Balmain. UCSF, San Francisco, CA. The selection and specificity of mutations occurring in genes that drive cancer is a highly complex process that remains poorly understood for even the most well studied oncogenes. The specific pattern of RAS mutations across different cancers is thought to be modulated by differences in carcinogen exposure and metabolism, local DNA features and repair mechanisms, tissue-specific expression patterns and functional differences of the RAS isoforms1. Less is known about functional differences between the various activating mutations in RAS that may lead to selection of particular mutants in mouse or human cancers. Studies of mouse cancer models have provided evidence for strong genetic background effects on allele-specificity of Ras mutations2 as well as on selection of particular mutations at Kras codon 61 in lung adenomas3. We have also recently demonstrated a major role of germline Kras status in mutation selection during initiation, where carcinogen-induced lung tumors from Kras WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous mice carry mostly (92%) Q61L mutations4. In a M. Musculus (Mm) x M. Spretus (Ms) backcrossed population of HrasKO mice treated with DMBA/TPA, a model of Kras-driven tumorigenesis in the skin, we observed a wide spectrum of Kras mutations. Interestingly, genotype at the Kras locus significantly influenced Kras mutation specificity, with a switch from predominantly G13R mutations in Mm/Mm carcinomas to G12 and Q61L mutations in Mm/Ms carcinomas. Importantly, total Kras expression driven by the Mm Kras allele is higher than that driven by the Ms allele in the skin, and Kras mutations occur with high specificity for the Mm allele, suggesting that Q61L and G12 mutations may be suppressed by higher expression of the WT Kras allele in Kras Mm/Mm animals. We hypothesize that the ability of WT Kras to suppress certain mutants may be dependent on upstream activation by EGFR, and that this phenomenon may underlie the divergent responses to EGFR inhibition of colorectal cancer patients harboring Kras G12 versus G13 mutations5. Experiments to test this in mice by extended EGFR inhibition following carcinogen treatment are ongoing. References 1. Prior, I. A., Lewis, P. D. & Mattos, C. A comprehensive survey of Ras mutations in cancer. Cancer Res 72, 2457-2467 (2012). 2. To, M. D. et al. A functional switch from lung cancer resistance to susceptibility at the Pas1 locus in Kras2LA2 mice. Nat. Genet. 38, 926-30 (2006). 3. Dwyer-Nield, L. D. et al. Epistatic interactions govern chemicallyinduced lung tumor susceptibility and Kras mutation site in murine C57BL/6J-ChrA/J chromosome substitution strains. Int J Cancer 126, 125-132 (2010). 4. Westcott, P. M. K. et al. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer. Nature (2014). Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 89 Late-Breaking Poster Session: Tumor Biology 1 doi:10.1038/nature13898 5. De Roock, W. et al. Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 304, 1812-20 (2010). Poster Section 41 Poster Board 7 LB-135 E-cadherin as a buffer for mutated B-catenin: A genetic comparison of Apc and B-catenin mutations in vivo. David J. Huels, Rachel A. Ridgway, Owen J. Sansom. Beatson Institute for Cancer Research, Glasgow, United Kingdom. Wnt deregulation is a characteristic of many cancers, especially Colorectal Cancer (CRC). However, only the intestine harbours mutations in the tumor suppressor gene (TSG) Adenomatous Polyposis Coli (APC) which can be found in around ~80% of CRC patients. Other cancers (e.g. hepatocellular carcinoma, solid pseudopapillary cancer) achieve Wnt deregulation by specific mutations of B-catenin (CTNNB1). Using genetic approaches in the mouse intestine we compared mutations in APC and B-catenin in the level of Wnt activation, their kinetics and their ability to transform the intestinal epithelium in vitro and in vivo. Deregulation of Wnt by mutation of B-catenin is much slower than by loss of APC, and only gradually increases Wnt activity. In contrast to loss of APC, Wnt deregulation by mutation of B-catenin is almost completely restricted to the small intestine of mice, and not the colon. We have evidence that the restriction of the Bcatenin phenotype is due to different E-cadherin levels in the small intestine compared to the colon. Mutation of B-catenin in addition to reduction of E-cadherin levels synergises dramatically in vivo and is now able to transform the colon epithelium. We show that Ecadherin at the adherens junctions acts as buffer for the mutated Bcatenin and slows down its accumulation in the cytoplasm. These data shows a clear restriction of B-catenin mediated tumor initiation by E-cadherin. Interestingly, the other cancer types (hepatocellular carcinoma, solid pseudopapillary cancer) show a similar negative relationship of B-catenin mutation and E-cadherin expression. Poster Section 41 Poster Board 8 LB-136 The role of ER chaperone GRP94 in endometrial cancer progression. Jieli Shen, Yvonne G. Lin, Louis Dubeau, Amy S. Lee. University of Southern California, Los Angeles, CA. Endometrial cancer is the most prevalent gynecologic cancer in the United States and accounts for nearly 50,000 incident cases annually. One of the most common mutations detected in the more prevalent Type 1 endometrial cancer is the loss of the tumor suppressor gene, Pten (phosphatase and tensin homolog). Pten mutations are involved in a wide variety of human cancers, including >60% of endometrial cancer. The glucose-regulated protein 94 (GRP94), also known as glycoprotein 96 (gp96), is a major chaperone protein in the ER. The client proteins of GPR94 include important regulators of cell growth and cell adhesion such as IGF-1, Toll-like receptors and integrins. GRP94 is known to play major roles in immune modulation. GRP94 also possesses antiapoptotic properties. GRP94 elevation has been reported in a variety of human cancers and has been implicated in cancer development. Previously, GRP94 knockout in multiple myeloma reduced cell proliferation and survival, through inhibiting Wnt-LRPsurvivin pathway. GRP94 knockout in macrophages reduced inflammatory colon tumorigenesis. On the other hand, GRP94 knockout in hepatocytes resulted in liver progenitor cell proliferation and acceleration of liver cancer, associating with repopulation of GRP94-positive hepatocytes. Therefore, the role of GRP94 in cancer appears to be context-dependent due to its multifaceted functions. Currently, little is known about GRP94 expression and function in endometrial cancer. We examined GRP94 expression in human endometrial cancer tissues and robust GRP94 expression was observed. In human endometrial cancer cell line, GRP94 knockdown by siRNA led to decreased proliferation and cell migration. To examine the requirement of GRP94 in the initiation and development of endometrial cancer, we utilized the Progesterone Receptor-Cre to specifically delete genes in the mouse endometrium. Before studying GRP94 in endometrial cancer, we first determined the effect of GRP94 depletion in normal uteri. Surprisingly, GRP94 single knockout uteri showed squamous cell metaplasia, confirmed by expression of K14 and p63, and reduction in size. Next, we created the biallelic mutant mice with concurrent deletion of Pten and Grp94 in the endometrium. We monitored the progress of endometrial cancer development, with mice bearing Pten knockout alone serving as the positive controls. As expected, Pten single knockout mice develop endometrial cancer by 4 week and initiate invasion into myometrium by 8 week. Analysis of the mice uteri indicated that GRP94 deficiency delayed the onset of endometrial cancer, associating with altered cell adhesion properties and pro-oncogenic signaling pathways. This study expands our understanding of GRP94 function in the progression of solid tumors and provides the first evidence that GRP94 could be a target for combating endometrial cancer. Poster Section 41 Poster Board 9 LB-137 Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer. Bilal B. Hafeez, Louise Meske, Anupama Singh, Ashok Singh, Weixiong Zhong, Patricia Powers, Manorama John, Anne Griep, Ajit Verma. University of Wisconsin-Madison, Madison, WI. Evidence from our laboratory and others indicate that PKCε is a transforming oncogene and a predictive biomarker of various human cancers including prostate, breast, head and neck, lungs, brain, bladder and cutaneous squamous cell carcinoma (SCC). However, a precise in vivo link of PKCε and its downstream signaling components to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice using cre-lox technology. To do so, we generated a targeting vector in which Exon 4 of the PKCε gene was flanked by LoxP sites. This vector was used to generate mice carrying two floxed alleles of PKCε (PKCεLoxP/LoxP mice) by standard gene knockout methodology. Homozygous PKCεLoxP/LoxP mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostrate, the PKCεLoxP/LoxP mice were bred to prostate specific cre (PB-Cre4+). Western blot and immunohistochemical analyses showed inhibition of PKCε protein level in the prostate of PKCε-KO mice. However, no change in the PKCε protein level was observed in the spleen, liver and lungs of PKCε-KO mice. Also, PKCε deletion in prostate did not affect the levels of other PKC isoforms (PKCα, PKCβII, and PKCς). No significant difference was observed in the prostate weight of PKCεLoxP/LoxP and PKCε-KO mice. Histopathological analyses of prostate from both PKCεLoxP/LoxP and prostate PKCε-KO mice showed normal pathology in the PKCε-KO prostate. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. In this experiment, TRAMP mouse tumor cells (TRAMPC1, 2X106) were implanted in the prostate. Mice were sacrificed at 6 weeks postimplantation. Results demonstrated a significant (P<0.05) decrease in the growth of prostate tumor weight in PKCε-KO mice compared to wild type. To determine a role for PKCε in the epidermis, PKCεLoxP/LoxP mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of ultraviolet radiation (UVR) exposure (2 kJ/m2)-induced Stat3 and AKT phosphorylation, the molecular events essential for UVRinduced development of SCC. In summary, our novel PKCεLoxP/LoxP mice will be useful to define the functional role and molecular mechanism of PKCε linked to cancer induction, American Association for Cancer Research • AACR ANNUAL MEETING 2015 89 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 90 Late-Breaking Poster Session: Tumor Biology 1 progression and metastasis. (Support: NIH Grants CA35368 and CA102431). Poster Section 41 Poster Board 10 LB-138 Use of cerebral organoids to model pediatric gliomas with H3.3K27M and H3.3G34R. Amin Ismail. New York University-Pace University, New York, NY. Glioblastoma (GBM) is the most devastating brain cancer with dismal prognosis both in adults and children and accounts for 16% of primary brain tumors. Despite aggressive therapeutic approaches, the majority of patients die within one year after diagnosis. Giving the limitation of current GBM mouse models, which do not reflect the biological properties of human tumors, in particular, tumor heterogeneity and pathogenesis, there is a growing need for new models. Current in vitro 3D models involve the use of hydrogel scaffolds and cell lines, which do not represent a viable developmental context where the tumor mass interacts with and invades the surrounding brain tissues. Starting with pluripotent human embryonic stem cells we reproduced a 3D tissue structure (so-called cerebral organoid) representing a developing human brain in vitro. We engineered these mini-brains to express oncogenic hits responsible for the derivation of tumors in pediatric GBM patients. Pediatric GBM is driven by a combination of epigenetic and genetic hits including the expression of a mutant histone H3 variant H3F3A (mutated at K27M or G34R) or a gain of function mutant isocitrate dehydrogenase IDH1R132H in combination with the loss of the histone remodeling factor ATRX and/or p53 tumor suppressor. We used a combination of these oncogenic hits to create a model of tumor engineering representing the first 3D human tissue model of GBM. This model combines the accurate multi-lineage differentiation and physiology of human brain with the easiness of manipulation of in vitro systems. H&E staining of the modified organoids shows remarkable dysplasia reminiscent of human primary glioma. Different genetic hits result in different type of dysplasia with IDH1R132H showing distinct morphology, suggestive of a broad range of applicability to model oncogenic-dependence in pediatric GBM. Cerebral organoids may also be used as a model to study tumor invasion starting with glioma stem-like cells (GSCs, which are believed to be the tumor-initiating cells in GBM). Indeed 923 GSCs (which are not invasive in mice brain) quickly and spontaneously (within few days) invade a co-cultured cerebral organoid. This study is the first to engineer a human CNS tumor in vitro. The model carries a tremendous potential to uncover the pathogenesis and initiation of brain cancer in pediatric settings. Poster Section 41 Poster Board 11 LB-139 Tissue stem cell specific enhancer element identifies two types of stem cells in the corpus epithelium of the stomach. Junichi Matsuo,1 Shunichi Kimura,1 Cai Ping Koh,1 Md Zakir Hossain,1 Akihiro Yamamura,1 Kazuyoshi Kohu,1 Michiaki Unno,2 Jimmy Bok Yan So,1 Feng Zhu,1 Supriya Srivastava,1 Teh Meng,3 Nicholas Barker,4 Khay Guan Yeoh,1 Motomi Osato,1 Yoshiaki Ito1. 1 National University of Singapore, Singapore, Singapore; 2Tohoku Univerisity, Sendai, Japan; 3National University Health System, Singapore, Singapore; 4A*STAR, Singapore, Singapore. The activity of a previously identified hematopoietic stem cell marker, Runx1 enhancer element (eR1) (Ng et al, Stem Cells 28, 1869-1881, 2010), was found to mark tissue stem cells of multiple organs. In corpus of stomach, stem cells are known to reside at isthmus, upper part of gastric unit. They are undifferentiated cells. However, molecular approach to these stem cells has not been described. Recently, fully differentiated Pepsinogen expressing chief cells at the bottom of gastric unit were shown to have tissue regeneration activity, and these cell were named reserve stem cells (Stange et al, Cell 155, 357-368, 2013). In our study, eR1 was shown to mark stem cells of both types. Lineage tracing experiments demonstrated that both types of stem cells 90 continuously gave rise to mature cells to maintain the gastric unit. Manipulation of gene expression in a stem cell-specific manner in the stomach, especially in undifferentiated stem cells, is a long sought-after approach to study gastric carcinogenesis. We crossed transgenic mouse carrying eR1-CreERT2 with LSL-K-rasG12D mice. After tamoxifen treatment, rapid differentiation from the stem cells in the isthmus was observed, mainly into Muc5ac+ cells (surface epithelial cells). As a result, pseudo-pyloric metaplasia was induced which is similar to that observed in human gastritis. Acidproducing parietal cells were eliminated during this process. Rapid generation of Muc5ac+ cells and other phenotype are similar, but not identical, to the phenotype described in Menetrier disease which is known to be caused by excessive production of TGF-α and hyper-stimulation of EGF receptor (Reviewed in Coffey et al, J Clin Invest. 117:70-80, 2007). Menetrier disease is considered to be pre-malignant state. Activation of K-rasG12D in eR1+ chief cells also induced metaplastic lesions. In this case, pepsinogen producing chief cells robustly expressed the marker of mucous neck cells that are considered to be a precursor to chief cells. Therefore, chief cells appeared to be de-differentiated to precursor cells and acquired the stem cell property. We are using eR1 to study step-wise carcinogenesis in stomach and other organs. Poster Section 41 Poster Board 12 LB-140 Investigating cancer stem cells in glioblastoma initiation and recurrence. Xuanhua Xie,1 Xiuping Zhou,2 German B. Sánchez,3 Luis F. Parada1. 1 UT Southwestern Medical Center at Dallas, Dallas, TX; 2Nervous System Diseases, Xuzhou Medical College, Xuzhou, China; 3 Departamento de Biología Celular, Facultat de Ciències Biològiques, València, Spain. In this study, I examine the unique features of quiescent cancer stem cells (CSCs) in glioblastoma initiation and recurrence in genetically engineered mouse models. A nestin-TK-GFP transgene was used to label CSCs with GFP and to render them susceptible to ganciclovir (GCV) treatment in a fully penetrant mouse model of GBM (M7: hGFAP-Cre; Nf1fl/+; p53fl/fl; Ptenfl). Using foodmediated GCV delivery (GCV chow) to optimize the drug delivery, Doublecortin-positive and NG2-positive cells derived from the nestin-TK-GFP transgene labeled neural stem cells were significantly reduced after six weeks. In addition, on GCV chow the tumor-bearing mice containing the nestin-TK-GFP transgene live significantly longer than the ones without the transgene. Isolation and transplantation of the GFP+/GFP- tumor cells demonstrated that the GFP+ cells have enhanced tumorigenic potential over the GFP- cells. These results support the hypothesis that the nestinTK-GFP transgene targets the CSCs that are responsible for the sustained GBM growth. In the M7 model, the hGFAP transgene has a broader expression, and thus broader Cre targeting, than the nestin transgene. To eradicate the problem of differing scopes of Cre expression versus targeting of CSCs, I generated a new transgene (CGD: nestin-CreERT2-H2BeGFP-hDTR) that enables Cre-mediated recombination, specific cell labeling and targeted cell ablation. The CGD is designed to express a fusion protein that consists of CreERT2, H2B-eGFP, and hDTR (human diphtheria toxin receptor). Containing P2A linkers between the three gene cassettes, the fusion protein can be efficiently cleaved into three active. I have screened for a transgenic line that can induce GBM formation upon tamoxifen induction. Six to eight week-old mice with the following genetic configuration: CGD; Nf1fl/+; p53fl/+; Ptenfl/+, were given one dose of tamoxifen and all mice died of high grade gliomas three to six months later. The analysis of the CGD-induced tumors demonstrated that the CGD transgene labels tumor cells that are relatively quiescent, compared to the highly proliferative Ki67+ cells. Thus our CGD transgene labels putative CSCs within the tumor. In this model, the same cells initiating tumor formation (Creexpressing) are also expressing H2B-eGFP and hDTR. Thus, all the tumor cells are derived from the CGD transgene and all the CSCs should be labeled with the H2B-eGFP protein. This dramatically Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 91 Late-Breaking Poster Session: Tumor Biology 1 facilitates the sorting of the tumor-initiating cells. Transplantation of the GFP+/GFP- cells in these tumors revealed that the quiescent GFP+ cells are more tumorigenic than the proliferating GFP- cells. Furthermore, diphtheria toxin-mediated ablation of the hDTRexpressing cells significantly elongates the lives of tumor-bearing mice. To mimic the endogenous environment for GBM growth, wildtype mice were also used to prove the differential tumorigenic ability of the GFP+/- cells. These results validate the essential roles of CSCs in GBM initiation and recurrence. Poster Section 41 Poster Board 13 LB-141 Specific and potent silencing of K-Ras by asymmetric silencing RNA (aiRNA) reveals addiction of cancer stem cells to mutant K-Ras amplification. Jun Oishi, Hiroki Umehara, Nithya Jesuraj, Jelena Barbulovic, Xiangao Sun, Chiang J. Li. Boston Biomedical, Inc., Cambridge, MA. K-Ras, the first oncogene identified in human cancer, is mutated in about 30% of human solid tumors. K-Ras protein, a small membrane-bound GTP-binding protein, acts as a molecular switch to transduce cell proliferation signals. Activating mutations of K-Ras lock the Ras protein into the hyper-active GTP-bound state, resulting in the activation of numerous signaling pathways that control cell survival and proliferation. Ras is also an important oncoprotein in many cancers where it is not mutated since Ras can be functionally activated through aberrant activation of other signal transduction elements. Activated K-Ras proteins are, therefore, found in a large proportion of all human cancers, and occupy a central position of interest. Hypermalignant cancer cells, termed cancer stem cells (CSCs), that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties. These stemness-high cancer cells are hypothesized to be fundamentally responsible for cancer metastasis and relapse. Furthermore, a number of stemness genes, such as beta-catenin, nanog, Sox2, Oct3/4 have been implicated in cancer cell stemness. The role of oncogenes such as K-Ras in cancer cell stemness, however, is not clear. To elucidate the role of K-Ras in the maintenance of cancer cell stemness, we employed asymmetric silencing RNA technology (aiRNA) which is able to silence target genes with high potency and precision. Moreover, aiRNA technology can be readily applied to CSCs. Here we report, to our surprise, that CSCs are not simply addicted to activating mutations of K-Ras, or activation of downstream regulators of the Ras pathway. However, CSCs with amplified mutant K-Ras are highly sensitive to K-Ras silencing. Moreover, the DNA copy number of the mutant K-Ras directly predicts the sensitivity of CSCs to K-Ras silencing. Our studies suggest that amplified mutated K-Ras is required for the maintenance of malignancy and cancer cell stemness, which may have significant implications for understanding the connections between oncogenes and cancer cell stemness, and for developing cancer stem cell inhibitors. Poster Section 41 Poster Board 14 LB-142 Functional role of DNA methyltransferase1 (DNMT1) in regulation of mammary stem/progenitor and cancer stem cells. Rajneesh Pathania,1 Sabarish Ramachandran,2 Puttur Prasad,1 Vadivel Ganapathy,2 Muthusamy Thangaraju1. 1Georgia Regents University, Augusta, GA; 2Texas Tech University, Lubbock, TX. Tumor propagation is the hallmark feature of the cancer stem/tumor propagating cells. Several genetic and epigenetic components are involved in regulation of this process; however, DNA methylation provides a potential epigenetic mechanism for the cellular memory, which needed to preserve the tumorigenic potential through repeated cell divisions. Further, DNA methylation plays a critical role in stem/progenitor cell maintenance wherein the DNMT proteins get enriched in undifferentiated cells and thereby it retains the regenerative capacity while suppressing differentiation. However, the precise role of DNMTs in maintaining stem/progenitor and tumorigenic phenotype in constantly replenished organ, like mammary glands and mammary tumor is not yet known. Here we show that Dnmt1 is required for mammary gland outgrowth and terminal end bud development and that mammary-gland specific Dnmt1 deletion in mice leads to significant reduction in mammary stem/progenitor cell formation. Interestingly, Dnmt1 deletion almost completely abolishes Neu-Tg- and C3(1)-SV40-Tg- driven mammary tumor formation and metastasis. This phenomenon is associated with significant reduction in cancer stem cell (CSC) formation. Similar observations were also recapitulated using pharmacological inhibitors of Dnmts in Neu-Tg mice. To unravel the cause of tumorigenicity of tumor propagating cells, we used genome-wide methylation and RNA sequence approach and find that DNA methylation plays a vital role in regulation of abnormal self-renewal by hypermethylating genes that are involved in development and cell commitment pathways; thereby leading to immortality and autonomous growth to the tumor propagating cells. Overall, our studies provide the first in vivo evidence that DNMT1 is indispensable for mammary stem, progenitor and cancer stem cell formation and that functional inactivation of this gene drastically reduces mammary tumor formation even in the aggressive triplenegative breast cancer subtype. Furthermore, we identified ISL1 as a functional target of DNMT1 in tumor progenitor cells, and stable expression of ISL1 induces apoptosis in cancer stem cells. Thus, DNMT1 specific inhibitors could have a great impact on eradication of cancer stem cells and associated disease recurrence, and ISL1 hypermethylation status could be used as a prognostic marker for early breast cancer diagnosis. Poster Section 41 Poster Board 15 LB-143 DNp63 governs metastatic outgrowth of breast cancer stem cells. Alice Turdo,1 Simone Di Franco,1 Antonina Benfante,1 Maria Luisa Colorito,1 Marco Bonanno,1 Miriam Gaggianesi,1 Daniela Barcaroli,2 Francesco Dieli,1 Jan Paul Medema,3 Vincenzo De Laurenzi,2 Giorgio Stassi,1 Matilde Todaro1. 1University of Palermo, Palermo, Italy; 2University, Chieti, Italy; 3University of Amsterdam, Amsterdam, Netherlands. Background: In the human mammary gland p63 isoforms control mammary epithelial cells fate and DNp63, which lacks the amino-terminal trans-activation domain, counterbalances the fulllength isoform TAp63 to allow maturation of epithelia and the maintenance of the myoepithelial/basal cells features. Mammary gland tissue’s adult stem cells retain self-renewal and multi-lineage differentiation ability and, by acquiring a malignant behavior, are in charge of tumor seeding. A correlation between Cancer Stem Cells (CSCs) in primary lesions and increased metastatic dissemination has been reported, however, still little is known about the unique profile of distant metastasis’ cell origin. Herein, we show for the first time that breast CSCs (BCSCs) expressing DNp63 are capable to generate metastasis in a preclinical model, while TAp63 can be solely tumorigenic and unable to serial xenografting. We anticipate novel farsighted implications for a prognostic role of p63 and for its targeting to prevent tumor growth and metastatic disease. Methods: BCSCs were freshly isolated from the histological uninvolved resection of breast cancer tissues. Obtained cells were plated in ultra-low flask in serum-free media in presence of bFGF and EGF. Cells were treated with 1mmol/l doxorubicin hydrochloride. Synthetic genes encoding DNp63 and TAp63 were inserted into the p-TWEEN EGFP lentiviral expression vector. Stable p63 knockdown was produced by lentiviral transduction of the pLentilox 3.7 vector carrying shp63 and scramble sequences. For the tumorigenic and metastagenic assay BCSCs (3x105) were suspended in matrigel and injected orthotopically or in the subrenal capsule of NOD/SCID mice, respectively. Tumors were measured with a caliper each week and volume was calculated by the formula: π/6 x larger diameter x (smaller diameter)2. Metastasis formation was followed over time and visualized by in vivo imaging. American Association for Cancer Research • AACR ANNUAL MEETING 2015 91 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 92 Late-Breaking Poster Session: Tumor Biology 1 Summary: Herein, we demonstrate that luminal and basal BCSCs differ in the content of p63 isoforms, DNp63expression was higher in the stem-like compartment of basal BCSCs, compared to TAp63 mainly expressed in bulk primary cells. DNp63 increased the G0-G1 phase in both luminal and basal BCSCs, enhanced doxorubicin resistance and induced a mesenchymal switch. Despite both TAp63 and DNp63 overexpressing BCSCs formed subcutaneous xenografts in NOD/SCID mice, DNp63 cells exclusively retain capabilities of serial xenografting. Thus, suggesting that exogenous expression of TAp63 could promote transition from stem cells to progenitor cells. In a metastatic preclinical model TAp63 overexpression hampered metastatic potential of BCSCs, while DNp63 overexpressing cells gained capabilities to colonize distant organs such as lung and kidney. Conclusions: These findings state DNp63 as a major regulator of stemness and invasiveness in malignant breast tissues and offer new insights on p63 role as a prognostic biomarker and therapeutic target. Poster Section 41 Poster Board 16 LB-144 Derivation of a model of cancer stem cell from human induced pluripotent stem cells. Tomonari Kasai,1 Kenta Hoshikawa,1 Shuto Takejiri,1 Masashi Ikeda,1 Kazuki Kumon,1 Anna Sanchez Calle,1 Arun Vaidyanath,1 Akifumi Mizutani,1 Chen Ling,2 Masaharu Seno1. 1Okayama University, Okayama, Japan; 2Tianjin Central Hospital of Gynecology Obstetrics, Tianjin, China. The existence of cancer stem cell (CSC) has been considered as one of the important reason as to why patients have a poor prognosis. However, heterotopic transplantation of embryonic stem cells and induced pluripotent stem cells has been shown to form teratoma, but not malignant teratoma. Since the microenvironment niche is playing a significant role for the proper differentiation of stem cells, the cancerous niche should drive stem cells into malignant cells in vivo. According to this hypothesis, we tried to generate cancer cells from human induced pluripotent stem (hiPS) cells. For the conversion into CSC, the conditioned medium from different human cancer cell lines was collected from confluent dishes and filtered using 0.22 micrometer filter. Then, hiPS cells, without MEF feeder cells, were maintained in the conditioned medium (CM) in the ratio of 1:1. The medium was changed every day with CM for 4 weeks. hiPS cells with the complete medium were used as control. For transplantation studies, 10^4 cells were suspended in HBSS and were xenotransplantated into NOD-SCID mice. After 3 months, tumors were excised and fixed in 10% neutral formalin buffer solution, or subjected to primary culture. The converted cells and primary cultured cells formed spheroids in suspension culture, and had tumorigenicity in vivo. The stemness of living cells was checked under fluorescent microscopy observation with rBC2LCN-FITC staining. The RNAs were extracted from converted cells and microarray analysis was perfomed. The RNA expression patterns of cell lines were visualized by sphered selforganizing map (sSOM) analysis. The sSOM analysis perfomed based upon various parameters shows the converted CSCs can be characterized into various cell types. Utilizing this method, we successfully established two different hiPS-CSC lines using CM from A172 and RERF-LC-KJ. The comprehensive understanding of cancer could be realized as the heterogeneity of cancer tissues is clarified and their component cells are identified. This study will lead to the development of the true personalized therapy of cancer in the future. Poster Section 41 Poster Board 17 LB-145 Pten deletion in SOX9+ cells synergizes with hepatic injury to drive tumor development in mouse liver. Ni Zeng,1 Anketse Kassa,1 Janel Kopp,2 Lina He,1 Maike Sander,2 Bangyan Stiles1. 1University of Southern California, Los Angeles, CA; 2University of California San Diego, San Diego, CA. Liver cancer is an extremely deadly disease ranked as the third 92 most common cancer and the second leading cause of male cancer-related death worldwide. Among primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the two most frequent subtypes, accounting for 85-95% of the total liver cancer cases, and both types of liver cancer have been found to originate from the expansion and differentiation of tumor initiating cells (TICs). Thus, it is critically important to understand mechanisms that regulate liver TICs activation. By utilizing a Pten(loxp/loxp); Albumin-Cre+ mouse liver cancer model (L-PKO), our previous studies have confirmed the activation of TICs during liver tumorigenesis. We also found that SOX9 was expressed in both HCC and CC, suggesting that SOX9 might serve as a liver TICs marker. We thus used the Pten(loxp/loxp); SOX9-CreER+ mouse model (TIC-PKO) to study the role of PTEN in TICs during liver tumorigenesis. Liver tumor development was observed in TICPKO mice at the age of 12 months old, and immunohistochemical analysis revealed that the tumors were heterogeneous, indicating TICs were involved in the tumorigenesis process. In addition, treatment of DDC, which is a liver toxin and thus causes hepatocyte death, dramatically increased the incidence and led to the early onset of liver tumorigenesis in the mutant but not the control mice, suggesting that liver injury served as an essential microenvironmental niche in promoting liver tumor development. Together, our observations suggested that the PTEN signaling is critical for TICs regulation and Pten deletion synergizes with liver injury to drive both TICs activation and liver tumorigenesis. Poster Section 41 Poster Board 18 LB-146 TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells. Naoki Oshimori,1 Daniel Oristian,1 Elaine Fuchs2. 1Rockefeller University, New York, NY; 2HHMI/Rockefeller University, New York, NY. Among the most common and life-threatening cancers worldwide, squamous cell carcinoma (SCC) exhibit high rates of tumor recurrence following anti-cancer therapy. Subsets of cancer stem cells (CSCs) often escape anti-cancer therapeutics and promote recurrence. However, its sources and mechanisms that generate tumor heterogeneity and therapy-resistant cell population are largely unknown. Tumor microenvironment may drive intratumor heterogeneity by transmitting signaling factors, oxygen and metabolites to tumor cells depending on their proximity to the local sources. While the hypothesis is attractive, experimental evidence is lacking, and non-genetic mechanisms that drive functional heterogeneity remain largely unknown. As a potential non-genetic factor, we focused on TGF-β because of its multiple roles in tumor progression. Here we devise a functional reporter system to monitor, track and modify TGF-β signaling in mouse skin SCC in vivo. Using this approach, we found that perivascular TGF-β in the tumor microenvironment generates heterogeneity in TGF-β signaling in neighboring CSCs. This heterogeneity is functionally important: small subsets of TGF-β-responding CSCs proliferate more slowly than their non-responding counterparts. They also exhibit invasive morphology and a malignant differentiation program compared to their non-responding neighbors. By lineage tracing, we show that although TGF-β-responding CSCs clonally expand more slowly they gain a growth advantage in a remarkable ability to escape cisplatininduced apoptosis. We show that indeed it is their progenies that make a substantial contribution in tumor recurrence. Surprisingly, the slower proliferating state of this subset of CSCs within the cancer correlated with but did not confer the survival advantage to anti-cancer drugs. Using transcriptomic, biochemical and genetic analyses, we unravel a novel mechanism by which heterogeneity in the tumor microenvironment allows a subset of CSCs to respond to TGF-β, and evade anti-cancer drugs. Our findings also show that TGF-β established ability to suppress proliferation and promote invasion and metastasis do not happen sequentially, but rather simultaneously. This new work build Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 93 Late-Breaking Poster Session: Tumor Biology 1 upon the roles of this factor in tumor progression, and sets an important paradigm for a non-genetic factor that produces tumor heterogeneity. Poster Section 41 Poster Board 19 LB-147 Colonic crypt neuroendocrine cells regulate colonic stem cells during colorectal cancer development. Shirin R. Modarai, Lynn M. Opdenaker, Bruce M. Boman. University of Delaware, Newark, DE. Our studies on tissues from colon cancer patients show that in the development of CRC, stem cells (SC) overpopulation underlies tumor initiation and progression. Specifically, we have involved studying patients with Familial Adenomatous Polyposis (FAP) during CRC development, which carry cancer-predisposing germline mutations in the adenomatous polyposis coli (APC) gene. These results indicate that SC overpopulation caused by APC mutations is the driver mechanism. However, how the APC mutations cause SC overpopulation needs clarification. Similar to SCs, Neuroendocrine cells (NECs) comprise ~1% of the total cells in the colon and both reside in the crypt stem cell niche in the colonic epithelium. NECs are involved in autocrine and paracrine signaling to help regulate the function of surrounding cells. This leads to an overall goal of this project, to study the relationship between NECs and SCs in young-onset CRC, especially those individuals with a genetic predisposition to CRC. Our overall hypothesis is that NECs play a key role in maintaining the SC population in the normal colon and, aberrant signaling from NECs contributes to tumor initiation and increased stemness. To begin to investigate underlying mechanisms, we studied human colon tissues with varying APC genotypes: (i) normal crypts (homozygous-wildtype APC); (ii) normal-appearing FAP crypts (heterozygous-mutant APC); (iii) adenomatous FAP crypts (homozygous-mutant APC); (iv) CRCs (homozygous-mutant APC& other mutations). Preliminary data from our laboratory has shown that in colon cancer progression: (i) there is a decrease in the number of cells that co-express both SC and NEC markers (ii) there is a decrease in the total number of NECs, and (iii) there is an increase in the total number of SCs. Furthermore, we have established an in vitro co-culture model system to study regulation of SCs by NECs. Results from our model show that SSTR1 and GLP-2R NEC signaling plays a role in modulating colonic SC populations. This in vitro model system can help analyze the interaction between SCs and NECs during CRC progression. Taken together, our findings indicate that: (1) NE factors contribute to colonic SC regulation via an autocrine or paracrine mechanism in normal colon, and (2) dysregulation of these mechanisms contributes to colon cancer development. Using our established in vitro model system, we can track the changes in the SC and NEC population size, based on different conditions of anchorage independent growth and presence of potential therapeutic agents. American Association for Cancer Research • AACR ANNUAL MEETING 2015 93 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 94 Late-Breaking Poster Session: Molecular and Cellular Biology 3 Late-Breaking Poster Session Tuesday, April 21, 2015 8:00 AM-12:00 PM Poster Section 39 Late-Breaking Research: Molecular and Cellular Biology 3 Poster Section 39 Poster Board 1 LB-149 A novel strategy to inhibit CpG island hypermethylation and restore BRCA1 expression. Yoo Jeong Han,1 Jing Zhang,1 Aleix Prat,2 Toshio Yoshimatsu,1 Maria Gomez-Vega,1 John Kwon,1 Charles M. Perou,3 Prasanth Kannanganattu,4 Olufunmilayo I. Olopade1. 1University of Chicago, Chicago, IL; 2Vall d´Hebron Institute of Oncology, Barcelona, Spain; 3 University of North Carolina, Chapel Hill, NC; 4University of Illinois at Urbana-Champaign, Urbana, IL. BRCA1 promoter methylation is observed in 20-60% of sporadic triple negative breast cancer (TNBC) and may be an important mechanism contributing to the loss of BRCA1 function in sporadic TNBC and other cancers with low BRCA1 expression. Demethylation and consequent reactivation of tumor suppressor genes are rational approaches being used in the treatment of cancer. However, currently available nucleoside-based DNMT inhibitors affect genome-wide DNA methylation and cannot specifically target tumor suppressor genes like BRCA1. Here, we describe a novel approach to modulate local DNA methylation by silencing a neighboring long non-coding RNA (lncRNA) which tethers DNMT1 at the BRCA1 genomic locus. The human genomic region encompassing the BRCA1 gene is complex and includes two protein coding genes (BRCA1 and NBR1), a non-coding RNA gene (NBR2), and a pseudogene of BRCA1 (BRCA1P1), within a ~170kb region of chromosome 17q21. The BRCA1 gene on the minus strand is located head-to-head with NBR2 on the plus strand, whereas BRCA1P1 on the minus strand is located head-to-head with NBR1 on the plus strand. The promoter between the BRCA1 and NBR2 genes and that between the BRCA1P1 and NBR1 genes are bidirectional, expressing transcripts in opposite directions through convergent transcription. The BRCA1P1 pseudogene was generated by a recent evolutionary event: partial duplication of BRCA1 gene followed by insertion of a processed pseudogene of RPLP1. It expresses a chimeric lncRNA retained in nuclei. Interference with the nuclear expression of BRCA1P1 lncRNA using a specific anti-sense oligonucleotide (ASO) decreased the promoter methylation of BRCA1 and increased BRCA1 expression. RNA immunoprecipitation (RIP) assays revealed DNMT1 interactions with BRCA1 mRNA and BRCA1P1 lncRNA at the locus. Chromosome conformation capture (3C) will assess whether there is a long-range interaction between the BRCA1 and BRCA1P1 promoters through DNMT1 and mediators. Our data support a long-range cis-regulation of BRCA1 expression by neighboring BRCA1P1 lncRNA through an interaction with DNMT1 at the locus. Depleting BRCA1P1 with ASO could be developed as a therapeutic method to inhibit BRCA1 promoter methylation and restore BRCA1 expression. Poster Section 39 Poster Board 2 LB-150 Genetic depletion of DNMT1 reveals a DNMT1 threshold controlling DNA methylation in human cells. Yi Cai, Hsing-chen Tsai, Ray-whay Yen, Limin Xia, Yang Zhang, Stephen Baylin. Johns Hopkins University, Baltimore, MD. Altered DNA methylation patterns are a central feature of most types of human cancer cells. However, the exact roles of DNA methyltransferases (DNMTs) in human DNA methylation maintenance are not well characterized. The long-established model that DNA methyltransferase DNMT1 maintains most DNA methylation in human cells has been challenged by multiple DNMT1 knockdown/knockout studies in the past decade. A potential role for de novo DNA methyltransferases DNMT3A and DNMT3B in maintaining DNA methylation in cancer cells has been suggested but is also not precisely known. 94 To investigate the above issues, we have utilized both genetic recombination and CRISPR technology to deplete DNMTs individually or in combination in human colorectal cancer cell line HCT116. Previously we created a strong HCT116 DNMT1 hypomorph (1KO) by genetic recombination. Although 1KO cells lost about 85% of DNMT1 protein, they still maintained the epigenetic silencing of tumor suppressor genes and showed only 20% decrease in overall DNA methylation. We now show that further depletion of DNMT1 in 1KO cells, to less than 1% of wild type HCT116 cells, resulted in elimination of most DNA methylation and reactivation of key tumor suppressor genes including p16. In addition, we found that DNA methylation in HCT116 cells is controlled by different DNMT1 thresholds. Surprisingly, when we disrupted DNMT3A or DNMT3B with CRISPR in HCT116 cells, we see altered DNA methylation patterns involving both gains and losses of DNA methylation. Since abnormal DNA hypermethylation in cancer often associates with epigenetic gene silencing, this observation is especially relevant for the function of DNMT3A mutations in acute myeloid leukemia. Finally, we demonstrated that depletion of DNMT1-targeting protein UHRF1 efficiently reduced DNA methylation in human colon cancer cells. Our results not only defined the distinct roles of individual DNMTs in human cancer cell DNA methylation maintenance but also suggested UHRF1 inhibition as an alternative approach to reverse abnormal DNA hypermethylation in human cancer cells. Poster Section 39 Poster Board 3 LB-151 Methylation-mediated silencing of TIMP-3 facilitates oral cancer metastasis by modulating binding of SP1 transcription factor. Shun-Fa Yang,1 Chun-Wen Su,1 Chiao-Wen Lin2. 1Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; 2 Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan. Tissue inhibitor of metalloproteinase (TIMP)-3, a member of the TIMP family, is the only substance that can bind with the ECM and its main role is regulating the cell cycle and cancer cell progression. However, little is known about whether abnormal expression and promoter methylation of TIMP-3 facilitates oral cancer metastasis. In this study, DNA methylation levels of the TIMP-3 CpG islands were assessed using bisulfite DNA sequencing and methylationspecific PCR. The results shown that expression of TIMP-3 is decreased in most oral cancer tissues compared with adjacent normal tissues (p<0.001). DNA methylation analysis showed a hypermethylation of TIMP-3 gene in oral cancer cell lines and in oral cancer tumors. Furthermore, suppression of TIMP-3 transcription by DNA methylation involves inhibition of transcription factor SP1 binding to the TIMP-3 promoter. Functional analyses revealed that the stable overexpression of TIMP-3 reduced the migration ability in oral cancer cells (p<0.001). Moreover, the expression levels of the epithelial markers E-cadherin were increased, while the expression levels of the mesenchymal markers vimentin and fibronectin were decreased in cells overexpressing TIMP-3. In conclusion, these results suggest that suppression of TIMP-3 by DNA methylation contributes to oral cancer metastasis. Poster Section 39 Poster Board 4 LB-152 A new role for ERα: Gene silencing via DNA methylation. Eric A. Ariazi, John C. Taylor, Emmanuelle Nicolas, Michael J. Slifker, Jeff Boyd. Fox Chase Cancer Center, Philadelphia, PA. As a master transcription factor, estrogen receptor-α (ERα) regulates expression of a multitude of genes. We hypothesized that ERα may also regulate gene expression via DNA methylation since methylation of specific CpG sites associates with ERα-positive status in human breast cancer. We tested this hypothesis by identifying genes normally silenced in ERα-positive T47D and MCF7 luminal breast cancer cell lines but which became expressed (or derepressed) upon exposure to the demethylating agent decitabine Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 95 Late-Breaking Poster Session: Molecular and Cellular Biology 3 and antagonizing ERα activity. These genes were identified using a combination of Agilent gene expression microarrays and assessed according to the Significance Analysis of Microarrays (SAM) method at a 2-fold change and 5% FDR. Selected genes were validated by pyrosequencing to quantitate CpG methylation, lentiviral reintroduction of ERα, real-time PCR and immunoblotting. In a shortterm study, T47D cells were treated with decitabine and the ER antagonist fulvestrant (FUL) for 96 h. Array analysis identified 87 genes that were derepressed by decitabine and FUL, of which 31 (36%) were markers of the basal subtype of breast cancer. In longterm studies (8 - 36 weeks), MCF-7 and T47D cells were FULtreated or estrogen-deprived (ED) to derive multiple antihormone-resistant cell lines (MCF-7/FUL, T47D/FUL, and 2 independent T47D/ED), all of which lost ≥ 95% of ERα. Comparing all of the resistant cell lines and decitabine-treated T47D cells by array analysis revealed that 58 genes were up-regulated by both loss of ERα and decitabine treatment, 35 (60%) of which were basal markers. Two basal breast cancer subtype markers, LCN2 and IFI27 which are involved in epithelial-mesenchymal transition and interferon signaling, respectively, were selected for verification. First, LCN2 and IFI27 expression increased ~132- and 343-fold, respectively, while their CpG methylation levels significantly decreased upon ERα loss in all T47D antihormone-resistant cell lines. Second, LCN2 and IFI27 expression decreased while their methylation levels increased upon estrogen re-exposure or lentiviral ERα re-introduction in the T47D/ED cell lines. Moreover, high CpG methylation levels of LCN2 and IFI27 directly associated with ERαpositive status but inversely correlated with their expression levels across a panel of 12 breast cancer cell lines. Therefore, ERα targets specific genes, such as basal markers, for DNA methylationmediated silencing. Mechanistically, ERα-dependent DNA methylation may occur as a consequence of long-term transcriptional repression. Although not well appreciated, estrogenbound ERα represses transcription of more genes than it activates. Thus, ERα may direct DNA methylation by interacting with multicomponent corepressor complexes, which could then recruit DNA methyltransferases to target promoters. Moreover, ERαdependent silencing of basal markers may promote the prognostically more favorable luminal breast cancer subtype. Poster Section 39 Poster Board 5 LB-153 In vivo visualization of the targeted DNA methylation. Yao-Li Chen,1 Shou-Tung Chen,2 Chun-Chun Chung,3 Chia-Chen Hsu,3 Ping-Yi Lin,1 Wan-Ling Chuang,1 Yi-Ju Chu,3 Yu-Wei Leu,3 Shu-Huei Hsiao3. 1Transplant Medicine & Surgery Research Centre, Changhua Christian Hospital, Changhua, Taiwan; 2Comprehensive Breast Cancer Center, Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan; 3National Chung Cheng University, Chia-Yi, Taiwan. DNA methylation is a dominant silencing epigenetic modification that is inheritable in somatic cells. The recruitment of DNA methylation was proven to be able to reshape the cell physiology and cause tumorigenesis. The reversible nature of the methylation modification also makes the DNA methylation a therapeutic target and the monitoring of the methylation/demethylation dynamics in vivo is thus important for the development of demethylation agents. A targeted DNA methylation method and a two-component reporter system were combined to monitor the increase and demolishment of DNA methylation in vivo. The two constructs of the reporter systems are: target gene promoter (like HIC1 and ENSA) linked with the Tet repressor gene; the other is the Tet operator linked with the reporter like EGFP (in cell), IFP and iRFP. Both constructs were cotransfected into cells like mesenchymal stem cells, MDA-MB-231 and HCT116, and stable clones with both constructs were isolated and validated. Targeted DNA methylation method was used to transfect the isolated cell and, as the increase of DNA methylation, the reporters expressed. The increased methylation was then quenched after the adding of demethylation agents. To test this system in vivo, cells with both constructs were injected into the immune-deficient mice and the tumors grew afterward. Targeted DNA methylation was performed in vivo and the increase of methylation/expression of reporter genes were observed by fluorescence-based tomography. This combined system is also effective to monitor the demethylation effects of the demethylation agents. (Supported by: NSC- 102-2320-B-194-003-MY3 and NSC102-2314-B-371- 003-MY3, MOST Taiwan) Poster Section 39 Poster Board 6 LB-154 Genome-wide analysis identified differentially methylated regions in head and neck squamous cell carcinoma anatomic subsites. Bianca L. Rivera,1 Oluwasina Folawiyo,2 Nitesh Turaga,2 Francesca Pirini,2 Ricardo Lopez,1 Roger Vazquez,1 Rafael Guerrero,2 Adriana Baez3. 1University of Puerto Rico-Rio Piedras Campus, San Juan, PR; 2Johns Hopkins University, School of Medicine, Baltimore, MD; 3 University of Puerto Rico, School of Medicine, San Juan, PR. Introduction: Several groups have identified hundreds of genes differentially methylated in head and neck squamous cell carcinoma (HNSCC) using genome-wide technologies. However, there is a paucity of studies examining differential methylation in HNSCC anatomic subsites. Objective: Our principal objective is to determine DNA methylation differences between Head and Neck Squamous Cell Carcinoma (HNSCC) anatomic subsite: larynx, oral cavity and pharynx. Methods: DNA was extracted from 24 HNSCC samples and 10 normal oral epithelium tissue samples of Puerto Rican Hispanic patients. To unveil the methylome of HNSCC anatomic subsites genomic DNA was enriched with Methylated DNA immunoprecipitation prior to labeling and hybridization to genomewide oligonucleotide tilting arrays (3 X 720K Human CpG IslandPlus-Promoter Array, Roche/NimbleGen). Differentially methylated regions were identified with CHARM using the bumphunter algorithm. Downstream analyses were performed to identify differences in the methylation landscape of the three anatomic subsites: larynx, oral cavity and pharynx. Results: When datasets were analyzed by anatomic site using DAVID v6.7 we identified 2565 differentially methylated genes common to the three subsites. We also identified 738 differentially methylated CpG loci in laryngeal cancer, 889 differentially methylated CpG loci in oral cavity and 363 differentially methylated CpG loci in pharyngeal cancer. We identified 11 key genes which were uniquely methylated in the larynx (CDH1, PAX1, BID, SMAD4, FGF19, BMP2, ITGA2, RXRA, IGFBP1, CDKN2D and RBL2). BCL2, AKT2, TRAF3, STAT5B, PTCH1, WNT9B, AXIN2, DVBL2, and APC were differentially methylated in oral cavity. In the pharynx, a comparatively lower number of differentially methylated genes were detected including B-catenin, RAF, BRAF, ATR, CTNNA1, FOXO1 and MED15. Conclusion: We found evidence that HNSCC subsites (larynx, oral cavity and pharynx) manifest different methylation patterns that may be consistent with the clinical expression of the disease. A panel of differentially methylated genes identified will be further tested in a large cohort of HNSCC samples to determine their utility as prognostic biomarkers of HNSCC according to subsite. Poster Section 39 Poster Board 7 LB-155 Epigenetic profiling of chemotherapy sensitivity. Zachary A. Gurard-Levin,1 Vera Pancaldi,2 Laurence OW Wilson,1 Elisabetta Marangoni,1 Sergio Roman-Roman,1 Alfonso Valencia,2 Paul Cottu,1 Genevieve Almouzni1. 1Institut Curie, Paris, France; 2 CNIO, Madrid, Spain. Many anti-cancer drugs act by directly binding or modifying DNA, or through indirect binding during processes that rely on the chromatin architecture. Mis-regulation of the expression of epigenetic factors may perturb chromatin organization and contribute to drug resistance mechanisms. Here, we aim to American Association for Cancer Research • AACR ANNUAL MEETING 2015 95 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 96 Late-Breaking Poster Session: Molecular and Cellular Biology 3 investigate the power of chromatin regulators and epigenetic factors as predictive markers for the efficacy of single chemotherapy drugs. We exploit transcriptome data from breast cancer cell lines and a validation set comprised of breast cancer patient derived xenograft (PDX) models that are either sensitive or resistant to individual chemotherapy agents, to correlate mRNA expression of chromatin regulators to drug efficacy. We then use Random Forests, a non-parametric machine learning method, to model the cell lines’ response to different compounds. The model is then used to predict the patients’ response to each drug, identifying which genes contribute the most to the prediction. We then compare our predictive gene signature to commercially available prognostic kits. Random Forest analysis revealed a 28-gene signature (termed EPOCH28) containing several histone chaperones, histone variants, histone modifying enzymes, among other chromatin regulators, which is highly predictive of drug efficacy. Indeed, our model accurately predicts 11 of 14 resistant PDX models and 15 out of 17 sensitive PDX models (~ 84%), with an average confidence level of over 73%. Importantly, we find that the EPOCH28 gene signature is specific, and is thus not a general prognostic signature for patients who will benefit from any chemotherapy. In line with this, the EPOCH28 outperforms the breast cancer prognostic gene signatures of Mammaprint and Oncotype DX, suggesting that while these commercial tests may help guide clinical decisions, it will be critical to consider additional factors that can help identify which drug will be most beneficial to an individual patient. The integration of chromatin regulators as clinical biomarkers, in particular in the context of predictive markers for the response to a single drug, will help guide clinical decisions and treatment options for breast cancer. Importantly, our approach is general and thus can be applied to other chemotherapy drugs, including those in clinical trials to develop companion diagnostics, and for other cancer types. Poster Section 39 Poster Board 8 LB-156 Increased Notch1 expression suppressed the growth but promoted the stemness of gastric cancer cells. Chia-Chen Chiu,1 Chien-Ju Lo,1 Chien-Heng Shen,2 Chung-Kuang Lu,2 Jian-Liang Chou,2 Pei-Yi Chu,3 Shu-Huei Hsiao,1 Min-Jen Tseng,1 Yu-Wei Leu,1 Chia-Chen Hsu1. 1National Chung Cheng Univ., Chia-Yi, Taiwan; 2Chang Gung Memorial Hospital, Chia-Yi, Taiwan; 3St. Martin De Porres Hospital, Chia-Yi, Taiwan. To explore if the Notch pathway were abnormally regulated during gastric tumorigenesis, abnormal DLL1 promoter methylation and inversely correlated mRNA expression were detected in cultured cell lines and patient samples. Elevated DLL1 expression was observed in gastric lesion like gastritis or erosion, but decreased DLL1 expression was detected in all four stages of gastric cancers (n=44). To identify the effector genes and construct the possible Notch signaling network in gastric cancer, Myc-tagged Notch1 was overexpressed in gastric cancer cell line followed by the chromatin-immuno-precipitation sequencing (ChIP-seq) analysis. In ChIP-seq analysis, antibodies against Notch1, Rbpjκ, and Myc were used to identify the Notch1 targets while antibodies against trimethylated histone 3 at lysine 4 (H3K4me3) or lysine 27 (H3K27me3) were used to detect the associated epigenetic states. We found that the Notch1-overexpression suppressed the gastric tumoral growth, and the target loci-associated H3K4me3 (active chromatin) were reduced while the association with H3K27me3 (suppressive chromatin) were increased. Overall, the H3K4me3 and H3K27me3 combined bivalent marks were increased after Notch1overexpression, and this was correlated with promoted stemness as indicated by the increased Oct4 and CD44 (reported gastric cancer stem cell like marker) expression. EZH2, a Polycomb protein, was found to be associated with Rbpjκ which further links the Notch signaling together with the maintenance of cell stemness. Therefore, Notch signaling might be critical for tumor formation in gastric cancer. (Supported by: NSC-102-2320-B-194-003-MY3 and 96 NSC-102-2314-B-371-003-MY3, MOST Taiwan) Poster Section 39 Poster Board 9 LB-157 Demonstration of the usefulness of an epigenetic cancer risk marker by a multicenter prospective cohort study. Masahiro Maeda,1 Kiyoshi Asada,1 Takeshi Nakajima,2 Taichi Shimazu,3 Nobutake Yamamichi,4 Takao Maekita,5 Chizu Yokoi,6 Ichiro Oda,2 Takayuki Ando,1 Takeichi Yoshida,7 Sohachi Nanjo,1 Mitsuhiro Fujishiro,4 Takuji Gotoda,8 Masao Ichinose,7 Toshikazu Ushijima1. 1National Cancer Center Research Institute, Tokyo, Japan; 2National Cancer Center Hospital, Tokyo, Japan; 3Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo, Japan; 4University of Tokyo, Tokyo, Japan; 5 Wakayama, Wakayama, Japan; 6National Center for Global Health and Medicine, Tokyo, Japan; 7Wakayama Medical University, Wakayama, Japan; 8Tokyo Medical University, Tokyo, Japan. An epigenetic cancer risk marker is a promising cancer risk marker that can reflect past exposure to various environmental factors, such as chronic inflammation, unlike single nucleotide polymorphism cancer risk markers. Cross-sectional studies have shown that the degree of accumulation of aberrant DNA methylation in normal-appearing tissues was associated with risk of some types of cancer, especially gastric cancer [Ushijima et al., Clin Cancer Res,2,143,2012]. In this study, we aimed to demonstrate that cancer risk can be predicted by an epigenetic cancer risk marker. For this purpose, we decided to predict the risk of metachronous gastric cancer after endoscopic resection because we could expect a sufficient number of events in three years. 826 patients with early gastric cancer, aged 40-80 years, who had undergone endoscopic resection were enrolled. If the patients were infected with Helicobacter. pylori (H. pylori), a potent inducer of aberrant DNA methylation, eradication was performed. For all enrolled patients, methylation levels of three preselected genes, miR-124a-3, EMX1 and NKX6-1, were measured by quantitative methylation-specific PCR. Patients were followed up annually by endoscopy to detect a metachronous gastric cancer. Authentic metachronous gastric cancers were defined as cancers excluding those detected within 1 year after the enrollment. 782 of 826 patients had at least one follow-up, with a median follow-up of 2.97 years. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1-2, 2-3 and ≥3 years after the enrollment, respectively. The highest quartile of the miR-124a-3 methylation level had a significantly high HR in univariate analysis (95% CI) [2.17 (1.07 to 4.41); p=0.032] and adjusted HR in multivariate analysis [2.30 (1.03 to 5.10); p=0.042] of developing authentic metachronous gastric cancers. Similar trends were observed for EMX1 and NKX6-1. This study, for the first time, demonstrated the usefulness of an epigenetic cancer risk marker by a multicenter prospective cohort study [Asada et al., Gut,1,171,2014]. It is speculated that DNA methylation is accumulated both in stem and differentiated cells of gastric mucosa, and that methylation in stem cells can permanently persist, even after H. pylori infection discontinues, and that DNA methylation accumulated in stem cells is correlated with cancer risk [Ushijima et al., J Gastroenterol,3,161,2006]. Poster Section 39 Poster Board 10 LB-158 P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC). Rebecca D. Greenspan, Nithya Krishnan, Carl Morrison, Angela Omilian, Wiam Bshara, Amber Worral, Kristopher Attwood, Donald L. Trump, Anna Woloszynska-Read, Candace S. Johnson. Roswell Park Cancer Institute, Buffalo, NY. Intrinsic and/or acquired resistance to cisplatin is a major obstacle in the treatment of advanced bladder cancer (BC). It is important to find markers that predict tumor responsiveness to cisplatin-based chemotherapy. P73, a p53 homologue, plays an important role in cisplatin sensitivity. TAp73, a major isoform of p73, Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 97 Late-Breaking Poster Session: Molecular and Cellular Biology 3 utilizes an extrinsic promoter (P1) and is pro-apoptotic. We utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples and found 9 distinct CpGs in the P1 promoter region that were hypermethylated in tumors compared to adjacent normal tissues (p<.05). To determine the functional link between promoter methylation and transcriptional regulation of TAp73, we treated BC cell lines (253J, HT1376, and T24) with DNA hypomethylating agent decitabine (DAC) at various doses (0,1,5 µM). We found that a 1020% decrease in methylation across 5 interrogated CpG sites resulted in a two fold increase in TAp73 expression. We hypothesize that demethylation of the P1 promoter increases TAp73 expression, sensitizing BC cells to cisplatin. To test this, T24 cells were pretreated with DAC (2µM), followed by cisplatin treatment (0.5µg/mL). Clonogenicity of T24 was measured, as well as changes in p73 methylation and expression. Results show a decrease in clonogenic potential following combination therapy. T24 treated with DAC alone or in combination with cisplatin showed a 15% decrease in P1 promoter methylation, while the same was not seen in cells treated with cisplatin alone. Treatment with DAC and cisplatin did not have a clear effect on TAp73 mRNA expression. We are investigating whether TAp73 protein expression reflects the effects of DAC. To determine the clinical value of TAp73 protein expression we utilized tissue microarrays (TMAs) of 354 BC patients. Immunohistochemical staining was assigned H-score indicating intensity of TAp73 expression and percentage of positive nuclei. Results showed lower TAp73 expression to be associated with patient samples of higher tumor grade and staging (p<.001), suggesting that TAp73 expression is lost as the disease progresses from superficial to advanced BC. Consequently, low TAp73 protein expression was associated with shorter overall survival (p<.05). To further investigate clinical utility of TAp73 promoter methylation and expression, we analyzed tumor samples from 24 patients treated with cisplatin in a neoadjuvant setting. Results showed hypermethylation in tumor compared with non-tumor samples (p<.05), but no significant difference in mRNA expression was observed. Studies continue to focus on understanding the mechanism of cisplatin resistance in the context of epigenetic dysregulation and utilization of DAC as a potentiating agent of cispatin-based chemotherapy in muscle invasive BC. Supported by NCI grants CA067267 and CA016056. Poster Section 39 Poster Board 11 LB-159 Methylation of BRCA1 gene in blood is not inherited via maternal germ line and may predispose to triple-negative or medullary breast cancer. Tomasz K. Wojdacz,1 Satish Gupta,2 Katarzyna Jaworska-Bieniek,2 Florencia Harari,3 Steven Narod,4 Karin Broberg,3 Lise Lotte Hansen,1 Jan Lubinski,2 Anna Jakubowska2. 1Aarhus University, Aarhus, Denmark; 2Pomeranian Medical University, Szczecin, Poland; 3Karolinska Institute, Stockholm, Sweden; 4Womens College Research Institute University of Toronto, Toronto, Ontario, Canada. Methylation of the BRCA1 gene in peripheral blood (epimutation) has been associated with pathology of early onset breast cancer (1). This epimuataion was suggested to be of constitutional origin and hence can potentially be transmitted to the next generation as previously reported for MLH1 gene (2). We used methylation sensitive high resolution melting technique (MS-HRM) (3) to measure methylation of BRCA1 gene in blood samples from 226 healthy women from the Andean region in northern Argentina. In total 29 (13%) of the women showed detectable methylation of this gene. The analyses of mother-daughter pairs (n=6) in this study, showed discordant methylation of BRCA1 between generations, with mothers that tested positive having daughters that tested negative for BRCA1 methylation, and vice versa. This indicates that the BRCA1 epimutation is not transmitted from mother to daughters via the maternal germline. Furthermore we conducted a casecontrol study of 66 breast cancer cases and 36 unaffected controls of polish women selected from registry of International Hereditary Cancer Centre (IHCC), Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland. Cases were triple-negative or of medullary histology, or both; 30 carried a constitutional BRCA1 mutation and 36 did not carry a mutation. Methylation of the BRCA1 promoter was detected in 15 of 66 cases and in 2 of 36 controls (OR 5.0, p = 0.03). Methylation was present in 15 of 36 women with breast cancer and without germline BRCA1 mutation, but in none of 30 women with breast cancer and a germline mutation (p < 0.01). The association between methylation and breast cancer was restricted to women with no constitutional BRCA1 mutation (OR 12.1, p = 0.0006) (4). In conclusion it is unlikely that epimutation of BRCA1 is inherited through maternal germ line and this epimutation may be a marker of increased susceptibility to triple-negative or medullary breast cancer. References 1.. Wong EM. et al, Constitutional methylation of the BRCA1 promoter is specifically associated with BRCA1 mutationassociated pathology in early-onset breast cancer. Cancer Prev Res (Phila). 2011 Jan;4(1):23-33. 2.. Hitchins MP, et al, Inheritance of a cancer-associated MLH1 germ-line epimutation. N Engl J Med. 2007 Feb 15;356(7):697-705. 3.. Wojdacz TK, Dobrovic A, Hansen LL,. Methylation-sensitive high-resolution melting. Nat Protoc. 2008;3(12):1903-8. 4.. Gupta S et al, Methylation of the BRCA1 promoter in peripheral blood DNA is associated with triple-negative and medullary breast cancer. Breast Cancer Res Treat. Breast Cancer Res Treat. 2014 Dec;148(3):615-22. Poster Section 39 Poster Board 12 LB-160 HDAC9 expression is deregulated in malignant B-cell lymphomas in particular in diffuse large B-cell lymphoma and mantle cell lymphoma. Elena Cubedo,1 Veronica Gil,2 Chae Hwa Kim,1 German Campuzano-Zuluaga,1 Nitin Kumar Agarwal,1 Louise Howell,2 Kevin R Petrie,2 Francisco Vega,1 Arthur Zelent1. 1University of Miami, Miami, FL; 2The Institute of Cancer Research, Sutton, United Kingdom. Histone Deacetylase 9 (HDAC9) is a class IIa chromatinmodifying enzyme that, within the hematopoietic system, is preferentially expressed in the B-cell lineage. In our previous works, in order to identify HDAC9 function in the B cell lineage we developed mice that constitutively expressed human HDAC9 from early stages of B-cell development, under the control of the immunoglobulin heavy chain (IgH) enhancer. These mice developed lymphoproliferative disorders, including indolent Marginal Zone Lymphoma (MZL) and more aggressive post-Germinal Center (GC) lymphomas, demonstrating an oncogenic role for HDAC9 in B-cells. In order to examine the relationship between diseases observed in the mouse model and human primary lymphoma, we have examined, using immunohistochemistry (IHC) the expression of full length HDAC9 isoform in a panel of various B-cell malignancies from human tumor samples. The study group included 59 nonHodgkin lymphomas (NHL), and 3 classical HL. Non-HL consisted of 34 diffuse large B cell lymphoma (DLBCL), 9 follicular lymphoma (FL), 5 marginal zone lymphoma (MZL), 6 mantle cell lymphoma (MCL), and 2 small lymphocytic lymphomas (SLL). HDAC9 expression was assessed by IHC using tissue microarray and/or routine tissue sections. Protein expression was scored as negative (0), low (1), or high (2) depending on the staining signal intensity. Expression of HDAC9 in the nuclei of the tumor cells was compared with that seen in adenocarcinoma cells; if equal or higher, then expression of HDAC9 was considered high and if lower, then expression of HDAC9 was considered low. Five reactive lymph nodes were studied to assess the baseline expression of HDAC9. Rectal adenocarcinomas were used as positive controls. In reactive lymph nodes, HDAC9 was weakly expressed in a subset of germinal center cells, a subset of lymphoid cells in the paracortex as well as in endothelial cells. HDAC9 expression was detected in all subsets of B-cell American Association for Cancer Research • AACR ANNUAL MEETING 2015 97 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 98 Late-Breaking Poster Session: Molecular and Cellular Biology 3 lymphomas analyzed and in most cases with a level of expression higher than those seen in reactive lymph nodes. DLBCL and MCL tumors had the highest frequency of high HDAC9 expression among the B-cell lymphomas analyzed, 77 and 83% (Fisher’s exact test P=1.0), respectively. No differences in HDAC9 expression were detected in DLBCL of GC and non-GC type. In contrast, most (69%) of the low-grade B cell lymphomas showed no or lower expression of HDAC9 (Fisher’s exact test P=0.004; as compared to DLBCL). Classical HL showed frequently low-expression of HDAC9 in the tumor cells. In summary, HDAC9 is frequently expressed in B-cell lymphomas with the highest level of expression found in the most aggressive lymphomas such as DLBCL and MCL. These findings support the biological role of HDAC9 in the pathobiology of aggressive B cell neoplasms and highlight the need to further study HDAC9 function in these malignancies as well as its importance as a therapeutic target. Poster Section 39 Poster Board 13 LB-161 Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells. Young Seok Ju, Jose Tubio, William Mifsud, Beiyuan Fu, ICGC Prostate Cancer, Bone Cancer, Breast Cancer Working Groups, Fengtang Yang, Peter Campbell, Michael Stratton. Wellcome Trust Sanger Institute, Cambridge, United Kingdom. Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm and the mitochondrial double membrane. Despite these physical barriers we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements and the features of the fusion fragments indicate that non-homologous end joining and/or replication-dependent DNA double strand break repair are the dominant mechanism involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. Poster Section 39 Poster Board 14 LB-162 Oxidative damage disrupts telomere integrity and leads to cell senescence. Rong Tan, Luxi Sun, Ying Gao, Li Lan. University of Pittsburgh, Pittsburgh, PA. Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising less than one percent of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence and cell death in U2OS, HeLa, and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including signal-free ends and sister-chromatid fusions, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining 98 telomere integrity upon oxidative damage. Poster Section 39 Poster Board 15 LB-163 Pan-cancer analysis of the causes and consequences of Intra-tumor heterogeneity. Noemi Andor,1 Trevor A. Graham,2 Athena C. Aktipis,3 Claudia Petritsch,4 Hanlee P. Ji,5 Carlo C. Maley3. 1Stanford, San Francisco, CA; 2Evolution and Cancer Laboratory, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, London, United Kingdom; 3Center for Evolution and Cancer, University of California San Francisco, San Francisco, CA; 4Department of Neurological Surgery, University of California San Francisco, San Francisco, CA; 5 Stanford, Palo Alto, CA. Tumors are typically mosaics of mutant clones that have evolved from a common ancestral cell. This intra-tumor heterogeneity (ITH) is thought to drive both neoplastic progression and acquired therapeutic resistance. Availability of just one sample per tumor and moderate sequencing depth have limited systematic analysis of ITH during previous TCGA pan-cancer analyses, confining the study of ITH to a small numbers of tumor samples and cancer types. The molecular and histopathological causes of ITH and its prognostic significance have thus far remained uncertain. To overcome these limitations, we used an analysis method called EXPANDS that estimates the proportion of cells harboring specific mutations from exome sequencing data, as well as other methods that quantify ITH. We extrapolate the number of genetically diverse clonal subpopulations in 1,165 primary tumors among 12 different cancer types from TCGA and investigate mechanisms underpinning ITH as well as the correlation of ITH and genomic instability with prognosis. Lastly we validate the prognostic significance of genomic instability in an independent, high-density SNP-array dataset consisting of 2010 tumor samples, across seven additional cancer types. We found evidence of ITH in the vast majority of tumors. Mutations in driver genes tended to have a characteristic clone size, suggesting differential fitness effects of those mutations. The detection of a mutation in a driver gene that typically appears in a small clone was a predictor of poor survival. Driver gene mutations, tumor microenvironment composition and prevalence of copy number gains were significantly associated with increased ITH. In general, the prevalence of copy number alterations was a universal biomarker of prognosis: tumors with intermediate copy number burden (50 to 75% of the genome affected by copy number alterations) progressed faster than tumors with higher copy number burden, independent of cancer type. Chemo-radiation therapy administration was more efficient in decelerating tumor progression among patients with intermediate copy number burden than among patients with low or high copy number burden. These results were validated and confirmed in the independent SNP-array dataset. This study suggests a tradeoff exists between the costs and benefits of genomic instability that impacts both the evolvability and fitness of the tumor cell population. In the future, this tradeoff might be exploited to improve survival. Poster Section 39 Poster Board 16 LB-164 Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells. Dario Palmieri, Mario Scarpa, Anna Tessari, Rexhep Uka, Foued Amari, Cindy Lee, Timothy Richmond, Tyler Sheetz, Jeffrey Parvin, Thomas Ludwig, Carlo M. Croce, Vincenzo Coppola. The Ohio State University, Columbus, OH. Currently, most of the anti-neoplastic treatments for lung cancer patients are based on DNA-damaging cytotoxic therapies such as platinum-containing compounds and radiotherapy. Therefore, a better understanding of the molecular mechanisms involved in the cellular response to genotoxic stresses is required in order to improve the clinical management of lung cancer. Ran Binding Protein 9 (RanBP9, also known as RanBP-M) is a ubiquitous and evolutionary conserved scaffold protein that shuttles Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 99 Late-Breaking Poster Session: Molecular and Cellular Biology 3 between the nucleus and the cytoplasm, and interacts with many major players in tumor biology. However, its biological functions are not well studied and still debated. Here, we show that RanBP9 interacts with and is phosphorylated by ATM, one of the apical activators of the DNA damage response (DDR) following DNA Double-Strand Breaks (DSBs). Following ATM-dependent phosphorylation, RanBP9 rapidly accumulates into the nucleus of lung cancer cells. By using three different lung cancer cell lines (A549, H460, and H1299), we found that stable silencing of RanBP9 (ShRanBP9) significantly affects the DDR. In fact, stable ShRanBP9 clones display a delayed and/or reduced activation of key components of the cellular response to Ionizing Radiation (IR), including ATM, Chk2, H2AX-γ and p53. Accordingly, abrogation of RanBP9 expression significantly affected Homology-Directed repair of damaged DNA. On the other hand, stable silencing of RanBP9 results in increased IR-induced senescence and apoptosis. In summary, here we present evidence that RanBP9 is a novel mediator of the cellular DDR, whose recruitment into the nucleus upon IR is dependent on ATM kinase activity. In turn, nuclear RanBP9 participates to the efficient activation of cellular DDR. On the contrary, its absence hampers the repair of damaged DNA following DSBs, resulting in enhanced lung cancer sensitivity to genotoxic stresses. Taken together, our findings suggest that targeting RanBP9 might represent a new potential approach to increase sensitivity of lung cancer cells to genotoxic anti-neoplastic treatments. Poster Section 39 Poster Board 17 LB-165 Antiproliferative effects of AZD6738 and the inhibition of DDR activity. Hee Jun Kim,1 Ahrum Min,2 Seock-Ah Im,3 Hyemin Jang,2 Miso Lee,2 Seongyeong Kim,2 Jungen Kim,2 Kyung-Hun Lee,3 Sae-Won Han,3 Tae-Yong Kim,3 Do-Youn Oh,3 Tae-You Kim,3 Yung-Jue Bang3. 1 Chung-Ang University Hospital, Cancer Research Institute, Seoul National University, Seoul, Republic of Korea; 2Cancer Research Institute, Seoul National University, Seoul, Republic of Korea; 3 Cancer Research Institute, Seoul National University, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea. Background: Anti-tumor effect of DNA damaging agents was reduced by the activation of DNA damage repair (DDR), which was led to resistance. Therefore, the inhibition of DNA repair could lead to induce the accumulation of errors which is becoming an attractive strategy. The ataxia telangiectasia and Rad3-related (ATR) proteins play a role of sensor for DNA damage, which induces homologous recombination-dependent repair. ATR is a master regulator of DDR, signaling to control DNA replication, DNA repair, and apoptosis. Therefore, the ATR pathway might be useful target for new drug development and it is important that the effects of many current cancer treatments are modulated by DDR. Materials and Methods: The growth inhibitory effects of ATR inhibitor, AZD6738 were studied on human breast cancer cell lines using MTT assay. Cell cycle analysis and western blotting were also performed to determine molecular changes. Immunofluorescence assay and comet assay were conducted to understand the action mechanisms of AZD6738. Results: This research identified the anti-proliferative effects and the inhibition of DDR activity by AZD6738 on human breast cancer cell lines. AZD6738 induces cell cycle arrest and apoptosis, which impaired DDR function and promoted cell death by damage accumulation. Results of MTT showed that the heterogeneous response and two cell lines were chosen for the focus on the HER2positive breast cell lines: SKBR-3 and BT-474. In sensitive cell line, SKBR-3 cell, the expression of phosphorylated CHK1 was reduced with the other repair markers; RAD51, MRE11 and ERCC1 as opposed to less sensitive breast cancer cell BT-474. The decreased functional CHK1 leads to the accumulation of DNA damage due to homologous recombination inactivation. And it was also identified that ATR inhibitor played a role of sensitizer to increase the efficacy of cytotoxic chemotherapeutic agents, cisplatin and paclitaxel in breast cancer cell lines. Conclusion: Understanding the antitumor efficacy and the mechanisms of ATR inhibitor in the breast cancer cell lines open up the possibility of future clinical trial targeting DNA damage repair in breast cancer Poster Section 39 Poster Board 18 LB-166 PARP inhibition effects on endocrine therapy and resistance in estrogen receptor positive (ER+) breast cancer models. Agostina Nardone,1 Amit Goldstein,1 Martin J. Shea,1 Tamika Mithchell,1 Xiaoyong Fu,1 Carmine De Angelis,1 Huizhong Hu,1 Xiaowei Xu,1 Mahitha Rajendran,1 Mark O’Connor,2 Gershon Locker,2 Susan Hilsenbeck,1 Kent Osborne,1 Rachel Schiff1. 1Baylor College of Medicine, Houston, TX; 2AstraZeneca, Macclesfield, United Kingdom. Background: Poly [ADP-ribose] polymerase (PARP) is an important mediator of DNA damage repair. Preclinical and clinical studies have indicated PARP inhibitors (i) as promising agents in DNA-repair-defective cancers, especially in the presence of BRCA1/2 alterations. Recent evidence also suggests a role for additional DNA damage-independent functions of PARP, involving transcriptional and epigenetic regulation. Particularly, PARP-1 can interact with the estrogen receptor (ER) and modulate its transcriptional activity in vascular smooth muscle cells, and PARPi can circumvent endocrine resistance in prostate cancer cells. However, the effect of PARPi on the efficacy of endocrine therapy (EndoTx) in ER+ breast cancer is unknown. Here we aimed to establish these effects using the PARPi olaparib (AZD2281, Olap) in our preclinical models. Methods: The effects of Olap (0.01-1µM) alone or in combination with EndoTx [estrogen deprivation (ED) or tamoxifen (Tam)] were tested in vitro in three ER+ parental cell lines (MCF7, ZR75-1, and T47D; BRCAwt ) and their endocrine resistant (R) derivatives. Clonogenic assays were used to assess Olap-induced changes in cell growth. In vivo, ovariectomized nude mice bearing 200 mm3 tumors growing in the presence of estrogen supplementation (E2 pellets), were randomized to either continued E2 (control), ED, or ED+Tam; all ± oral Olap (100mg/kg daily). Results: In MCF7 parental cells in vitro, Olap, in a dose dependent manner, inhibited E2-stimulated growth and increased the growth inhibitory effect of EndoTx. In addition, at a clinically relevant dose (1µM), Olap significantly inhibited the growth of MCF7 EndoR cell derivatives. Similar results were also observed in the other 2 ER+ cell models. In vivo, long-term Olap-treatment in the presence of EndoTx (Tam ~200days, and ED ~300days) was well tolerated and no apparent drug-related toxicity was observed. Olap did not affect the growth of E2-stimulated MCF7 xenografts. In contrast, Olap enhanced sensitivity to Tam by delaying time to tumor progression (TTP; size doubling) from a median of 95 days to 155 days (p=0.03), and numerically but not significantly reduced time to tumor regression (TTR; size halving) from 138 to 51 days (p=0.2). Olap also numerically accelerated median time to tumor regression in the presence of ED (34 ED vs. 25 days ED+Olap; p=0.23), but had no effect on TTP (P=0.36). Conclusion: Our results suggest a potential role for Olap in enhancing EndoTx efficacy and circumventing resistance in the absence of BRCA mutations . The absence of any toxicity and negative interaction by adding Olap to EndoTx in the in vivo experiment supports the inclusion of ER+ breast cancer patients in clinical trials using PARPi. Further studies are warranted to better understand the biology of PARP and the efficacy of PARPi in ER+ breast cancer, especially in the presence of EndoTx and resistance. Poster Section 39 Poster Board 19 LB-167 The role of Sirt1 in tumorigenesis. Natalie Shunxiang Ren. NIH\NIEHS, Durham, NC. American Association for Cancer Research • AACR ANNUAL MEETING 2015 99 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 100 Late-Breaking Poster Session: Molecular and Cellular Biology 3 SIRT1 is a highly conserved NAD+-dependent protein deacetylase. Homologs of SIRT1 in lower model organisms are able to delay the aging process in response to nutrients. Although the SIRT1 protein deacetylase activities have been shown to regulate numerous transcription factors and co-factors (eg. PCG-1, p53), modulate cellular metabolism and promote cell survival and stress resistance, the role of SIRT1 in cancer metabolism, growth and survival is still highly controversial. Colorectal cancer is one of the most common malignancies in the world. However, there are few studies revealing the clinical relevance of the expression of SIRT1 and related markers in colorectal cancer with human tissue. Although recent studies have revealed that SIRT1 plays an important role in colorectal tumorigenesis, the exact role of SIRT1 in colorectal tumorigenesis is still controversial. Thus far, our data demonstrates that knocking out SIRT1 induces the acetylation of p53 and alters cellular metabolism. Deletion of SIRT1 in MEFs decreases glutamine uptake and increases cell death by trigging apoptosis pathways. As a result, SIRT1 KO MEFs fails to induce tumors in a xenograft model in nude mice. We have recently generated a mouse colon cancer model on SIRT1 intestine knockout mouse strains (SIRT1 iKO) and discovered that SIRT1 iKO mice display a reduced rate of tumorigenesis. To further confirm the role of SIRT1 on colon cancer, we propose to analyze the association of SIRT1 expression and the prognosis in colorectal cancer. Poster Section 39 Poster Board 20 LB-168 Comprehensive genomic characterization of small cell lung cancer. Julie George,1 Jing Shan Lim,2 Martin Peifer,1 Julien Sage,2 Roman Thomas1. 1Department of Translational Genomics, University of Cologne, Cologne, Germany; 2Departments of Pediatrics and Genetics, Stanford University, Stanford, CA. Small cell lung cancer (SCLC) is a highly aggressive, neuroendocrine tumor of the lung that accounts for 15-20% of all lung cancer malignancies. SCLC patients are usually diagnosed with an extensive stage of a tumor, which complicates further surgical resections. The current standard of care is a platinumbased chemotherapy and targeted therapies have not yet been identified for the treatment of SCLC patients. We sought to comprehensively characterize the genomic alterations in SCLC to identify novel candidate targets for therapy. To this end, we performed whole genome sequencing on 110 tumor-normal pairs and transcriptome sequencing on 80 primary tumors. Following the paradigm of classical Knudson tumor suppressors, TP53 and RB1 were detected with somatic, bi-allelic genomic alterations, thus emphasizing the loss of both tumor suppressors as an obligatory event in SCLC. Two cases did not carry genomic alterations in TP53 and RB1, but instead were found to undergo chromothripsis; genomic rearrangements between chromosome 3 and 11 led to the upregulation of Cyclin D1, thus revealing an alternative mechanism of Rb1 deregulation. Additionally, SCLC tumors were found to harbor complex genomic rearrangements of the RB1 and TP53 family members RBL1 (p107), RBL2 (p130) and TP73, the latter resulting in the oncogenic splice variant TP73Δex2/3. SCLC tumors revealed focal amplifications of MYC transcription factors and FGFR1 in 10% and 6 % of the cases analyzed, respectively. Additionally, low frequent alterations were detected in SCLC, such as focal amplifications of IRS2, a candidate oncogene involved in the IGF1R tyrosine kinase signaling pathway. A few SCLC cases were found to harbor mutations with possible immediate therapeutic implications; including mutations in BRAF, KIT and PIK3CA. The integrative study for significantly mutated genes confirmed that the histone acetyl transferases CREBBP and EP300 were inactivated in 25 % of the tumors(1). Additionally, SCLC tumors showed frequent alterations of centrosomal proteins and in 25% of 100 the cases inactivating mutations of NOTCH family genes. The transcriptional profile of SCLC tumors confirmed a high expression of neuroendocrine markers and of the NOTCH pathway regulator Dlk1, pointing to the notion that NOTCH functions as a tumor suppressor in SCLC tumors. We sought to analyze the impact of Notch re-activation in a preclinical SCLC mouse model by introducing the Notch intracellular domain (NICD) in mice with conditional lung epithelial triple knock-out (TKO) of Trp53, Rb1 and Rbl2(2). NICD expression dramatically reduced the number of tumors arising in TKO mice and led to an extended survival. Additionally, Notch activation abrogated the expression of neuroendocrine markers. In summary, this whole genome sequencing analysis provides a comprehensive understanding of the genomic nature of SCLC tumors endorsing universal bi-allelic loss of TP53 and RB1 as key events in the pathogenesis of SCLC. This study further emphasizes NOTCH as a tumor suppressor and regulator of neuroendocrine differentiation in SCLC tumors. Furthermore, this large-scale study uncovers several biological key processes and candidate therapeutic targets in this deadly form of cancer. 1. Peifer, M. et al. Nat. Genet. 44, 1104-10 (2012). 2. Schaffer, B. E. et al. Cancer Res. 70, 3877-83 (2010). Poster Section 39 Poster Board 21 LB-169 Multidimensional genomic dissection of chromosome 9p in glioma. David M. Roy,1 Logan A. Walsh,2 Alexis Desrichard,2 Jianjiong Gao,2 Promita Bose,2 Jason T. Huse,2 William Lee,2 Timothy A. Chan2. 1 Weilll Cornell Medical College, New York, NY; 2Memorial Sloan Kettering Cancer Center, New York, NY. Background: Characterization of focal somatic copy number alterations (SCNAs) has led to the identification of many cancer genes, yet similar investigations of arm-level SCNAs remain challenging. The identity of driver genes within these broad SCNAs remains a critical unanswered question in cancer genetics. One of the most frequent arm-level SCNAs is 9p loss, which contains the tumor suppressor gene (TSG) CDKN2A. It is also believed that other TSGs exist on 9p, though their identity has yet to be revealed. Methods: We analyzed every arm-level SCNA in 28 cancer types from The Cancer Genome Atlas (TCGA) to clarify the relative impact of 9p loss across cancer. We also performed a multi-tiered genomic dissection of chromosome 9p in 540 patients from 3 independent lower grade glioma (LGG) datasets (TCGA, REMBRANDT, MSKCC) to pinpoint genetic loci that are tied to tumor aggressiveness and poor survival. Focal and arm-level SCNAs were determined by GISTIC2.0. As per recently proposed criteria, 3 LGG subtypes were clustered by IDH mutation and 1p/19q deletion status. Survival analyses were performed using logrank tests or Cox regression. Results: We found that chromosome 9p loss is one of the most frequent and prognostic arm-level events across 28 TCGA cancer types. Of these, the strongest 9p survival association was found in LGG. We performed a large-scale associations test of 376 important clinical and molecular variables and revealed that 9p loss was only associated with 6q or 9q loss, and 9p loss alone was sufficient to predict poor prognosis. On subtype-specific analysis, 9p loss predicted worse overall survival (OS) in both IDH mutant LGG subtypes but not IDH wild-type (IDHwt). To identify underlying drivers on 9p, we identified 87 genes most frequently targeted by 9p loss. Additional genetic events were rare except for homozygous deletion (HD) at 9p21.3, which contains CDKN2A. In contrast, mRNA/miRNA expression at all gene loci was much more variable and poorly correlated to copy number status. Therefore, pan-LGG and subtype-specific gene expression analyses were used to identify several drivers of tumor aggressiveness, notably KLHL9, MTAP, PLAA, and PTPRD. Although CDKN2A HD was linked to worse OS in a pan-LGG analysis, this event significantly cooccurred with the LGG IDHwt subtype, a group with GBM-like Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 101 Late-Breaking Poster Session: Molecular and Cellular Biology 3 survival outcomes. Further, CDKN2A copy number status and expression did not predict worse OS in any subtype. Discussion: We characterize the nature of 9p loss in LGG, pinpoint genes involved in worse survival, and redefine the role of the tumor suppressor CDKN2A. These results provide new critical insight into long-standing questions about the nature of chromosome 9p loss and establish a framework for examining other arm-level SCNAs in cancer. in GIST and can increase our understanding of the underlying cancer biology as well as suggesting treatment possibilities for this malignancy. We acknowledge the contributions of the NIH WildType GIST clinic investigators to this project. References 1. Killian JK, Miettinen M, Walker RL, Wang Y, Zhu YJ, Waterfall JJ, et al. Recurrent epimutation of SDHC in gastrointestinal stromal tumors. Sci Transl Med. 2014;6:268ra177. Poster Section 39 Poster Board 22 LB-170 Single cell sequencing identifies clonal stasis and punctuated copy number evolution in triple-negative breast cancer patients. Ruli Gao,1 Alexander Davis,1 Emi Sei,1 Xiuqing Shi,2 Anna Unruh,1 Jill Waters,3 Amy (Hong) Zhang,1 Funda Meric-Bernstam,1 Nicholas Navin1. 1University of Texas MD Anderson Cancer Center, Houston, TX; 2Chinese Medical College, Beijing, China; 3Illumina Inc., San Diego, CA. Intratumor heterogeneity is widely reported in many human tumors including breast cancer. However most studies to date have been limited to genomic analysis of bulk tumor tissues and cytogenetic analysis. Here, we applied single cell copy number profiling to delineate the clonal substructure of triple-negative (ER-, PR-, Her2-) breast cancer (TNBC). We applied Single Nucleus Sequencing (SNS) to profile genomic copy number in 1000 single cells from 11 TNBC patients. Our data show that copy number profiles within the tumor are highly stable, belonging to a few (N=13) major clonal subpopulations that share similar amplifications and deletions,. In addition to the clonal cells, we also identified diverse copy number profiles that belong to two classifications: 1) replicating cells, or 2) apoptotic cells. We also find evidence of rare diploid cells that harbor non-clonal amplification and deletions and may play an important role in aneuploidy initiation. Our data suggests a model of punctuated genome evolution and clonal stasis, in which aneuploidy occurs early in tumor evolution, followed by one or more stable clonal expansion to form the tumor mass. These data challenge the paradigm of gradual copy number evolution and extensive copy number diversity in triple-negative breast cancer, and suggest that that amplifications and deletions are ideal markers for personalized cancer therapy. Poster Section 39 Poster Board 24 LB-172 Novel candidate oncogenic drivers in pineoblastoma. Matija Snuderl,1 Kasthuri Kannan,1 Olga Aminova,1 Igor Dolgalev,1 Adriana Heguy,1 Arline Faustin,1 David Zagzag,1 Sharon L. Gardner,1 Jeffrey C. Allen,1 Jeffrey H. Wisoff,1 David Capper,2 Volker Hovestadt,2 Sama Ahsan,3 Charles Eberhart,3 Stefan M. Pfister,2 David T. W. Jones,2 Matthias A. Karajannis1. 1NYU Langone Medical Center, New York, NY; 2German Cancer Research Center, Heidelberg, Germany; 3Johns Hopkins Hospital, Baltimore, MD. Introduction: Pineoblastoma (PB) is one of the rarest and most aggressive brain tumors of childhood . PB is considered a “primitive neuroectodermal tumor” (PNET) based on histology, and commonly treated using treatment protocols developed for medulloblastoma; however the survival remains poor. A subset of PBs may occur in the setting of germline mutations involving DICER1 or RB1, but no next-generation sequencing studies have been published on PB to date, and the genetic drivers of sporadic PB remain unknown. Methods: Twenty-one tumor samples with a histological diagnosis of PB (including recurrent/metastatic samples) from 15 patients were included in this study. Matching germline DNA was available from 2 patients. We performed genome-wide methylation array profiling (Illumina Infinium 450k) on all samples, as well as whole-genome (for samples with matching germline DNA) or wholeexome sequence analysis. Fluorescence in situ hybridization (FISH) and digital droplet PCR (ddPCR) was performed to confirm select focal somatic gains. Results: 14/18 samples from 9/13 patients analyzed by 450k profiling had a methylation signature similar to previously profiled PBs from a reference cohort. Samples from 4 patients were found to be more consistent with a diagnosis of embryonal tumor with multilayered rosettes (ETMR) - like tumor (non 19q amplified), papillary tumor of the pineal region, or pineal parenchymal tumor of intermediate differentiation, respectively. No mutations in DICER1 or RB1 were found. Homozygous deletions in DROSHA were found in tumors from 3 PB patients. In addition, we identified novel recurrent somatic gains involving chromosomal region 1q21 that were confirmed by FISH and ddPCR in 4/5 PB patients. Conclusion: Our studies revealed multiple candidate drivers of oncogenesis in PB. We identified novel homozygous deletions in DROSHA, a nuclease involved in microRNA processing. We also identified novel, highly recurrent somatic focal gains involving chromosomal region 1q21, which has been linked to brain growth, autism and schizophrenia, but not previously associated with cancer. Poster Section 39 Poster Board 23 LB-171 Collaborating mutations in gastrointestinal stromal tumor. Joshua Waterfall, J. Keith Killian, Yuelin Jack Zhu, C. Christopher Lau, Markku Miettinen, Lee J. Helman, Paul S. Meltzer. NCI/NIH, Bethesda, MD. Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor of the gastrointestinal tract, is driven most commonly by activating mutations in the receptor tyrosine kinases KIT or PDGFRA. Alternatively, inactivating mutations of genes for the succinate dehydrogenase complex (SDH) constitute a distinct oncogenic mechanism in GIST. Using integrated genomic analyses we have recently reported that SDH deficiency in GIST may also arise from epigenomic inactivation of SDHC, reflected by coupled DNA methylation and expression silencing (1). SDHC epimutation is particularly frequent in, though not restricted to, patients with the cancer predisposition syndrome Carney Triad. It is well established that these primary driver mechanisms correlate with unique clinical, molecular, and epidemiological profiles. In order to identify collaborating oncogenic pathways associated with specific GIST subtypes we have extended our integrated genomic profiling of over 70 tumors enriched for SDH deficient cases. The primary focus is sequencing a panel of 200 cancer related genes for both somatic and inherited variants. We also include RNA expression arrays, DNA methylation arrays, and SNP arrays in this analysis. We have identified mutations in several known cancer associated genes including PTEN, KRAS, ARID1A, and TP53. In conclusion, collaborating mutations and deregulated pathways are identifiable Poster Section 39 Poster Board 25 LB-173 The Thailand initiative in genomics and expression research for liver cancer (TIGER-LC): Defining novel subtypes of hepatocellular carcinoma and cholangiocarcinoma. The TIGER-LC Consortium, Jittiporn Chaisaingmongkol,1 Anuradha Budhu,1 Hien Dang,1 Siritida Rabibhadana,2 Benjarath Pupacdi,2 Marshonna Forgues,1 Vajarabhongsa Bhudhisawasdi,3 Nirush Lertprasertsuke,4 Anon Chotirosniramit,4 Chawalit Pairojkul,3 Chirayu U. Auewarakul,5 Thaniya Sricharunrat,5 Kannika Phornphutkul,6 Suleeporn Sangrajrang,7 Maggie Cam,1 Ping He,8 Stephen M. Hewitt,1 Xiaolin Wu,1 Snorri S. Thorgeirsson,1 Paul S. Meltzer,1 Christopher A. Loffredo,9 Robert H. Wiltrout,1 Curtis C. Harris,1 Chulabhorn Mahidol,2 Mathuros Ruchirawat,2 Xin W. Wang1. 1 National Cancer Institute, Bethesda, MD; 2Chulabhorn Research Institute, Bangkok, Thailand; 3Khon Kaen University, Khon Kaen, American Association for Cancer Research • AACR ANNUAL MEETING 2015 101 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 102 Late-Breaking Poster Session: Molecular and Cellular Biology 3 Thailand; 4Chiang Mai University, Chiang Mai, Thailand; 5 Chulabhorn Hospital, Bangkok, Thailand; 6Rajavej hospital, Chiang Mai, Thailand; 7National Cancer Institute, BANGKOK, Thailand; 8 FDA, Bethesda, MD; 9Georgetown University Medical Center, Washington, DC. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent two major histological cancer subtypes confined within the liver. They are clinically and biologically heterogeneous, and are highly resistant to treatment, which makes them the second most lethal cancer for men in the world. In Thailand, liver cancer represents the primary cause of cancer-related death and is a major health problem, especially in north-eastern area of Thailand where liver fluke (O. viverrini) is endemic and approximately 70% of liver cancers are CCA. While HBV and HCV are major etiological factors for HCC globally, liver fluke infection is a major etiological factor for CCA in Thailand. These unique risk factor patterns provide an opportunity to study cancer heterogeneity and its unique tumor biology. The Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC) consortium was established to identify genomic and expression factors that may modify HCC and CCA susceptibility and progression. In a Phase I study, we determined molecular subtypes of HCC and CCA. We performed genomic profiling of 398 surgical specimens derived from 199 liver cancer patients. We employed the Affymetrix Human Transcriptome Array 2.0 to examine transcriptome profiles. Unsupervised Consensus Clustering (cCluster), Subclass Mapping (SM) and Gene Set Enrichment Analysis (GSEA) algorithms were used to analyze transcriptome data. The results were validated in 247 Asian HCC cases and 104 Caucasian CCA cases. We found that the Thai HCC cases consisted of 3 stable subgroups (C1-C3), while the Thai CCA cases contained 4 stable subgroups (C1-C4) based on gene expression patterns determined by cCluster. SM analysis revealed that CCA-C1 and HCC-C1 subtypes shared a similar gene expression matrix, as did CCA-C2 and HCC-C2 for a separate pattern. Interestingly, patients in both CCA-C1 and HCC-C1 had a poor prognosis, while those in CCA-C2 and HCC-C2 had a good prognosis. These prognostic subtypes were validated in an independent Asian HCC cohort but not in a Caucasian CCA cohort. GSEA revealed that among 17 significantly altered canonical pathways in the C1 subtype, 8 are related to mitotic checkpoint signaling. In contrast, the main signaling pathways associated with the C2 subtype were related to cytokine and chemokine signaling. We found that certain mitotic checkpoint genes are highly activated only in C1, but not in the C2 subtype. These results are consistent with the hypothesis that CCA and HCC from Asian populations consist of molecularly-similar tumor subgroups with similar prognostic impacts and unique tumor biology and that the C1 subtype may be sensitive to mitotic checkpoint blockage. Our ability to rigorously classify and validate both HCC and CCA using these tools may represent a new avenue for the development of targeted therapeutic interventions. Poster Section 39 Poster Board 26 LB-174 Examining the genomic differences between upper and lower tract urothelial carcinomas. Sasinya N. Scott,1 John P. Sfakianos,2 Eugene K. Cha,1 Gopa Iyer,1 Emily C. Zabor,1 Philip H. Kim,1 A. A. Hakimi,1 Irina Ostrovnaya,1 Ricardo Ramirez,1 Aphrothiti J. Hanrahan,1 Neil Desai,1 Qinghu Ren,1 Arony Sun,1 Jonathan E. Rosenberg,1 Guido Dalbagni,1 Dean F. Bajorin,1 Michael F. Berger,1 Bernard H. Bochner,1 Hikmat AlAhmadie,1 David B. Solit,1 Jonathan A. Coleman1. 1Memorial Sloan Kettering Cancer Center, New York, NY; 2Mount Sinai, New York, NY. Background: Urothelial carcinomas are capable of arising at multiple sites within the urinary tract. About 90% of cases originate in the urinary bladder (lower tract) and about 10% of cases emerge in the pelvis or ureter (upper tract). While these sites have similar histologic appearances, there are differences in their epidemiologic, clinical and pathologic characteristics, suggesting they may 102 represent two distinct diseases. Previous studies have observed a more aggressive disease from patients with upper tract urothelial carcinoma (UTUC) versus patients with urothelial carcinoma of the bladder (UCB). Our aim was to examine whether the clinicopathological differences between upper and lower tract urothelial tumors are the result of differences in their scope of somatic genetic alterations. Methods: Tumors and matched germline DNA from 59 patients with high-grade UTUC and 102 patients with high-grade UCB were extracted. The genomic profiles of these patients were analyzed and compared using a custom hybridization capture-based sequencing assay to identify point mutations, small insertions, deletions and copy number alterations of 230 cancer-associated genes. Results: Average next-generation sequencing coverage for high-grade UTUC (674x) and UCB (762x) tumors. The comparison between the high-grade UTUC and UCB tumors identified significant differences in the prevalence of somatic alterations between these cohorts. Alterations in oncogene HRAS (13.6% UTUC vs. 1.0% UCB, p=0.001) were more common in high-grade UTUC. Another oncogene FGFR3 (35.6% UTUC vs. 21.6% UCB, p=0.065) was not significantly different between the UTUC and UCB cohorts. Genes identified as significantly less frequently altered in UTUC compared to UCB tumors included tumor suppressor genes TP53 (25.4% vs. 57.8%, p<0.001), RB1 (0.0% vs. 18.6%, p<0.001), and ARID1A (13.6% vs. 27.5%, p=0.05). Conclusions: While the genes with somatic alterations in upper and lower tract urothelial tumors were similar, we did identify significant differences in the prevalence of several recurrently altered genes including TP53, RB1, HRAS and ARID1A between UTUC and UCB cohorts. These findings may account for the divergence in clinical outcomes observed between these two disease sites and the high prevalence of actionable genomic targets may assist in the development of novel therapeutic approaches for these diseases. Poster Section 39 Poster Board 27 LB-175 Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in breast cancer. Mariacarla Andreozzi,1 Princy Francis,1 Elizabeth Lenkiewicz,1 Mia Champion,1 Brady Laughlin,2 Karen Anderson,3 Heather Cunliffe,4 Ann E. McCullough,1 Michael T. Barrett,1 Barbara Pockaj1. 1Mayo Clinic Arizona, Scottsdale, AZ; 2Arizona State University, Tempe, AZ; 3 Mayo Clinic Arizona,Arizona State University, Scottsdale, AZ; 4 University of Otago, Dunedin, New Zealand. Background: Breast tumors exhibit intratumor heterogeneity resulting in targeted therapy resistance and other challenges in disease management. To address the sources of heterogeneity, we performed a unique, in-depth analysis of clonal architecture in primary chemoradiation-naïve breast cancers. We combined DNA content-based flow cytometry and ploidy analysis with aCGH and whole exome next-generation sequencing (NGS) in multiple biopsies from the tumors and involved lymph nodes (LNs). Material and Methods: We sorted nuclei from distinct populations of diploid, tetraploid, and aneuploid cells in surgical tumor samples from three chemoradiation-naïve patients. Each sorted tumor cell population was interrogated with aCGH and whole exome NGS. In Patient #1, we sorted and interrogated the genomes of tumor populations from 12 fresh frozen sections morphologically mapped from within a HER2+, ER+, PR- primary invasive ductal carcinoma (IDC) of histological grade 3 with LN involvement and 23 sections from 2 out of 5 LNs. In Patient #2, 11 morphologically mapped fresh frozen sections were analyzed from a grade 2, ER+, PR+, HER2+, BRCA2 mutant LN- IDC. In parallel, matching samples were processed for IHC assays. In Patient #3 biopsies from a grade 2 ER+, PR+ primary tumor were profiled. Results: The 18 primary and LN biopsies from Patient #1 fell into 6 distinct ploidy groups albeit with aberrant but homogenous aCGH profiles, characterized by SARC amplification and Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 103 Late-Breaking Poster Session: Molecular and Cellular Biology 3 homozygous deletions in ROBO1 and ROBO2. In contrast a dominant ploidy was identified throughout 10 biopsies in Patient #2 but with heterogeneous aCGH profiles. Patient #3 had a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Mutation profiles obtained through exome sequencing further confirmed that ploidy was the main driver in Patient #1 whereas copy number aberrations played the key role in Patient #2 with the BRCA2 mutation (R3129X). A dendrogram based on exome variant calls of the aneuploid populations in Patient #1 strongly correlated with ploidy group and further revealed the specific clonal population characterized by a 5N ploidy and homozygous mutations in TP53 and PIK3CA as the progenitor to the ploidies present in the distant LNs. Strikingly, patients #1 and #2 were HER2 wild type across their clonal populations, contradicting IHC staining in a single core biopsy. Conclusions: Rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying clonal populations and then interrogating their genomes, provides an objective method of exploring the sources and clinical significance of tumor heterogeneity. Our approach of clonal analysis has broad implications in the study of tumor heterogeneity and the identification of drivers in breast and other solid tumors that can advance more effective treatment and clinical management of patients with this disease. Poster Section 39 Poster Board 28 LB-176 Identification of mutations in histone modification genes in Hodgkin lymphoma. Winnie S. Liang,1 Bodour Salhia,1 Adrienne Helland,1 Shobana Sekar,1 Ignacio Garrido-Laguna,2 Michelle Fanale,2 Yasuhiro Oki,2 Jason R. Westin,2 R. Eric Davis,2 Funda Meric-Bernstam,2 Filip Janku2. 1Translational Genomics, Phoenix, AZ; 2The University of Texas MD Anderson Cancer Center, Houston, TX. Background: Understanding genetic aberrations in cancer has led to discoveries of new targets for cancer therapies. The genomic landscape of Hodgkin lymphoma (HL) has not been fully described. Methods: We performed targeted next generation sequencing (NGS) on 13 archival tumor samples from patients with relapsed/refractory HL treated in the phase I clinical trial with the mTOR inhibitor sirolimus and the HDAC inhibitor vorinostat using the Foundation One NGS panel (Foundation One, Foundation Medicine, MA). Subsequently, we performed whole exome and RNA sequencing on pre- and post-treatment tumor biopsies from 3 patients treated in the same study. Results: In archival samples from 13 HL patients tested using the Foundation One panel, a total of 21 gene aberrations across 13 genes were detected; 12 (92%) tumor samples had mutations in genes involved in immune response, apoptosis, and cell proliferation pathways (SOCS1, PIM1, MCL1, BRCA1, TP53, TNFAIP3, B2M, XPO1, BCL6) and 7 (54%) samples had mutations in DNA repair pathway genes (TP53, BRCA1, ATM, PIM1). In addition, whole exome and RNA sequencing of pre- and posttreatment tumor and germline (peripheral blood mononuclear cells) samples from HL patients treated with sirolimus and vorinostat (complete response, n=1; stable disease for 3 months, n=1; progression, n=1) identified missense point mutations in key histone modification genes not included in the Foundation One panel, including HDAC8 (histone deacetylase 8), JMJD1C (jumonji domain containing 1C), and KDM2A (lysine-specific demethylase 2A) in the patient who progressed on therapy. This same patient additionally had a B2M missense mutation (M1R) affecting the same residue as the B2M event (M1I) identified in the archival cohort. Furthermore, there was a trend towards increased burden of molecular aberrations (median, 67 aberrations) in pre- and post- tumor samples of the patient who progressed compared to the other 2 patients (median, 6 aberrations), who did not progress. Conclusion: While analysis of additional HL specimens is needed, our data suggest that testing for molecular aberrations with NGS is feasible and somatic missense mutations in HDAC8, JMJD1C, and KDM2A may be associated with lack of clinical response to sirolimus and vorinostat. Poster Section 39 Poster Board 29 LB-177 Novel approaches for improving interpretation and predictive models of comparative genomic hybridization data. Daniel Rotroff, Matthew Breen, Alison Motsinger-Reif. North Carolina State University, Raleigh, NC. As costs of genome wide analyses decline and become more accessible, their use in both human and animal cancer studies are generating increasing information regarding underlying cancer etiology. Comparative genomic hybridization (CGH) is providing valuable information relating copy number aberrations (CNAs) to cancer mechanisms and clinical outcomes. However, challenges exist to interpreting and fully utilizing these data. First, without matched tumor and healthy tissue samples from individuals, distinguishing naturally occurring copy number variations (CNVs) from CNAs is difficult. Second, the large search space of genome wide analyses makes finding combinations of CNAs with improved predictive potential compared to single CNAs challenging. Here we provide novel methods to address these challenges associated with CGH data. Many new resources (e.g. The Cancer Genome Atlas (TCGA)), are making large volumes of genomic data publically accessible. However, most datasets do not have matched normal and tumor tissue samples between subjects. We tested matched normal and tissue samples from 30 patients with colorectal, lung, and pancreatic cancer and compared CNVs and CNAs to findings in larger, non-matched samples in TCGA. Even with limited matched samples, this approach allows for the differentiation of CNVs from CNAs discovered in analyses of non-matched samples. In some cases, combinations of CNAs can provide improved predictive capability compared to any single CNA. However, it is computationally intractable to exhaustively test combinations of CNAs in a genome-wide study. To address this limitation, we use a novel approach for CNA feature reduction that minimizes the variance within CNA segments across subjects, and Random Forest Ensemble Classification. This approach provides CNA combinations with balanced accuracies of 83.5% and 94.9 % for distinguishing 52 cases of canine ALL/AML and 71 cases of BCLL/T-CLL, respectively. These two approaches address frequent limitations in the interpretation CGH data. Better distinguishing CNVs and interrogating CNA combinations, can provide additional information about the role of CNAs in disease mechanisms and improve treatment decisions. Poster Section 39 Poster Board 30 LB-178 Genomic analysis identifies a subset of laryngeal tumors with poor prognosis. Suraj Peri,1 Michael J. Slifker,1 Ranee Mehra,1 Barbara Burtness,2 Eric Ross,1 Erica Golemis1. 1Fox Chase Cancer Center, Philadelphia, PA; 2Yale Cancer Center, Yale School of Medicine, Yale University, New Haven, CT. Squamous cell carcinoma of head and neck (HNSCC) is a highly heterogeneous tumor type with aggressive behavior. Clinical outcomes depend on tumor site, exposure to carcinogens or Human Papillomavirus (HPV) and other prognostic factors. Patients with HPV+ tumors, primarily arising in the oropharynx, have better survival outcomes than those with HPV- tumors. Using comprehensive genomic data resources, an integrative analysis to characterize these tumors by defining molecular classes and estimating their prognostic relevance has not been done. HNSCC data, from the TCGA repository representing non-silent mutations, copy number variations, gene-level read counts, and methylation were obtained. Data from 256 patients was used for which all four types of data were available. The level 3 copy number segmentation values were deconvoluted to obtain unique set of chromosomal regions. For gene-level read count matrices, the top 2000 genes were selected based on Median Absolute Deviation on RPKM values. In the case of methylation, top 2000 logit American Association for Cancer Research • AACR ANNUAL MEETING 2015 103 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 104 Late-Breaking Poster Session: Molecular and Cellular Biology 3 transformed ß-values were used. The processed data was then clustered using an integrative clustering methodology. Kaplan-Meier plots and log-rank tests were computed to compare survival distributions among clusters. Fisher’s exact tests were used to test association of clinical factors with cluster membership. We performed clustering independently on all HNSCC samples and on laryngeal tumor samples alone. Integrative clustering identified 5 clusters and they can be defined based on tumor site and HPV status. While 74% of HPV+ cases grouped into one cluster; over 80% of oral tumors were distributed into two other clusters; and 64% of the laryngeal tumors cases were segregated into two clusters. We found significant differences in overall survival among these clusters (p = 0.00007). Based on the observation that laryngeal tumors showed different patterns of survival in distinct clusters, we repeated clustering on 104 laryngeal samples alone. This analysis resulted in 2 clusters that recapitulated the survival differences shown in the first analysis (p = 0.0086). We observed distinguishing differences in methylation and expression among the two larynx tumor classes. The 2-year survival estimates for the two clusters were 45% and 75%, and no significant differences in age, gender, tumor stage, smoking status, or alcohol status were observed. Survival differences between these clusters (based on genomic and epigenomic measurements) suggest the possibility that this clustering model can predict survival in larynx cancer, independent of TNM stage. We have identified two classes of laryngeal tumors based on molecular profiles with substantially different clinical outcomes. These results provide a genomic signature that could lead to prognostic and therapeutic intervention for a debilitating malignancy. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 105 Late-Breaking Poster Session: Epidemiology Late-Breaking Poster Session Tuesday, April 21, 2015 8:00 AM-12:00 PM Poster Section 40 Late-Breaking Research: Epidemiology Poster Section 40 Poster Board 1 LB-179 Expression of estrogen receptor, progesterone receptor, and ki67 in normal breast tissue and subsequent risk of breast cancer. Hannah Oh,1 A Heather Eliassen,2 Molin Wang,3 Stephanie A. Smith-Warner,4 Andrew H. Beck,5 Stuart J. Schnitt,5 Laura C. Collins,5 James L. Connolly,5 Laleh Montaser-Kouhsari,5 Rulla M. Tamimi2. 1Department of Epidemiology, Harvard School of Public Health; Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School (Current: National Cancer Institute, Division of Cancer Epidemiology & Genetics, Rockville, MD), Boston, MA; 2Department of Epidemiology, Harvard School of Public Health; Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; 3Department of Biostatistics, Harvard School of Public Health; Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; 4 Department of Nutrition, Harvard School of Public Health, Boston, MA; 5Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA. Background: Biological activity, including potential carcinogenic effects, of estrogen and progesterone in breast tissue is primarily mediated by their receptors in the tissue. Ki67 is a marker of cell cycle activation. We examined the associations of estrogen receptor (ER), progesterone receptor (PR), and Ki67 expression in normal breast tissue from benign biopsies with subsequent breast cancer risk. Methods: We conducted an analysis among 385 women (90 cases, 295 controls) with benign breast disease (BBD) in a nested case-control study within the Nurses’ Health Study (NHS) and the NHSII. Tissue microarrays (TMA) were constructed using cores obtained from benign biopsies containing normal terminal duct lobular units (TDLU). Immunohistochemical staining for ER, PR, and Ki67 was performed on sections cut from the TMAs. Staining results were interpreted by computational image analysis which scored the percentage of positively stained cells for each marker. Unconditional logistic regression models, adjusting for matching factors and benign lesion subtype, were used to estimate odds ratios (OR) for developing subsequent breast cancer by tertiles of marker expression. Results: ER and Ki67 expression (highest vs. lowest tertiles) in normal breast TDLUs was not significantly associated with subsequent breast cancer risk (≥14.7 vs. <7.3 % ER-positive cells: OR=0.55, 95% CI=0.21-1.44, p-trend=0.85; ≥6.2 vs. < 2.4 % Ki67positive cells: OR=1.75, 95% CI=0.87-3.50, p-trend=0.15). PR expression was suggestively positively associated with breast cancer risk (≥9 vs. <4%: OR=2.08, 95% CI=1.00-4.31, ptrend=0.06); the positive association was significant among women who were premenopausal at BBD biopsy (OR=3.55, 95% CI=1.289.87, p-trend=0.03). Conclusion: PR expression in normal breast tissue was significantly positively associated with subsequent breast cancer risk in premenopausal women. Although we did not observe significant results with ER and Ki67, we cannot exclude associations given the limited power in this study. These findings may contribute to understanding of breast cancer biology and may suggest new targets for breast cancer risk assessment and prevention. However, further studies are required to confirm these results. Poster Section 40 Poster Board 2 LB-180 Metabolomic changes in response to chronic stress in healthy mice. Shelley S. Tworoger,1 Elizabeth M. Poole,2 Guillermo Armaiz Pena,3 Laura Kubzansky,1 Susan E. Hankinson,4 Anil K. Sood,3 Chirag Patel,5 Clary Clish6. 1Harvard T.H. Chan School of Public Health, Boston, MA; 2Brigham and Women’s Hospital, Boston, MA; 3MD Anderson Cancer Center, Houston, TX; 4University of Massachusetts Amherst, Amherst, MA; 5Harvard Medical School, Boston, MA; 6Broad Institute, Cambridge, MA. Background: Animal models of ovarian cancer suggest that chronic stress and subsequent prolonged distress can increase tumor aggressiveness. While specific stress hormones, such as norepinephrine, appear to have a direct influence on tumors, chronic stress may cause other metabolic perturbations important for cancer development. Here, we searched for potential metabolic biomarkers associated with chronic stress using preclinical models. Methods: We induced stress with a daily restraint protocol, and assessed the impact of daily restraint versus control in 19 healthy, female C57 black mice on plasma and ovarian tissue metabolomic profiles. At 12 weeks old, mice were randomly assigned to three weeks of daily restraint (2 hr/day; n=10) or normal care (n=9). At the end of the experiment, a necropsy was performed and plasma and ovarian tissues were obtained. Metabolomic profiling was conducted using LC-MS-MS, and resulted in peak information for 248 identified metabolites and over 18,000 unidentified peaks. Results: For the identified metabolites, a Q-Q plot showed a significant deviation from the expected p-value distribution under the null hypothesis for both plasma and tissue when comparing stressed versus non-stressed animals. Triacylglycerols (TAGs), as a group, differed between stressed versus non-stressed animals in both ovary and plasma, although the specific TAGs that were altered and the direction of association appeared to differ by tissue type. Putrescine was significantly higher in stressed versus nonstressed animals in both plasma and tissue. Further, multiple phosphatidylcholines (PCs) and their downstream metabolites, lyso-PCs, differed in plasma of stressed versus non-stressed animals. When considering metabolites that significantly differed in plasma between conditions (FDR<5%), stressed and non-stressed animals clustered together with the exception of one non-stressed animal that clustered with the stressed animals. Similar results were observed for tissue, with 2 stressed and 1-non-stressed animals clustering with the other group. The median correlation between tissue and plasma metabolites was -0.01 for stressed animals and 0.21 for control animals. Conclusions: Our results suggest that lipid metabolism, which is often dysregulated in cancer cells, may be altered by exposure to stress in healthy animals. Putrescine, which is created by the breakdown of amino acids and is important for cell proliferation, was increased by stress. Overall, our results suggest that chronic stress can lead to metabolic changes at a macro (plasma) and tissue specific level. Additional analyses will characterize specific metabolites altered by stress, detail relationships between tissue and plasma metabolites, and assess correlations with corticosterone. Poster Section 40 Poster Board 3 LB-181 Oral HPV DNA detection and subsequent risk of head and neck cancers in two prospective cohorts. Ilir Agalliu,1 Zigui Chen,1 Tao Wang,1 Rebecca Ludvigsen,2 Lauren Teras,2 Aimee R. Kreimer,3 Richard B. Hayes,4 Susan Gapstur,2 Robert D. Burk1. 1Albert Einstein College of Medicine, Bronx, NY; 2 American Cancer Society, Atlanta, GA; 3NCI/NIH, Bethesda, MD; 4 New York University, New York, NY. Background: Alpha HPV16 detection in the oral cavity is associated with head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal cancer. However, there have been no prospective studies examining the temporal relationship between oral HPV detection and subsequent risk of HNSCC. Moreover, American Association for Cancer Research • AACR ANNUAL MEETING 2015 105 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 106 Late-Breaking Poster Session: Epidemiology recent data indicates that the oral cavity contains a plethora of HPV types in addition to alpha HPVs (e.g. beta and/or gamma HPVs), but their association with risk of HNSCC is unknown. Methods: We examined prospective associations between alpha, beta and gamma HPVs and risk of HNSCC, using a nested case-control design among >120,000 participants with available mouthwash samples in the American Cancer Society (ACS) Cancer Prevention Study-II Nutrition Cohort (CPS-II) and the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. A total of N=132 incident cases of HNSCC (oropharyngeal, oral and laryngeal SCCs) were identified during an average 3.94 years of follow-up (range 0.02-9.0) in both cohorts. Three controls per case (N=396) were selected using incidence density sampling, with matching on age (±2 years), race/ethnicity, gender, and time since mouthwash collection (±3 months). Detection of HPV DNA in mouthwash samples was carried out using (1) a novel nextgeneration sequencing assay designed to detect all HPV types, (2) the MY09/MY11 assay targeting alpha-HPV types, and (3) a RTPCR specific for HPV16. Associations of alpha, beta and gamma HPVs with risk of HNSCC were evaluated using conditional logistic regression models for matched risk sets to estimate odds ratios (OR) and 95% confidence intervals (CI), adjusting for smoking and alcohol as well as alpha HPV16 for beta and gamma HPVs. Results: The prevalence of oral HPV16 was 1.8% in controls. HPV16 detection was associated with a 7.1-fold higher risk of overall HNSCC (95% CI 2.2-22.6); the risk was highest for oropharyngeal cancer (OR=22.41, 95% CI 1.8 - 276.7), while no excess was found for oral cavity or larynx cancers. There were no associations between other alpha HPVs and risk of HNSCC. Oral prevalence of any beta or gamma HPVs was 59.4% and 38.2%, respectively in controls. Detection of any beta HPV (OR=1.74, p=0.05) or any gamma HPV (OR=2.11, p=0.005) was also associated with risk of HNSCC, with several beta (β1 HPV5, β2 HPV17, β2 HPV38) and gamma (γ11, γ12) HPVs having statistically significant increased risks (ORs from 3.92 to 7.36). In regard to tumor location, β1HPV5 was associated with oropharyngeal (OR=7.42, p=0.05), oral cavity (OR=5.34, p=0.01) and laryngeal cancers (OR=2.71, p=0.05); while β2 HPV38 was associated with oropharyngeal cancer (OR=7.28, p=0.02) only. Gamma HPV species groups 11 and 12 were associated with both oral cancer (OR=7.47, p=0.03; and OR=6.71, p=0.01, respectively) and laryngeal cancers (OR=7.49, p=0.04 and OR=5.31, p=0.03). Conclusion: This study is the first to demonstrate that alpha HPV16 detection precedes the incidence of oropharyngeal cancers. Risks identified with other HPV types from gamma11 and 12 species as well as beta HPV5, previously associated with skin cancer, suggests a broader role for HPVs in HNSCC etiology. Readily-collected oral wash samples provide a strong prospective marker for oropharyngeal cancer and, with the incorporation of other HPV types, may indicate risk for a broader spectrum of HNSCC. Poster Section 40 Poster Board 4 LB-182 Improved survival among lung cancer patients taking antidepressants. Adriana Zingone,1 Derek Brown,1 Elise Bowman,1 Oscar Vidal,1 Joel Neal,2 Julien Sage,2 Brid M. Ryan1. 1National Cancer Institute, Bethesda, MD; 2Stanford University, Stanford, CA. This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2015 Official Press Program. It will be posted online following its presentation. 106 Poster Section 40 Poster Board 5 LB-183 Metformin use does not increase survival of pancreatic cancer patients: A cautionary lesson. Roongruedee Chaiteerakij,1 David B. Zhen,2 Patrick A. Burch,3 Kari G. Chaffee,4 William R. Bamlet,4 Ann L. Oberg,4 Lewis R. Roberts,1 Gloria M. Petersen5. 1Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN; 2Division of Hematology/Oncology, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, MI; 3Division of Medical Oncology, Mayo Clinic College of Medicine, Rochester, MN; 4 Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic College of Medicine, Rochester, MN; 5Department of Health Sciences Research, Division of Epidemiology, Mayo Clinic College of Medicine, Rochester, MN. This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2015 Official Press Program. It will be posted online following its presentation. Poster Section 40 Poster Board 6 LB-184 Risk factors for lung cancer among survivors of nonHodgkin lymphoma. Clara Lam,1 Rochelle Curtis,1 Graca Dores,2 Eric Engels,1 Neil Caporaso,1 Aaron Polliack,3 Joan Warren,1 Heather Young,4 Paul Levine,4 Angelo Elmi,4 Joseph Fraumeni,1 Margaret Tucker,1 Lindsay Morton1. 1National Cancer Institute, Rockville, MD; 2Oklahoma City Veterans Affairs Health Care System, Oklahoma City, OK; 3 Hadassah University Hospital, Jerusalem, Israel; 4The George Washington University, Washington, DC. Background: Previous studies have reported that non-Hodgkin lymphoma (NHL) survivors have increased risk of developing lung cancer; however, risks associated with specific treatments and immune-related risk factors have not been quantified. Methods: We assessed second lung cancer risk among 44,870 1-year survivors of first primary NHL diagnosed at ages 66-84 years during 1992-2009 from the Surveillance, Epidemiology, and End Results (SEER)-Medicare data linkage. Information on potential risk factors, including NHL treatments, infections, and immune-related medical conditions, was derived from Medicare claims. Results: A total of 700 second lung cancers were diagnosed among NHL survivors, including 259 after chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 127 after follicular lymphoma (FL), 116 after diffuse large B-cell lymphoma (DLBCL), and 137 after other NHL subtypes. Lung cancer risks were significantly increased among CLL/SLL survivors who received fludarabine-containing chemotherapy without rituximab (Cox regression hazard ratio [HR]=2.28, 95% confidence interval [CI]=1.48-3.52). Elevated risks were also seen among DLBCL survivors who received cyclophosphamide-containing chemotherapy (without rituximab HR=2.13, 95%CI=1.01-4.47; with rituximab HR=1.82, 95%CI=0.87-3.80). After adjusting for history of smoking, lower airway respiratory infections, particularly pneumonia, were associated with increased risks of second lung cancer after CLL/SLL (HR=2.21, 95%CI=1.65-2.96), DLBCL (HR=1.90, 95%CI=1.26-2.87), and FL ( HR=1.98, 95%CI=1.313.00). The pattern of lung cancer risks associated with autoimmune conditions was complex, with increased risks seen for B-cell activating autoimmune conditions among DLBCL survivors (HR=1.63, 95%CI=1.00-2.66) and T-cell activating autoimmune conditions among survivors of other NHL subtypes (HR=1.56, 95%CI=1.07-2.27). In contrast, T-cell activating autoimmune conditions were significantly inversely associated with lung cancer among FL survivors (HR=0.57, 95%CI=0.34-0.96), and no associations with autoimmune disease were identified among CLL/SLL survivors. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 107 Late-Breaking Poster Session: Epidemiology Conclusion: We report for the first time that chemotherapy used in treatment of NHL, infections, and autoimmune diseases are associated with the risk of developing lung cancer after NHL. Further research is needed to confirm the observed associations and understand the underlying carcinogenic mechanisms. Poster Section 40 Poster Board 7 LB-185 The effects of plasma folate and other B vitamins on breast cancer risk in BRCA1 and BRCA2 mutation carriers. Shana J. Kim,1 Anna Zuchniak,1 Young-In Kim,1 Yvonne Lamers,2 Joanne Kotsopoulos,1 Steven Narod1. 1University of Toronto, Toronto, Ontario, Canada; 2University of British Columbia, Vancouver, British Columbia, Canada. Background: Women who inherit a deleterious BRCA mutation face a high lifetime risk of developing breast cancer, estimated at 80% compared with 11% in the general population. Current prevention options are limited to prophylactic surgery (removal of the breast and/or ovaries) and/or chemoprevention. Nonsurgical prevention options including dietary and lifestyle recommendations are highly desired by this population but have yet to be elucidated. Folate and other B-vitamins involved in folate metabolism are of particular interest given their essential roles in DNA synthesis and maintaining genomic stability, the reported dual effects on carcinogenesis (promoting or preventing), and the high levels of intake due to mandatory food fortification and supplement use. Given the heightened predisposition for cancer development among BRCA mutation carriers, clarifying the role of these nutrients on breast cancer development in this population is of extreme importance. Objectives: To prospectively investigate the relationship between plasma folate, B12 and B6 levels and the risk of breast cancer in women with a BRCA1 or BRCA2 mutation. Methods: We included 169 Canadian women who provided a blood sample at enrollment and had no history of cancer from an ongoing international cohort of BRCA mutation carriers, the Risk Factor Analysis of Hereditary Breast and Ovarian Cancer Study. Baseline and biennial follow-up questionnaires collected relevant information regarding family, reproductive and medical histories, selected lifestyle factors, and disease incidence. Plasma folate and B12 levels were determined with microbiological microtitre assays, methylmalonic acid (MMA, a functional biomarker of vitamin B12) was quantified using reversed-phase LC-tandem mass spectrometry, and pyridoxal 5’phosphate (PLP) measuring active B6 was determined with a nonradioactive apo-enzymatic assay. Cox proportional hazards models were used to estimate relative risks and 95% confidence intervals. Results: During a mean follow-up of 8 years, 21 women were diagnosed with invasive breast cancer. At baseline, the participants had a mean age of 52.7 ± 12.2 years and BMI of 25.0 ± 5.0 kg/m². The mean plasma folate level was 17.8 ng/mL ± 11.9, ranging from 0.24 to 68.5 ng/mL. Unaffected women had an average plasma folate level of 21.7 ng/mL compared to 19.2 ng/mL in affected women (P = 0.46). Plasma B12 and B6 assays are currently underway. Results from statistical analyses are expected in March 2015. Impact: To our knowledge, this represents the first prospective study evaluating the effect of folate and other B vitamins on the risk of breast cancer among women with a BRCA1 or BRCA2 mutation. The results from this study will help develop safe and targeted recommendations for prevention in genetically predisposed women. Funding: Canadian Institutes of Health Research, Canadian Gene Cure Foundation. Poster Section 40 Poster Board 8 LB-186 Fiber intake modulates the effect of alcohol on breast cancer. Isabelle Romieu. International Agency for Res. on Cancer, Lyon, France. Alcohol intake has been related to an increase risk of breast cancer while dietary fiber intake has been inversely correlated to breast cancer risk. A beneficial effect of fiber on ethanol carcinogenesis through their impact on estrogen levels is still controversial. The purpose of this study is to investigate the role of dietary fiber as potential modifying factors of the association of alcohol to breast cancer using data from the European Prospective Investigation into Cancer and Nutrition (EPIC) study. A prospective observational study of 334,850 women aged 35-70 years at baseline enrolled in the ten countries of the EPIC study and followed up for 11.0 years on average was conducted. Information on fiber and alcohol intake at baseline and average lifetime alcohol intake were calculated from country-specific dietary and lifestyle questionnaires. Hazard ratios (HR) and 95% confidence intervals (CI) of developing invasive breast cancer according to different levels of alcohol and fiber intake were calculated. During 3,670,439 person-years, 11,576 incident breast cancer cases were diagnosed. Among women with low intake of fiber (<18.5 g/day), the risk of breast cancer per 10g/day of alcohol intake was 1.057 (1.032;1.082) while among women with high intake of fiber (>24.2 g/day) the risk of breast cancer was 1.012 (0.983;0.1.04) (Test for interaction p=0.01). This modulating effect was stronger for fiber from vegetables and for estrogen positive tumors. Our results suggest that fiber intake may modulate the positive association of alcohol intake to breast cancer risk. Women should be advised to control their alcohol intake, and increase their intake of fiber. Poster Section 40 Poster Board 10 LB-188 Epigenome-wide study in prediagnostic samples from the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort: Differentially methylated microRNAs in subjects who developed breast cancer. Alessio Naccarati,1 Silvia Polidoro,1 Giovanni Fiorito,1 Francesca Cordero,2 Giulio Ferrero,2 Gianluca Campanella,3 Carlotta Sacerdote,4 Amalia Mattiello,5 Salvatore Panico,5 Giovanna Masala,6 Domenico Palli,6 Claudia Agnoli,7 Vittorio Krogh,7 Graziella Frasca,8 Rosario Tumino,8 Paolo Vineis9. 1Human Genetics Foundation, Turin, Italy; 2University of Torino, Turin, Italy; 3Imperial College London, London, United Kingdom; 4AO Citta’ della Salute e della Scienza,University of Torino, Turin, Italy; 5Federico II University, Naples, Italy; 6Cancer Research and Prevention Institute – ISPO, Florence, Italy; 7Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 8Cancer Registry ASP, Ragusa, Italy; 9Imperial College, London, United Kingdom. The crosstalk between microRNAs (miRNAs) and other epigenetic factors may lead to novel hypotheses about carcinogenesis identifying new targets for research. Since a single miRNA can regulate multiple downstream target genes, its altered expression may potentially be a very sensitive biomarker to detect early malignant transformation and improve diagnosis and prognosis. In the current study, we used a genome-wide approach to test the hypothesis that altered methylation of miRNA encoding genes may be related to dietary and lifestyle factors and may be associated with deregulated mature miRNA expression ultimately leading to cancer. In a case-control study nested in a prospective cohort (EPICItaly), we analysed DNA methylation levels of miRNA encoding genes (2,191 CpG probes related to 517 genes) that are present in the Infinium Human Methylation450 BeadChip array in prediagnostic peripheral white blood cells of subjects who developed Colorectal Cancer (CRC, n=159) and Breast Cancer (BC, n=166) and matched subjects who remained clinically healthy. In the whole cohort, several differentially methylated miRNA genes were observed in association with age, sex, smoking habits and physical activity. Interestingly, in the case-control study, 8 differentially methylated miRNAs were identified in subjects who went on to develop BC (miR-328, miR675, miR-1307, miR-1286, miR-1275, miR-1910, miR-24-1, and miR-548a-1; all Bonferroni-adjusted p-values < 0.05). No significant associations were found with CRC. Assuming that altered methylation of miRNAs may be present before diagnosis, it may represent a biomarker for early detection or risk of cancer and may help to understand the cascade of events preceding tumor onset. American Association for Cancer Research • AACR ANNUAL MEETING 2015 107 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 108 Late-Breaking Poster Session: Epidemiology Poster Section 40 Poster Board 11 LB-189 Genetic Epidemiology of Lung Cancer Consortium: genome-wide association study of familial lung cancer cases. Diana M. Toledo,1 Susan M. Pinney,2 Diptasri Mandal,3 Mariza de Andrade,4 Elena Kupert,2 Jennifer Franks,1 Colette Gaba,5 Claire L. Simpson,1 Ming You,6 Marshall W. Anderson,2 Joan E. BaileyWilson,7 Christopher I. Amos,1 Ann Schwartz8. 1Dartmouth College, Hanover, NH; 2University of Cincinnati, Cincinnati, OH; 3LSU Medical School, New Orleans, New Orleans, LA; 4Mayo Clinic, Rochester, MN; 5University of Toledo, Toledo, OH; 6Medical College of Wisconsin, Milwaukee, WI; 7NHGRI, Baltimore, MD; 8Karmanos Cancer Center, Detroit, MI. Several studies have identified common genetic factors that influence susceptibility to lung cancer but no large studies have systematically studied familial lung cancers. The Genetic Epidemiology of Lung Cancer Consortium completed a genomewide association study (GWAS) using samples from 757 cases and 796 controls, all of European American ancestry, and frequency matched on age and sex. Genotyping was performed by the Center for Inherited Disease Research (CIDR) using an Illumina Human OmniExpressExome-8v1 array. The most significantly associated SNPs were identified in the cases as a whole, and were also stratified by histology (adenocarcinoma and squamous cell carcinoma) and family history (0-1 and 2-5 affected family members). Analysis of all cases and controls identified a large number of SNPs around the CFTR gene that are strongly associated with lung cancer risk. Upon imputation with European Ancestry 1000 genomes data, the most significant SNP in this region is rs43034 (p= 3.89E-07, OR = 0.657) in the promoter region. In addition, an exonic missense SNP, rs213950 (p=6.32E-07, OR=0.663), and an intronic SNP with a possible splicing effect, rs213935 (p=5.20E-07, OR=0.660) were also identified as possible protective alleles. Analysis by histological subtype identified several rare and exonic SNPs that were specific by histology. We highlight two SNPs, rs114719990 (p=1.194E-04, OR=336.8, SETD2, c.3229A>G, p.Thr1077Ala) and rs199710487 (p=1.124E-04, OR=366, NTN5, c.656G>A, p.Arg219Gln), that were identified as strongly associated SNPs in cases with histologically confirmed adenocarcinoma, and two SNPs, rs144465058 (p=1.029E-04, OR=484.6, ANK3, c.6555G>T, p.Gln2185His) and rs140216112 (p=1.087E-04, OR=476, GRIK4, c.1013C>G, p.Ala338Gly), that were identified as top SNPs in cases with histologically confirmed squamous cell carcinoma. The SETD2 gene is thought to be a tumor suppressor gene and variants in this gene have previously been associated with leukemia and glioma (Zhu X et al. 2014, Fontebasso AM et al. 2013), and may now have implications in lung cancer, specifically adenocarcinoma development. In conclusion, we identified SNPs in the CFTR gene that may have a protective effect on lung cancer and several rare and exonic variants that were specific to histology subtype. Poster Section 40 Poster Board 12 LB-190 Ethnic differences in the relationship between diabetes, adiposity measures and breast cancer-specific mortality. Avonne E. Connor,1 Kala Visvanathan,1 Kathy Baumgartner,2 Richard N. Baumgartner,2 Stephanie Boone,2 Lisa M. Hines,3 Anna R. Giuliano,4 Esther M. John,5 Roger K. Wolff,6 Martha L. Slattery6. 1 Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 2 University of Louisville, Louisville, KY; 3University of Colorado, Colorado Springs, Colorado Springs, CO; 4H.Lee Moffit Cancer Center & Research Institute, Tampa, FL; 5Cancer Prevention Institute of California, Fremont, CA; 6University of Utah, Salt Lake City, UT. Background: Type 2 diabetes and obesity are modifiable factors that are associated with breast cancer (BC) specific and all-cause (AC) mortality among women with BC. Few studies have examined the role of adiposity in the association between diabetes and BC outcomes, particularly among Hispanic women who have high prevalence of obesity and diabetes. We hypothesized that adiposity mediates the association between diabetes and risk of BC-specific and AC mortality in BC survivors from the Breast Cancer Health Disparities Study. Methods: The study population included 2,478 women (1,180 Hispanics, 1,298 non-Hispanic whites (NHW)) from the San Francisco Bay Area, New Mexico, Utah, Colorado and Arizona with incident, invasive breast cancer diagnosed between April 1995 and April 2002 or between October 1999 and May 2004, depending on study site. Information on diabetes and lifestyle factors was collected by inperson interview, as well as self-reported weight and height during the referent year, and measured waist and hip circumferences. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated by Cox proportional hazards regression models for overall associations and by ethnicity. Covariates of interests included ethnicity, age at diagnosis, breast cancer stage, tumor phenotype, study site, education, body mass index (BMI), waist circumference (WC), waist-hip ratio (WHR), diet, physical activity, smoking and alcohol use. Results: The median follow-up time was 10.8 years since BC diagnosis, with a total of 466 deaths from any cause. Overall, in multivariable models physician diagnosis of diabetes was significantly associated with BC-specific (HR, 1.69; 95% CI 1.142.51) and AC mortality (HR, 1.62; 95% CI 1.22-2.19). The association was stronger among Hispanic women for BC-specific mortality (HR, 1.83; 95% CI 1.11-3.03) compared to NHWs (HR, 1.53; 95% CI 0.792.95). Further adjustment for BMI, WC and WHR attenuated the point estimates for AC and BC-specific mortality, and the degree of attenuation varied by ethnicity and adiposity measure. For example, WHR was the only measure that significantly attenuated the point estimate among Hispanic women for BC-specific mortality (by 15%). Weight gain between age 30 and referent year was also evaluated; diabetics who gained 25 kg or more had a significant increase in BCspecific mortality (HR, 5.04; 95% CI 1.69-15.07) compared to nondiabetic women. Lastly, BC-specific mortality was significant among women diagnosed with diabetes within five years of BC diagnosis (HR, 2.33, 95% CI 1.31-4.13), compared to non-diabetic women. Conclusions: Diabetes was associated with BC-specific mortality, particularly among Hispanic women and abdominal adiposity based on WHR was a significant mediator of this association. Future studies of diabetes and BC outcomes among diverse populations should examine changes in WHR among Hispanic BC survivors. Poster Section 40 Poster Board 13 LB-191 Association of anti-estrogen therapy in breast cancer patients and subsequent risk of rheumatoid arthritis: An electronic health record study. Matthew K. Breitenstein,1 Ming-Fen Ho,1 Richard M. Weinshilboum,2 James R. Cerhan,2 Jyotishman Pathak,2 Tim Bongartz,2 Liewei Wang,2 James N. Ingle2. 1Mayo Clinic, Rochester, MN; 2Mayo Clinic College of Medicine, Rochester, MN. Background: The use of anti-estrogen (AE) therapies (selective estrogen receptor modulators [SERMs], aromatase inhibitors [AIs]) in 108 Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 109 Late-Breaking Poster Session: Epidemiology breast cancer (BC) patients was recently associated with risk of developing rheumatoid arthritis (RA) (J Rheum 2015;42:55-9). We attempted to replicate and extend these results using electronic health record (EHR) enhanced cancer registry data that assessed specific AE therapies and also accounted for potential confounding by age, smoking status, and chemotherapy use. Methods: Using cancer registry and EHR data, we identified a cohort of BC patients who were newly diagnosed and treated at Mayo Clinic Rochester from 1998-2011 and had no previous diagnosis of RA. Smoking status at diagnosis, BC treatments and RA at diagnosis and during follow-up were identified using validated EHR-based algorithms. Hazard ratios (HRs) and 95% confidence intervals (CI) from a multivariate Cox model were used to estimate the association of AE use with risk of RA, controlling for age, smoking status at BC diagnosis, and BC chemotherapy use. Results: The analytic cohort of 9,244 newly diagnosed BC patients treated and followed at Mayo Clinic had a median age at diagnosis of 59 years (range 18-97); 32% were smokers; and 29% were treated with chemotherapy. During a median follow-up of 49 months (range 1-191), 19 patients developed RA. Compared to BC patients not receiving any AE therapy, those receiving AI monotherapy, but not SERM monotherapy or both SERMs and AIs, were at significantly increased risk of developing RA (Table 1). Conclusions: Our findings indicate that the use of AIs for BC therapy, but not SERMs or SERMs followed by AIs, is associated with increased risk for development of RA, when controlling for smoking status and use of chemotherapy. While needing replication, these findings suggest a specific role for AIs in RA development and suggest pathways that could be targeted to prevent this potential treatment complication. Poster Section 40 Poster Board 14 LB-192 A single catastrophic genomic event is required for the development of osteosarcoma. Sven Bilke, Paul Meltzer. National Cancer Inst., Bethesda, MD. Over sixty years ago, the groundbreaking works by Nordling1, Armitage and Doll2 found a deep connection between the age dependent incidence rates of cancer with elementary mutational processes in cancer. However, their method to estimate the number of driver mutations from the characteristic increase of adult cancer risk with patient age implicitly assumes tissue homeostasis. It can therefore not be used for developmental cancers emerging from growing tissues. Osteosarcoma (OS), a bone cancer most frequently occurring during adolescence, is one prominent example. Here we combine a model of bone growth and maintenance with somatic mutation theory. Based on age dependent stature3` and morphometric data, we find that our model accurately reproduces epidemiological OS incidence rates4`. We conclude that the OS risk can be explained by the cell cycle rate in the osteoblast lineage. Other risk factors, such as endocrine and paracrine signaling have only a weak, if any, impact. The strict association with the cell cycle rate, as opposed to wall clock time, suggests that environmental factors play no dominant role either. In other words, with few exceptions such as genetic syndromes or unusually strong environmental factors such as radiation in cancer treatment, the OS risk is dominated by pure chance. We find that the OS incidence rate is strictly proportional to the age dependent cell cycle rate. This result implies that there is only a single rate limiting factor in the etiology of the disease. This result is a remarkable as it suggests that the disease is not necessarily initiated within a stem cell because the Hayflick limit set by telomere shortening can not suppress single-hit-malignancies. Our results are not only important for a better understanding of the disease and the design of future sequencing efforts. But they also inform development of treatment strategies to reduce the mortality caused by this still deadly disease. 1. Nordling, C. O. C. A New Theory on the Cancer-inducing Mechanism. Br. J. Cancer 7, 68¨C72 (1953). 2. Armitage, P. & Doll, R. The age distribution of cancer and a multistage theory of carcinogenesis. Br. J. Cancer VIII, 1¨C12 (1954). 3. CDC Height Data. at 4. Cancer incidence in five continents, vol. I to VIII. CancerBase No. 7. (Lyon: IARC). Poster Section 40 Poster Board 15 LB-193 Prediagnostic breast cancer metabolites in Mexican Americans: a nested case control study. Sara S. Strom, Hua Zhao, Abenaa Brewster, Yuko Yamamura. UT MD Anderson Cancer Center, Houston, TX. Breast cancer (BrCa) is the most common invasive cancer in Hispanic women. Although at decreased risk, Hispanic women are diagnosed at younger ages and with more aggressive disease than non-Hispanic Whites. Early detection is key in BrCa survival; however, there are no pre-diagnostic circulating biomarkers for early detection. This pilot nested case-control study based on our Mexican-American Cohort (MAC) study evaluates the role of global metabolic expression in predicting BrCa risk. MAC includes ~23,000 participants in the Houston area with baseline blood specimens and epidemiological data. Using mass spectrometrybased global protein expression, 435 plasma metabolites were analyzed by Metabolon in 30 invasive BrCa cases (14 pre- (preM) and 16 post-menopausal (postM)) diagnosed 1-5 years after enrollment and 40 healthy controls (matched to cases on age, menopausal status) and length of follow-up). Mean age of preM women was 37.6 years (range 32-43) compared to 63.4 for postM (range 50-81). Using principal component analysis, we evaluated differences by disease and menopausal status. We found 76 metabolites that differed significantly (P<0.05) between cases and controls among postM women compared to only 13 metabolites in preM women. Using a pathway-based approach, we found that the majority of case-control differences were in metabolites related to hormone, lipid and energy metabolism. The specific metabolites differed by menopausal status. In postM cases, levels of steroid hormones epiandrosterone sulfate, androsterone sulfate, and 5alpha-androstan-3beta,17beta-diol disulfate were higher than in controls; among preM cases, steroid hormones 5-alpha-pregnan3beta,20alpha-diol disulfate and 5-alpha-pregnan-3alpha,20betadiol disulfate 1 levels were higher. PostM cases had significantly (P<0.05) lower levels in fatty acids valerylcarnitine and butyrylcarnitine and higher levels of monoacylglycerols and glycerol 3-phosphate (G3P) than controls; there were no significant differences in these metabolites in preM cases and controls. The statistically significant (P<0.05) decreases in 3-phosphoglycerate, pyruvate, sarcosine (N-methylglycine), citrate, and malate which are all associated with glycolysis and the tricarboxylic acid cycle found in cases compared to controls irrespective of menopausal status, suggest energy metabolism may be associated with BrCa risk. In summary, our findings suggest: (1) case-control differences in plasma levels of specific metabolites associated with key functional pathways; (2) the specific metabolites and number of relevant metabolites differs by menopausal status; and (3) these differences American Association for Cancer Research • AACR ANNUAL MEETING 2015 109 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 110 Late-Breaking Poster Session: Epidemiology are detectable years prior to diagnosis. Future studies exploring these associations with tumor hormone receptor status, dietary intake and anthropometric measurements will aid in further identifying key pre-diagnostic plasma markers for BrCa. Poster Section 40 Poster Board 16 LB-194 Cesarean delivery and risk of childhood leukemia: findings from the Childhood Leukemia International Consortium (CLIC). Erin Marcotte,1 Thomas Thomopoulos,2 Jacqueline Clavel,3 John Dockerty,4 Sameera Ezzat,5 Stephen S. Francis,6 Claire InfanteRivard,7 Corrado Magnani,8 Catherine Metayer,6 Ana Maria Mora,9 Beth A. Mueller,10 Wafaa M. Rashed,5 Michael E. Scheurer,11 Joachim Schuz,12 Catharina Wesseling,9 Alkistis Skalkidou,2 Eleni Petridou,2 Logan Spector1. 1University of Minnesota, Minneapolis, MN; 2University of Athens, Athens, Greece; 3Inserm, Villejuif, France; 4University of Otago, Dunedin, New Zealand; 5Menoufiya University, Cairo, Egypt; 6University of California, Berkeley, Berkeley, CA; 7McGill University, Montreal, Quebec, Canada; 8Universita del Piemonte Orientale, Novara, Italy; 9Universidad Nacional, Heredia, Costa Rica; 10Fred Hutchinson Cancer Research Center, Seattle, WA; 11Baylor College of Medicine, Houston, TX; 12International Agency for Research on Cancer, Lyon, France. Introduction: Recent meta-analyses have reported modest but significant associations between birth by cesarean delivery (CD) and subsequent risk of immune-related disorders. An association of CD with childhood leukemia has not been established, although two recent case-control studies showed an increased risk of acute lymphoblastic leukemia (ALL) among young children born by CD, and elective CD (E-CD) in particular. Methods: We pooled data from 12 case-control studies in the Childhood Leukemia International Consortium. We analyzed CD overall and according to indications that likely resulted in E-CD (multiple birth and previous CD). Odds ratios (OR) and 95% confidence intervals (CIs) for risk of ALL and acute myeloid leukemia (AML) were estimated using multivariable logistic regression, adjusting for child’s birth weight, sex, age, ethnicity, parental education, maternal age, and study center. Results: Delivery method was known for 8017 ALL cases, 659 AML cases, and 21273 controls. Among three studies with information on indication for CD, data were available for 3677 ALL cases, 114 AML cases, and 3929 controls (Table 1; see next page). The association between CD and ALL (pooled OR: 1.06 [95% CI: 0.99, 1.14]) was not statistically significant, whereas birth by E-CD was associated with an increased risk of ALL (pooled OR: 1.27 [95% CI: 1.06, 1.52]). Subgroup analysis by immunophenotype revealed an association between E-CD and B-ALL (pooled OR: 1.28 [95% CI: 1.04, 1.57]), but not T-ALL. Pooled ORs for AML were 1.02 (95% CI: 0.82, 1.27) for overall CD and 1.39 (95% CI: 0.76, 2.53) for E-CD. Conclusions: Findings derived from a pooled analysis of data from several countries suggest a higher risk of childhood ALL following E-CD. More comprehensive assessment of the indications for E-CD in consortia studies will allow investigators to further explore the potential for confounding by indication. If this association is causal, maladaptive immune activation due to lack of stress response before birth and differential colonization of the microbiome in children born by E-CD should be considered as potential mechanisms. 110 Poster Section 40 Poster Board 17 LB-195 Supporting cancer epidemiology research through cohort registration: NCI’s cancer epidemiology cohort descriptive database. Crystal Lane,1 Amy Kennedy,1 Michelle Brotzman,2 Amy Miller,2 Gabriel Lai,1 Muin Khoury,3 Daniela Seminara1. 1National Cancer Institute, Rockville, MD; 2Westat, Rockville, MD; 3Centers for Disease Control and Prevention, Atlanta, GA. Carefully designed observational studies have recognized value and efficiency in determining associations between exposures and outcomes and thereby evaluating health care. In particular, longitudinal cohorts have served as an essential research infrastructure for studies producing a wealth of information on disease etiology and prevention. The onset of the ‘omics age has compelled many funding agencies to foster collaborations across existing large cohorts, to design studies large enough to address the dynamic and interactive nature of the factors underlying common diseases. There is currently an ongoing discussion in the scientific community of whether or not observational studies should be registered, and if so what the scientific and practical benefits of the implementation would be. Cohorts are the backbone of observational studies, and making their descriptive data rapidly available to the scientific community through a centralized descriptive database is the first step necessary to increase transparency, scientific quality and collaboration. We have created the Cancer Epidemiology Descriptive Cohort Database (CEDCD) to assist the research community in identifying and accessing population resources, with the intention of maximizing the efficiency and effectiveness of existing cohorts and targeting areas of needs when establishing new ones. This public database includes many details, including cohort profiles and investigator contact information, study design and eligibility criteria, enrollment numbers broken down by race/ethnicity/gender, number and types of biospecimens and cancer diagnoses, policies, protocols and questionnaires, publications and funded research projects, and links to the cohort websites and related resources. The initial phase of this database will include cohorts focusing on cancer as their primary outcome, specifically those funded by NCI’s EGRP and members of NCI’s Cohort Consortium that have more than 10,000 subjects enrolled (healthy individuals or cancer survivors). The CEDCD is serving as a model for a parallel world-wide cohort registration project. The creation of a querable web-based tools for direct population of this descriptive database, which will provide a valuable resource for the scientific community in the planning and design of large, cooperative observational studies is in progress. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 111 Late-Breaking Poster Session: Epidemiology American Association for Cancer Research • AACR ANNUAL MEETING 2015 111 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 112 Late-Breaking Poster Session: Tumor Biology 2 Late-Breaking Poster Session Tuesday, April 21, 2015 8:00 AM-12:00 PM Poster Section 41 Late-Breaking Research: Tumor Biology 2 Poster Section 41 Poster Board 1 LB-197 Circulating rare cells enable highly efficient cancer detection. Eva Obermayr,1 Elisabeth Maritschnegg,2 Paul Speiser,2 Christian Singer,2 Eva M. Schuster,2 Sabine Danzinger,2 Nina Pecha,2 Robert Zeillinger2. 1Ludwig Boltzmann Cluster Translational Oncology, Vienna, Austria; 2Medical University of Vienna, Vienna, Austria. Objectives of the Study: We intended to develop a protocol combining a novel micro-fluidic enrichment technology and RTqPCR for the molecular analysis of CTCs. Recently we identified CTC-specific mRNA markers allowing CTC detection in 29% of breast cancer patients and in 24.5% of ovarian cancer patients at diagnosis. However, the detection of cancer cells was hampered by the large number of contaminating leukocytes. The necessary use of cut-off values for positivity reduced the specificity of the downstream PCR assay. By improving the purity of cancer cells and PCR analysis we sought to increase both sensitivity and specificity of the diagnostic procedure. Methodology: For reducing the blood sample volume (up to 20ml) density gradient centrifugation was used when needed. The samples were passed through a micro-fluidic disposable cassette, which captures tumor cells based on their less deformable nature and larger size compared to blood cells. Lysis of the captured cells was done directly in the cassette and total RNA was extracted. After a cDNA pre-amplification step gene expression levels of leukocyte-specific and CTC-related markers were measured using RT-qPCR. The efficiency of the combined protocol was assessed in blood samples taken from patients with primary and recurrent malignant diseases. Results: 7 out of 13 pre-selected RNA markers were not detectable in blood samples from 11 healthy volunteers. In cancer patients we observed measurable gene expression of at least one out of these 7 RNA markers. 5 out of 5 breast cancer (primary disease: N=1; metastatic: N=4), 9 out of 10 primary and 4 out of 8 relapsed ovarian cancer patients were classified correctly by the test. The most striking finding is the detection rate of 90% for primary ovarian cancer at a specificity of 100%. Conclusions: The enrichment of rare cells from blood samples using micro-fluidics results in a highly pure cell population. This enables the application of extremely sensitive methods, like RTqPCR to specifically detect rare events. By combining a novel micro-fluidic cell enrichment and molecular analysis we have taken a major step forward, which allows the implementation of liquid biopsies in cancer detection studies and as a companion diagnostic in clinical trials. Poster Section 41 Poster Board 2 LB-198 Molecular drivers of metabolic reprogramming during EMT. Zi Qiang Teo, Ming Keat Sng, Jeremy CHAN, Nguan Soon Tan. Nanyang Technological University, Singapore, Singapore. The reprogrammed cancer metabolism is an emerging cancer hallmark pervasive in many cancers and is fundamental to neoplastic progression. Many key cancer-promoting pathways are known to converge on the regulation of cellular metabolism. Besides providing basic sustenance to fuel cancer cells limitless replicative potential, emerging data reveals critical roles for the reprogammed metabolism during maglignant progression. Metastasis is often regarded as the end-stage of malignancy and majority of cancer mortality is attributed to the metastatic dissemination of the malignant cancer cells. The initiation of metastasis has been pinpointed as pivotal event, without which, the malignant cancer cells loses their motility and invasive capabilities 112 for distal dissemination. This initiation event highly resembles epithelial-mesenchymal transition (EMT) process that occurs during embryogenesis and wound healing. Microenvironmental signals such as hypoxia and the inflammatory milleu that initiate EMT, interestingly, are also shown to be critical in rewiring cancer metabolism. Thus, it is conceivable that the manipulation of cancer metabolism can impact EMT. Furthermore, whether such metabolic reprogramming is a functional pre-requisite for the initiation of EMT remains unclear. Using three distinct in vitro EMT models, we show that EMT is an energy-demanding process. Comparative microarray analysis of the EMT models reveals that genes involved in fatty acid, glucose and ROS metabolism are altered. We went further to show that cancer cells undergoing EMT, derives much of the ATP/energy from glucose and lipid metabolism. Importantly, we identified several key molecular drivers that coordinate EMT and cellular bioenergetics, which is validated by our in vivo EMT model. Poster Section 41 Poster Board 4 LB-200 Loss of the scaffold protein Kibra in a mouse model of triple-negative breast cancer. Jennifer France Knight,1 Tina Gruosso,1 Danielle Angeline de Verteuil,1 Sadiq Saleh,1 Robert Lesurf,1 Hong Zhao,1 Ryan Davis,2 Dongmei Zuo,1 Robert Cardiff,2 Jeffrey Gregg,2 Michael Hallett,1 Morag Park1. 1Goodman Cancer Research Center, Montreal, Quebec, Canada; 2University of California at Davis, Davis, CA. Introduction: Targeted therapies in breast cancer rely on tumor cell expression of Estrogen and/or HER2 receptors. Triple negative breast cancers (TNBCs), lacking these receptors, have no targeted therapies. We have developed a preclinical murine model expressing a naturally occurring oncogenic variant of the Met receptor in the mammary gland combined with loss of function of the tumor suppressor p53 (MMTV-Met;Trp53fl/+;Cre). This model recapitulates many features of TNBC at the level of gene expression, pathological markers and genomic alterations. Notably, this model spontaneously undergoes loss of a genomic region syntenic with human chromosome 5q which is lost in up to 70% of human TNBCs (Turner et al., 2010). This provides an opportunity for us to identify and study potential driver genes for this breast cancer subtype. Experimental Procedures: Approximately 80% of MMTVMet;Trp53fl/+;Cre mammary tumors have a mesenchymal pathology that recapitulates key features of TNBC. Array CGH profiling showed a consistent loss of chromosome 11:31.3558.2Mb, syntenic with human 5q31.1 and 5q33.1-35.3. Analysis of mouse model expression profiling data confirmed decreased expression of 83 genes within this region. Comparison with human breast cancers within the TCGA dataset further highlighted 14 of these genes with significantly decreased expression in humans with the TNBC subtypes ‘Basal’ and ‘Claudin-low’. One of these genes, WWC1, undergoes heterozygous deletion in 49% of human Basal breast cancers (TCGA, Nature 2012) and has also been associated with Claudin-low cancers (Moleirinho et al., 2013). WWC1, also known as Kibra, encodes a scaffold protein that positively regulates the Hippo tumor suppressor pathway. We used both Kibra knockdown and over-expression to understand the functional consequences of Kibra-loss. Results: MMTV-Met solid carcinoma cells retain both Kibra expression and epithelial characteristics typical of human luminal breast cancers (Ponzo et al., 2009). Kibra depletion by siRNA in these cells led to weakening of cell-cell junctions and colony dispersal, as well as decreased cortical actin and increased membrane protrusions, consistent with epithelial-to-mesenchymal transition (EMT). Mice injected with Kibra shRNA-tumor cells developed secondary lesions at sites distant to the mammary gland. By comparison, re-expression of Kibra in MMTVMet;Trp53fl/+ cell lines and human TNBC cells led to morphological changes consistent with an EMT reversal, including loss of actin rich protrusions. Furthermore, re-expressing Kibra impaired Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 113 Late-Breaking Poster Session: Tumor Biology 2 proliferation in vitro and decreased tumorigenicity in vivo. Globally, our results strongly suggest that Kibra loss plays a significant role in TNBC development and metastasis and is a candidate driver gene for Chr5q loss. Poster Section 41 Poster Board 5 LB-201 Functional role for tumor heterogeneity: Paracrine signaling between tumor subclones of mouse SCLC promotes metastasis. Min Chul Kwon, Natalie Proost, Kate Sutherland, John Zevenhoven, Anton Berns. Netherlands Cancer Institute, Amsterdam, Netherlands. Tumor heterogeneity might not only lead to pools of cells with a different resistance profile to therapy, the heterogeneity can also create a unique tumor-microenvironment. Earlier we have shown that clonal evolution in mouse SCLC can result in subclones with either neuro-endocrine (NE) and non-NE features. Co-grafting of such subclones endow the NE tumor cells with metastatic potential. To address the underlying mechanism we conducted a series of in vivo graft experiments and found that non-cell autonomous paracrine signaling is required in the subcutaneously grafted tumor to permit dissemination. The expression of transcription factor Pea3 in NE cells was identified as a functional downstream target of this paracrine interaction between the tumor subclones. Fgf2 secreted by non-NE subclones causes the expression of Pea3 in the NE cells through activation of Mapk pathway thereby temporarily potentiating its invasive capacity and facilitating intravasation into the circulation. These findings reveal a cooperating role of tumor cell subclones in mouse SCLC. Our data indicate that inhibition of the Fgf2/Mapk/Pea3 axis might be exploited to impair growth and metastatic spread of SCLC. Given the similarity in phenotypic tumor cell heterogenenity and signaling aberrations between mouse and human SCLC, inhibition of this signaling axis is therefore worth exploring in human SCLC. Poster Section 41 Poster Board 6 LB-202 14-3-3ζ turns TGF-β’s function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2. Jia Xu,1 Sunil Acharya,1 Ozgur Sahin,1 Lin Zhang,1 Frank J. Lowery,1 Aysegul A. Sahin,1 Xiang H.-f. Zhang,2 Mien-Chie Hung,1 Dihua Yu1. 1 UT MD Anderson Cancer Center, Houston, TX; 2Baylor College of Medicine, Houston, TX. TGF-β manifests multifunctional and dichotomous roles in different stages of cancer progression. In premalignant cells, TGF-β is primarily a tumor suppressor that inhibits cell proliferation or induces apoptosis. In the later stages of cancer progression, however, TGF-β functions as a metastasis promoter by inducing epithelial-mesenchymal transition (EMT), leading to increased invasion of cancer cells, and also by inducing genes that facilitate metastatic colonization of secondary organ sites. Although the opposing functions of TGF-β in early- versus late-stage cancer have been known for decades, how and when TGF-β switches its functional roles are long-standing questions with no clear answer. It has been postulated that the dichotomous functions of TGF-β are dictated by different partners of its downstream Smads. However, it is unclear how Smad partners are changed in different stages of cancer. We have investigated how Smad partners are changed in different stages of breast cancer development in 2D and 3D cell cultures by both hypothesis-driven and unbiased approaches, in mouse models of bone metastasis, by bioinformatics analysis of breast cancer TCGA data, and by immunohistochemistry analysis of different stages of human breast diseases/cancers. Together, our data demonstrated that 14-3-3ζ overexpression induces contextual changes of Smad partners. Specifically, 14-3-3ζ 1) switches off TGF-β’s cytostatic tumor suppressor function during the early stage (ADH) of breast cancer development by cytoplasmic sequestration of YAP1, leading to reduced 14-3-3σ transcription, which results in destabilization of p53, a Smad determinant for p21 transactivation in pre-malignant cells, and consequently decreased p21 expression; 2) switches on TGF-β’s metastasis promoter function during breast cancer progression (DCIS/IDC) by blocking Gli2 binding with its E3 ligase β-TrCP, thus stabilizing Gli2, a Smad determinant for PTHrP transactivation in cancer cells, leading to bone metastasis (Cancer Cell, In Press, 2015). The critical role of TGF-β in cancer, especially in the process of metastasis, has spurred the development of TGF-β-targeting agents as cancer therapeutics. Disappointingly, many of the current TGF-βtargeting drugs showed limited clinical efficacy. Considering the opposing functions of TGF-β in cancer development, general inhibition of the TGF-β pathway may have deleterious consequence. Based on our new findings, the 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as a) biomarkers and b) therapeutic targets of TGF-β-mediated cancer progression and metastasis. Poster Section 41 Poster Board 7 LB-203 Obesity promotes resistance to anti-VEGF therapy in breast cancer via pro-inflammatory and angiogenic pathways. Joao Incio,1 Daniel McManus,1 Priya Suboj,1 Nuh Rahbari,2 Shan M. Chin,1 Suboj Babycutty,1 Trupti Vardan-Kaur,1 Yuhui Huang,1 Keehoon Jung,1 Dan Duda,1 Raquel Soares,3 Dai Fukumura,1 Rakesh K. Jain1. 1Harvard Medical School/MGH, Boston, MA; 2 Department of Surgery, Technical University Dresden, Dresden, Germany; 3Department of Biochemistry, Faculty of Medicine, Porto University, Porto, Portugal. Background: Most breast cancer (BC) patients are overweight or obese at the time of diagnosis. Obesity is associated with increased risk, recurrence, and worse prognosis of BC. It has been shown that obesity associates with worse outcome in metastatic kidney or colon cancer treated with bevacizumab. If and how excess body weight contributes to the failure of anti-VEGF therapy in BC is unknown. Results: Here we found that diet-induced obesity promoted resistance to anti-VEGF therapy in two syngeneic mouse breast cancer models. The effects of anti-VEGF therapy on tumor growth and metastasis, VEGF downstream signaling pathways and vessel density were significantly attenuated in obese mice. Under obesity condition, intra-tumor adipocytes increased. These adipocyte-rich regions in breast cancers were hypoxic and overexpress IL-6 or FGF-2 by adipocytes, fibroblasts, and myeloid cells. In IL-6 overexpressing obese breast cancer model (E0771), neutralization of IL-6, either genetically or pharmacologically, abrogated the obesity-induced resistance to anti-VEGF therapy seen in both primary and metastasis sites. This occurred due to a reversion of the obesity-augmented STAT3 signaling and cell proliferation, of hypoxia via vessel normalization, and of immunosuppression. In another breast cancer model (MCaIV), which overexpress FGF-2 under obesity, anti-FGF receptor antibody restored tumor sensitivity to anti-VEGF treatment in obesity. Conclusion: Our findings indicate that obesity promotes resistance to anti-VEGF therapy in breast cancer via the production of pro-inflammatory and angiogenic factors that circumvent the loss of VEGF signaling. Poster Section 41 Poster Board 8 LB-204 Reprogramming inflammatory monocytes to mediate anti-tumor activity in pancreatic carcinoma. Kristen B. Long, Whitney L. Gladney, Graham M. Tooker, Gregory L. Beatty. University of Pennsylvania, Philadelphia, PA. In cancer, increased mobilization of inflammatory monocytes from the bone marrow into the peripheral blood correlates inversely with patient survival. Peripheral blood inflammatory monocytes, which express CCR2, are recruited to tumor tissue by the chemokine CCL2 where they then differentiate into macrophages and support tumor development, growth, and metastasis. While neutralization of CCL2 can block inflammatory monocyte recruitment and inhibit metastasis, this approach has recently been American Association for Cancer Research • AACR ANNUAL MEETING 2015 113 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 114 Late-Breaking Poster Session: Tumor Biology 2 shown to be at risk for lethal outcomes if therapy is interrupted. An alternative approach to inhibiting inflammatory monocyte recruitment to tumors is to reprogram monocytes with anti-tumor activity. Using the KrasG12D/+; Trp53R172H/+; Pdx-1 Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC), we report that systemic immune activation induced with an agonist CD40 monoclonal antibody can redirect Gr-1+ inflammatory monocytes to infiltrate the tumor microenvironment and degrade tumor-associated fibrosis leading to tumor regression. Extra-tumoral macrophages were found to be necessary for mobilization of inflammatory monocytes from the bone marrow into the peripheral blood. In addition, systemic IFN-γ released in response to anti-CD40 therapy was necessary for reprogramming inflammatory monocytes with anti-tumor activity. Inflammatory monocytes responding to IFN-γ displayed a distinct matrix metalloproteinase gene expression profile necessary for selective degradation of extracellular matrix proteins, including type I collagen and fibronectin, which define fibrosis in PDAC. Therefore, although inflammatory monocytes are commonly associated with pro-tumor activity, our findings demonstrate that they can also be reprogrammed with potent anti-tumor properties. Poster Section 41 Poster Board 9 LB-205 Myeloid cells are required to establish an immunesuppressive regulatory network in pancreatic cancer by activating the PD-1/PD-L1 checkpoint. Yaqing Zhang, Esha Mathew, Flor Mendez, Kevin Flannagan, Diane Simeone, Marina Pasca Di Magliano. University of Michigan, Ann Arbor, MI. Background: Pancreatic cancer is characterized by the accumulation of a fibro-inflammatory stroma. Myeloid cells are a predominant population within the stroma. Different myeloid cell subsets have been correlated with tumor promotion and unmasking of anti-tumor immunity. Objective: The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer, and to understand the relationship between myeloid cells and T cell infiltration and activity within the pancreatic cancer microenvironment. Methods: Primary mouse pancreatic cancer cells were transplanted in CD11b-DTR mice. CD11b+ cells (most myeloid cell populations) were depleted by Diphtheria Toxin treatment; either at the time of tumor implantation or after tumors had formed. Results: Depletion of myeloid cells delayed tumor initiation. In pre-established tumors, myeloid cell depletion resulted in arrest of growth or tumor regression. We observed that tumor regression was dependent myeloid cell-mediated blockade of CD8+ T cell anti-tumor activity. We investigated the mechanism of this inhibition. We found that myeloid cells regulate expression of the immune checkpoint ligand Programmed death-ligand 1 (PD-L1) in the tumor cells in an EGFR/MAPK dependent manner. Conclusions: Our results show that myeloid cells regulate a complex network of signals that ensure immune suppression within the pancreatic cancer microenvironment. Moreover, we show that depletion of the myeloid cell population is sufficient to restore antitumor immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer. Poster Section 41 Poster Board 10 LB-206 The role of fibroblast activation protein (FAP) in lung tumorigenesis. Diana Avery, Michelle Jacob, Lisa Chang, Leslie Todd, Ellen Pure. University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA. The desmoplastic reaction, characterized by the recruitment and differentiation of a heterogeneous population of carcinomaassociated stromal cells (CASCs) as well as extensive extracellular matrix (ECM) deposition and remodeling, regulates tumorigenesis via complex and incompletely understood mechanisms. In virtually 114 all epithelial-derived tumors, CASCs selectively express FAP, a cell surface serine protease that promotes tumorigenesis. We investigated the mechanisms that govern FAP expression as well as the mechanisms by which FAP regulates the intratumoral desmoplastic reaction and, consequently, lung tumor progression. A screen of soluble factors revealed (1) that the inducible expression and activity of FAP in lung fibroblasts depended on ECM components and (2) differential requirements for induction of FAP and αSMA (a canonical marker of myofibroblasts). Unlike TGFβ-mediated induction of αSMA expression, TGFβ-mediated induction of FAP expression depended on a collagen-rich substratum. These experiments underscored the fundamental role of ECM in mediating fibroblast differentiation. We further delineated bidirectional regulatory mechanisms between activated fibroblasts and ECM by studying FAP-dependent changes to ECM. Using twophoton second harmonic generation imaging, we found that activated FAP-/- lung fibroblasts deposited ECMs with a significant twofold enhancement of fibrillar collagen accumulation compared to WT fibroblast-derived ECMs. Immunoblots illustrated differences in type I collagen fragmentation in WT versus FAP-/- fibroblastderived ECMs, substantiating prior evidence that FAP plays a role in the ordered proteolytic processing of fibrillar collagen. In addition, collagen contraction assays demonstrated that FAP negatively regulates fibroblast-mediated contraction of type I collagen. Collectively, these experiments highlight the importance of FAP in orchestrating fibroblast-dependent remodeling of intratumoral ECM composition and architecture. Lastly, by analyzing tumor burden in the spontaneous KrasG12D latent allele model, we found that FAP-/mice had significantly smaller lung tumors than WT mice. However, the multiplicity of lung tumors did not differ between WT and FAP-/mice. These results indicate that FAP promotes progression, rather than initiation, of lung tumorigenesis. Current analyses are studying differences in intratumoral ECM in this model and how these changes to lung CASC-derived ECM influence tumor cell proliferation. In summary, we have delineated bidirectional regulatory mechanisms between FAP and ECM as well as FAPdependent regulation of the desmoplastic reaction and lung tumor progression. These studies further clarify the mechanisms by which the microenvironment promotes tumorigenesis and validate FAP as a potential therapeutic target in the treatment of cancer. Poster Section 41 Poster Board 11 LB-207 ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell typespecific manner. Md. Ruhul Amin, Yuiko Morita-Fujimura, Shuntaro Ikawa. Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Japan. Introduction: Despite apparent resection of tumors, breast cancer patients often suffer relapse years after due to remnant dormant tumor cells. Nevertheless, the molecular mechanism regulating dormancy of breast cancer cell is still unclear. On the other hand, relapsed tumor is generally believed to be derived from cancer stem cells retaining normal stem cell property, which can undergo quiescence. ΔNp63α, an N-terminally truncated isoform of p51/p63 protein was shown to play crucial roles in the maintenance of stem cells within mammary epithelium. Furthermore, both tumor suppressive and oncogenic roles of ΔNp63α have been reported in mammary carcinogenesis. Thus, we investigated the role of ΔNp63α in the regulation of normal and malignant breast cells. Method: MCF10A, normal human mammary epithelial cells and breast cancer cell lines of different subtypes were introduced with doxycycline-inducible constructs of ΔNp63α. These cells were subjected to cell cycle analysis, western blot, BrdU-Ki-67 immunostaining. Stemness was determined by mammosphere formation assay combined with flow cytometry analysis of cell surface markers. Drug response was studied using MTT assay. Also luminal breast cancer cell line, MCF7, was subjected to combined mRNA-miRNA microarray analysis. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 115 Late-Breaking Poster Session: Tumor Biology 2 Results: The induction of ΔNp63α in MCF7 luminal breast cancer cell line led the cells to acquire phenotype typical to quiescent luminal progenitor-like cells. Furthermore, the BMP and Wnt signaling pathways emerged as regulators of ΔNp63α-induced quiescence by microarray analysis. Interestingly, these quiescent cells exhibited down-regulation of BRCA1-dependent DNA repair pathways making them more sensitive to DNA damaging drug, Doxorubicin, but more resistant to anti-mitotic drug, Paclitaxel. Conversely, induction of ΔNp63α in MCF10A normal basal cells resulted in increased cell proliferation and expansion of mammary stem-like cells. ΔNp63α expression also led to the increase of cells expressing breast cancer stem cell markers in both MCF7 and MCF10A cells. However, induction of ΔNp63α affected neither cell proliferation nor the increase of mammary stem- or luminal progenitor-like cells in more aggressive luminal (T47D) and basal (MDA-MB-231) breast cancer cells containing mutant p53. This result interestingly suggested a cell type-specific function of ΔNp63α. We also identified a microRNA network that might regulate quiescence in ΔNp63α-expressing breast cancer cells which will be presented at the meeting in detail. Conclusion: Our results suggest that the role of ΔNp63α in regulating quiescence, stem or progenitor activity in normal and breast cancer cells is cell type-specific. Given the heterogeneity in the cellular origin of breast cancer subtypes, ΔNp63α-directed chemotherapeutic strategy might be instructive to target therapeutically resistant breast cancer cells. Poster Section 41 Poster Board 12 LB-208 Open air fluorescence imaging of tumors using a new, hands-free translational imaging system. Jeffrey A. Meganck, Kristine Vasquez, Jeffrey Peterson, Chris Condron, Ali Behrooz, Josh Kempner, Alexandra de Lille, Yiyong Tan, Pete Harvey, Hongyan Gu, Paul Kennedy, Mathew Roxo, Ilias Faqir, Yan Zhang, Leo Mirkin, Peter Miller, Wael Yared. PerkinElmer, Hopkinton, MA. Intraoperative tumor resection currently relies on the ability of a surgeon to visually detect and/or palpate the tumor and tumor margins. Small tumor nodules can be missed or tumor margins may be inadequately removed, resulting in the need for secondary treatment. Intraoperative fluorescence imaging can help improve the initial resection and, therefore, both improve outcomes and reduce cost. Unconjugated fluorescent dyes have been previously used for this type of study to identify tumors in first-in-human studies. However, dyes conjugated to a targeting moiety have better specificity for the tumor itself and provide better guidance for the surgeon to locate the tumor and remove margins. The new Solaris imaging system is an open air fluorescence imaging instrument designed specifically for intraoperative imaging in small to large animals. The system supports 4 different fluorescence channels to image common dyes (e.g. indocyanine green [ICG] and Fluorescein isothiocyanate [FITC]) and more unique near-infrared (NIR) fluorescent dyes. All of these can be imaged in ambient light to achieve sensitivities of 10 nM for single, long exposures and 10-100 nM for videos. The imaging head is attached to an adjustable arm so that it be can be positioned 75 cm above the object plane, far enough to be considered outside of the sterile field. The imaging head also has two cameras for simultaneous fluorescence and bright field (color) imaging; these images can be overlaid in the software. Single, long exposure images acquired from 2 different wavelengths can be overlaid to enable multiplexing and improve tumor identification; previously published studies have shown this to be useful for sentinel lymph node detection. For FITC, a custom liquid crystal tunable filter (LCTF) is included in the system in tandem with spectral unmixing software to separate tissue autofluorescence from fluorescence emitted by the dye. Although this system is designed for larger animals, proof of concept intraoperative tumor resection has been performed in rodents. Subcutaneous tumors have been resected while imaging mice injected with either the targeted agent IntegriSense™ 680 or the activatable agent ProSense® 750. To investigate a more challenging model, tumor cell lines have also been implanted intrasplenically in rats. After injection with either the targeted agent BombesinRSense™ 680 or the activatable agent MMPSense® 750, deep tissue tumors can be identified intraoperatively and removed. Both the residual tumor bed (in vivo) and the resected tumors (ex vivo) can be imaged to confirm complete resection. In addition, although there are depth limitations due to the absorption and scattering of light in tissue, images acquired using a rat osteoarthritis model also provides insight into the ability to detect targeted fluorescence agents non-invasively. These results suggest that intraoperative resection of tumors detected with both targeted and activatable fluorescent agents is feasible using the new Solaris imaging system. Poster Section 41 Poster Board 13 LB-209 Neuroblastoma is biphasic with classical neuroepithelial cells and chemoresistant mesenchymal cells controlled by PRRX1-NOTCH signaling. Tim van Groningen, Natalia E. Nowakowska, Nurdan Akogul, Marloes Broekmans, Johannes Bras, Jan Booij, Marli E. Ebus, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Peter van Sluis, Jan Koster, Bart A. Westerman, Godelieve A. Tytgat, Rogier Versteeg, Johan van Nes. Academic Medical Center, Amsterdam, Netherlands. Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We observed that new neuroblastoma cell lines cultured in defined medium always include two phenotypically divergent cell types. Whole genome sequencing showed that both types were genetically identical. One cell type has a neuro-epithelial (NE) phenotype and expresses all classical and diagnostically used neuroblastoma markers. The other type has a mesenchymal (MES) character, lacks all neuroblastoma markers and is highly motile. MES cells are more chemo-resistant in vitro as compared to NE cells. Immunohistochemistry (IHC) of primary neuroblastoma detected a small fraction of MES cells in most tumors. However, MES cells were strongly enriched in surgically removed postchemotherapy samples. Moreover, neuroblastoma patients that had been tumor-free for several years but relapsed, also showed a strong accumulation of MES type cells in their relapses as compared to the primary tumors. As these data suggest a major role for this new neuroblastoma cell type in development of therapy-resistant relapses, we analyzed their key regulatory pathways. In multiple cell line models, the homeobox gene PRRX1 was identified as a master regulator that converted the NE phenotype in a MES phenotype. PRRX1 concomitantly induced a chemo-resistant phenotype in vitro. PRRX1 activated a cascade of MEK, NOTCH and PDGFRβ signaling. Also NOTCH was able to induce the mesenchymal phenotype, as well as chemo-resistance. Analysis of the PRRX1induced downstream signaling pathway identified several drugable key-players, like MEK and PDGFRβ. Targeting them with smallmolecule inhibitors specifically killed MES cells in vitro. Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells have very different characteristics, but can transdifferentiate into each other. MES cells strongly accumulate after chemo-therapy and in relapses. They may survive classical therapy and seed relapses, that ultimately become heterogeneous again. Targeted elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. Poster Section 41 Poster Board 14 LB-210 Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Martin Peifer,1 Frederik Roels,2 Falk Hertwig,2 Roopika Menon,3 Andrea Kraemer,2 Reinhard Buettner,1 Sven Perner,3 Alexander American Association for Cancer Research • AACR ANNUAL MEETING 2015 115 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 116 Late-Breaking Poster Session: Tumor Biology 2 Schramm,4 Johannes H. Schulte,4 Frank Westermann,5 Roman K. Thomas,1 Matthias Fischer2. 1University of Cologne, Cologne, Germany; 2University Hospital of Cologne, Cologne, Germany; 3 University Hospital of Bonn, Bonn, Germany; 4University Hospital of Essen, Essen, Germany; 5German Cancer Research Center, Heidelberg, Germany. Neuroblastoma is a malignant pediatric tumor of the sympathetic nervous system. While roughly half of these tumors regress spontaneously or are cured by limited therapy, high-risk neuroblastomas have an unfavorable clinical course, despite intensive multimodal treatment. The genetic basis of the various clinical subtypes of the disease has remained largely elusive. To gain a better understanding of the genetic events that may drive neuroblastoma tumorigenesis, we here performed whole-genome sequencing of 42 primary neuroblastomas (high-risk, n=25; lowrisk, n=17). We identified genomic rearrangements affecting chromosome 5p15.22 in a 50 kb region centromeric of the human telomerase reverse transcriptase gene (TERT) in 8 tumors. The rearrangements occurred only in high-risk neuroblastomas (8/25, 32%) in mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumor type. In an Independent validation cohort of 14 high-risk neuroblastomas, we detected rearrangements of the TERT locus in 4 additional samples. The structure of the rearrangements varied greatly, including balanced translocations, low-level copy number gains, focal amplifications and chromothripsis. Independent of the copy number at this region, all alterations consistently induced massive transcriptional up-regulation of TERT and of three additional genes located in close proximity to the chromosomal breakpoint. By contrast, MYCN-amplified tumors showed only upregulation of TERT itself, suggesting that both MYCN amplification and TERT rearrangements converge on TERT activation. Supporting a functional role of TERT, both MYCN-amplified neuroblastoma cell lines and cell lines bearing TERT rearrangements exhibited elevated TERT expression and enzymatic telomerase activity in comparison to cell lines without these aberrations. Our findings show that remodeling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma, and places telomerase activation in the center of transformation in a large fraction of these tumors. More broadly, our findings provide a mechanistic basis for molecular diagnosis and therapy of this deadly pediatric tumor entity. Poster Section 41 Poster Board 15 LB-211 NUP98-PHF23 is a novel fusion gene in pediatric cytogenetically normal acute myeloid leukemia. Marco Togni,1 Riccardo Masetti,1 Martina Pigazzi,2 Annalisa Astolfi,3 Daniele Zama,1 Valentina Indio,3 Salvatore Serravalle,1 Elena Manara,2 Valeria Bisio,2 Sergio Rutella,4 Franca Fagioli,5 Giuseppe Basso,2 Andrea Pession,1 Franco Locatelli6. 1Department of Pediatrics, “Lalla Seràgnoli”, Hematology- Oncology Unit, University of Bologna, Bologna, Italy; 2Department of Paediatric Haematology, University of Padova, Padova, Italy; 3Giorgio Prodi Cancer Research Centre, University of Bologna, Bologna, Italy; 4 Department of Haematology, Catholic University Medical School, Roma, Italy; 5Department of Pediatric Hematology-Oncology, Ospedale Regina Margherita, Torino, Italy; 6Department of Pediatric Hematology-Oncology, IRCCS Ospedale Bambino Gesù University of Pavia, Roma, Italy. Introduction and Aim: Childhood cytogenetically normal acute myeloid leukemia (CN-AML) is a subgroup of pediatric AML lacking any known cytogenetic and abnormality. CN-AML accounts for 20% of pediatric AML and exhibits a very heterogeneous response to treatment and, consequently, variable outcome. With the aim of providing new insights into the molecular lesions of this subset of AML, we analyzed, through RNA-seq, 13 cases of pediatric CNAML, corroborating our findings in an independent cohort of 152 AML patients enrolled in the AIEOP AML 2002/01 clinical trial. Methods: Paired-end RNA-seq (75x2) was performed on 116 PoliA(+) RNA extracted from blasts at diagnosis. ChimeraScan, deFuse and FusionMap algorithms were used for chimeric transcript detection. Results: An alteration that was present in 2 out 13 cases was a chimeric transcript involving the genes nucleoporin 98kDa (NUP98) and PHD finger protein 23 (PHF23), resulting from a cryptic translocation t(11;17)(p15;p13). Both patients showed an in-frame fusion between exon 13 of NUP98 and exon 4 of PHF23 that was confirmed by RT-PCR analysis and Sanger sequencing. To date, the cryptic translocation t(11;17)(p15;p13) was never described before in pediatric AML and has been reported only once in an adult AML patient (Reader, et al. Leukemia 2007). In both our patients the same breakpoint in PHF23 gene at the beginning of exon 4 was present, which was different from that present in the adult patient. To assess the incidence of NUP98-PHF23 fusion in pediatric CNAML, then we studied by RT-PCR and Sanger sequencing a validation cohort of 152 CN-AML children negative for the most common recurrent cytogenetic and molecular lesions, i.e. those involving MLL, CBFB, NPM1 and FLT3. Overall, 2 out of the 152 CN-AML cases were found positive for NUP98-PHF23, demonstrating that this genomic aberrancy is not rare in pediatric CN-AML (tentative frequency 2.4%). Fluorescence in situ hybridization analysis confirmed the cryptic chromosomal translocation t(7;11)(p15;p13) leading to the fusion between NUP98 and PHF23 in all cases. Conclusions: Lately, genome-wide approaches have been widely used to investigate the mutational landscape of CN-AML in adults. However, the genetic profile of childhood CN-AML still needs to be defined. Here, for the first time, we report the identification of a NUP98-PHF23 chimeric transcript in pediatric CN-AML. Together with recently published data demonstrating that NUP98-PHF23 promoted leukemogenesis and the observation that treatment with inhibitors of PHD-domain-mediated H3K4me3 binding, such as disulfiram (an FDA-approved drug), could selectively killed cells expressing NUP98-PHF23 (Gough, et al Cancer Discovery 2014), our findings enforce the role of epigenetic regulators in pediatric AML and suggest screening for this fusion gene at diagnosis in CN-AML pediatric patients. Poster Section 41 Poster Board 16 LB-212 Treehouse Childhood Cancer Project: a resource for sharing and multiple cohort analysis of pediatric cancer genomics data. Olena Morozova,1 Yulia Newton,1 Melissa Cline,1 Jingchun Zhu,1 Katrina Learned,1 Josh Stuart,1 Sofie Salama,1 Robert Arceci,2 David Haussler1. 1University of California Santa Cruz, Santa Cruz, CA; 2 Phoenix Children’s Hospital, Phoenix, AZ. Deep sequencing of adult and pediatric tumors revealed that different cancers share common genetic mutations. Aside from sequence mutation, gene expression, copy number, and epigenetic mechanisms contribute to tumorigenesis, and integrating this information may reveal more aberrant signaling pathways than analysis of mutations alone. Significantly, agents targeting specific pathways may be effective against multiple malignancies, regardless of the mechanisms of pathway deregulation. These observations suggest that pediatric cancer patients may benefit from targeted therapies developed for adults. Since the development of pediatric-cancer-specific therapies is hindered by the limited involvement of pharmaceutical companies and small patient cohorts, repositioning drugs designed for adult tumors remains the fastest and most effective way to bring new treatment options to pediatric cancer patients While pediatric tumors have been characterized by genomewide technologies, the data from these studies are typically underutilized beyond the initial single cohort, single data type analyses. Consequently, we still lack a comprehensive picture of the molecular pathways that contribute to pediatric cancer in each patient, especially those that can be targeted in the clinic. Integrating multiple datasets is essential for assembling large Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 117 Late-Breaking Poster Session: Tumor Biology 2 enough patient cohorts to achieve an understanding of cancerdriving molecular aberrations in individual patients. The Treehouse Childhood Cancer Project consolidates gene expression, mutation and copy number datasets under the UCSC Cancer Genomics Browser (https://genome-cancer.ucsc.edu), and currently contains data from over 1000 pediatric tumors from TARGET and other studies. Treehouse enables mining these data alongside the data from adult cancers studied by The Cancer Genome Atlas consortium (TCGA). This is accomplished using bioinformatics tools developed for the TCGA Pan-Cancer Analysis Working Group and aimed at identifying situations where a subset of pediatric tumors may be driven by similar molecular pathways as adult tumors. We have assembled a consortium of researchers who plan to both contribute data to the Treehouse platform and apply Treehouse data in their analyses. These include John Maris (Children’s Hospital of Philadelphia), Michael Taylor (Hospital for Sick Children, Toronto), Poul Sorensen (University of British Columbia), Timothy Triche (Children’s Hospital Los Angeles), Soheil Meshinchi (Fred Hutchinson Cancer Research Center), Doug Hawkins (Seattle Children’s Hospital), Javed Khan (NIH Center for Cancer Research), Ching Lao (Texas Children’s Hospital), Leonard Sender (UC Irvine, Children’s Hospital of Orange County), Alejandro Sweet-Cordero (Stanford School of Medicine), and D.W. Parsons (Baylor College of Medicine). In this submission, we demonstrate the utility of the Treehouse resource by analyzing the neuroblastoma TARGET cohort in the context of adult TCGA cancers. This work presents a proof of concept that cross-cancer multiple cohort analysis can lead to new insights into pediatric malignancies. Poster Section 41 Poster Board 17 LB-213 Combination of epigenetic modifiers achieves complete remission in xenograft models of pediatric acute myeloid leukemia. Anilkumar Gopalakrishnapillai, E Anders Kolb, Sonali P. Barwe. Nemours/A. I. duPont Hospital for Children, Wilmington, DE. Acute myeloid leukemia (AML) is the second most-common form of leukemia in children. Although, the 5-year survival rate for children with AML is estimated at 64%, nearly half of the patients have refractory disease. Novel targeted therapeutics with minimal side effects are required to improve the outcome in kids with refractory disease. One class of molecular targeted therapeutics that shows specific anti-leukemic effects while sparing normal hematopoeitic progenitor cells includes inhibitors of epigenetic modifications such as DNA methylation and histone deacetylation. Although DNA methyltransferase (DNMT) inhibitors are currently on clinical trials for AML, their efficacy as single agents is limited and the mechanism of action is unknown. This limited efficacy could be due to co-regulatory effects of DNA and histone modifications on gene expression. Therefore, DNA hypomethylating agents when used in combination with other epigenetically active agents such as histone deacetylase (HDAC) inhibitors should achieve greater efficacy by optimal re-expression of tumor suppressor genes silenced in AML. Our data shows a synergistic induction of cytotoxicity upon treatment with azacytidine (DNMT inhibitor) and panobinostat (HDAC inhibitor) with combination indices ranging from 0.6 to 0.8 in a variety of pediatric AML cell lines bearing distinct chromosomal abnormalities. Furthermore, immune-deficient (NSG-B2m) mice transplanted with MV4;11 cells via the tail vein were treated i.p. with 20 doses of maximally tolerated dose of 2.5 mg/Kg azacytidine and/or 2.5 mg/Kg panobinostat over a period of 33 days. The mice treated with azacytidine or panobinostat increased median survival by 26 and 6 days respectively. However, mice treated with both drugs showed a drastic reduction in leukemic burden leading to complete remission till the end of their life (around 2 years). Reduced leukemic burden and prolonged survival was also observed in AML-193 xenografted mice treated with the drug combination. In an effort to understand the mechanism of synergism between azacytidine and panobinostat, an Infinium Human Methylation 450K Bead Chip array was performed to identify methylation status of 400,000 CpG islands spanning promoter regions and island shores. Azacytidine treatment reduced the number of hypermethylated CpG islands by 30%, while panobinostat had no effect. However, treatment with drug combination did not change the number of hypermethylated CpG islands indicating that the observed synergy both in vitro and in vivo might result from specific activation of a subset of gene targets. Identification and validation of targets that mediate these synergistic effects is in progress to improve the selection of patients most likely to benefit from combination therapy. Poster Section 41 Poster Board 18 LB-214 DNAJB1-PRKACA chimera increases Aurora kinase A expression in fibrolamellar hepatocellular carcinoma. Irene Isabel P. Lim,1 Emily A. Greene-Colozzi,2 Jennifer M. Murphy,1 Todd E. Heaton,1 Sanford M. Simon,2 Michael P. LaQuaglia1. 1 Memorial Sloan Kettering Cancer Center, New York, NY; 2 Rockefeller University, New York, NY. Purpose: Treatment of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver cancer affecting the pediatric and adolescent population, is limited to surgical resection with no agents or therapeutic modalities available for unresectable disease. There has been recent interest in using Aurora kinase A (AURKA) inhibitors, with a clinical trial evaluating its efficacy in FL-HCC pending. Previously, we described a consistent single deletion of approximately 400 kB in chromosome 19 that results in a chimera between the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A, PRKACA, in all FL-HCC tumor tissue studied. Furthermore, comparative ribonucleic acid sequence analysis has demonstrated that expression of AURKA, a regulator of cytokinesis and mitosis, is increased in FL-HCC tumor tissue compared with adjacent normal tissue. We hypothesize that the DNAJB1-PRKACA chimera is responsible for changes in AURKA expression in FLHCC. Methods: The DNAJB1-PRKACA chimera was cloned into a lentiviral vector with a fluorescent reporter. Two stable cell lines (HeLa and a hepatocyte cell line, Huh-7) were transduced with a lentivirus containing either an empty multicloning site (control) or the DNAJB1-PRKACA chimera. Transduction was confirmed by the presence of a positive epifluorescent signal and immunoblots showing expression of the chimeric protein. Microarray data were evaluated for changes in AURKA expression while immunoblots were analyzed for the presence of AURKA in all transduced cells. Results: Microarray analysis showed an increase in AURKA transcripts in cells stably expressing the DNAJB1-PRKACA chimera. Compared to cells transduced with the control lentivirus, HeLa and Huh-7 cells transduced with the DNAJB1-PRKACA chimera demonstrated an increase in AURKA on immunoblots. Levels of a loading control, actin, were equivalent, indicating equal levels of protein examined between samples. Conclusion: Expression of the DNAJB1-PRKACA chimera results in an increase in AURKA. Upregulation of AURKA, in turn, may be responsible for subsequent tumor formation in FL-HCC. This finding further supports the DNAJB1-PRKACA chimera’s role in the pathogenesis of FL-HCC and provides a potential therapeutic target for a disease that has no effective treatment besides surgery. Poster Section 41 Poster Board 19 LB-215 Epigenetic loss-of-function BRCA1 mediates tumor cure by single dose radiotherapy. Cecile G. CAMPAGNE, Tin H. Thin, John D. Fuller, Ellen Ackerstaff, Jason A. Koutcher, Adriana Haimovitz-Friedman, Simon N. Powell, Richard N. Kolesnick, Zvi Fuks. MSKCC, New York, NY. The mechanism of tumor cure by ionizing radiation is regarded tumor cell autonomous, effected by misrepair of radiation-induced DNA double strand breaks (DSBs), mainly via the function of error prone non-homologous end joining (NHEJ). Genomic instability yields mitosis-dependent buildup of potentially lethal chromosomal American Association for Cancer Research • AACR ANNUAL MEETING 2015 117 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 118 Late-Breaking Poster Session: Tumor Biology 2 aberrations, with repeated radiation exposures required for tumor ablation. Here we show that at the high dose range (>10Gy), single dose radiotherapy (SDRT) engages an alternative dual target model, inducing in addition to DSBs also an early wave of acid sphingomyelinase (ASMase)-mediated microcirculatory ischemia/reperfusion injury. DSB repair was analyzed in situ by quantitative assessment of the time-dependent buildup and resolution of ionizing radiationinduced foci (IRIF) of specific NHEJ or homology-driven repair (HDR) mediators. Effect of SDRT on the tumor microvasculature was assessed by dynamic contrast-enhanced magnetic resonance imaging. Engagement of microvascular dysfunction in DSB repair was assessed using ASMase-deficient mice, refractory to vascular endothelial injury. Western blot analysis of Small Ubiquitin-like Modifiers (SUMO) in tumor extracts and studies of SUMO conjugating enzymes IRIF in situ were used to evaluate effects of SDRT on SUMOylation. SDRT concomitantly induces DSBs in tumor cells and an early wave (<1 hour) of ASMase-mediated microcirculatory ischemia/reperfusion, synthetically coupling parenchymal tumor cell 118 DNA damage response to re-program tumor lethality. Ischemia/reperfusion induced in reperfused tumor clonogens an oxidative stress, dysfunctioning therein SUMO conjugating enzymes that are critically required for assembly of the inherent DSB repair codex, leading to catastrophic reprograming of DSB repair. While Ku- and 53BP1-mediated NHEJ were not affected, although 53BP1 resolution was delayed, HDR was aborted. We show that SUMO 2/3 dysfunction, specifically induced post reperfusion, impairs recruitment of RAP80, BRCA1, RPA and RAD51 into DSB repair foci. The epigenetic loss-of-function BRCA1/HDR diverted DSB repair to an aberrant lethal NHEJ pathway, yielding massive lethal chromosomal aberrations, reproductive cell death and tumor cure. The dual target microvascular/tumor clonogen model, described here, which functions exclusively at high-dose radiation exposures, constitutes a functional alternative to the classical single target mechanism operating at the low dose range, and provides new targets for modulation of the radiation response, with a potential for yielding new options for tumor ablation in the clinical management of human cancer. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 119 Late-Breaking Poster Session: Immunology Late-Breaking Poster Session Tuesday, April 21, 2015 1:00 PM-5:00 PM Poster Section 39 Late-Breaking Research: Immunology Poster Section 39 Poster Board 1 LB-217 PD-1 suppresses antibody responses to Tn+ tumors. Marcela A. Leyva, Karen M. Haas. Wake Forest School of Medicine, Winston Salem, NC. Cancer cells often have aberrant glycosylation of surface molecules leading to glycan antigens that are not typically found on normal cells. These antigens are referred to as tumor associated carbohydrate antigens (TACAs) and are a promising target for anticancer vaccines. One such TACA is the Thomsen-nouvelle (Tn) antigen, which is expressed by up to 90% of adenocarcinomas. The mechanisms regulating the B cell response to Tn and other TACAs are not well understood. This knowledge is essential for the development of vaccines to elicit protective immune responses to tumors bearing TACAs. PD-1 (programmed cell death 1) is a member of the B7:CD28 superfamily of receptors found on antigen-activated B and T cells. It negatively regulates antigen receptor signaling and thereby limits adaptive immune responses, including the anti-tumor T cell response. However, virtually nothing is known about the role of PD1 in regulating B cell responses to tumors. In this study, we tested the hypothesis that PD-1 suppresses B cell production of Tnspecific antibodies and thereby suppresses protective humoral immunity against Tn+ tumors. Our results show that PD-1-/- mice immunized with a Tn-bearing mucin (desialyated bovine submaxillary mucin) have increased Tn+ tumor-reactive IgM and IgG antibodies, relative to wild type mice. PD-1-/- mice immunized with the Tn+ mucin had significantly increased survival relative to wild type mice following challenge with a Tn+ bearing mammary tumor (TA3-Ha). Survival in PD-1-/- mice was dependent on the presence of B cells. Therefore, our results show that PD-1 plays a critical role in suppressing the protective anti-tumor B cell response elicited by Tn-bearing antigen immunization. This work was supported by NIH/NCI F31 CA183567-01 fellowship to M.A.L. and an American Cancer Society Research Scholar Grant (RSG-12-170-01-LIB) to K.M.H. Poster Section 39 Poster Board 2 LB-218 Protein arginine methyltransferase inhibition of malignant gliomas leads to restored chemokine expression and enhanced immune effector function. Fengting Yan, Yeshavanth Banasavadi-Siddegowda, John T. Patton, Mark Lustberg, Xin Wu, Balveen Kaur, Rober A. Baiocchi. Ohio State University, Columbus, OH. Patients with Glioblastoma Multiforme (GBM) face a poor prognosis despite multimodal therapy, thus, there is an unmet need for discovery of novel therapeutic targets and approaches. The immune-privileged nature of the central nervous system and downmodulation of cytokines and chemokines in GBM tumors led us to explore epigenetic approaches to restore expression of immunerelevant genes. PRMT5 is a type II arginine methyltransferase that catalyzes symmetric dimethylation of arginine residues on histone proteins leading to transcriptional repression. Our previous work showed PRMT5 overexpression correlates with poor clinical outcome of GBM patients and that PRMT5 silencing inhibited tumor growth in vitro and in vivo. Microarray transcriptional studies following PRMT5 depletion identified potential PRMT5 target genes interferoninducible protein 10 (CXCL10) and interferon-inducible T cell α chemoattractant (CXCL11). Increased secretion of CXCL10 and CXCL11 has been shown to facilitate homing of innate and adaptive immune-effector populations that promote anti-GBM activity in vivo. Thus we further investigated the significance of restored CXCL10 and CXLC11 with PRMT5 inhibition in GBM. CXCL10 protein expression was found to be significantly lower in GBM tumors (N=15) by immunochemistry staining, compared with grade I gliomas (N=7) (p=0.0001). PRMT5 knockdown led to increased mRNA and protein expression of CXCL10 and CXCL11. Chromatin immunoprecipitation (ChIP) experiments identified CXCL10 and CXCL11 promoters to be directly targeted by PRMT5 repressive complexes. Previous work has shown that PRMT5 associates with other co-repressor molecules including HDAC2, DNA methyltransferase 3a (DNMT3a) and methyl binding domain protein 2 (MBD2). ChIP studies confirmed that PRMT5, HDAC2, DNMT3a and MBD2 proteins were all recruited to the promoters of CXCL10 and CXCL11. Interestingly, when two patient-derived GBM cell lines were transfected with siRNA targeting PRMT5, recruitment of all co-repressor proteins, PRMT5, HDAC2, DNMT3a, and MBD2 was lost on CXCL10 and CXCL11 promoters. These findings suggest that PRMT5 is a master transcriptional repressor that plays a central role in coordinating and assembling co-repressor chromatin remodeling complexes on PRMT5 target gene promoters. PRMT5 silencing resulted in secretion of biologically relevant levels of CXCL10 and CXCL11 in culture medium that promoted immune cell recruitment in transwell migration assays toward target glioma cells. Our data suggests that in addition to restoring regulatory and tumor suppressor gene expression that promotes direct GBM cell death, PRMT5 silencing may enhance immune-mediated anti-tumor activity. These findings further justify the development experimental therapeutic strategies targeting PRMT5 to modulate activity of immune-relevant genes. Poster Section 39 Poster Board 3 LB-219 Interleukin-8 promotes generation of M2 macrophages and arginase-producing granulocytes: Prognostic significance of interleukin-8 and infiltration of immune cells positive for CD163 and CD66b in tumor tissues in patients with oral squamous cell carcinoma. Masato Okamoto,1 Yohei Fujita,2 Hiroyuki Goda,2 Koh-ichi Nakashiro,2 Hiroyuki Hamakawa2. 1Department of Advanced Immunotherapeutics, Kitasato University School of Pharmacy, Tokyo, Japan; 2Ehime University Graduate School of Medicine, Ehime, Japan. Biomarkers are essential for improving the effectiveness of treatments for patients with cancer. The immunological status in a tumor microenvironment is closely associated with the prognosis of cancer patients. Here we investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in 50 patients with resectable oral squamous cell carcinoma (OSCC). We found that the relapse-free survival (RFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p=0.001). The tumor expression of IL-8, the infiltration of CD163-positive cells, and of CD66b-positive cells in the tumor tissues were correlated with the serum IL-8 level (p=0.033, p=0.038 and p=0.044, respectively), and they were associated with poor clinical outcome (p=0.007, p=0.002 and p=0.001 respectively, in DFS) in all patients. A multivariate analysis revealed that N status, tumor IL-8 expression, CD163 and CD66b infiltration significantly affected the DFS of the patients. A further analysis suggested that the combination of N status with serum IL-8, tumor IL-8, CD163 and CD66b infiltration in the tumor tissues may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Finally, the in vitro experiments clearly demonstrated that IL-8 enhanced the generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10 which suppress anti-tumor immunity. Furthermore, IL-8 increased the generation of arginase-producing, CD66b-overexpressing cells from peripheral blood polymorphonuclear cells. These findings indicate that IL-8 may be involved in poor clinical outcomes via the generation of CD163-positive M2 macrophages and CD66boverexpressing, arginase-producing granulocytes, and that these American Association for Cancer Research • AACR ANNUAL MEETING 2015 119 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 120 Late-Breaking Poster Session: Immunology factors may have prognostic value as well as may be new targets for treatment in patients with resectable OSCC. Poster Section 39 Poster Board 4 LB-220 Minimal asbestos exposure in germline BAP1 heterozygous mice is associated with deregulated inflammatory response and increased risk of mesothelioma. Andrea Napolitano. University of Hawaii Cancer Center, Honolulu, HI. Germline BAP1 mutations predispose to several cancers, in particular malignant mesothelioma (MM). MM pathogenesis is generally associated to professional exposure to asbestos. However, to date we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. We hypothesized that germline BAP1 mutations might influence the asbestos-induced inflammatory response that is linked to asbestos carcinogenesis, thereby increasing the risk of developing mesothelioma after even minimal exposure. In a set of short-term experiments, we intraperitoneally injected BAP1+/- and wild-type littermates with low doses of asbestos fibers and analyzed the inflammatory response both at a cellular and humoral level. In a long-term experiment following a similar protocol, we assessed the incidence of MM in mice with and without germline BAP1 mutations and their survival. We found that, compared to their wild type littermates, BAP1+/mice exposed to low doses of asbestos fibers showed significant alterations of the peritoneal inflammatory response. In particular, we observed significantly higher levels of pro-tumorigenic alternatively polarized M2 macrophages, and lower levels of several chemokines and cytokines. Consistent with these data, BAP1+/- mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos, and shorter survival. Our findings suggest that minimal exposure to carcinogenic fibers may significantly increase the risk of malignant mesothelioma in genetically predisposed individuals carrying germline BAP1 mutations, possibly via alterations of the inflammatory response. [NOTE: Due to a scheduling confilct, the abstract below has been moved to the following poster session: Adoptive Cell Therapies; Tuesday, April 21; 8:00 am-12:00 PM; Poster Section 12; Poster Board 28.] LB-221 Diversity of tumor infiltrating lymphocyte recognition of diverse mutated antigens in cutaneous melanoma. Jessica S. Crystal, Todd Prickett, Yong-Chen Lu, Jared J. Gartner, Maria Parkhurst, Alena Gros, Yong F. Li, Mona El-Gamil, Steven A. Rosenberg, Paul F. Robbins. NIH/NCI, Bethesda, MD. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate long term tumor regression in some patients with metastatic melanoma. Recent observations suggest that autologous melanoma TIL administered to patients in adoptive T cell therapy (ACT) protocols may frequently recognize multiple tumor-specific somatic mutations, findings that have been facilitated by advances in whole exome sequencing and RNAseq methods. In an attempt to evaluate the antigenic diversity of TIL and gain some insights into the potential association between the recognition of mutated antigens and clinical responses to TIL therapy, we analyzed between 7 and 30 individual cultures derived from resected melanoma tumor fragments and pooled populations of administered TIL from each of 5 patients for their ability to recognize mutated antigens expressed by patients’ autologous tumors. Two of the patients who were evaluated exhibited durable complete tumor regressions, 1 exhibited a partial response, and 2 were non-responders to ACT. Screening assays were carried out by evaluating the interferon gamma release stimulated by the coculture of autologous patient TIL with autologous dendritic cells or EBV transformed B cells transfected with mini-genes encoding mutated antigens identified by exomic sequencing of patient tumors. Using this approach, we identified 10, 3, and 2 mutated 120 antigens targeted by TIL from the 3 patients who responded to ACT. The TIL that were administered to 1 of the non-responders appeared to recognize at least 4 mutated antigens, whereas TIL administered to the second non-responder failed to recognize any of the mutated minigenes that were tested. Immuno-dominant mutated antigens that were recognized by the majority of the evaluated TIL fragment cultures, as well as the bulk population of infusion TIL, could be identified in each of the 4 patients in this study for whom mutated antigen targets could be identified. In addition, 1 or more sub-dominant mutated antigens that were recognized by one or a relatively small percentage of the screened TIL fragment cultures were identified from each of these same 4 patients. Future studies will be directed at developing methods for enriching T cells reactive with mutated epitopes from TIL or peripheral blood in an attempt to enhance the effectiveness of ACT for the treatment of patients with metastatic melanoma and that will hopefully lead to the development of effective therapies for the treatment of patients with other malignancies. Poster Section 39 Poster Board 6 LB-222 Long-term subclonal evolution of CLL from immune selective pressure after allogeneic stem cell transplant and donor lymphocyte infusion. Haven R. Garber, Hannah Beird, Yu Cao, Jianhua Zhang, Rachel Sargent, Pei Lin, Sahil Seth, Xingzhi Song, Huandong Sun, Xizeng Mao, Lisa St John, Karen Clise-Dwyer, Gheath Alatrash, P. Andrew Futreal, Jeffrey J. Molldrem. UT MD Anderson Cancer Center, Houston, TX. Next-generation sequencing (NGS) has revealed that the malignant subclones comprising a patient’s cancer can possess tremendous genetic heterogeneity at different sites of disease and over time. In leukemia, chemotherapy can hasten subclonal evolution allowing for rare leukemic subclones with aggressive driver mutations to gain a competitive advantage and to predominate at relapse, often portending an inferior treatment response. The impact of immunotherapy on subclonal evolution is less well studied. To determine the effects of allogeneic stem cell transplant (alloSCT) and donor lymphocyte infusion (DLI) on subclonal evolution, we performed whole exome sequencing (WES) on longitudinal peripheral blood and bone marrow from 4 patients with CLL. Specifically, timepoints analyzed included pre-transplant, post-transplant relapse, and post-DLI relapse over a period of up to 8.5 years. B-CLL cells (CD19+CD5+) and normal T cells (CD3+) were sort-purified by fluorescence-activated cell sorting prior to genomic DNA extraction. Libraries for WES were constructed and sequenced to an average depth of 300x on an Illumina HiSeq 2000 using 76 bp paired-end reads. Somatic single nucleotide variants (sSNVs) and indels were called using MuTect and Pindel, respectively, and copy number changes were assessed using an inhouse algorithm. In general, these patients had more nonsynonymous mutations per pre-alloSCT sample than reported in other CLL NGS studies (average 30.3; range 8-45), likely related to the significant amount of pre-transplant therapies. Heterogeneous patterns of linear and branched subclonal evolution were seen after alloSCT and DLI in both responders and non-responders. Mutations in several candidate CLL driver genes were seen in this cohort, including SF3B1, SAMHD1, BCOR, EGR2, TP53, and DDX3X. Interestingly, sSNVs in multiple recurrently mutated CLL or cancer census genes (e.g. MAP2K1) rose to levels of detection only after alloSCT or DLI, suggesting they may play a role in immune evasion. In addition, several subclonal genetic variants, including missense mutations in FAM126B and LTBP3, were no longer detected after alloSCT or DLI and may thus represent potential neoantigens. In one treatment-refractory patient, a somatic nonsynonymous clonal CHEK2 mutation was found in 8 longitudinal samples and may represent a novel unique driver mutation. Finally, in one patient who experienced a durable complete remission after DLI, concurrent CLL WES and T-cell receptor beta chain CDR3 NGS was performed, which demonstrated a rapidly evolving T-cell repertoire Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 121 Late-Breaking Poster Session: Immunology at the time of complete remission after DLI. For CLL, alloSCT and DLI offer a potentially curative treatment strategy and a better understanding of the genes that confer susceptibility or resistance to these immunotherapies may help unlock the mechanisms that underlie these durable responses. Poster Section 39 Poster Board 8 LB-224 Nab-paclitaxel and agonist CD40 mAb combination therapy induces tumor-associated macrophage polarization switching in pancreatic cancer. Jane E. Cullis,1 Despina Siolas,1 Anirban Maitra,2 Dafna Bar-Sagi1. 1 NYU Langone Medical Center, New York, NY; 2UT MD Anderson Cancer Center, Houston, TX. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive tumor stroma that is composed of immune cells, vascular cells, fibroblasts and extracellular matrix components (Erkan et al., 2012). This increased desmoplasia correlates with decreased survival and has been shown to mediate chemoresistance (von Hoff et al., 2009). Macrophages within the stromal compartment have been shown to play tumor-promoting roles by switching from their classical immunostimulatory function (‘M1’) to an immunosuppressive state (‘M2’) (Yoshikawa et al., 2012). Nab-Paclitaxel (nab-Ptx) is an albumin-bound form of Paclitaxel that, in combination with Gemcitabine, is currently accepted as the standard chemotherapeutic regimen for pancreatic cancer. However, pre-clinical and clinical studies have suggested that nabPtx may exert its effects by altering the tumor stroma (Desai et al., 2006, von Hoff et al., 2011). Here, we show that tumor-associated macrophages uptake high levels of nab-Ptx in an orthotopic model of PDAC via macropinocytosis. Eighty to ninety percent of macrophages within the tumor internalize fluorescently labeled nabPtx ex vivo, as compared to thirty to forty percent of macrophages from the spleens of the same animals. These data suggest that M2like macrophages within the tumor microenvironment preferentially macropinocytose nab-Ptx. To analyze the potential consequence of nab-Ptx internalization on macrophages, we treated the RAW macrophage cell line with Ptx alone or in combination with the immunostimulatory cytokine IFN gamma (IFNγ). Prolonged exposure (48h) of RAW cells to Ptx induced an increase in the M1 marker iNOS, with the combination of low dose IFNγ and Ptx resulting in a synergistic increase in iNOS expression with accelerated kinetics (12h). Moreover, Ptx alone or in combination with IFNγ was able to revert IL-4-induced expression of the M2 marker Arginase 1. These findings suggest that high levels of nab-Ptx internalization by M2 macrophages may repolarize them to an M1, immunostimulatory state. Our in vitro studies suggest that the potential effect of nab-Ptx and Gemcitabine on macrophage polarization in vivo may be enhanced by the presence of an additional immunostimulatory signal. The agonist CD40 monoclonal antibody (mAb) is a member on the TNFα receptor superfamily that has been shown to induce immune cell activation and therapeutic efficacy in human and mouse models of PDAC (Beatty et al., 2013). We therefore examined the effect of combining CD40 mAb with nab-Ptx and Gemcitabine treatment on macrophage phenotype in an orthotopic model of PDAC. Our studies to date show that the induction of both an increase in M1 marker (MHCII and CD86) expression and a decrease in M2 marker expression (CD206) in pancreas tumor-associated macrophages requires the triple combination therapy. Together, these data suggest that the internalization of nab-Ptx by tumor-associated macrophages in combination with immune activating signals like CD40 mAb may be required to restore their M1-like, tumor cell cytotoxic functions. CD40 mAb and nab-Ptx combination therapy may therefore enable the effective targeting of the pancreatic tumor stroma, resulting in enhanced therapeutic benefit in PDAC. Poster Section 39 Poster Board 9 LB-225 Imprime PGG modulates the function of monocytederived M2 macrophages and dendritic cells to drive T-cell expansion. Anissa SH Chan, Xiaohong Qiu, Adria Jonas, Takashi Kangas, Nadine R. Ottoson, Nandita Bose. Biothera, Eagan, MN. Imprime PGG is a soluble, yeast β-1,3/1,6 glucan currently in phase 3 clinical trial for the treatment of cancer in conjunction with complement-activating, therapeutic monoclonal antibodies (e.g. cetuximab). Imprime PGG is a pathogen- associated molecular pattern (PAMP) that complexes with endogenous anti-β-glucan antibodies, then binds and primes innate immune cells (including neutrophils and monocytes) to kill antibody-targeted cancer cells via a complement receptor 3-dependent mechanism. The early response to a PAMP requires innate immune effector cells (neutrophils, monocytes, macrophages, dendritic cells) and is a prerequisite for subsequent activation of adaptive immune effector cells. Given that macrophages and dendritic cells are the two key antigen presenting cell types that bridge innate and adaptive immunity, the objective of this study was to evaluate the phenotypic and functional effect of Imprime PGG on human monocyte-derived macrophages and dendritic cells (MoDC). Monocytes enriched from Imprime PGG- or vehicle-treated whole blood were cultured in media containing the appropriate cytokines for differentiation of the different cell types: GM-CSF for M1 macrohages; M-CSF for M2 macrophages; M-CSF plus IL-4 for M2a macrophages; and GMCSF plus IL-4 for dendritic cells. Although Imprime PGG treatment did not affect the expression of CD206, CD209, CD163, HLA-DR, CD80, and CD86 on M1 macrophages, Imprime PGG treatment of whole blood did elicit a substantial reduction in surface expression of the scavenger receptor CD163 on M2 and M2a macrophages. Further, both M2 and M2a macrophages derived from Imprime PGG-treated whole blood substantially enhanced CD3/CD28stimulated CD4 T cell proliferation and IFNγ production, whereas those from vehicle-treated whole blood did not. MoDC from Imprime PGG-treated whole blood showed increased surface expression of the maturation and co-stimulatory markers CD80, CD83, CD86 as well as HLA-DR. Furthermore, these MoDC also showed enhanced function in an allogeneic mixed lymphocyte reaction, triggering increased CD4 and CD8 T cell expansion and increased IFNγ production versus MoDC from vehicle treated whole blood. Imprime PGG’s ability to enhance M2 mediated T cell proliferation and MoDC maturation was maintained even in the presence of tumor conditioned media. These results demonstrate that Imprime PGG treatment drives a coordinated immune response, modulating the function of M2 macrophages and MoDC, enabling the expansion of CD4 and CD8 T cell effector cells, and driving Th1 polarization. These data thereby suggest the potential of combining Imprime PGG treatment with the modalities that relieve tumor-mediated T cell immunosuppression. Poster Section 39 Poster Board 10 LB-226 Depletion of kynurenine using an engineered therapeutic enzyme potently inhibits cancer immune checkpoints both as a monotherapy and in combination with anti-PD-1. Everett Stone, Nicholas Marshall, Moses Donkor, Kendra Triplett, John Blazek, Todd Triplett, Lauren Ehrlich, George Georgiou. University of Texas at Austin, Austin, TX. Introduction: The kynurenine pathway is of key importance for immune suppression in cancer, a function exerted primarily via kynurenine as an AhR ligand that potently impairs adaptive immune responses. Inhibition of kynurenine (Kyn) synthesis, mediated by IDO1, TDO (and possibly IDO2) is of great interest for cancer immune checkpoint inhibition, with 3-4 IDO1 inhibitors currently in clinical development. However: (1) IDO1 inhibition has marginal anti-cancer effects as a monotherapy; (2) IDO1 inhibitors only block one of the two major pathways for Kynureine synthesis (the other American Association for Cancer Research • AACR ANNUAL MEETING 2015 121 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 122 Late-Breaking Poster Session: Immunology being via TDO) and (3) IDO1 inhibition does generally not impact the serum concentration of Kynurenine and thus no pharmacodynamic marker is available. Experimental Methods: We postulated that administration of a therapeutic enzyme (Kynureninase) that can degrade Kyn into nontoxic and immunologically inactive metabolites (Alanine and anthranilic acid) may be able to: (a) potently relieve cancer immune suppression. An extensive protein engineering campaign was carried out to develop a Kynureninase suitable for therapeutic administration which was then PEGylated (Kynase-PEG) to enable long circulation persistence. Kynase-PEG was evaluated in the wellestablished B16-OVA melanoma model in wild type C57BL/6J mice as a monotherapy and in combination with anti-PD-1 administration. Quantitation of tumor size/regression, histology, as well as flow cytometric analyses assessing lymphocytes in the spleen, tumor (TILs) and tumor-draining lymph nodes (dLNs) was used to evaluate efficacy. Summary: Administration of KYNase-PEG in B16-OVA melanoma allografts in C57BL/6J mice reduced serum Kyn levels and resulted in significant tumor growth retardation and extended survival in a manner indistinguishable from that observed with immune checkpoint inhibitors anti-PD1 (clone RMP1-14) or antiCTLA-4 (clone 9D9) antibodies. KYNase-PEG administration did not display anti-tumor activity in NOD-scid Il2Rγ-/- mice or in IDO1-/mice revealing that the function of the enzyme is dependent on adaptive immune responses and also on the function of stromal IDO1. Consistent with the hypothesis that depletion of Kyn relieves immune inhibition, we observed a marked increase in CD8+ TILs expressing Gzm B + IFNγ, enhanced proliferation of CD4+ and CD8+ T cells in the tumor dLNs as well as changes in tumor neovascularization. Importantly, combination of KYNase-PEG and anti-PD-1 administration resulted in complete tumor eradication in 60% of the animals (n=10 per group) for >100 days, with all the surviving animals completely rejecting tumors following rechallenge. For comparison, anti-PD-1 treatment alone, only led to 20% long-term survival in this model. Conclusions: We demonstrated that unlike IDO1 inhibitors, KYNase-PEG displays a significant anti-tumor efficacy as a monotherapy as well as excellent synergism with anti-PD1. The observed increase in tumor infiltration and proliferation of cytotoxic CD8+ T cells argues that the mechanism of action of Kynase-PEG acts by relieving the immune suppression that normally occurs in the tumor microenvironment as a consequence of Kyn accumulation. Thus, KYNase-PEG represent a “first in class” immune checkpoint enzyme. Progress in developing a clinical candidate enzyme will be discussed. Poster Section 39 Poster Board 11 LB-227 Novel synthetic vesicle for rapid in vivo expansion of CD8 T cells can significantly improve checkpoint inhibitor therapy. Adrienne V. Li,1 Jackson K. Eby,1 Peter C. DeMuth,1 Darrell J. Irvine2. 1 Vedantra Pharmaceuticals, Inc, Cambridge, MA; 2MIT, Cambridge, MA. Harnessing the immune system for cancer treatment offers many advantages over traditional methods as the immune system can generate tumor-specific killer cells and long-term memory cells to prevent recurrence. The success of recent clinical trials using T cell therapies has proven the potential of immunotherapy, but the ex vivo generation of T cells in these trials is costly and labor-intensive. Here, we developed wholly synthetic, pathogen-mimicking nanocapsules, Interbilayer-Crosslinked Multilamellar Vesicles (ICMVs), to deliver antigens to the lymphoid system, which prime large tumor-specific CD8 T cell populations in vivo. ICMVs use bilayer-to-bilayer crosslinks in the walls of multi-lamellar lipid vesicles to create an easily manufacturable, highly stable virus-like particle to present antigens and/or adjuvants to immune cells. ICMV carrying HPV16 E6 and E7 full-length proteins were administered alone or co-encapsulated with TLR agonists to mice. CD8 T cell 122 responses were assessed by tetramer staining and intracellular cytokine staining (ICS) was done to assay functionality. Efficacy of ICMV treatment was demonstrated by monitoring tumor size and survival in C57BL/6 mice bearing a lethal dose of TC-1 tumor cells. ICMVs induced unprecedented antigen-specific CD8 T cell frequencies against HPV whole proteins even without added adjuvant. Repeat doses of ICMVs boosted CD8 responses resulting in 30% of the total CD8 T cell population in blood being E7-specific while treatment with free protein elicited <1% E7-specific cells, indicating that ICMVs promote strong cross-presentation of antigens. When ICMVs were combined with anti-PD-1 to treat tumor-bearing mice, 10-15% of CD8 T cells produced IFNγ+ in response to either E6 or E7 antigens, while treatment with anti-PD-1 alone elicited <1% IFNγ+ T cells. Significant tumor regression was seen with ICMV+anti-PD-1 treatment, doubling overall survival time while anti-PD-1 treatment alone delayed tumor growth only slightly. Similar results were seen when ICMV were combined with various immunomodulators including anti-PD-L1, anti-CTLA-4 and antiCD40. These results indicate that the ICMV-induced expansion of antigen-specific CD8 T cells can significantly improve the therapeutic efficacy of checkpoint inhibitors. A triple therapy of ICMV + checkpoint inhibitor + Treg inhibiting drugs is now being assessed and eradication of >85% of established tumors was detected in preliminary experiments. These findings demonstrate the potential of ICMVs as a novel cancer immunotherapy platform for the in vivo generation of CD8 T cells. ICMV are a cost effective, easily manufactured and versatile strategy to generate T cells for immunotherapy. Poster Section 39 Poster Board 12 LB-228 Imprime PGG treatment elicits a coordinated antitumor immune response that triggers enhanced expression of PD-L1 on tumor cells as well as monocyte-derived macrophages and dendritic cells. Nandita Bose, Anissa SH Chan, Adria Jonas, Xiaohong Qiu, Nadine R. Ottoson, Takashi Kangas, Jeremy R. Graff. Biothera, Eagan, MN. Immune checkpoint inhibitors, including anti-PD-1 and anti-PDL1 antibodies are emerging as an important therapeutic modality in NSCLC as well as other cancers, as these therapies block the tumor-induced T cell suppression. Translational studies from clinical trials with PD-1 and PD-L1 targeted therapies have demonstrated that patients whose cancers show PD-L1 on the surface of tumor cells and infiltrating immune cells, or PD-1 on T cells (i.e. “adaptive immune resistance”) derived greatest benefit from these therapies. It has therefore been suggested that the efficacy of these anti-PD1/PD-L1 immunotherapies could be enhanced by combinations with agents that can adaptively induce PD-L1 expression as a consequence of de novo immune responses within the tumor microenvironment. Here we show that Imprime PGG, a yeast β1,3/1,6 glucan currently in phase 3 development in combination with complement-activating monoclonal antibodies (e.g. cetuximab), can elicit a coordinated, anti-cancer immune response that prompts a tumor response akin to adaptive immune resistance. Monocytes derived from human whole blood treated with Imprime PGG or vehicle were cultured in media with the appropriate cytokines to foster differentiation of M2 macrophages (M-CSF), M2a macrophages (M-CSF plus IL-4) or dendritic cells (GM-CSF plus IL-4). At the end of differentiation, both M2 and M2a macrophages showed lower surface expression of the scavenger receptor CD163 and elevated levels of surface PD-L1. Imprime PGG treatment also enhanced the ability of both M2 and M2a macrophages to augment CD3/ CD28-stimulated expansion of effector T cells and increased production of IFNγ. Imprime PGG similarly affected monocyte-derived dendritic cells (MoDC), eliciting increased surface expression of the maturation and antigen presentation markers CD80, CD83, CD86, HLA-DR and PD-L1. Furthermore, the MoDC increased CD4 and CD8 T cell proliferation and IFNγ production in an allogeneic mixed lymphocyte reaction. After confirming the ability of Imprime PGG to drive Th1 polarizing Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 123 Late-Breaking Poster Session: Immunology immunity, the supernatants from the M2/Mo-DC and T cell cocultures were incubated with cell lines from numerous cancer types, including NSCLC, breast, pancreatic, colon, and B cell lymphoma. PD-L1 expression was substantially upregulated on A549 (NSCLC), MiaPaCa2 (pancreatic), and SKBR3 (breast) cancer cell lines but not on the colon cancer cell line (HT-29). These results demonstrate that Imprime PGG has the potential to drive PD-L1-upregulating adaptive immune responses by modulating the function of M2 macrophages and MoDC and suggest that further studies to evaluate potential combinatorial approaches of Imprime PGG with immune checkpoint inhibitors are warranted. Poster Section 39 Poster Board 13 LB-229 Agonistic antibodies to costimulatory molecules, OX40 and GITR, significantly enhance the antitumor efficacy of Listeria monocytogenes (Lm-LLO)-based immunotherapy. Mikayel Mkrtichyan,1 Rajeev Shrimali,1 Shamim Ahmad,1 Rasha Abu Eid,1 Zuzana Berrong,1 Anu Wallecha,2 Robert Petit,2 Samir N. Khleif1. 1Georgia Regents University, Augusta, GA; 2Advaxis Inc., Princeton, NJ. Combinational anticancer immune therapies that target multiple tumor-mediated suppressive mechanisms or enhance effector immunity are emerging as promising strategies for cancer treatment. Recently, we demonstrated that the combination of Listeria monocytogenes (Lm-LLO)-based immunotherapy (ADXS11001) and anti-PD-1 antibody results in improved anti-tumor therapeutic and immune efficacy. Interestingly, regardless of the presence of an antigen or treatment with anti-PD-1 antibody, treatment with the Lm-LLO alone significantly inhibited the suppressive arm of the immune system, including regulatory T cells and myeloid-derived suppressor cells. We therefore hypothesized that combination of ADXS11-001 with agonistic antibodies against co-stimulatory molecules will lead to further improvement of immune and therapeutic efficacy as a result of the combined downregulation of the suppressive arm and the enhancement of the effector arm. Here we demonstrate that combining Lm-LLO-based immunotherapy with anti-OX40 or anti-GITR antibodies lead to significant inhibition of tumor growth and prolonged survival of animals. The complete regression of established TC-1 tumors occurs in 40% and 60% of animals treated with ADXS11-001 in combination with anti-OX40 and anti-GITR antibodies, respectively. We show that this therapeutic potency enhancement is due to a significant increase in antigen-specific immune responses along with ADXS11-001-mediated inhibition of suppressor cells. Thus, we believe that simultaneous stimulation of T cells while inhibiting the suppressor cells, specifically using Lm-LLO-based immunotherapy combination with agonistic anti-co-stimulatory molecule antibody is a feasible and translatable approach that can lead to overall enhancement of the efficacy of an anti-tumor immunotherapy. Poster Section 39 Poster Board 14 LB-230 SOCS5 mediates defective function of monocytederived dendritic cells in patients with chronic lymphocytic leukemia. Patricia A. Toniolo,1 Suhu Liu,1 Sarah R. Walker,1 Jose Alexandre M. Barbuto,2 David A. Frank1. 1Dana-Farber Cancer Institute, Brookline, MA; 2University of Sao Paulo, Sao Paulo, Brazil. Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. This clinically heterogeneous disease is characterized by the progressive accumulation of monoclonal B cells co-expressing CD5, CD19, CD20 and CD23 in the peripheral blood and in the primary and secondary lymphoid organs. Additionally, a range of immune cells are altered in patients with CLL. The resulting immunologic defects predispose patients to severe recurrent infections, which is the major life-threatening complication associated with CLL. Functional impairment of dendritic cells (DCs) in CLL not only diminishes the response to microorganisms, but it also allows for tumor escape from immune control. Understanding the mechanism for this effect can provide insight into the molecular regulation of DC function and may also suggest therapeutic strategies to reverse the immunosuppressive effect of tumors on DCs. To elucidate the mechanism for DC dysfunction in patients with CLL, we focused on signal transduction pathways that regulate the expression of genes necessary for the immune response. We found that monocytes from CLL patients have a decrease in IL-4-induced activation of the transcription factor STAT6, which prevents the phenotypic and functional maturation of DCs. This does not reflect a generalized defect in cytokine-induced signal transduction, as the activation of the related transcription factor STAT5 in response to GM-CSF is unaffected. Monocyte-derived DCs from CLL patients display low levels of HLA-DR, costimulatory molecules and CD83, and reduced secretion of pro-inflammatory cytokines. These changes are associated with low expression of TLR4 and related molecules, which decrease the ability of these cells to respond to stimulus afforded by LPS, impairing their complete maturation. Consequently, monocyte-derived DCs from CLL patients have decreased ability to induce proliferation of T-cells and display an increased induction of immune-suppressive regulatory T cell. Although monocytes from CLL patients exhibit high IL-4R expression, activation of the downstream transcription factor STAT6 is inhibited because of increased expression of the negative regulator SOCS5. It is known that CLL cells produce IL-10, leading to elevated serum levels of this cytokine. IL-10-treatment of monocytes from healthy donors mimics the alteration in signaling observed in patients, through enhanced STAT3-dependent expression of SOCS5, which inhibits STAT6 activation and leads to defective DC differentiation. These findings suggest that SOCS5 can mediate the impaired function of DCs in CLL patients, and may be a new potential therapeutic target for reversing cancerassociated immune suppression. Poster Section 39 Poster Board 15 LB-231 A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors. Donald A. Bergstrom, Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Michael DeVit, Mao Yin, Laura Poling, Joshua D. Thomas, Dmitry Gumerov, Dongmei Xiao, Elena Ter-Ovanesyan, LiuLiang Qin, Alex Uttard, Alex Johnson, Timothy B. Lowinger. Mersana Therapeutics, Cambridge, MA. Antibody-drug conjugates are effective in the treatment of HER2-amplified breast cancer and Hodgkin’s lymphoma, but current ADC technologies have faced limitations expanding the addressable patient population and target space. Ado-trastuzumab emtansine (T-DM1) is an ADC with 3-4 cytotoxic drugs per antibody that was recently approved for HER2 IHC 3+ or HER2-amplified breast cancer. Even within this high HER2-expressing population, several studies have now shown greater T-DM1 benefit in patients with HER2 mRNA expression above the median. These data suggest the need for more potent anti-HER2 ADCs to maximize benefit for HER2 IHC 3+ or amplified patients, and to extend HER2 ADC therapy to low HER2-expressing patients (HER2 IHC 1+/2+). XMT-1522 is an anti-HER2 ADC that uses a novel, human antiHER2 antibody optimized for cytotoxic payload delivery, and is noncompetitive with trastuzumab or pertuzumab for HER2 binding. Each antibody is conjugated to ~15 proprietary auristatin molecules using Fleximer, a biodegradable hydrophilic polymer. XMT-1522 shows nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell, and is typically 1-3 logs more potent than T-DM1 across a panel of 25 tumor cell lines. In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well-tolerated doses. In the high HER2-expressing N87 gastric cancer model (800,000 HER2 receptors/cell), complete regressions are achieved with a single 1 mg/kg dose of XMT-1522, while 10 mg/kg T-DM1 is required for comparable activity. In the American Association for Cancer Research • AACR ANNUAL MEETING 2015 123 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 124 Late-Breaking Poster Session: Immunology same model, the XMT-1522/trastuzumab/pertuzumab triple combination results in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis. In the low HER2-expressing JIMT-1 breast cancer (79,000 HER2/cell) and SNU5 gastric cancer (22,000 HER2/cell) models, complete regressions are achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively, while T-DM1 is inactive at doses ≥10 mg/kg. In non-human primates XMT-1522 demonstrates good stability of drug conjugate in plasma with t1/2 ~5 days (comparable to antibody t1/2) and minimal exposure to free payload. Despite the high potency of XMT-1522 in low HER2 tumor models, there is no XMT-1522-related toxicity observed in critical HER2-expressing tissues including heart and lung. The preclinical data support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2-directed therapies are not indicated. Furthermore, combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2driven tumors. Poster Section 39 Poster Board 16 LB-232 BiTE antibody constructs can mediate bystander tumor cell killing. Sandra L. Ross,1 Marika Mulen,1 Patricia L. McElroy,1 Julie Lofgren,1 Gordon Moody,1 Patrick A. Baeuerle,2 Angela Coxon,1 Tara L. Arvedson1. 1Amgen, Thousand Oaks, CA; 2Amgen, Munich, Germany. Recent clinical data demonstrate the significance of T cells in anti-tumor activity. For instance, the CD19/CD3 bispecific T cell engager (BiTE) blinatumomab is a proven means of harnessing T cells for cancer treatment. BiTE antibody constructs comprise an anti-CD3 scFv (single chain variable fragment) linked to an scFv binding a tumor-associated antigen (TAA). One potential challenge for TAA-targeted therapeutics is that treatment may only eliminate TAA-expressing tumor cells and heterogeneity of TAA expression becomes a potential means of resistance. To prevent escape of TAA-negative tumor cells, a treatment modality with a bystander effect on TAA-negative cells may be desirable. To evaluate the potential of BiTE antibodies to mediate bystander cell killing, mixtures of TAA-positive and -negative (bystander) cells were co-cultured with human T cells and the effect of BiTE antibodies tested. Lysis of TAA-expressing and bystander cells was evaluated using both imaging and viability assays. For this study, we used BiTE antibodies recognizing either epidermal growth factor receptor (EGFR) or CD33. In the presence of TAA-positive cells, T cells were activated and bystander cells lysed. In the absence of TAA-positive cells, bystander cells were not killed. Bystander cell lysis was also observed in a xenograft mouse model with subcutaneous tumors comprising EGFR-positive and -negative cancer cells, and human T-cells. The mechanism of BiTE-mediated bystander killing was further investigated. In the presence of TAA-positive cells, T cells released many cytokines, including IFN-γ and TNFα. However, exposure of bystander cells to just the soluble factors released by T cells did not induce their lysis, suggesting that a direct interaction between BiTE-activated T cells and bystander cells was required. BiTE treatment induced the expression on bystander cells of intercellular adhesion molecule 1 (ICAM-1), a protein involved in formation of cytolytic T cell synapses with target cells. ICAM-1 upregulation on bystander cells was also observed following exposure to recombinant IFN-γ and TNFα. These findings suggest that exposure of bystander cells to cytokines secreted by BiTE-activated T cells caused ICAM-1 expression on bystander cells leading to their improved attachment and cytolytic synapse formation. Blockade of ICAM-1 by an antibody partially protected bystander cells from lysis. Our data suggest a model where BiTE-activated T cells secrete cytokines that cause upregulation of ICAM-1 on TAA-negative cells. 124 This can then lead to T cell binding and T cell-induced bystander cell lysis. This mechanism is not expected to cause systemic cell death because only those cells proximal to the activated T cell in the tumor environment would be exposed to sufficiently high concentrations of ICAM-1-inducing cytokines. However, this locally confined bystander cell lysis may be sufficient to enable effective treatment of tumors that are heterogeneous for TAA expression. Poster Section 39 Poster Board 17 LB-233 Blockade of TGF-beta1 and 2 without TGF-beta3 blockade is sufficient to facilitate tumor vaccine efficacy. Masaki Terabe,1 Faith Robertson,1 Shingo Kato,1 Emma De Ravin,1 Katharine Clark,1 Amer M. Mizra,2 Jay A. Berzofsky1. 1National Cancer Institute/NIH, Bethesda, MD; 2Xoma Corp, Berkeley, CA. TGF-beta is one of the most potent immunosuppressive cytokines involved in the regulation of tumor immunity. TGF-beta has three isoforms, TGF-beta1, 2 and 3. It has been demonstrated in multiple mouse tumor models that blockade of all three isoforms of TGF-beta facilitates natural tumor immunosurveillance as well as tumor vaccine efficacy. Most studies on the roles of TGF-beta in immunology are on TGF-beta1, which has been demonstrated to induce Treg cells, IL-17-producing T cells and MDSCs. Although some studies reported immunosuppressive activities of TGF-beta2, the role of TGF-beta3 in immune regulation is not well understood. In this study we asked whether it is necessary to inhibit TGF-beta3 to enhance tumor immunity induced by a tumor vaccine in a syngeneic TC1 tumor model. When the tumor reached at least 5 mm in diameter, the mice were given a peptide-based vaccine targeting HPV16 E7, which is an oncogene expressed in TC1 cells. Although the vaccine alone had minimal effects on tumor growth, combination with an anti-TGF-beta antibody that neutralizes all three isoforms significantly delayed tumor growth. A similar effect was obtained with an antibody that neutralizes only TGF-beta1 and 2 but not TGF-beta3. Thus, it is not necessary to block TGF-beta3 to overcome immune suppression. Flow cytometric analysis of immune cells in tumor draining lymph nodes and tumors showed that there was no difference in the number of Treg and MDSCs between mice with/without treatment. The vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, but there was no further increase in combination with anti-TGF-beta. The vaccine also induced a significant number of T-bet-expressing T cells (both CD4 and CD8) in both tumor draining lymph nodes and tumors, and anti-TGF-beta, regardless of TGF-beta3 blockade, further increased the number of these cells. Together the results suggested that blockade of TGF-beta1 and 2 alone is sufficient to enhance therapeutic tumor vaccine efficacy by facilitating the induction of Th1 type T cells. With the notion that TGF-beta3 might be beneficial for patients in some cancers, developing TGF-beta antagonists that do not inhibit TGF-beta3 may be lead to better outcomes. Poster Section 39 Poster Board 18 LB-234 Poxvirus-based active immunotherapy synergizes with PD-1 plus LAG-3 immune checkpoint inhibition to enhance antitumor efficacy in preclinical models. Barbara Sennino, Susan P. Foy, Ryan B. Rountree, Tracy dela Cruz, Evan J. Gordon, Veronica Xavier, Felicia Kemp, Alex Franzusoff, James Breitmeyer, Stefanie J. Mandl. Bavarian Nordic Inc, Mountain View, CA. Treatment with poxvirus-based active immunotherapies shows evidence of robust immune responses against a variety of tumorassociated antigens in preclinical and clinical studies. Poxvirusbased immunotherapies in development include PSA-targeted PROSTVAC, now in Phase 3 clinical development; CV-301 (targeting CEA and MUC-1); as well as MVA-BN-HER2 and MVABN-Brachyury (targeting HER-2 and the transcription factor Brachyury, respectively). Evidence of robust and productive antitumor efficacy in preclinical models was accompanied by Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 125 Late-Breaking Poster Session: Immunology treatment-emergent infiltration of tumors by activated cytotoxic CD8 T cells producing high amounts of IFNγ. Treatment with immune checkpoint inhibitors such as anti-PD-1 antibodies is showing significant clinical benefit by re-activating dormant tumor-specific T cells. Furthermore, preclinical studies have shown further synergistic efficacy by combining PD-1 blockade with inhibition of LAG-3, which acts independently of PD1 to modulate T cell function. We hypothesized that poxvirus-based active immunotherapy may provide even greater improvements to patient outcome when used in combination with immune checkpoint blockade, by inducing new productive tumor-specific responses. This may be especially important in patients lacking an endogenous T cell response against their tumors. In therapeutic CT26-HER2 solid and metastatic tumor models, mice were administered MVA-BN-HER2 immunotherapy alone or in combination with anti-PD-1 and/or anti-LAG-3 antibodies. Synergistic benefit for anti-tumor efficacy was observed when combining MVA-BN-HER2 immunotherapy with anti-PD-1 alone, while combination with anti-LAG-3 alone had little effect. Notably, a further enhancement occurred when MVA-BN-HER2 immunotherapy was combined with PD-1 and LAG-3 blockade as shown by complete tumor regression in 20/20 mice. Subsequent rejection of HER-2 negative tumors 6 months after the original challenge revealed that immune responses were durable and included antigen spread to additional tumor antigens. Flow cytometric analysis demonstrated that tumor infiltrating lymphocytes (TILs) in untreated tumors were PD-1hi and LAG-3+, a more exhausted phenotype. Poxvirus-based immunotherapy led to the induction of activated TILs characterized by low to mid-levels of PD-1 expression. While PD-1 blockade prevented binding to PD-L1 it also caused an increase in LAG-3 expression on T cells. Together these data provide further rationale for why combination therapy of poxvirus-based immunotherapy with inhibition of PD-1 plus LAG-3 resulted in synergistic efficacy in preclinical tumor models. Overall these data demonstrate that combining complementary immune-based therapies such as poxvirus-based active immunotherapy and PD-1 plus LAG-3 immune checkpoint blockade result in synergistic anti-tumor efficacy. Poster Section 39 Poster Board 19 LB-235 Therapeutic human papillomavirus vaccine design based on epitopes identified on the tumor cell surface by mass spectrometry. Stephanie Hoppe, Renata Blatnik, Julia P. Schessner, Lisa Dressler, Alina Steinbach, Jan Winter, Martin Wuehl, Alexandra Klevenz, Hadeel Khallouf, Angelika B. Riemer. German Cancer Research Center (DKFZ), Heidelberg, Germany. Detailed knowledge about T cell epitopes, which are bona fide presented on the surface of human papillomavirus (HPV)transformed cells, is essential for rational design of therapeutic HPV vaccines. HPV affects the cellular antigen processing machinery, thus not every epitope derived from viral proteins is presented on human leukocyte antigen (HLA) molecules. Even the presented epitopes are displayed in low abundance. In this study, we developed a highly sensitive nano-UPLC-ESIMS3 multiple-reaction monitoring mass spectrometry (MS) approach for direct detection of low-abundant epitopes on the cell surface. Several web-based prediction algorithms were used to predict prospective epitopes from the HPV16 E6 and E7 proteins. These candidate epitopes were tested for actual HLA binding in cellular binding assays. The presence of binding peptides on HPV16-transformed cells was analyzed by our MS technology. Immunogenicity of candidate peptides was assessed in vitro with short-term and long-term T cell line experiments, generated from peripheral blood mononuclear cells of healthy donors, and in vivo for a selected HLA-A2-restricted epitope in HLA-A2/DR-1 transgenic mice. To ensure >95% population coverage, prospective HPV epitopes were predicted for the HLA supertypes HLA-A1, A2, A3, A11, A24, B7, and B15. Close to 500 peptides were tested in competition-based binding assays and multiple novel binders were identified. MS epitope detection was first established for HLA-A2 epitopes, and is now extended to the other supertypes. In the immunogenicity assays, several peptides induced robust IFN-γ responses. In conclusion, several new HPV16 E6 and E7 epitopes were identified and validated in this study. They represent promising candidates for inclusion into a therapeutic HPV vaccine. Validated epitopes are the basis of rational therapeutic vaccine design and are also important for immunomonitoring purposes. Poster Section 39 Poster Board 20 LB-236 Imprime PGG conjugated directly to protein enables cross-presentation of antigen that generates multifunctional cytotoxic T cells. Ross B. Fulton, Steven Leonardo, Kyle Michel, Lindsay Wurst, Trinda Phelon, Mike Danielson, Keith Gorden. Biothera, Inc., Eagan, MN. Imprime PGG is a soluble yeast-derived β-1,3/1,6 glucan innate immune cell modulator that is in phase 3 and multiple phase 2 clinical trials in combination with complement activating monoclonal antibodies (e.g. bevacizumab, cetuximab). We have previously shown that Imprime binds to complement receptors and modulates the function of a variety of immune cells, including monocytes, neutrophils, and B cells. We have also demonstrated that Imprime PGG binds to various subsets of dendritic cells (DCs) and can cause intermediate upregulation of MHC class II and the co-stimulatory molecules CD80/86 critical for antigen presentation and T cell activation. Based on these findings, we hypothesized that Imprime PGG conjugated to a protein would efficiently deliver antigen to DCs and prime a cytotoxic CD8+ T cell response. To test this hypothesis, we employed the model antigen chicken ovalbumin (OVA) in a C57BL/6 mouse model to examine the generation of OVA-specific CD8+ T cell responses. We covalently linked OVA to Imprime PGG to generate a β-glucan/protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8+ T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, OVA-specific CD8 T cells underwent vigorous expansion, upregulated the transcription factor Tbet, which is central to developing effector functions, and gained the ability to produce the cytokines IFN-γ, TNF-α and IL-2. By comparison, OVA alone did not generate a functional CD8+ T cell response and instead induced anergy. To determine if crosspresenting DCs are required for CD8+ T cell activation, we used mice deficient in the transcription factor Batf3, which selectively eliminates CD8α+ cross-presenting DCs. Following treatment with the Imprime-OVA conjugate in these Batf3-/- mice, the expansion of OVA-specific CD8+ T cells was more than 10-fold reduced compared to that in wild-type mice. Further, these cells failed to develop effector functions, indicating that CD8α+ cross-presenting DCs are crucial for this response. Together, these data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. Poster Section 39 Poster Board 21 LB-237 Vaccination increases the breadth and diversity of melanoma neoantigen-specific T cells in humans. Beatriz M. Carreno,1 Vincent Magrini,1 Michelle Becker-Hapak,1 Saghar Kaabinejadian,2 Jasreet Hundal,1 Allegra A. Petti,1 Amy Ly,1 Wen-Rong Lie,3 William H. Hildebrand,2 Elaine R. Mardis,1 Gerald P. Linette1. 1Washington University School of Medicine, Saint Louis, MO; 2University of Oklahoma, Oklahoma City, OK; 3EMD Millipore Corp., Billerica, MA. Melanoma genomes harbor somatic mutations that are caused by exposure to mutagens such as UV light. Tumor missense American Association for Cancer Research • AACR ANNUAL MEETING 2015 125 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 126 Late-Breaking Poster Session: Immunology mutations (MM), translated into amino acid substitutions (AAS), may provide a form of non-self that elicits tumor specific T cell immunity. Indeed, T cell immunity directed against tumor encoded AAS has been reported in humans with cutaneous melanoma, thus implicating MM as a source of patient specific (private) neoantigens. However, a systematic evaluation of these putative neoantigens as validated targets is lacking and it is unknown whether the immune response directed against tumor encoded AAS can be augmented by vaccination. Here we show that vaccination against melanoma AAS-encoding peptides augments naturally occurring T cell immunity and reveals new HLA class I restricted private neoantigens in patients with advanced melanoma. Neoantigen specific T cells recognized naturally processed and 126 presented antigen but failed to recognize wild type (non-mutated) antigen. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. As determined by TCRΒ CDR3 DNA sequencing, vaccination promotes a repertoire of neoantigen-specific T cells that is highly diverse in terms of both TCRΒ usage and clonal composition. No evidence of autoimmunity was apparent after vaccination. Our results demonstrate that tumor MM can be targeted as neoantigens and vaccination directed at tumor AAS somatic mutations broadens the antigenic breadth and clonal diversity of anti-tumor immunity. These findings provide a new paradigm for private neoantigen identification in neoplasia and may form the basis of personalized cancer immunotherapies targeting somatic mutations. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 127 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 Late-Breaking Poster Session Tuesday, April 21, 2015 1:00 PM-5:00 PM Poster Section 40 Late-Breaking Research: Experimental and Molecular Therapeutics 2 Poster Section 40 Poster Board 1 LB-239 The Hippo effector YAP promotes resistance to RAF and MEK targeted therapies. Luping Lin,1 Amit Sabnis,1 Elton Chan,1 Victor Olivas,1 Lindsay Cade,1 Evangelos Pazarentzos,1 Saurabh Asthana,1 Dana Neel,1 Jenny Jiacheng Yan,1 Xinyuan Lu,1 Luu Pham,1 Mingxue Wang,1 Niki Karachaliou,2 Maria G. Cao,2 Jose L. Manzano,2 Jose L. Ramirez,2 Jose M. Torres,3 Fiamma Buttitta,4 Charles M. Rudin,5 Eric A. Collisson,1 Alain Algazi,1 Eric Robinson,6 Iman Osman,6 Eva MunozCouselo,7 Javier Cortes,7 Dennie T. Frederick,8 Zachary A. Cooper,9 Martin McMahon,1 Antonio Marchetti,4 Rafael Rosell,2 Keith T. Flaherty,8 Jennifer A. Wargo,9 Trever G. Bivona1. 1University of California at San Francisco, san francisco, CA; 2Catalan Institute of Oncology “Hospital Germans Trias i Pujol” Badalona, Barcelona, Spain; 3Hospital Universitario de La Princesa, Madrid, Spain; 4 University Foundation, Chieti-Pescara, Chieti, Italy; 5Memorial Sloan Kettering Cancer Center, New York, NY; 6NYU Cancer Institute, New York, NY; 7Vall d’Hebron Institute of Oncology, Barcelona, Spain; 8MGH Comprehensive Cancer Center, Boston, MA; 9MD Anderson Cancer Center, Houston, TX. Resistance to RAF-MEK targeted therapy is a major clinical challenge. RAF-MEK inhibitors are initially but only transiently effective in some but not all BRAF mutant patients, and largely ineffective in RAS mutant patients because of resistance. Through a genetic screen in BRAF mutant tumor cells, we show that the Hippo pathway effector YAP acts as a parallel survival input to promote resistance to RAF-MEK inhibitor therapy. Combined YAP and RAFMEK inhibition was synthetically lethal not only in several BRAF mutant tumor types but also in RAS mutant tumors. Increased YAP in BRAFV600E patient tumors was a biomarker of worse initial response to RAF inhibition in patients, establishing the clinical relevance of our findings. Our data uncover YAP as a novel mechanism of resistance to RAF-MEK targeted therapy. The findings unveil the synthetic lethality of YAP and RAF-MEK cosuppression as a promising strategy to enhance response and patient survival. Poster Section 40 Poster Board 2 LB-240 SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer. Andreas Papadakis*,1 Chong Sun*,2 Theo Knijnenburg,3 Yibo Xue,1 Wipawadee Grernrum,2 Michael Hölzel,4 Wouter Nijkamp,2 Lodewyk Wessels,2 Roderick Beijersbergen,2 Rene Bernards#,2 Sidong Huang#1. 1McGill University, Goodman Cancer Centre, Montreal, Quebec, Canada; 2Netherlands Cancer Institute, Amsterdam, Netherlands; 3Institute for Systems Biology, Seattle, WA; 4University of Bonn, Bonn, Germany. Recurrent inactivating mutations in components of SWI/SNF chromatin-remodelling complexes have been identified across cancer types, supporting their roles as tumor suppressors in modulating oncogenic signalling pathways. We report here that SMARCE1 loss induces EGFR expression and confers resistance to MET and ALK inhibitors in non-small cell lung cancers (NSCLCs). We found that SMARCE1 binds to regulatory regions of the EGFR locus and suppresses EGFR transcription in part through regulating expression of Polycomb Repressive Complex component CBX2. Addition of the EGFR inhibitor gefitinib restores the sensitivity of SMARCE1 knockdown cells to MET and ALK inhibitors in NSCLCs. Our findings link SMARCE1 to EGFR oncogenic signalling and suggest targeted treatment options for SMARCE1 deficient tumors. Acknowledgements: *Equal contribution. #Corresponding authors. This work was supported by grants from Canadian Institutes of Health Research (CIHR) grant MOP-130540 (S.H.), Canada Research Chair (CRC), the European Research Council (ERC), the Dutch Cancer Society (KWF), the EU COLTHERES project and grants by the Netherlands Organization for Scientific Research (NWO) to the Cancer Genomics Center Netherlands (CGC.NL). Poster Section 40 Poster Board 3 LB-241 HGF promotes resistance to MET inhibitor AMG337 in MET-amplified gastric cancer cells. Marcia Gordon, Alexandra Croft, Chi-Ming Li, Sean Taylor, Sean R. Caenepeel, Kim Quon. Amgen Inc., Thousand Oaks, CA. The MET oncoprotein is a tyrosine kinase that is the only known receptor for its ligand, HGF (Hepatocyte Growth Factor). In approximately 5% of gastric cancers, MET is activated by focal amplification, rendering it constitutively active and ligand independent. MET-amplified cancer cell lines and xenografts are typically dependent on MET signaling for proliferation or survival. To test this dependency in patients, a number of small molecule MET inhibitors, including AMG337, are currently undergoing clinical evaluation for the treatment of MET-amplified tumors.Here we characterize resistance mechanisms to MET inhibitor AMG337 in vitro. We show that under standard culture conditions, resistance does not readily arise in MET-amplified gastric cell lines SNU620 and MKN45 treated with a therapeutic dose (>IC90) of AMG337. Surprisingly however, exogenous recombinant human HGF enables resistance to readily appear, with emergent cultures possessing resistance mutations in the MET activation loop at positions D1228 and Y1230. Phosphoprotein analysis shows that HGF increases autophosphorylation and activation of mutant MET sufficiently to drive cell proliferation even in the presence of AMG337.Notably, deep sequencing and clonal analysis of individually barcoded cells show that pre-existing mutant clones are likely present in cultures prior to drug treatment. However, such clones are unable to robustly proliferate in drug-containing media unless HGF is present. Based on copy number analysis, we propose that a single or small number of pre-existing mutant MET alleles in an individual cell is sufficient to drive resistance in the presence of exogenous HGF. In contrast, in HGF-negative media, a single mutant MET allele is insufficient and thus resistance does not readily arise unless gradual drug dose escalation protocols that select for increased mutant MET copy number are employed.Our results identify a key mechanism by which HGF can promote resistance to MET inhibitors in MET-amplified cancer cell lines. Because HGF is present in tumor microenvironments, combining anti-HGF antibodies with maximally-tolerated doses of small molecule MET inhibitors such as AMG337 may be beneficial in preventing resistance and in promoting durable responses. Poster Section 40 Poster Board 4 LB-242 PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies. Beata Pyrzynska, Kamil Bojarczuk, Marta Siernicka, Michal Dwojak, Malgorzata Bobrowicz, Nina Miazek, Piotr Zapala, Agnieszka Zagozdzon, Jakub Golab, Magdalena Winiarska. Medical University of Warsaw, Warsaw, Poland. The therapeutic strategies currently used in B cell malignancies include the treatment with monoclonal antibodies (rituximab and ofatumumab) directed against CD20 antigen. These antibodies specifically eliminate B cells by triggering indirect effector mechanisms of the immune system, like complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. In many patients, the reduced level of CD20 antigen on the surface of tumor B cells leads to the resistance to anti-CD20 therapy. The aim of this study was to explore the molecular mechanisms governing the regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to rituximab/ofatumumab therapy. We previously observed that CD20 mRNA expression is American Association for Cancer Research • AACR ANNUAL MEETING 2015 127 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 128 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 significantly affected by the SRC family inhibitors. Here, we report that also PTEN tumor suppressor is a negative regulator of CD20 expression. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. Overexpression of wild-type PTEN (but not the phosphatasedeficient mutant) strongly affected the promoter activity and the expression of CD20, leading to decreased binding of rituximab and ofatumumab and increased resistance of tumor cells to complement-dependent cytotoxicity. Using the truncated versions of the CD20 promoter we identified a particular region (-313/-198) as the major region sensitive to PTEN overexpression. We found that the negative regulation of CD20 promoter activity by PTEN was mediated by inhibition of AKT signaling. We observed that the overexpression of constitutively active AKT1 (CA-AKT1) overcame the negative effect of PTEN and sensitized cells to rituximab/ofatumumab treatment. The results of our studies indicate that PTEN status in tumor cells should therefore be considered when analyzing the mechanisms of resistance of B cell malignancies to anti-CD20 therapies. Poster Section 40 Poster Board 5 LB-243 The ErbB3-targeting antibody MM-121 (seribantumab) reverses heregulin-driven resistance to multiple chemotherapies on tumor cell growth. Kristina Masson, Viara Grantcharova, Olga Burenkova, Marisa Wainszelbaum, Sergio Iadevaia, Sharlene Adams, Andreas Raue, Akos Czibere, Birgit Schoeberl, Gavin MacBeath. Merrimack Pharmaceuticals, Cambridge, MA. Purpose: Heregulin-mediated activation of the human epidermal growth factor receptor 3 (HER3/ErbB3) is required for the growth and survival of many epithelial cancers. This signaling pathway is also emerging as a mechanism of resistance to targeted agents and chemotherapies. MM-121 (seribantumab) is an investigational human monoclonal anti-ErbB3 antibody that has previously been shown to effectively block ligand-dependent activation of ErbB3 in a range of tumors, and has demonstrated clinical activity in biomarker positive patients in several Phase II trials. The purpose of this study was to examine in three indications of interest, the ability of heregulin to induce resistance to standard chemotherapies and the reversal of this effect by MM-121. Such systematic evaluation of different combinations can serve as a guide for the future clinical development of MM-121. Methods: To assess the effect of heregulin and MM-121 on chemotherapies in cancer cells, we conducted a high throughput proliferation screen in 3D cultures. A panel of 60 cell lines of relevant clinical indications (ovarian, breast and lung cancer) was selected and tested for the sensitivity to respective standard-ofcare chemotherapies in the absence or presence of exogenously added heregulin. Using these data, we analyzed the rescuing capacity of heregulin and the MM-121 combination’s sensitivity, and selected representative combinations for in vivo models. Results: We show that in a large panel of cancer cell lines the presence of heregulin can induce resistance to multiple chemotherapies with very different mechanisms of action. The combination of MM-121 with any one of these chemotherapies can reverse the heregulin-meditated rescue and provide an additive treatment effect at therapeutically relevant doses achieved in the clinic. These results were further validated in xenograft mouse models of all three indications, using representative chemotherapies and doses. In addition, biomarker analysis revealed that ErbB3 receptor levels largely determine responsiveness to heregulin and MM-121. Conclusions: MM-121 is an anti-ErbB3 antibody designed to block ligand-mediated signaling, and currently in clinical development. The results presented here demonstrate the role of heregulin in reducing the sensitivity of tumors to standard-of-care chemotherapies, and the effect of ErbB3 pathway inhibition across indications. 128 Poster Section 40 Poster Board 6 LB-244 Amino acid deprivation selectively targets multidrugresistant breast cancer cells. Catherine A. Del Vecchio, Yuxiong Feng, Ethan S. Sokol, Erik J. Tillman, Sandhya Sanduja, Ferenc Reinhardt, Piyush B. Gupta. MIT Whitehead Institute for Biomed. Research, Cambridge, MA. Tumor recurrence and metastasis underlie the majority of cancer-related deaths. Cancer cells that recur or metastasize are often both de-differentiated and multidrug resistant, but the mechanistic basis for this has been poorly understood. We have recently shown that de-differentiation promotes multidrug resistance by activating Nrf2, which stimulates transcription of drug efflux pumps and enzymes that scavenge reactive oxygen species (ROS). De-differentiation activates Nrf2 by a non-canonical mechanism involving its phosphorylation by the ER membrane kinase PERK. PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy, and inhibiting this signaling axis re-sensitizes de-differentiated cancer cells to treatment. To further explore this pathway we profiled the effects of PERK inhibition on global gene expression in both differentiated and de-differentiated cells upon treatment with chemotherapy. This analysis showed that PERK inhibition results in an amino acid deprivation phenotype, and suggested that de-differentiated cells may be sensitive to perturbations in amino acid availability. Consistent with this, we found that the aminopeptidase inhibitor Tosedostat was selectively toxic to de-differentiated breast cancer cells when given in combination with chemotherapy. Our findings identify a novel vulnerability of therapy-resistant breast cancer cells, and suggest that targeting amino acid availability in combination with chemotherapy could be an effective treatment for aggressive breast cancers that are multidrug resistant. Poster Section 40 Poster Board 7 LB-245 Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy. Ehsan Malek, Mohamed Abdel Malek, James J. Driscoll. University of Cincinnati, Cincinnati, OH. The molecular events that dictate the initiation, progression and therapeutic response in multiple myeloma (MM) remain incompletely understood. The ubiquitin (Ub) ligase Skp1-Cullin-1Skp2 (SCFSkp2) complex promotes proliferation by inducing proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1. Skp2 overexpression and reduced p27 levels are frequent events in human cancers and are associated with poor prognosis. Methods: Prospective pharmacogenomics was performed using next generation exome sequencing gene expression profiles (GEPs) compiled from the APEX/SUMMIT phase III clinical trial deposited in dataset GSE9782. 255 relapsed MM patients were randomized to bortezomib (BTZ) vs. dexamethasone (DEX) alone and progression-free (PFS) and overall survival (OS) then correlated with the pre-treatment GEPs. RPMI8226 cells were exposed for 8 months to successively escalated doses of the proteasome inhibitors BTZ, carfilzomib or ixazomib to generate chemoresistant cell lines. Side population (SP) cells were isolated from MMCLs and patients’ samples by flow cytometry sorting based upon Hoechst 33342 staining. Results: Pharmacogenomic correlation of GEPs with clinical response revealed hyperexpression of CUL1 and SKP2 in tumors from MM patients that did not respond to BTZ. CUL1 and SKP2 hyperexpression was correlated with significantly reduced PFS and OS after treatment with BTZ but not with DEX. Cullin-1 and Skp2 were elevated in cells generated with acquired PI-resistance. CUL1 or SKP2 genetic ablation significantly enhanced the sensitivity of chemoresistant cells to PIs. A high-throughput assay was then employed using GFP-tagged p27 to screen large databases and chemical libraries in order to identify novel lead compounds that inhibited the Skp2-dependent ubiquitination of p27. Treatment with DT204, a novel lead compound identified here, reduced p27 ubiquitination, its accumulation in myeloma cells and impaired Skp2 Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 129 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 association with Cullin-1 and Cks1, a critical rate-limiting effector of Skp2 activity. The anti-myeloma effect of DT204 was diminished using a phosphorylation-defective p27 mutant. Co-treatment with DT204 enhanced the anti-myeloma effect of BTZ against MM cell lines, patient tumor cells, stem cells and the tumor-initiating clonogenic side population (SP). The in-vivo efficacy and survival benefit of DT204 as monotherapy or in combination with BTZ will be reported. Conclusion: Taken together, the results demonstrate that pharmacologics to selectively disrupt the SCFSkp2 complex-p27 axis within the UPS holds promise to overcome chemoresistance with therapeutic benefit for myeloma patients. Skp2, Cullin1 can be utilized as biomarkers to select MM patients who will benefit from early experimental therapeutics of Skp2-inhibitors. Poster Section 40 Poster Board 8 LB-246 Evaluating the role of admixture in cancer therapy via in vitro drug response and multivariate genome-wide associations. John Jack,1 Tammy M. Havener,2 Howard L. McLeod,3 Alison A. Motsinger-Reif,1 Matthew Foster2. 1North Carolina State University, Raleigh, NC; 2University of North Carolina, Chapel Hill, NC; 3Moffit Cancer Center, Tampa, FL. It is well established that the majority of drugs/doses to combat diseases and disorders do not exhibit uniform effects for individuals. Indeed, the relationship between ethnicity, admixture or genetic ancestry and drug response is not fully understood. To explore this relationship, we used the lymphoblastoid cell line model to investigate dose-dependent drug response across a broad group of chemotherapeutic drugs spanning 7 drug families including nucleosides, tyrosine kinase inhibitors and DNA alkylating agents. Cell lines from 589 individuals were tested at 6 different concentrations for 28 chemotherapeutic drugs. Moreover, dosedependent cytotoxic effects were measured for each drug with samples from two distinct cohorts: Hispanic and nonHispanic/Caucasian. Genome-wide and phenotypic data from these individuals were used in univariate (IC50) and multivariate analyses to explore the relationship between ethnicity and drug response. Both univariate and multivariate models indicated that the majority of drugs tested showed highly significant correlations between selfreported ethnicity and drug responses. The software package ADMIXTURE was used to estimate genetic ancestry per individual. Using these estimates in lieu of self-reported ethnicity, we found highly significant correlations between percent admixture and drug response for the majority of drugs tested. Genome wide association analyses were carried out on each drug for each cohort independently as well as the combined data. From the association analyses, we were able to generate a number of interesting hypotheses on the role of particular genetic variants in variability of drug responses. 10 out of 28 drugs indicated significant associations in the Hispanic cohort. For the combined association analysis, 9 drugs had significant associations. Among the association findings, for the Hispanic cohort analysis, we were able to recapitulate previous association analysis work (in nonHispanic/Caucasian) showing the role of variants in the gene, MGMT, and the susceptibility to temozolomide treatment. Remarkably, we were able to confirm the association result independently in a Hispanic and non-Hispanic cohort linking temozolomde response in Lymphoblastoid cells to variants in MGMT. To further understand the biological significance or the clinical impact of all of the association results, additional studies involving gene suppression techniques (e.g., knockdown models) and/or clinical trial gene candidate studies are warranted. Poster Section 40 Poster Board 9 LB-247 Querying the RAS genomic network with siRNAs and and flow cytometry: Automatic, multidimensional phenotyping of 135 cancer cell lines by Gaussian mixture fitting and expectation maximization. Arnaud Amzallag,1 Tina L. Yuan,2 Rachel Bagni,3 Ming Yi,3 Robert Stephens,3 Sridhar Ramawamy,1 Frank McCormick,2 Cyril H. Benes1. 1Massachusetts General Hospital, Charlestown, MA; 2 University of California, San Francisco, CA; 3Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD. To discover novel therapeutic modalities and genomic predictors of response, large screen utilizing small molecules or sh/siRNA are performed on increasingly large collections of cancer cell lines. However these screens suffer from two main limitations: 1) the off-target effects of the probes 2) the coarse measurement of the cellular response that cannot distinguish between different outcomes such as proliferation block and apoptosis. Here we profile 50 lung cancer cell lines using highly specific combinations of siRNAs against effector nodes of KRAS, and measured several characteristics of phenotypic response by flow cytometry including viability, level of reactive oxygen species and cell membrane integrity. For each assay [node-cell line], typically 25,000 events were measured. We often observed multi modal distributions following node silencing. We used the expectation maximization algorithm to fit the different cell populations induced by the gene silencing. This allows us to automatically extract the proportion of dying cells, and also provides estimates of the cell growth impairment. Assessment of replicates shows that our results are highly reproducible, provide an accurate estimate of the proportion of dying cells, and reveal the complexity of multi-modal response of KRAS cancer cell lines to perturbation of key signaling nodes. Using the genomic profiling and pharmaceutical screen of the same cell lines, we reveal the association of specific node silencing with 1) the basal genomic state of the cell lines 2) the activity of a panel of drugs with the goal of identifying new targeting modalities and patient selection strategies for KRAS mutant cancers. Poster Section 40 Poster Board 10 LB-248 Protein arginine methyltransferase 5 (PRMT5) inhibition as a therapeutic strategy in B-cell lymphoma. Olena Barbash,1 Sarah Gerhart,1 David Soong,1 Christine Thompson,1 Rocio Montes de Oca,1 Ping Zhang,1 Charles McHugh,1 Kristy Kuplast,2 Christina Majer,2 Richard Chesworth,2 Jesse Smith,2 Robert Copeland,2 Elayne Penebre,2 Kenneth Duncan,2 Neil Johnson,1 Chris Carpenter,1 Ryan Kruger1. 1GSK, Collegeville, PA; 2Epizyme, Boston, MA. PRMT5 is responsible for symmetric dimethylation of arginine residues in glycine and arginine rich (GAR) motifs on a variety of cytosolic and nuclear proteins including histones, spliceosome components, regulators of translation, transcription factors, kinases and others. PRMT5 driven methylation of some of these proteins has been implicated in tumorigenesis. For example, PRMT5 deposits repressive marks on histones and silences a subset of tumor suppressor genes, such as RB and ST7. PRMT5 methylation of non-histone substrates (such as E2F1 and p53) also contributes to cancer cell growth and death. PRMT5 driven methylation of spliceosome subunits and components of translational machinery has been well described but its connection to PRMT5’s role in cancer has not been established. We have identified first-in-class small molecules that are highly potent, selective, reversible inhibitors of PRMT5. Cellular mechanistic studies revealed that PRMT5 inhibition decreases symmetric arginine dimethylation on a variety of cellular proteins including spliceosome components, histones and transcription factors. PRMT5 inhibition leads to gene expression and splicing changes ultimately resulting in the induction of p53 in lymphoma cell lines. In addition to impacting the p53 pathway, PRMT5 inhibition leads to attenuation of the expression of cell cycle related genes, genes involved in ribosome and spliceosome homeostasis, as well as genes important for cellular metabolism. PRMT5 inhibitor attenuates proliferation and induces cell death in a subset of mantle cell and diffuse large B-cell lymphoma cell lines and inhibits tumor growth in xenograft models of mantle cell lymphoma. These data underline the potential of American Association for Cancer Research • AACR ANNUAL MEETING 2015 129 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 130 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 PRMT5 inhibitors as a therapeutic strategy in mantle cell and diffuse large B-cell lymphoma. Poster Section 40 Poster Board 11 LB-249 TDG, a dual genomic and epigenomic regulator, as a novel antimelanoma target. Rossella Tricarico,1 Pietro Mancuso,2 Vikram Bhattacharjee,1 Neil Beeharry,1 Emmanuelle Nicolas,1 Margret Einarson,1 Laura Cosentino,1 Irwin Davidson,3 Lionel Larue,4 Robert W. Sobol,5 Timothy J. Yen,1 Alfonso Bellacosa1. 1Fox Chase Cancer Center, Philadelphia, PA; 2Fox Chase Cancer Center, Philadelphia, PA and University of Siena, Siena, Italy; 3Institute of Genetics and Molecular and Cellular Biology, Illkirch, France; 4Institut Curie, Orsay, France; 5 Mitchell Cancer Institute, University of South Alabama, Mobile, AL. Melanoma is an aggressive cancer resistant to treatment, whose incidence has increased over the past two decades. Although the majority of melanoma cases are cured after surgical excision of the primary tumor, metastases occur frequently, and the metastatic form of the disease has a poor prognosis and is highly resistant to all current forms of therapy. Thus, new prognostic factors and advanced therapeutic strategies are urgently needed. We recently reported that the base excision repair protein Thymine DNA Glycosylase (TDG) has dual roles in safeguarding the genome and the epigenome (Cell 146:67, 2011). TDG not only protects CpG sites from spontaneous deamination of 5methylcytosine and cytosine (genomic stability), but importantly, at the epigenomic level, acts in a DNA demethylation pathway that converts 5-methylcytosine to cytosine (epigenomic stability). Specifically TDG removes the novel bases 5-formylcytosine and 5carboxylcytosine, demethylation intermediates produced by the upstream TET dioxygenases. TET alterations have been recently found in melanoma and correlate with poor prognosis. Moreover, TDG sequence variants in melanoma are reported in the TCGA database. For these reasons, we began studying the functional significance of TDG in melanoma. We reasoned that the two nonredundant (genomic and epigenomic) functions of TDG may represent a vulnerability of tumor cells and be exploited as novel drug targets for cancer treatment, because targeting TDG would achieve the dual effect of impairing DNA repair and disrupting the epigenetic state of the cancer cell. We found that reduced TDG levels correlate with tumorigenic melanomas and therefore TDG inhibition might further promote aggressiveness. Unexpectedly, however, TDG knockdown in melanoma lines caused cell cycle arrest, senescence and ultimately cell death. Senescence and cell death induced by TDG knockdown occurred without apparent activation of the DNA damage response, based on absence of H2AX phosphorylation. These in vitro findings were confirmed in vivo, as TDG knockdown in melanoma lines blocked tumor formation in xenografts. Given its potential as a novel therapeutic target, we conducted a pilot high-throughput screen and identified first-generation TDG chemical inhibitors. Two compounds were confirmed to inhibit TDG repair activity in vitro by radioactive-based glycosylase assay. Importantly, both inhibitors also blocked TDG demethylase function in cells, as evidenced by increased staining intensity of 5carboxylcytosine. Both compounds inhibited proliferation (by clonogenic, MTT and Xcelligence assays) of melanoma cell lines in the micromolar range and could synergize with alkylating agents and other anti-melanoma drugs. Thus, while reduced TDG levels may be part of the tumorigenesis process, limited levels of TDG are essential for melanoma viability. Therefore, TDG inhibition may represent a novel approach for melanoma treatment. 130 Poster Section 40 Poster Board 12 LB-250 Induction of c-Cbl is a novel mechanism for the anticancer effect of HDAC inhibitor in lung cancer. Chen Ching-Chow, Tzu-Tang Wei, Yu-Chin Lin, Pei-Hua Lin, JinYuan Shih, Chia-Wei Chou. National Taiwan University, Taipei, Taiwan. Here we found loss of c-Cbl, an E3 ligase, expression in Nonsmall cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment. Poster Section 40 Poster Board 13 LB-251 Histone deacetylase inhibition for the treatment of epithelioid sarcoma; novel cross talk between epigenetic components. Gonzalo Lopez,1 Yechun Song,2 Dennis Ruder,3 Chad Creighton Creighton,4 Svetlana Bolshakov,3 Xiaoli Zhang,1 Dina Lev,5 Raphael Pollock1. 1The Ohio State University, Columbus, OH; 2Zunyi Medical College, Guizhou, China; 3MD Anderson, Houston, TX; 4Baylor College of Medicine, Houston, TX; 5Sheba Medical Center, Tel Aviv, Israel. Objective: Enhanced knowledge of epithelioid sarcoma (ES) molecular determinants will hopefully aid the development of urgently needed targeted therapies. The hallmark loss of INI1 causes a disruption in epigenetics, potentially contributing to the development of the disease. Recently, we have demonstrated that HDAC inhibitors (HDACi) to have efficacious value for the treatment various genetically complex sarcomas. Here, we aim to evaluate the potential therapeutic efficacy of HDACi in ES. Methods: Three ES cell lines were used for this study: Epi-544, HS-ES, VAESBJ. MTS and clonogenic assays were utilized to evaluate the effect of HDACi on cell line growth. Cell cycle FACS and annexin V PI/FACS analysis with WB for cleaved caspase 3 were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. In vivo growth effects of HDACi were evaluated using SCID mouse ES xenograft models. An Illumina gene array was used to evaluate HDACi-induced gene change. Results: HDACi inhibited ES cell growth and colony formation. HDACi induced G2 cell cycle arrest and enhanced apoptosis in all ES cell lines tested. Similarly, HDACi abrogated ES xenograft growth and increased apoptosis in vivo. Due to the HDACi mechanisms of function, one of the major consequences of this therapy is gene expression modulation. Using an Illumina gene array, we sought to identify genes responsible for the in vitro and in vivo anti-ES effects observed. Array analysis showed Survivin be significantly down-regulated in response to HDACi treatment. HDACi-induced decrease of Survivin was confirmed via WB. For enrichment analysis of our genes set with published annotated gene signatures, the Molecular Signatures Database (MSigDB) was used. MSigDB analysis identified a curated gene set of regulated genes after EZH2 knockdown (KD) with significant commonality of our gene array. We demonstrated HDACi-induced decrease of EZH2. EZH2 KD resulted in reduced cell growth and increased apoptosis in VAESBJ cells. Conclusion: HDACs play a role in the progression of ES, where HDACi abrogate ES cell growth and enhance apoptosis in vitro and in vivo. Gene array analysis revealed various genes modulated by HDACi that may contribute to the progression of the disease. AntiES effects may be in part through the HDACi-induced repression of EZH2. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 131 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 Poster Section 40 Poster Board 14 LB-252 IDH mutations are promising targets for acute myeloid leukemia. Yoko Ogawara,1 Hironori Matsunaga,2 Takahiko Seki,2 Yukino Machida,1 Kazushi Araki,2 Issay Kitabayashi1. 1Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan; 2R&D Division, Daiichi Sankyo Co., Ltd, Tokyo, Japan. Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to αketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KGdependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDHdependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that IDH2/R140Q decreased the level of 5hmC and the expression of differentiation-inducing genes, including Ebf1, Spib and Pax5. Gene expression analysis revealed that IDH2/R140Q activated the hypoxia pathway and the expression of Meis1. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML. Poster Section 40 Poster Board 15 LB-253 Inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis. Youzhi Li, Harry A. Rogoff, Sarah Keates, Yuan Gao, Sylaja Murikipudi, Keith Mikule, David Leggett, Wei Li, Arthur Pardee, Chiang J. Li. Boston Biomedical, Inc., Cambridge, MA. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drugsenstivity, and their potential to metastasize and cause relapse. Subpopulations of cancer cells with extremely high tumorigenic potential have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties termed cancer stem cells. These stemness-high cancer cells are extremely tumorigenic and are resistant to conventional therapeutics due to activation of pro-survival and anti-apoptotic pathways, overexpression of drug efflux pumps, and increased DNA repair capacity. Moreover, chemotherapy and radiation have been found to induce stemness genes in cancer cells, converting stemness-low cancer cells to stemness-high cancer cells. Such highly tumorigenic and drug-resistant stemness-high cancer stem cells are, therefore, likely to be “left-over” following chemotherapy or radiotherapy and ultimately responsible for relapse. We hypothesized that cancer stemness inhibition is sufficient to suppress metastasis and relapse. Stemness, initially defined by the expression of stem cells genes, is a property shared by embryonic stem cells and adult stem cells. It has been demonstrated that the gene expression profiles of cancer stem cells more closely resemble embryonic stem cells than adult stem cells, suggesting the feasibility to identify molecular targets that are required for cancer stemness, but not (or less so) by normal adult stem cells. Through gene-silencing approaches, we have identified Stat3 as critically important for maintaining cancer stemness, yet largely dispensable for adult stem cells. Here we show that BBI608, a small molecule identified by its ability to inhibit gene-transcription driven by Stat3 and cancer cell stemness properties, displays anticancer properties that are highly different from chemotherapeutics agents. Stemness-high cancer cells enriched by multiple techniques are resistance to chemotherapeutics, yet highly sensitive to the stemness inhibitor BBI608. Blockade of spherogenesis and reduction of stemness gene expression by BBI608 were observed in stemness-high cancer cells isolated from a variety of cancer types. While treatment of xenografted tumor models with chemotherapeutics enriched stemness-high cancer cells, BBI608 induced significant depletion of stemness-high populations in vivo. Moreover, the inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis in xenografted human cancers in mice. These data demonstrate targeting cancer stemness as an effective way to suppress cancer relapse and metastasis. Poster Section 40 Poster Board 16 LB-254 High-throughput drug matrix screen with SMAC (second mitochondria-derived activator of caspases)-mimetic birinapant in high-grade serous ovarian cancer cell lines identifies synergism. Kristen Bunch,1 Ian Goldlust,2 Craig Thomas,2 Lidia Hernandez,3 Rajarshi Guha,2 Christina Annunziata3. 1Walter Reed National Military Medical Center, Bethesda, MD; 2National Institutes of Health, National Center for Advancing Translational Sciences, Bethesda, MD; 3National Cancer Institute, Women’s Malignancies Branch, Bethesda, MD. Background: Inhibitors of apoptosis (IAPs) are attractive anticancer therapeutic targets as a mechanism to sensitize cancer cells to apoptosis. IAPs are overexpressed in ovarian cancer and portend a poor prognosis. SMAC- mimetics are IAP antagonists that have shown in vitro and in vivo antitumor activity in ovarian cancer, but preclinical and clinical studies suggest SMAC mimetics may be more effective in combination therapies. Methods: Birinapant (B) was initially screened in combination with each of 1,912 compounds using an automated acoustic drug dispensation system in 6x6 matrices. Seventy compounds were chosen from the initial screen, based on synergistic characteristics, and verified in 10x10 matrices. The most clinically rational drug combinations with B were further evaluated in 8 high-grade serous ovarian cancer cell lines (CAOV3, CAOV4, OV-90, OVCAR4, OVCAR5, OVCAR8, PEO1, PEO4), focusing on 3 synergistic (docetaxel, panobinostat, volasertib) and 2 antagonistic combinations (cisplatin, olaparib) for exploration of mechanism. Combination index was determined using Compusyn software. Results: Specific classes of drugs were identified to have synergism with B, including taxanes, HDAC inhibitors, and PLK1 inhibitors, while platinum agents and PARP inhibitors were found to be antagonistic. Four of the 8 cell lines were sensitive to B. Caspase-Glo 3/7 assay was performed in B-sensitive cell lines and confirmed increased caspase-3 activity in the combination American Association for Cancer Research • AACR ANNUAL MEETING 2015 131 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 132 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 treatments. Ongoing studies include genomic analysis, mechanistic interrogation, and in vivo xenograft studies. Conclusions: B is a promising novel therapy for use in combination treatment for ovarian, fallopian tube, or primary peritoneal cancers. Our findings provide insight on optimal therapies to combine with B, and constitute a foundation for further evaluation of predictive and pharmacodynamic biomarkers to select the patient population most likely to respond to this targeted therapeutic approach. Poster Section 40 Poster Board 17 LB-255 Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows enhanced antitumor activity in combination with the PARP inhibitor, olaparib, in models of triple-negative breast cancer. Helene Marijon,1 Sigal Gery,1 Sivan Elloul,2 Sharon Y. Friedlander,2 TJ Unger,2 Robert Carlson,2 Sharon Shacham,2 Michael Kauffman,2 Harold P. Koeffler1. 1Cedars-Sinai Medical Center, Los Angeles, CA; 2 Karyopharm Therapeutics Inc, Newton, MA. Background: Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound currently in Phase I and II clinical trails for the treatment of hematological and solid malignancies. Selinexor blocks the key nuclear export protein XPO1 to force nuclear retention of tumor suppressor proteins (TSPs), including p53, BRCA1/2, pRB and FOXO3A. Olaparib is an FDA approved therapy for BRCA1/2 mutated ovarian cancer, which inhibits Poly-ADPRibose Polymerase (PARP) and prevents DNA damage repair. Furthermore, olaparib is being evaluated for the treatment of Triple Negative Breast Cancer (TNBC). We hypothesized that selinexor would restore genomic surveillance through nuclear accumulation of wild type BRCA1 and therefore combination treatment with olaparib would prevent DNA damage repair to amplify cancer cell death. Methods: The effects of selinexor alone or in combination with olaparib were tested on a panel of 7 TNBC cell lines using MTT and soft-agar colony formation assays in parallel with FACS analysis. In vivo efficacy of single-agent or combination therapy was evaluated using an MDA-MB-468 (BRCA1 wild type, TNBC) xenograft model. Combination index (CI) values were determined using the CompuSyn software and treatment was considered synergistic when CI<1. Results: The median IC50 values for selinexor and olaparib were 1.88 μM (range: 0.27 μM to >10 μM) and 92.6 μM (range: 17.5 μM to >300 μM), respectively. Combination treatment led to synergistic inhibition of proliferation in the 7 TNBC cell lines evaluated. The median CI tested on the panel of cell lines was 0.68 (ranging from 0.4 to 0.96). FACS analysis revealed an additive effect of the selinexor and olaparib combination on S-phase inhibition and G2 arrest in BRCA1 mutated and wild type cells. Furthermore, AnnexinV/PI staining showed an additive effect on TNBC cell apoptosis regardless of BRCA1 mutational status. In the MDA-MB468 xenograft model, 75% tumor growth inhibition (TGI) was observed in the combination group by day 22 compared to 55% and 35% TGI for single-agent selinexor and olaparib, respectively. Conclusion: Selinexor and olaparib in combination act synergistically to induce apoptosis in TNBC cells and amplify antitumor effects in a TNBC xenograft model. These data provide a rationale supporting the study of selinexor/olaparib combination in clinical trials. Poster Section 40 Poster Board 18 LB-256 Optimal clinical dose-finding strategies: Translational preclinical pharmacokineitcs, pharmacodynamics, and efficacy analysis of HM61713, an orally selective EGFR mutant inhibitor. Jooyun Byun, Taehun Song, Donghyun Kim, Junhyeng Son, Kwang-Ok Lee, Jaeho Lee, Yong Hoon Kim, Young-Mi Lee, Kwee Hyun Suh. Hanmi Pharm.Co. Ltd, Seoul, Republic of Korea. The first-generation of EGFR1 inhibitors (Gefitinib and Erlotinib) has significant clinical benefits in NSCLC caused by activating 132 mutations, but the efficacy of these agents is often limited due to the emergence of drug resistance conferred by a gatekeeper residue, T790M. HM61713 is a third-generation EGFR tyrosine kinase inhibitor that has been evaluated as a novel therapeutic agent for the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. HM61713 is an orally active and a novel EGFR mutant selective inhibitor which is potent on resistance mutation (T790M) without affecting EGFR wild type at efficacious dose level. HM61713 showed an anti-cancer activity in several EGFR mutant lung cancer cell lines including T790M mutation harboring cell line. Integrated pharmacokinetic - pharmacodynamic - xenograft tumor model (PK-PD-XTG) was used to characterize the relationship between HM61713 plasma concentration and tumor growth inhibition (TGI) in H1975 (T790M mutation) xenograft model. Simple one-compartment model applied re-absorption compartment with first-order absorption/elimination was used to describe HM61713 plasma concentration-time profiles. Biophase distribution model with baseline inhibition Emax equation was applied to characterize the PD marker (p-EGFR) and tumor volume shrinkage was explained by michaelis-menten kinetics of p-EGFR. Estimated in vivo IC50 value of p-EGFR inhibition (%) based on plasma free concentration in xenograft mice was 1.14 ng/mL. The human PD marker response curve and the tumor growth inhibition plot were obtained by replacing the mice PK to human PK in our developed model based on the hypothesis that translation provides a good relationship of surrogate mice tumor PD to human tumor regression corresponding human PK. According to our simulated curves, we predicted appropriate human active dose from 300 to 800 mg/man and it would be an efficacious dose in patients with NSCLC harboring the EGFR activating and also with T790M resistant mutation. Currently, HM61713 is undergoing in clinical trial phase I/II in NSCLC (ClinicalTrials.gov, NCT01588145). Poster Section 40 Poster Board 19 LB-257 Discovery and characterization of novel, highly potent and selective USP7 inhibitors. Gerald Gavory,1 Colin O’dowd,1 Keeva McClelland,1 Ewa Odrzywol,1 Alan Brown,1 Stephanie Burton,1 Oliver Barker,1 Frank Burkamp,1 Matt Helm,1 Iain James,1 Jakub Flasz,2 Elias Arkoudis,2 Tim Harrison1. 1Almac Discovery, Craigavon, United Kingdom; 2Queen’s University Belfast, Belfast, United Kingdom. Over the past decade, protein ubiquitination has emerged as an important post-translational modification with roles in a plethora of cellular processes, and dysregulation in the ubiquitin proteasome system pathway (UPS) has been implicated in multiple human disorders including cancer. Ubiquitin specific proteases (USPs) are cysteine proteases that catalyse the de-ubiquitination of numerous protein substrates including tumor suppressors and oncogenes, hence regulating their levels and/or functions. USPs therefore represent a fast growing and attractive target class for pharmacological intervention. USP7 in particular has attracted considerable attention for its implications in multiple key oncogenic pathways including most notably MDM2/p53, PTEN and DNA damage. As part of a focussed effort towards targeting USPs, fragment screening was performed against a panel of family members, including USP7. Hits were identified by surface plasmon resonance and validated using orthogonal biophysical techniques (NMR, thermophoresis). Subsequent hit expansion identified molecules for which high-resolution co-crystal structures have been solved providing unique opportunities for structure-based design. Medicinal chemistry optimisation has yielded a series of novel, reversible and potent USP7 inhibitors (e.g. IC50 < 10 nM) with excellent selectivity profiles against deubiquitinating (DUBs) and other non-related enzymes. These inhibitors are cell-permeable and also exhibit potent target engagement in cells (e.g. EC50 < 30 nM). In line with the mechanism of action, further cellular profiling has demonstrated effects on p53, p21 and MDM2 levels in a Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 133 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 concentration-dependant manner. From a translational viewpoint, initial studies aimed at identifying cell lines sensitive to these inhibitors will also be discussed. In summary, we report the discovery and detailed biochemical and cellular profiling of novel, potent and selective inhibitors of USP7. These molecules have drug-like properties and may provide opportunities for the development of new anticancer therapeutics. Poster Section 40 Poster Board 20 LB-258 The NAE inhibitor pevonedistat (MLN4924) interacts with the HDAC inhibitor belinostat via disruption of the intra-S checkpoint and both HR and NHEJ DNA repair in AML cells. Liang Zhou, Shuang Chen, Yu Zhang, Yun Leng, Lihong Li, Hui Lin, Maciej Kmieciak, Kathryn A. Rizzo, Catherine I. Dumur, Andrea Ferreira-Gonzalez, Yun Dai, Steven Grant. VCU Massey Cancer Center, Richmond, VA. Protein degradation via the ubiquitin proteasome system (UPS) is regulated by cullin-RING E3-ligases (CRLs). The first-in-class investigational Nedd8-activating enzyme (NAE) inhibitor MLN4924 (pevonedistat) blocks CRL activity by inhibiting their neddylation, thereby disrupting the turnover of a subset of proteins with broad functions associated with cancer cell growth and survival pathways, including NF-κB, DNA replication, and DNA damage checkpoints, among others. Such findings provide a rationale for combining MLN4924 with HDAC inhibitors (HDACIs e.g., the recently FDAapproved belinostat) that interrupt DNA replication, checkpoints and repair. Notably, both single-agent MLN4924 and HDACIs (e.g., pracinostat) have induced CRs in AML patients. Consequently, interactions between MLN4924 and belinostat were investigated in AML. MLN4924/belinostat co-administration synergistically induced apoptosis in AML cells with various genetic backgrounds (e.g., wtor mutant- p53 or FLT3-ITD), while p53 knock-down or enforced FLT3-ITD expression increased sensitivity to this regimen. MLN4924 blocked belinostat-induced NF-κB activation and belinostat coadministration up-regulated Bim, while Bim shRNA knockdown abrogated lethality. Following MLN4924/belinostat co-exposure, an RT2 Array revealed a distinct expression profile of DNA damage response (DDR) network genes. Significantly, belinostat diminished MLN4924-induced ATR/Chk1/Wee1 activation and Cdt1 upregulation, abrogating the intra-S checkpoint. Notably, shRNA knockdown of Chk1 or Wee1 significantly sensitized cells to MLN4924 in the absence of belinostat. Moreover, belinostat also blocked MLN4924-mediated up-regulation of multiple proteins involved in HR (e.g., CtIP, BRCA1 S1524 and S1423 phosphorylation, FANCD2-L), while co-administration increased H3K56 and H4K16 acetylation, indicating defective NHEJ repair. As a consequence, co-treatment induced robust DSBs, chromosome pulverization, and apoptosis. This regimen sharply induced apoptosis in primary MDS/AML blasts (n = 47) bearing various poor-prognostic mutations, defined by NGS using the Cancer Hotspot Panel v2, as well as CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells (n = 25), but was minimally toxic to normal CD34+ cells. Finally, MLN4924/belinostat cotreatment profoundly diminished tumor burden and prolonged survival in both s.c. and i.v. AML xenograft models, with minimal toxicity, via the PD events observed in vitro. Collectively, these findings indicate that MLN4924 and HDACIs interact reciprocally to disable the DDR and promote DNA damage and cell death in AML cells, including those with unfavorable genetic features and primitive progenitors, while sparing their normal counterparts. This strategy warrants further consideration in AML. Poster Section 40 Poster Board 21 LB-259 The potent and selective Axl/Mer dual inhibitor ONO9330547, shows promising single agent activity in non-small cell lung cancer (NSCLC). Kohei Tanaka, Toshio Yoshizawa, Tomoko Yasuhiro, Ryu Fujikawa, Tomoya Koike, Kazuhito Kawabata. Ono Pharmaceutical Co.,Ltd, Osaka, Japan. Purpose: Axl and Mer are members of the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family and they play an important role in regulating cell proliferation, survival, migration and cytokine production. Axl/Mer are known to be over-expressed in various types of haematological and solid tumor cancers and have been reported as poor prognostic factors. ONO-9330547 is a novel, small molecule Axl/Mer dual inhibitor, which potently inhibits phosphorylation of both Axl (P-Axl) and Mer (P-Mer), with higher selectivity against other TKs in a cell-based assay. We have previously reported that 3 mg/kg twice daily dosing of ONO9330547 for 24 days in an AML xenograft model, induced complete remission (Yasuhiro ASH 2014). Axl also appears to play a key role in epithelial-to-mesenchymal transitions (EMTs), which is involved in metastases and drug resistance in solid tumor such as NSCLC and breast cancer. Here, we investigated anti-tumor activity of ONO9330547 in NSCLC cell lines. Methods: Phosphorylated proteins were detected by Western blotting. In a two-dimensional (2D) assay, cells were treated with ONO-9330547 at concentrations up to 1μmol/L for 72 hours. Cell viability was determined by WST-8 Assay. In a clonogenic assay, cells in agarose solution were plated and the medium containing a ONO-9330547 were overlaid. After 17-21 days incubation, colonies were fixed, stained and counted. Luciferase-expressing NSCLC cell lines were injected intravenously into female SCID mice to mimic a model of disease metastases. After implantation, ONO-9330547 was administered orally at 1 or 10 mg/kg once daily (QD) for 41 days. For detecting tumor cells, D-luciferin solution was administered and total photon counts were measured once a week by IVIS imaging system. Results: ONO-9330547 strongly inhibited both P-Axl and the downstream signaling P-Akt upon Axl stimulation in NSCLC cells with an IC50 of 0.49 and 1.5 nmol/L respectively, while inhibiting cell growth partially in 2D assay. Interestingly, in a clonogenic assay, which is a common method to monitor anchorage-independent growth of cells, ONO-9330547 strongly inhibited colony growth of three NSCLC cell lines, NCI-H1299, A549, NCI-H229 (EGFR inhibitor-resistant cells) with IC50 of 4.4, 19 and 58 nmol/L, respectively. In a NSCLC model of metastases, the treatment of ONO-9330547 at dose of 1 and 10 mg/kg QD for 41 days resulted in significant inhibition (75 and 84%) of metastases in lungs compared with vehicle treated mice. Conclusion: ONO-9330547 is a highly potent dual Axl/Mer inhibitor with evidence of efficacy in NSCLC cells. Additional work to investigate the activity of ONO-9330547 alone and in combination with other therapeutic agents in other types of solid tumors are currently underway. Poster Section 40 Poster Board 22 LB-260 A small molecule Myc inhibitor has therapeutic effects on mouse pancreatic and other cancers. Dimitris Stellas,1 Matthias Szabolcs,2 Sanjay Koul,3 Zhe Li,4 Alexander Polyzos,1 Constantinos Anagnostopoulos,1 Zoe Cournia,1 Constantin Tamvakopoulos,1 Apostolos Klinakis,1 Argiris Efstratiadis1. 1Biomedical Research Foundation, Academy of Athens, Athens, Greece; 2Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY; 3 Division of Hematology/Oncology (SK), Columbia University Medical Center, New York, NY; 4Regeneron Research Laboratories, Tarrytown, NY. Mouse models faithfully simulating human cancer are valuable for genetic identification of potential drug-targets but, among them, the most advantageous for practical use in subsequent preclinical testing of candidate therapeutic regimes are those exhibiting rapid tumor development. Considering that pancreatic ductal adenocarcinoma (PDA) is the deadliest of the major malignancies and an unresolved clinical problem, and that a KRAS mutation (predominantly in codon 12, such as KRASG12D; KRAS*) occurs with high frequency (~90%) in PDA cases, we sought to develop a mouse PDA model that would exhibit high tumor incidence and American Association for Cancer Research • AACR ANNUAL MEETING 2015 133 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 134 Late-Breaking Poster Session: Experimental and Molecular Therapeutics 2 short latency by ectopic overexpression of Kras*. This transgenic modification caused by two weeks postpartum the appearance of pancreatic intraepithelial neoplasia (PanIN), which evolved into invasive PDA within a week later, and resulted in a moribund condition at one month of age. Interestingly, however, this aggressive form of pancreatic tumorigenesis was effectively prevented by genetic ablation of Myc specifically in the pancreas On the basis of this observation, causally demonstrating that Kras* oncogenicity fully depends on unperturbed Myc activity, we tested with positive results the effective treatment of our mouse PDA model by using orally-administered Mycro3, a small-molecule inhibitor of Myc-Max dimerization. PET/CT image analysis demonstrated marked shrinkage of PDA, while immunohistochemical analyses showed an increase in cancer cell apoptosis and reduction in cell proliferation. Tumor growth was also drastically attenuated in Mycro3-treated NOD/SCID mice carrying orthotopic or heterotopic xenografts of human pancreatic cancer cells. In addition, Mycro3 was efficacious in treating xenografts generated either heterotopically or orthotopically (by tail-injection) using a human lung carcinoma cell line (KRASG12D) and also in treating KRAS-driven mouse mammary tumors. These results provide strong justification for eventual clinical evaluation of antiMyc drugs as potential chemotherapeutic agents for the treatment of PDA and other Myc-dependent cancers. Poster Section 40 Poster Board 23 LB-261 Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. Cecile Geuijen,1 Eric Rovers,1 Tristan Gallenne,1 David MaussangDetaille,1 Arjen Kramer,1 Nellie Nieuwenhuizen,1 Carina Clements,1 Katinka van Zoest,1 Roy Nijhuis,1 Therese Visser,1 Renate Den Blanken-Smit,1 Willem Bartelink,1 Vanessa Zondag-van der Zande,1 Linda Kaldenberg,1 Pieter-Fokko van Loo,1 Rob Roovers,1 Robert Doornbos,1 Leo Price,2 Stefan Braam,3 Setareh van Driel,1 Lex Bakker,1 Ton Logtenberg,1 John de Kruif,1 Mark Throsby1. 1Merus, Utrecht, Netherlands; 2Ocello, Leiden, Netherlands; 3Pluriomics, Leiden, Netherlands. Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the 134 HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 135 Late-Breaking Poster Session: Prevention Research Late-Breaking Poster Session Wednesday, April 22, 2015 8:00 AM-12:00 PM Poster Section 40 Late-Breaking Research: Prevention Research Poster Section 40 Poster Board 1 LB-263 Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues. Chad W. Skaer,1 Pan Pan,1 Matthew Young,2 Hsin Tzu Wang,1 Kiyoko Oshima,1 Yi-Wen Huang,1 Gary D. Stoner,1 John Lechner,1 Li-Shu Wang1. 1Medical College of Wisconsin, Milwaukee, WI; 2 National Cancer Institute, Frederick, MD. In our previous work, a diet containing 5% freeze-dried black raspberries (BRB) was shown to significantly inhibit colorectal cancer in ApcMin mice. Studies in recent years have shown that metabolomics may help to elucidate the anti-cancer effects of dietary factors. Initially, therefore, we evaluated the effects of black raspberries on the metabolomic profiles of C57BL/6J mice, the mouse strain from which ApcMin mice were derived. In this study, we report the effects of the BRB diet on the metabolomic profiles from C57BL/6J mice. After 8 weeks on a 5% BRB or control AIN76A diet, feces, liver, and colon mucosa tissues from 9 BRB-fed mice and 7 control-diet fed mice were collected and profiled using mass spectrometry. A total of 424 named metabolites were observed in the liver, feces, and colon mucosa, with 115 metabolites whose levels were significantly different between BRB and control-diet mice (P<0.05). Of these 115 differentiated metabolites, 41 were altered in the colon mucosa, and 38 and 44 metabolites were different in the feces and liver, respectively. Almost all of the changed metabolites in the colon mucosa (36 of 41) were downregulated by the BRB diet, whereas in the feces and liver the majority were upregulated (58 of 82). Included in the upregulated metabolites were lipids and amino acids in the feces and liver tissue. Benzoate derivatives, the product of gut bacteria metabolism of BRB polyphenols, were also up in all tissues. Among the downregulated metabolites were lipids in the colon mucosa and lysolipids in the liver. The decreased levels of lipid metabolites, particularly fatty acids, in the colon mucosa of mice indicates that antagonizing fatty acid synthesis is a potential mechanism by which the BRB diet is altering energy utilization in the colon mucosa of mice. Poster Section 40 Poster Board 2 LB-264 Effects of dietary metformin/pioglitazone on lung adenoma formation in A/J mice. Donna Seabloom,1 Art Galbraith,1 Beverly Wuertz,1 Anna Haynes,1 Mark S. Miller,2 Vernon Steele,2 Lee Wattenberg,1 Frank G. Ondrey1. 1 University of Minnesota, Minneapolis, MN; 2Division of Cancer Prevention, NCI, Rockville, MD. Introduction: Metabolomics and cancer chemoprevention are important contemporary concepts in cancer prevention and gaining traction as potential clinical trial topics. Our group has worked with type II diabetes agents clinically in oral cancer prevention and clinically and preclinically in both lung and head and neck cancer. Both pioglitazone and metformin are type II diabetes agents, and both drugs have promising epidemiologic evidence for efficacy as solid tumor prevention agents. They are well tolerated clinically for many years in millions of patients worldwide with acceptable toxicity profiles. We hypothesize these agents would be suitable in the chemoprevention setting and tested this hypothesis in experimental lung carcinogenesis in a benzo[a]pyrene (B[a]P) mouse model. Materials and Methods: We utilized 224 seven week old female A/J mice. All groups received 3 weekly doses of B[a]P by oral intubation and were randomized into 8 groups of 27 mice per group based on weights. Experimental diets were started one week after the last dose of B[a]P. Metformin (12 mg/g and 10.2 mg/g) and/or pioglitazone (0.18mg/g) were administered in the diet for 18 weeks, whereas late stage inhibition diets were started at 8 weeks post carcinogen. Animals were continued on the feeding schedule, weighed weekly, and monitored for weight loss, behavior changes, rough hair coat, or other signs of ill health. Results: The average number of adenomas per animal was 17.44 +/- 1.39 (SEM) in the control group. At the early stage intervention the average number of adenomas was 5.00 +/- 0.69 (P<0.0001) in the highest dose of metformin, 6.15 +/- 0.57 (P<0.0001) for the low dose metformin, and 11.92 +/-1.00 (P=0.0023) for pioglitazone treatment. Metformin (low dose) and pioglitazone in combination resulted in 5.26 +/- 0.78 (P<0.0001) adenomas per animal. At the late stage intervention the average number of adenomas was 10.89 +/- 1.161 (P=0.0007) for the low dose metformin, and 14.42 +/-1.24 (P=0.1126, not significant) for pioglitazone treatment. Metformin (low dose) and pioglitazone in combination resulted in 9.77 +/- 0.85 (P<0.0001) adenomas per animal. The agents were well tolerated for the duration of the experiment from all physical appearance and other standard toxicities. However, animals treated with metformin had a 15% weight loss at the beginning of the experiment and completed the experiment with body weights 15% lower compared to nonmetformin treated animals. Conclusions: We conclude metformin and pioglitazone are promising agents for lung adenoma prevention in A/J mice. We also conclude dose adjustments of these drugs may require further optimization. Drugs of the thiazolidinedione class and sulfonylureas are powerful controllers of glucose metabolism. Although we did not have observable toxicity in the animals, it is likely glucose metabolism was altered and this contributed to the 15% differences in weight between the animal groups. Therefore, well designed metabolomic studies may be helpful in the future. Poster Section 40 Poster Board 3 LB-265 Comparison of oleanane triterpenoids and dimethyl fumarate in lung cancer. Ciric To, Darlene B. Royce, Charlotte R. Williams, Renee Risingsong, Michael B. Sporn, Karen T. Liby. Dartmouth College, Geisel School of Medicine, Hanover, NH. Lung cancer accounts for the highest number of cancer-related deaths in the United States, highlighting the need for better therapies. Nrf2 is an important therapeutic target as activation of this pathway detoxifies harmful insults and reduces oxidative stress. However, the role of Nrf2 in cancer biology is controversial. Protection against oxidative stress and inflammation can confer a survival advantage to tumor cells, leading to a poor prognosis, and constitutive activation of Nrf2 has been detected in numerous tumors. In our study, we examined the role of two clinically relevant classes of Nrf2 activators, the synthetic triterpenoids (CDDO-Im and CDDO-Me) and dimethyl fumarate (DMF) in mouse macrophages (Raw 264.7) and in VC1 lung cancer cells. Although both triterpenoids and DMF activated Nrf2, CDDO-Im and CDDOMe were more potent than DMF. Specifically, 25-50 nM CDDO-Im or CDDO-Me increased NQO1 and HO-1 expression 100-fold. Conversely, 10 μM of DMF was necessary to elicit the same effect. Additionally, 100 nM of CDDO-Im significantly (p<0.05) reduced ROS production induced by tert-butyl hydroperoxide by 69%, while 10 μM of DMF reduced ROS production by 44%. Moreover, nitric oxide production was significantly decreased by triterpenoids at nanomolar concentrations while DMF had a similar effect at micromolar concentrations (p<0.05). Using microarray analysis, we examined whether these Nrf2 activators target the same genes. Only 52 of 99 Nrf2 genes were targeted by all three compounds, and each drug targeted a unique subset of Nrf2 genes. Notably, CDDO-Im, CDDO-Me and DMF induced HO-1 expression by 9.1, 5.3 and 1.6 fold respectively. We then utilized Nrf2 knockout fibroblasts to confirm that induction of HO-1 expression was regulated via both Nrf2-dependent and Nrf2-independent pathways. To examine the effect of these Nrf2 activators in vivo, A/J mice were injected with vinyl carbamate to induce lung cancer. American Association for Cancer Research • AACR ANNUAL MEETING 2015 135 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 136 Late-Breaking Poster Session: Prevention Research Beginning one week after initiation, mice were fed drugs in diet for 15 weeks. CDDO-Me was the most effective drug in this model; it reduced the average number and size of tumors by 32% (2.2 ± 0.3) and 76% (0.09 ± 0.01), respectively, compared to controls (3.2 ± 0.2 and 0.4 ± 0.04; p<0.05). Average tumor burden was also reduced by 83% (0.2 ± 0.03), compared to the controls (1.2 ± 0.1; p<0.05). Additionally, the percentage of high-grade tumors significantly decreased from 52% in the controls to 31% in the CDDO-Me group (p<0.05). Though less potent, CDDO-Im had similar effects as CDDO-Me. In contrast, when mice were fed DMF in diet, the average number of tumors increased by 29% (4.1 ± 0.4) compared to controls (3.2± 0.2; p<0.05). The percentage of high-grade tumors in mice fed DMF diet also increased to 63% vs. 52% in the controls (p<0.05). These data indicate that DMF increased the severity of lung carcinogenesis in these mice. Collectively, our study suggests that although CDDO-Im, CDDO-Me and DMF all activate Nrf2, they target distinct genes, resulting in different effects for the prevention of lung cancer. Poster Section 40 Poster Board 4 LB-266 Suppression of colitis-associated colon carcinogenesis by the natural triterpenoid celastrol. Sung Hee Choi, Emily Baker, Byung-Gyu Kim, Gregory Tochtrop, John J. Letterio. Case Western Reserve University, Cleveland, OH. Celastrol is a natural triterpenoid isolated from the medicinal herb Triptyrgium wilfordii, commonly known as the Thunder of God vine. Celastrol has been shown to have a broad range of antiinflammatory and anti-cancer activities. Here we investigated whether celastrol administered as a dietary supplement can inhibit both colitis and colitis-associated colon cancer (CAC) in either genetic or chemically-induced CAC mouse models. As a genetic and spontaneous, inflammatory-driven intestinal neoplasia model, we generated a Smad4TKO / p27Kip1-/- (DKO) mouse in which a germ line p27Kip1 gene deletion (p27Kip1-/-) was combined with the T cell-restricted deletion of the Smad4 gene (Smad4TKO). All DKO mice developed spontaneous gastrointestinal inflammation and colon carcinoma by 3 months of age. Administration of celastrol as a dietary supplement (2 mg/day) to DKO mice increased survival and suppressed intestinal epithelial neoplasia by decreasing production of inflammatory mediators, including TNFalpha, IL-6, IL-1beta and iNOS. We investigated the chemopreventive activity of celastrol in the chemically induced CAC mouse model using dextran sulfate sodium (DSS)/azoxymethane (AOM). Mice (C57/BL6, ages 6-8 weeks) received intraperitoneal injections with 10 mg/kg AOM, followed by exposure to 2% DSS in drinking water for 7 days. We repeated 3 cycles of 2% DSS, with 10 days of normal drinking water provided between cycles. Mice were either maintained on control diet or given celastrol (2 mg/day) diet beginning with first administration of DSS and continuing until termination of the experiment. Mice receiving celastrol diet showed less loss in body weight compared with the control diet group and marked protection from AOM/DSS-induced colon cancer. The incidence and number of colonic tumors in mice in the celastrol diet group were also significantly lower than those in control diet group. These results were accompanied by decreased expression of iNOS and lower production of pro-inflammatory cytokines. Our data demonstrate the protective effects of celastrol in the chemoprevention of CAC, was and this effect correlated with a dampening of iNOS expression and with a decrease of inflammatory cytokine production. These results highlight the potential of celastrol as an effective agent for the chemoprevention of CAC in humans. Poster Section 40 Poster Board 5 LB-267 Role of VEGFR-1 signaling in obesity-induced tumor progression. Joao Incio,1 Joshua Tam,1 Nuh Rahbari,2 Priya Suboj,1 Daniel McManus,1 Shan Chin,1 Trupti Vardan-Kaur,1 Ana Batista,1 Suboj Babycutty,1 Keehoon Jung,1 Anna Khachatryan,1 Masabumi 136 Shibuya,3 Raquel Soares,4 Dan Duda,1 Rakesh K. Jain,1 Dai Fukumura1. 1Harvard Medical School/MGH, Boston, MA; 2 Department of Surgery, Dresden University of Technology, Dresden, Germany; 3Institute of Physiology and Medicine, Jobu University, Takasaki, Gunma, Japan; 4Department of Biochemistry, Faculty of Medicine, Porto University, Porto, Portugal. Background: Obesity associates with angiogenesis and increased macrophage infiltration in adipose tissues during weight gain. Whether these effects also occur in cancer to promote tumor progression in obese condition remains unclear. We have shown that the vascular endothelial growth factor receptor-1 (VEGFR-1) pathway can modulate tumor angiogenesis and recruitment of tumor-associated macrophages (TAMs). Here, we tested the emerging hypothesis that obesity enhances tumor progression and metastasis by augmenting angiogenesis and TAM recruitment via activation of the VEGFR-1 pathway. Methods: We used a high-fat diet-induced obesity model in either wild type (WT) or VEGFR-1 tyrosine kinase null (Flt1TK-/-) C57BL/6 mice. Then, we implanted orthotopically syngeneic pancreatic (PAN02) or breast (E0771) carcinomas. We evaluated the role of VEGFR-1 activity on systemic metabolism, tumor angiogenesis and immune environment, and tumor growth and metastasis. Results: Obesity increased p38-MAPK activation and TAM infiltration, tumor growth (p=0.001) and metastasis (p=0.035) in PAN02 tumors. VEGFR-1 inhibition reduced tumor growth (p=0.007) and metastasis (p=0.017) in obese but not lean mice. This was associated with a decreased p38-MAPK activity and a shift in TAM polarization towards the M1 phenotype with reduced secretion of pro-tumor cytokines, but no change in vascular density or number of TAMs. In the E0771 model, VEGFR-1 inhibition reduced MMP-9 expression and decreased lung metastatic burden (p=0.026) in obese mice. In addition to these tumor effects, VEGFR-1 inhibition reduced weight gain, but caused metabolic disorderhyperinsulinemia-during obesity. Combining metformin with VEGFR-1 inhibition not only prevented this metabolic alteration, but also by recruiting cytotoxic cells further decreased tumor growth in the PAN02 model (p=0.047). Conclusion: Inactivation of VEGFR-1 signaling prevents weight gain and obesity-induced acceleration of tumor progression in pancreatic and breast cancer models. Targeting VEGFR-1 signaling axis in combination with an anti-diabetic drug such as metformin might be a considerable cancer therapeutic option in the obese setting. Poster Section 40 Poster Board 6 LB-268 Black rapsberries induced protection in pancreatic cancer mouse models. Pan Pan, Chad Skaer, Hsin Tzu Wang, Susan Tsai, Kiyoko Oshima, Yi-Wen Huang, Gary Stoner, John Lechner, Li-Shu Wang. Medical College of Wisconsin, Milwaukee, WI. Background and Objectives: Pancreatic ductal adenocarcinoma (PDAC) is the 12th most common cancer worldwide with 338,000 new cases diagnosed in 2012. The estimated 5-year survival rate of diagnosed PDAC patients is less than 4%, making it the 7th leading cause of cancer death. Overexpression of epidermal growth factor (EGF) receptors and ErbB2 receptors has been associated with the development and progression of human PDAC. Diets rich in fruits and vegetables have been reported to reduce the risk of PDAC incidence and its recurrence. Our current study aimed to investigate the effects of dietary black raspberries (BRBs) on pancreatic carcinogenesis. . Methods: 4-5 weeks old pancreas-specific (Pdx-1-cre) mutant mice that carry constitutively-activated oncogene K-ras (G12D) and loss-of-function tumor-suppressor gene Trp53 (R172H), hereafter refers to KPC mice, were fed with control (n=26) or 5% BRB (n=29) diet. In addition, orthotopic pancreatic cancer xenografts were injected with luciferase transfected human Panc-1 cells. Four weeks after Panc-1 cells were injected, these mice were given Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 137 Late-Breaking Poster Session: Prevention Research control (n=8) or 5% BRB (n=19) diet for 6 weeks. Results: Kaplan-Meier survival analysis showed that BRBs prolonged survival of KPC mice with a medium survival of 124 days in comparison to 79 days from mice fed with control diet (p =0.03). Molecular studies suggest that BRBs decreased the activation of EGF receptor and ErbB2 receptor signaling pathways. Further, BRBs significantly suppressed growth of Panc-1 tumors and it was associated with decreased EGF receptor activation. Conclusion: These results suggest that BRBs might protect against carcinogenesis of PDAC through regulating EGF receptor and ErbB2 receptor signaling pathways. Further investigation of the downstream pathways of EGF receptor and ErbB2 receptor activation is warranted. Poster Section 40 Poster Board 7 LB-269 Nutritional effect on CD44 in head and neck cancer. Turki M. Almuhaimid, Faisal F. Alotaibi, Erika Rearegue, William Goodwin, Elizabeth Franzmann. University of Miami, Miami, FL. Head and neck (HN) cancer incidence accounts for more than 550,000 cases annually worldwide, with an estimated of 55,000 Americans developing head and neck cancer each year. There are multiple risk factors associated with HN cancer including smoking, alcohol consumption, human papillomavirus (HPV) infection, genetic predisposition, poor oral hygiene, periodontal disease and poor diet. According to World Health Organization (WHO), 35 - 55% of human cancers and approximately 15% of oropharyngeal cancers can be attributed to dietary deficiencies or imbalances. There is convincing evidence that we can reduce the risk of cancer, including that of oral cancer through improved nutrition. CD44 is a cancer stem cell marker associated with HN cancer and poor prognosis. Protein levels in saliva are also associated with HN cancer risk. This study will investigate the relationship between nutritional effect of a balanced diet which includes fruits and vegetables and levels of CD44 and salivary protein. 150 participants diagnosed with HN cancer and 150 controls were frequency matched for tobacco use, alcohol, age, gender, race and socioeconomic status. Participants completed a lifestyle questionnaire which included questions about fruit and vegetable consumption. Oral rinse CD44 and protein levels were measured in both groups. We did a preliminary investigation on the number of servings of juice, salad, fruits, carrots, potato and other vegetables within the cancer case and control groups to determine whether there was an association between these dietary variables and CD44 and protein levels. HN cancer cases who ate salad one or more times per week were found to have significantly lower CD44 levels than those who ate salad less frequently (4.22ng/ml verses 7.09 ng/ml p=0. 004). Since high CD44 levels are associated with worse prognosis, these findings suggest that dietary factors such as greater salad intake may be associated with better HN cancer prognosis. Poster Section 40 Poster Board 8 LB-270 Screening of novel phytochemical/s that can protect breast epithelial cells from PhIP induced cytotoxicity. Ashok K. Jain. Albany State University, Albany, GA. Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) is a known food carcinogen abundantly found in cooked meat. Several studies have shown that PhIP can induce tumors in breast, prostate and colon cells and rodent models. Metabolism of PhIP results in the formation of free radicals (ROS) and PhIP metabolites are known to produce DNA adduct and DNA strand breaks. The metabolism and mutational effects of PhIP are well defined. Phytochemicals are known to inhibit cytotoxic and genotoxic effects. Therefore, we hypothesized that the right combination of antioxidants and or phytochemical (naturally present in fruits, vegetables and spices) along with grilled meat should be capable of suppressing the PhIP induced cytotoxicity and development of breast cancer. Therefore, a model system using human breast epithelial cells (MCF 10A) was developed to mimic the cellular changes and test various antioxidants in presence or absence of PhIP. We have tested four vitamins (C, K3, D3, and E), Gingerol (6 and 10), N-acetyl cysteine, glutathione and curcumin at varying concentrations. The protective effect of these compounds was evaluated using cell viability assay, DCF assay to quantify ROS production, Comet assay to quantify the DNA damage and DNA adduct formation by immunofluorescence method. Results indicate that presence of these compounds improves cell viability as compared to PhIP treated group. However, curcumin showed significant differences and PhIP induced cell cytotoxicity was consistently reverted to normal in presence of Curcumin. Gene expression analysis using RT PCR technique indicates that curcumin interact via multi molecular targets. Hence, the present study suggests that Curcumin is a promising natural compound to regress the effect of PhIP induced cell cytotoxicity in breast cells. Poster Section 40 Poster Board 9 LB-271 Inulin inhibits free radical species in HCT 116 adenoma carcinoma and normal human HK (keratinocyte) cell line. Bene Akromaa Ekine-Afolabi, Sandra Appiah, Azra Pachenari, Lucy Ghali. Middlesex University, London, United Kingdom. Background: Bile acids have been implicated in oxidative damage via stimulation of reactive species including superoxide and nitric oxide. Superoxide and nitric oxide production are associated with inflammation. NF-κB upregulation provides a critical link between inflammation and cancer. It has also been shown that there is a possible correlation between total activity of nitric oxide synthase and p53 mutation frequency in lung adenocarcinoma. Inulin is an oligosaccharide, classified as a prebiotic, and shown to improve gut health when used in combination with probiotics. However, there is paucity of data on how prebiotics alone affect gut health and by which mechanism of action. Objectives: To evaluate the potential of inulin in reduction of reactive species and its mechanism of inhibiting cancer cells. Methods: HCT116 adenocarcinoma cells were treated with inulin at different concentrations and stopped at various time ranges (30 minutes - 24 hours). Modified Greiss reagent was used in the study of reactive nitrogen species (RNS). Nitro blue tetrazolium (NBT) assay (Modified), was used in the investigation of superoxide production (SOP) in HCT116 in the presence of deoxycholic acids (DC); a secondary bile acids. Inulin (5%-40%) significantly inhibited DC (100µM-500 µM), generated RNS in HCT 116 cell lines (p<0.05; n=6). The impact of inulin on RNS was observed to be dose and time-dependent. DC 100µM-300µM was observed to stimulate SOP in HCT116 (p<0.05; n=3). Inulin (30%) was observed to inhibit SOP in HCT116 (P<0.05). The impact of inulin on RS was not enhanced in the presence of probiotics. Probiotic (Bifidobacterium longum) inhibited nitric oxide production (p<0.05; n=3).The proliferation (Cyquant assay) of HCT116 was observed to be significantly influenced in the presence of DC; proliferation is inversely proportional to the concentration of DC; 100µM (p<0.05; n=3) Proliferation of HCT 116 is enhanced in the presence of low concentration of DC <150 µM, while cell death and morphological change into mesenchymal cells is enhanced with >DC150µM. 30% Inulin enhanced cell death of morphologically changed cells in the presence of DC >150µM, with complete cell death occurring at DC300µM.Inulin inhibits proliferation of HCT 116 cancer cells. Inulin inhibits nitric oxide induced by DC p<0.05(n=3) in normal human HK (keratinocytes) cells. Conclusions: Peroxynitrite is formed by nitric oxide and superoxide; and considered to be cytotoxic, causing DNA damage and leading to carcinogenesis via inactivation of tumor suppressor oncoprotein p53. Possible mechanism of action by which inulin could exert protective effect on colon cells could be via its antioxidant effect. Further work is underway investigating the effect of inulin and bile-acid on NF-κB, p53 and apoptosis in adenocarcinoma and normal dermal HK epithelial cells. American Association for Cancer Research • AACR ANNUAL MEETING 2015 137 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 138 Late-Breaking Poster Session: Prevention Research Poster Section 40 Poster Board 10 LB-272 Effect of smoking cessation on soluble CD44 levels in head and neck cancer. Faisal F. Alotaibi, Turki M. Almuhaimid, Erika Reategui, Elizabeth Franzmann, Jarrard Goodwin. University of Miami, Miami, FL. In the past year, an estimated 55,070 individuals were diagnosed with head and neck cancer and approximately 12,000 related deaths occurred. This disease is devastating to the patients and families it affects due to its significant impact on swallowing, speech and appearance. Tobacco, the single largest risk factor for head and neck cancer, accounts for an estimated 85% of head and neck cancers. Carcinogens found in tobacco smoke induce damage to DNA leading to mutations and DNA strand breaks. When not correctly repaired, overexpression of gene products including CD44, a cancer stem cell marker, occurs. CD44 can be cleaved into a soluble form (solCD44) that is found in body fluids. CD44 levels in oral rinses distinguish head and neck cancer patients from controls with around 62% sensitivity and 88% specificity. CD44 becomes abnormally expressed in dysplasia and elevated levels of solCD44 have been found prior to clinical evidence of disease, in some cases. In this study we investigate changes in solCD44 levels with smoking cessation. 150 smokers from a South Florida community known to be at high-risk for oral cancer were recruited and enrolled in smoking cessation programs. At baseline, questions on smoking status, frequency and history were administered to participants. Oral rinses and saliva samples were also collected to determine levels of solCD44 and cotinine, which was used to confirm quit status, using ELISA assays. One year after enrollment (± 3 weeks), samples and questionnaires were taken again from participants to identify quitters and measure their CD44 levels. Results were then computed and analyzed by paired t-test using SAS University Edition. So far, thirty-seven participants (24.6%) reported quitting smoking but only half of them (n=13) were confirmed by cotinine (<21). For confirmed quitters, the mean solCD44 dropped from 1.81 (SD=1.29) prior to quitting to 1.65 (SD=1.12) one year after enrollment. This association between solCD44 level and smoking cessation was not statistically significant (p=0.65). Four out of 13 quitters had elevated CD44 levels prior to quitting. Three of 4 with elevated levels experienced drops in their CD44 levels following smoking cessation, including 2 subjects whose CD44 level returned to the normal range. Based on this data, a larger number of quitters and/or an additional time points post quitting are needed as it might reveal more profound results. Such results would be critical, offering smoking cessation as a means for subjects with high levels of CD44 to decrease their risk of progression. Poster Section 40 Poster Board 11 LB-273 Molecular targeting Neu1 with oseltamivir phosphate abrogates tumor neovascularization, growth, and metastasis in mouse model of human triple-negative breast cancer. Myron R. Szewczuk, Fiona Haxho, Stephanie Allison, Farah Alghamdi, Lacey Brodhagen, Victoria Kuta, Samar Abdulkhalek, Ronald Neufeld. Queen’s University, Kingston, Ontario, Canada. Patients with triple-negative breast cancers (TNBCs) lacking the estrogen, progesterone, and epidermal growth factor (EGF) receptor-2 (HER2/neu) receptors have typical high grading, frequent relapses, and exhibit poorer outcomes or prognosis compared with the other subtypes. No current targeted therapies are effective for TNBC. Here, preclinical studies on the antitumor activity of oseltamivir phosphate (OP) monotherapy was investigated to identify its role in tumor neovascularization, growth, invasiveness, and long-term survival in RAGxCγ double mutant mouse model of human TNBC. Sialidase, WST-1, and immunohistochemistry assays were used to evaluate sialidase activity, cell viability, and expression levels of E-, and N-cadherins, and host CD31+/PECAM-1 cells in paraffin-embedded TNBC MDA-MB-231 tumors grown in these 138 mice. OP, anti-Neu1, and matrix metalloproteinase-9-specific inhibitor blocked Neu1 activity associated with EGF-stimulated MDA-MB-231 cells. OP treatment of MDA-MB-231 and MCF-7 cells and their stable tamoxifen-resistant clones reproducibly and dosedependently reduced the sialidase activity associated with EGFstimulated live cells and the cell viability after 72 hours of incubation. Combination of 1 µM cisplatin, 5-fluorouracil, paclitaxel, gemcitabine, or tamoxifen with OP in a dose-dependent manner significantly reduced cell viability at 24, 48, and 72 hrs when compared to the chemotherapeutic alone. Heterotopic xenografts of MDA-MB-231 tumors developed bloody tumor vascularization in these mice. OP treatment at 30 mg/kg daily I.P. treatment reduced tumor vascularization and growth rate with significant reduction in tumor weight and spread to the lungs compared with the untreated cohorts. OP treatment at 50 mg/kg completely ablated tumor vascularization, growth and spread to the lungs, with significant long-term survival for 180 days post-implantation, tumor shrinking, and no relapses after 56 days off-drug. OP 30 mg/kg cohort tumors had significantly reduced levels of human N-cadherins and host CD31+ endothelial cells with concomitant significant expression of E-cadherins compared to the untreated cohorts. These findings signify a novel treatment therapy for TNBC. Poster Section 40 Poster Board 12 LB-274 Transcriptional factor Snail and MMP-9 signaling axis amplifies Neu1-MMP-9 crosstalk on EGF receptor to regulate tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma. Myron R. Szewczuk, Samar Abdulkhalek, Olivia Geen, Lacey Brodhagen, Fiona Haxho, Farah Alghamdi, Stephanie Allison, Duncan Simmons, Leah O’Shea, Ronald Neufeld. Queen’s University, Kingston, Ontario, Canada. Snail is a transcriptional factor and repressor of E-cadherin. It is well known for its role in cellular invasion, epithelial to mesenchymal transition (EMT), tumor progression and metastases. shRNA lentiviral knockdown (KD) of Snail and its associated member Slug in human A2780 ovarian epithelial carcinoma cell line was investigated to identify its role in tumor neovascularization. Live cell sialidase, WST-1 and immunohistochemistry assays were used to evaluate sialidase activity, cell proliferation and the expression levels of tumor E-, N- and VE-cadherins, and host endothelial CD31+(PECAM-1) cells in A2780, A2780 Snail KD and A2780 Slug KD tumors grown in RAGxCγ double mutant mice. Oseltamivir phosphate (OP), anti-Neu1and MMP-9 specific inhibitor blocked Neu1 activity associated with epidermal growth factor (EGF) stimulated A2780 cells. Silencing Snail and Slug in A2780 cells abrogated the Neu1 activity following EGF stimulation of the cells compared to parental cells. OP treatment of A2780 and cisplatinresistant A2780cis cells reproducibly and dose-dependently abated the cell viability with a LD50 of 7 and 4μM, respectively. Heterotopic xenografts of A2780 tumors developed abnormal bloody tumor vasculature in these mice. Anti-tumor activity of OP treatment at 50 mg/kg daily I.P. did not significantly impede A2780 tumor growth rate in a time-to-progression, but significantly cause a reduction of lung metastases compared with the untreated and OP 30 mg/kg cohorts. Silencing Snail in A2780 cells, but not the Slug KD member completely abrogated the tumor vascularization, growth and spread to the lungs in these mice. A2780 and A2780 Slug KD tumors expressed high levels of human N- and VE-cadherins, and host CD31+ endothelial cells. These findings uncover a novel organizational signaling platform connecting the Snail-MMP-9 signaling axis in amplifying the Neu1-MMP-9 cross-talk in regulating EGF receptors, tumor neovascularization, growth and invasiveness. Poster Section 40 Poster Board 13 LB-275 Beneficial regulation of metabolic profiles by black raspberries in human colorectal cancer patients. Pan Pan,1 Chad Skaer,1 Steven Stirdivant,2 Matthew R. Young,3 Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 139 Late-Breaking Poster Session: Prevention Research Gary Stoner,1 John Lechner,1 Yi-Wen Huang,1 Li-Shu Wang1. 1 Medical College of Wisconsin, Milwaukee, WI; 2Metabolon, Inc, Durham, NC; 3National Cancer Institute, Rockville, MD. Objectives: Dietary intervention of freeze-dried black raspberries (BRBs) in a group of human colorectal cancer patients has demonstrated beneficial effects, including pro-apoptosis, antiproliferation and anti-angiogenesis. The aim of this study was to investigate BRB-mediated metabolite changes from this same cohort of patients. Methods: 28 colorectal cancer patients were given 60 g BRB powder daily for 1-9 weeks. Urine and plasma specimens were collected before and after BRB intervention. A comprehensive mass spectrometry-based nontargeted metabolomic analysis was conducted on each specimen. Results: A total of more than 400 metabolites were annotated in each specimen. Of these 69 and 58 metabolites were significantly changed by BRBs in urine and plasma, respectively. An increased level of 4-methylcatechol sulfate was correlated with a higher apoptotic marker (TUNEL) in post-BRB tumors, while a decreased level of N-acetylglycine was correlated with a lower cell proliferation marker (Ki67) in post-BRB tumors. Cis-aconitate and isocitrate, two tricarboxylic acid (TCA) cycle metabolites, were increased in postBRB urine. Fatty acid β-oxidation and synthesis were decreased in post-BRB plasma. In addition, BRB-derived polyphenols were absorbed and metabolized to various benzoate species, which were significantly increased in post-BRB specimens. Increased benzoate levels were positively correlated with enhanced amino acid and carbohydrate metabolism but were negatively correlated with decreased fatty acid metabolism. Conclusion: These results suggest that BRBs may induce systemic changes that affect energy generating pathways. This study supports the hypothesis that BRBs might be beneficial to colorectal cancer patients through altering multiple intrinsic metabolic pathways. Poster Section 40 Poster Board 14 LB-276 Effect of lay health worker intervention on complinace of hepatitis B vaccinations in Asian Americans: Randomizd control trial. Hee-Soon Juon,1 Frederic Kim,2 Carol Strong,3 Eunmi Park4. 1 Thomas Jefferson University, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA; 3National Cheng Kung University, Tainan City, Taiwan; 4Johns Hopkins University, Baltimore, MD. Background/Context: Asian Americans are at high risk with contracting the Hepatitis B virus (HBV). There are preventive measures with vaccinations that could be administered. However, there are several barriers that preclude Asian Americans from receiving the proper care. Purpose: To implement lay health worker (LHW) telephone intervention using randomized control trial. Setting: Asian American community-based organizations in the Baltimore-Washington Metropolitan area from April 2013 to March 2014. Participants: 232 Asian American adults who were unprotected and 18 years of age or older. Intervention: Calling those in the intervention group to remind to receive a 3-shot series for HBV vaccinations at month 1, 2, and 5. Data Collection/Analysis: In a parent study, 600 Asian Americans participated in study by completing pretest, reading photo novel, and doing free Hepatitis B screening. A week later, participants received the results (i.e., protected, unprotected, and infected). In this study, those unprotected was assigned to either intervention or control group. The intervention group received the LHW intervention. Seven months after the pretest, those unprotected (n=187) were followed by phone. The outcome measure was whether they had received a series of vaccines over 6 months with 3 groups (none, 1 or 2 vaccines, and completed). Multinomial logistic regressions were used. Results: We found that those in the intervention group were more likely to have 1 or more vaccines than the control group, compared to no vaccination group (OR=3.04, 95% CI, 1.16, 8.00). Also, those in the intervention group were more likely to complete a series of vaccinations than the control group, compared to no vaccination group (OR=7.29, 95% CI 3.39, 5.67). We also found that time is the most important barriers preventing them from seeking hepatitis B vaccinations. Conclusion: The LHW intervention was successful in increasing the chances to protect them from HBV infection in high risk group of Asian Americans. This study suggests that it is very critical to implement this culturally integrated LHW intervention program to reduce liver cancer health disparities among Asian Americans. Poster Section 40 Poster Board 15 LB-277 Socioeconomic status and the aggressiveness of prostate cancer among black males. Antoinette Percy-Laurry. National Cancer Institute, Rockville, MD. Prostate cancer, the leading non-skin malignant cancer among men, affects black men disproportionately. Black men are typically diagnosed at an earlier age and have a higher rate of aggressive tumors compared to other races. Black men have 58% higher risk of developing prostate cancer and almost 150% higher risk of mortality from prostate cancer, compared to white men. Little is known about the cause of the disease and uncovering the causes of the disparities remains challenging. One possible contributing factor are differences in socioeconomic status (SES).This study investigates the relationship of socioeconomic status on prostate cancer severity and mortality among black men. The purpose of the study is to determine whether prostate cancer risk, the aggressiveness of disease, and mortality differ by SES within the black race. Two datasets were used in this study. A total of 715 black men with a diagnosis of prostate cancer were studied using individuallevel data from the linked Surveillance, Epidemiology, and Ends Results National, Longitudinal and Mortality Study (SEER NLMS) database. Socioeconomic status was assessed and the stage and grade of prostate cancer from the medical histories of study participants were used to determined severity. Chi-square (X2) tests were used to assess bivariate relationships. Logistic regressions were used to model the likelihood of receiving a high or low stage and grade. The second dataset, the NLMS general population, was also accessed to obtain a larger sample of prostate cancer mortality cases which were analyzed using Cox proportional hazard regression model. There were 50,576 black men in this study sample. Study results showed minor differences in the severity of prostate cancer between low and high SES black men. Lower income was associated with higher grade. Poverty showed no association with aggressive prostate cancer. Education was the only variable that showed some significance for both stage and grade. Having some college seems to be protective; however, the association was weak. Within race differences between SES variables and mortality among black men were found. The lower the SES the less likely the mortality compared to those of higher SES. Higher risk of mortality among the less educated and those with low income persists. American Association for Cancer Research • AACR ANNUAL MEETING 2015 139 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:53 PM Page 140 Late-Breaking Poster Session: Prevention Research In conclusion, this study found that SES is not associated with the risk of severe prostate cancer incidence but is associated with mortality among black men. There seems to be a link among black men that may help to explain the disparities with other races. Commonalities in lifestyle factors such as environmental stressors and dietary choices may explain the onset of the disease across SES categories. Access to health education and healthcare for 140 preventive and treatment services is paramount for survival. In addition, much research is needed to assess other external factors such as discrimination, stress exposures and social norms that may play a part. Finally, attention should be focused on how to serve the black community to promote prostate health and mental health, and to motivate healthcare compliance. Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:54 PM Page 141 Late-Breaking Poster Session: Molecular and Cellular Biology 4 Late-Breaking Poster Session Wednesday, April 22, 2015 8:00 AM-12:00 PM Poster Section 41 Late-Breaking Research: Molecular and Cellular Biology 4 Poster Section 41 Poster Board 1 LB-279 Quantitative flux measurements of serum protein catabolism in PDAC and other KRAS-mutant cancers. Michel Nofal, Kevin Zhang, Josh Rabinowitz. Princeton University, Princeton, NJ. Activating KRAS mutations are prevalent in human cancer and are associated with poor prognosis. Among these malignancies is pancreatic ductal adenocarcinoma (PDAC), which is universally KRAS-driven and almost universally lethal. PDAC tumors are extremely fibrotic and hypovascularized, limiting perfusion of nutrients into the tumor, and tumor cells exhibit an altered metabolic program to support survival and proliferation in this environment. One metabolic activity upregulated in these tumor cells is the uptake and catabolism of serum protein. This process yields a substantial alternative source of amino acids and can support the proliferation of cultured cells lacking free essential amino acids. However, existing methods for assaying the uptake and degradation of intact protein provide qualitative outputs, and as such, do not yield accurate estimates of flux from serum protein to amino acid monomers. Here, we present a method for quantitative measurement of the catabolic production of amino acids from serum protein in cultured cells. By culturing cells in medium containing fully 13C-labeled glucose and amino acids supplemented with unlabeled albumin, we distinguish amino acids taken up as monomers from the medium from serum proteinderived amino acids. Using a variant of classical metabolic flux analysis, we derive flux estimates for serum protein catabolism from measurements of amino acid abundance and isotopic labeling over time. Our method is highly sensitive (i.e. can estimate “baseline” serum protein catabolism in KRAS wild-type cell lines) and yields precise estimates of amino acid influx from serum protein for all proteinogenic amino acids. We have measured serum protein catabolism in a variety of cultured cell lines and find that protein catabolism yields amino acids in comparable amounts to conventional uptake in various pancreatic and non-pancreatic lines. This approach enables estimation of protein catabolic flux in any cell line and can be used to assay the effects of various genetic and pharmacological perturbations on serum protein catabolism with high sensitivity and while accounting for growth rate differences. Poster Section 41 Poster Board 2 LB-280 Gene expression profiling of individual circulating tumor cells from non-small cell lung cancer (NSCLC) patients via integrated nanotechnologies. Seung-min Park,1 Dawson J. Wong,2 Chin Chun Ooi,2 Viswam S. Nair,1 Ophir Vermesh,1 Sang Hun Lee,3 Susie Suh,4 Luke P. Lee,3 Shan X. Wang,2 Sanjiv S. Gambhir1. 1Stanford University School of Medicine, Stanford, CA; 2Stanford University, Stanford, CA; 3UC Berkeley, Berkeley, CA; 4Case Western Reserve University School of Medicine, Cleveland, OH. Background: Circulating tumor cells (CTCs), defined as epithelial cells shed from a primary tumor into the bloodstream, are valuable prognostic, and possibly diagnostic, biomarkers that contain actionable genetic information for cancer treatment. Unfortunately, the rarity of CTCs in comparison to other blood components necessitates high-throughput separation technologies for efficient enrichment and practical downstream analysis. Moreover, genetic data extraction from CTCs currently suffers from a dearth of reliable analytical methods capable of handling low cell numbers. Technological innovations are urgently required to developing platforms that can help to optimize cancer management. Aim: To measure gene expression profiles of individual CTCs for cancer management via an integrated nanoscale platform. Methods: We have developed a protocol to effectively enrich rare cells via a magnetic sifting technology, whose methodology is based on using magnetic nanoparticles to tag CTCs in conjunction with magnetic filtration to enable high-throughput enrichment with release capability. This magnetic sifter offers 1) high capture efficiency at fast flow rates due to extreme field gradients at the pore edges, 2) high throughput due to the density of pores (~200 pores/mm2), 3) scalability via standard lithographic fabrication, and 4) harvesting of viable cells. For subsequent characterization, a robust nanowell-based assay was designed to circumvent experimental errors associated with ensemble measurements through detection of mRNA transcripts directly from single CTCs (using one-step RT-PCR). Using standard photolithography, 25,600 nanowells are positioned on top of polydimethyl-siloxane (PDMS) and designed for subsequent RT-PCR reaction from a single CTC. These massive single-cell arrays are able to isolate up to thousands of individual NSCLC cells to measure gene expression. Also, this device is easily interrogated by conventional fluorescence microscopy to detect a candidate panel of genes on CTCs that are relevant for cancer detection or therapy monitoring. Nanowell is innovative as a low-cost (PDMS-based), easily scalable (from currently 25k to more than 100k nanowells), adjunctive solution to existing diagnostic methods. Results and Discussion: To date, we have assayed 23 NSCLC patients using the MagSifter & Nanowell and detected CTCs with valid biomarkers (hTERT and cMET). Each 4-mL whole blood sample was processed within one working day using the current workflow. From these samples, individual CTCs were assessed for hTERT and cMet expression. Single CTCs displaying hTERT only, cMet only, and both were evident upon fluorescent imaging. Direct comparison with CTC enumeration confirms better sensitivity by Nanowell assay, since the Nanowell utilizes PCR amplification in fluorescence as a signal generator. We believe this is the first demonstration of ex vivo visualization of gene expression from individual lung cancer CTCs. Poster Section 41 Poster Board 3 LB-281 Development of a sensitive and quantitative PD-L1 immunoassay superior to IHC with application in human FFPE tissue samples. Gerald Wallweber, Ahmed Chenna, Roy Ravanera, David Stathas, Weidong Huang, Christos Petropoulos. Monogram Biosciences, South San Francisco, CA. Introduction: Cancer immunotherapy approaches and targets are rapidly expanding for the treatment of many different types of cancer. The programmed cell death-1 receptor (PD-1) and its ligand PD-L1 have garnered a great deal of interest lately, partially due to current therapeutic agents demonstrating a long and durable clinical response with low toxicities in several cancer types. Binding of the PD-1 receptor on activated T-cells to PD-L1 expressing tumor cells reduces T-cell activation, thereby evading an immune response against tumor progression. PD-L1 expression by immunohistochemistry (IHC) has been shown to be a prognostic and potential predictive biomarker for response to both anti-PD-L1 and anti-PD-1 therapy, however, currently the IHC assay is not standardized and the definitions used for positivity are variable and subjective due to a visual scoring system. A lack of sensitivity of the IHC assay may partially explain the observed response to therapy in patients whose tumors were identified as IHC=0/PD-L1 negative. In an attempt to offer a more sensitive and quantitative assay, we developed a PD-L1 protein expression assay using the VeraTag technology. The PD-L1 VeraTag assay utilizes the release of a unique fluorescent reporter (VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately and objectively quantify the amount of PD-L1 protein expression in FFPE samples. Methods: The anti-PD-L1 rabbit monoclonal antibody E1L3N (Cell Signaling Technologies) was utilized in the VeraTag assay together with a goat anti-rabbit secondary antibody conjugated to the VeraTag reporter. FFPE cancer cell line lines were used to optimize antigen retrieval, primary antibody concentration and American Association for Cancer Research • AACR ANNUAL MEETING 2015 141 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:54 PM Page 142 Late-Breaking Poster Session: Molecular and Cellular Biology 4 signal/background ratio, with emphasis on the lower end of the dynamic range. Results: VeraTag measurements of PD-L1 protein expression correlated to both IHC and PD-L1 gene expression (CCLE, R squared=0.7212) in FFPE cancer cell lines. The PD-L1 protein expression by VeraTag and IHC was compared across a group of FFPE squamous cell carcinoma of the head and neck (SCCHN) and HER2- and HER2+ breast samples. VeraTag assays for the measurement of the HER-family of receptors (HER1, HER2 and HER3 total, HER1-HER1 homodimer, HER2-HER3 heterodimer, phospho-HER3 and HER3-PI3 kinase complex) were evaluated for correlation to PD-L1 protein expression in these two cancer types. There was good agreement between the VeraTag and IHC measurements of PD-L1 protein expression, with the added advantage of the VeraTag assay providing an ~5-fold range of PDL1 expression within the IHC=0 category. Conclusions: We have developed a sensitive and quantitative measurement of PD-L1 protein expression in FFPE human SCCHN and breast cancer samples utilizing the VeraTag technology. Measurement of PD-L1 expression from clinical samples with the VeraTag assay is warranted. Poster Section 41 Poster Board 4 LB-282 Ex vivo 3d drug response profiling of primary human ovarian cancer differentiates treatment-naive and relapsed patients and molecular subtypes. Tessa Desrochers,1 Stephen Shuford,1 Christina Mattingly,1 Lillia Holmes,1 Matt Gevaert,1 Jeff Elder,2 David Orr,1 Christopher Corless,3 Larry Puls,2 Hal E. Crosswell1. 1KIYATEC, Inc., Greenville, SC; 2Greenville Health System, Greenville, SC; 3Knight Diagnostic Laboratory, OHSU, Portland, OR. Background: Epithelial ovarian cancer (EOC) affects nearly 22,000 women annually and is the leading cause of death from gynecologic cancer in the United States. Five year cure rates are <40% and approximately 14,000 will die each year. Large-scale efforts are currently underway to use molecular profiling via next generation sequencing (NGS) technology to guide treatment in cancer patients with poor prognosis, but limited application of NGS in ovarian cancer has been reported. In this report, our previously described Ex Vivo 3D Drug Response Profiling was used to identify response differences between newly diagnosed and relapsed ovarian cancer patients and correlated with NGS of primary tissue. Materials and Methods: Processing of Ovarian Cancers: Under informed consent, ovarian cancer samples were obtained and processed using standard mincing & digestion. 3D spheroids were developed and 3D perfused Ovarian Microtumors were cultured using the 3DKUBE™. Ex Vivo Testing and Analysis: Cultured cells were exposed to clinically relevant concentrations of cytotoxic or targeted agents. Relative IC50s and total percent inhibition were used for ranking compounds. Isolated DNA was sequenced in a CLIA laboratory using a 37-gene NGS panel (GeneTrails®) on the Ion Torrent PGM. Results: Tissues from both newly diagnosed, treatment naive subjects and relapsed subjects were obtained and processed after IRB-approved tissue consent was provided. Spheroid formation was uniform across all malignant tumor types. There was a statistical difference for 3D spheroids treated with Carboplatin formed from either naïve or relapse tissue. The relapse samples had a significantly higher median IC50 than did the naïve samples (70.8 vs. 17.4). This significant difference was not apparent in matched 2D treatment groups. Gemcitabine response varied across tissue type, but did not correlate with traditional biomarkers (i.e. hENT mRNA expression). NGS testing turn-around time was a median of 9 days (range 7-14). Ovarian 3D microtumors were successfully perfuse and tested with targeted agents guided by NGS results, as evidence by a tumor with a mutation of EGFR (p.P265T, clinical significance unknown) with both erlotinib and afatanib demonstrating activity (3.3uM and 0.7uM, respectively). Conclusions: EV3D DRP successfully differentiates carboplatin 142 response and 3D perfusion of microtumors permits ex vivo culture of primary ovarian samples for genotypic-phenotypic response determination. Clinical response data and clinical correlation is ongoing. EV3D DRP permits phenotypic drug response correlation with molecular profiling in real time and may be a clinically relevant functional assay for driver mutation identification and maximal patient response to targeted agent(s). Poster Section 41 Poster Board 5 LB-283 Human cancers harbor extensive subclonal mutations. Michael W. Schmitt,1 Edward J. Fox,1 Marc J. Prindle,1 Kate S. Reid – Bayliss,1 Pamela S. Becker,2 Lawrence A. Loeb1. 1University of Washington, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA. Recent advances in next-generation DNA sequencing (NGS) have revealed greater than expected mutational heterogeneity, not only between tumors of similar cancer type, but also within individual tumors. This mutational heterogeneity could serve as a reservoir for the emergence of new phenotypes, including resistance to therapy. While mutational diversity has been reported in many human cancers, the high error-rate of conventional NGS limits its ability to confidently resolve mutations present in less than 1.0 to 5% of the cells that comprise a tumor. These low-level subclonal mutations likely contribute to the rapid emergence of resistance to radiation and chemotherapy and the ability of cancer cells to invade adjacent tissues and to metastasize. In order to study subclonal mutations, we have developed a highly accurate sequencing protocol, termed Duplex Sequencing, which takes advantage of the double-stranded nature of DNA to increase the accuracy of DNA sequencing by more than 10,000fold; Duplex Sequencing has an unprecedented background error frequency of <5x10-8. Additionally, in order to analyze rare subclonal mutations in human diseases, we have also established a highly efficient, targeted capture method, which utilizes sequential rounds of hybridization to enrich targeted genes by >1,000,000fold, with up to 99.8% of resultant sequencing reads on target. Using these new methodologies, we have investigated the subclonal makeup of several cancers, including acute myeloid leukemia (AML), colon cancers (CRC), and glioblastoma (GBM). Upon targeting genes identified by NGS as drivers of clonal proliferation, we identified multiple subclonal mutations in each of these cancers, some with the potential to elicit drug resistance and others to drive tumor cell proliferation. We also investigated the five human replicative DNA polymerases. No reports have previously implicated mutations in these polymerases in either AML or GBM, nor in sporadic CRC with the exception of mutations in the exonuclease domain of DNA polymerase epsilon. However, our approach, combining targeted gene capture with Duplex Sequencing, revealed multiple subclonal mutations in the catalytic domains of all five replicative DNA polymerases in each of these tumors. The presence of subclonal mutations in the catalytic domains of replicative DNA polymerases supports the mutator phenotype hypothesis, which posits that an increased mutation rate is a driving force during early tumorigenesis. Furthermore, extrapolating the results on the number of subclonal mutations in target genes to the entire genome, our results indicate that sites harboring subclonal mutations are >100-fold more frequent than sites with clonal mutations. Thus, by the time a tumor is clinically diagnosed, every position in the genome could be mutated in at least one cell in the tumor; these subclones could be the reservoir for the emergence of new phenotypes and resistance to therapy. Poster Section 41 Poster Board 6 LB-284 Exome sequencing of high-risk Finnish hereditary breast and breast-ovarian cancer families. Kirsi M. Kuusisto,1 Tommi Rantapero,1 Minna Kankuri-Tammilehto,2 Matti Nykter,1 Satu-Leena Laasanen,3 Johanna Schleutker4. 1 University of Tampere, Tampere, Finland; 2Turku University Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts 03_AACR_2015_Abstracts_pp45_150_Layout 1 4/2/15 4:54 PM Page 143 Late-Breaking Poster Session: Molecular and Cellular Biology 4 Hospital, Turku, Finland; 3Tampere University Hospital, Tampere, Finland; 4University of Turku, Turku, Finland. Background: Breast cancer has a well-established genetic component. However, genetic predisposition factors remain mostly unresolved in high-risk hereditary breast cancer families that do not carry mutations in two major susceptibility genes, BRCA1 and BRCA2. In this study, we aimed to identify rare variants contributing to disease susceptibility in the high-risk Finnish BRCA1/2-negative hereditary breast or breast-ovarian cancer families by using familybased exome sequencing approach. Methods: Germline DNA of 37 individuals from 13 high-risk hereditary breast or breast-ovarian cancer families was analyzed using targeted exome capture and massively parallel sequencing. Priority was given for rare (minor allele frequency<0.05) coding functional variants including nonsynonymous single nucleotide variants (SNVs), frameshift indels as well as stoploss/stopgain and splice-site variants. Candidate variants were selected based on the computational pathogenicity predictions by several tools and gene pathway analyses. Moreover, segregation of the variants with the disease was studied in the families. Candidate variants were confirmed by Sanger sequencing and genotyped in additional cohorts of breast cancer patients and healthy controls. Results: Altogether 21,530 rare coding functional variants (97.0% nonsynonymous SNVs, 1.6% frameshift indels, 1.4% stoploss/stopgains variants) as well as 5 splice site variants were observed in 37 individuals. Of these, 93 novel variants, that harbor genes participating in the DNA damage response pathway, were prioritized. Furthermore, only variants that were predicted to be pathogenic, were considered and they were manually studied through literature. Altogether 20 candidate variants were selected for further segregation, validation, and genotyping experiments, which are currently ongoing. Conclusions: Identified rare functional variants especially in the DNA damage response pathway genes can explain a fraction of the genetic predisposition to breast and ovarian cancer in the high-risk Finnish families. Poster Section 41 Poster Board 7 LB-285 Computational pathology for predicting prostate cancer recurrence. Amit Sethi,1 Lingdao Sha,1 Ryan J. Deaton,1 Virgilia Macias,1 Andrew H. Beck,2 Peter H. Gann1. 1University of Illinois at Chicago, Chicago, IL; 2Harvard Medical School, Boston, MA. Background: Conventional methods for predicting prostate cancer (PCa) aggressiveness, which rely heavily on the Gleason score of architectural pattern, remain imperfect. We have developed an approach that combines machine vision and machine learning analysis of routine H&E stained tumors to discriminate recurrent from non-recurrent patients after prostatectomy, based on architectural, color, and nuclear patterns. Methods: We used a set of H&E-stained TMAs containing quadruplicate cores from 176 recurrent cases of PCa and 154 nonrecurrent controls, frequency matched on age, Gleason, pathological stage and race. Using software specifically adaptable for computational pathology with hierarchical object recognition (Definiens Developer XD®), we processed images of each tumor at two spatial scale levels: a larger scale for recognition of tissue compartments (epithelium, stroma, lumen and inflammation), and a smaller scale for recognition of normal and atypical nuclei in each of the compartments. Magnification of 20x was used for both levels. At the tissue level, we used multi-resolution segmentation to segment homogeneous superpixels, which were then classified using machine learning into epithelium, stroma. or ambiguous tissue classified as intermediate between epithelium and stroma. We used relative darkness in a spatial neighborhood, size, and shape criteria to segment typical and atypical nuclei. Relational features among objects within and between the tissue and nuclear levels were also created. The resultant feature library contained 884 base features and 3,551 total variables. We used several stages of iterative L1 penalized logistic regression and 5-fold cross-validation to reduce variables and obtain AUC estimates. Variable stability criteria applied across the folds further minimized model overfitting. Results: PSA, Gleason and CAPRA-s achieved AUCs < 0.60 for recurrence in this matched dataset. A fifth stage L1 model containing 18 histometric features had cross-validation AUC = 0.81 (95% CI: 0.76-0.85). A more permissive 74 feature model had AUC = 0.95 (95% CI: 0.93-0.97). Subgroup analysis restricted to Gleason 7 cases produced AUCs ranging from 0.79-0.93 with varying model sparseness. Including pre-surgical PSA, Gleason grade or CAPRA-s score did not change model performance. Notably, leading features were frequently derived from stromal or ambiguously stromal regions and involved nuclear shape or texture. Their independence from tumor grading reflected subject matching and lack of explicit accounting for nuclear or stromal characteristics in the Gleason criteria. Conclusion: A computational pathology approach, applied to discrimination of recurrent and non-recurrent PCa in routine H&E stains, shows considerable promise and appears capable of adding to the predictive power of Gleason and CAPRA scoring. We will conduct additional validation studies and extend this work to biopsy samples. Poster Section 41 Poster Board 8 LB-286 The patient-derived xenograft mouse model dilemma; a new solution for an old problem. Valentina Schneeberger, John Poirier, Charles Rudin. Memorial Sloan Kettering Cancer Center, New York, NY. Patient derived xenograft (PDX) mouse models have become an essential tool for cancer research. Our laboratory was one of the first to develop patient derived xenografts of multiple lung cancer types and has used these models extensively for therapeutic analysis in vivo. We previously demonstrated that lung PDXs more closely recapitulate patterns of gene expression found in primary tumors while revealing that traditional cell line xenografts are genetically divergent from the tumor of origin, and thus may not accurately model human disease. One disadvantage of PDX mouse models is the admixture of human tumor cells with murine stroma during growth in vivo. This phenomenon has important implications on genomic sequencing including wasteful sequencing of mouse DNA and false positive mutation calls in reads that map to both the human and mouse reference genomes. Bioinformatic approaches to mapping human reads obtained in the context of admixed mouse DNA can be helpful, but insufficient in the context of xenograft tissue with significant stromal content. We therefore evaluated immunomagnetic approaches to tumor cell purification prior to next generation sequencing. We tested the Miltenyi mouse cell depletion kit (MCD), the STEMCELL technologies EasySep EpCam positive selection kit and the Miltenyi EpCam positive selection kit for their ability to purify human tumor cells from mouse stroma using a quantitative PCR and fragment analysis based approach, using fluorescence activated cell sorting (FACS) as a gold standard for purity. In our hands, the Miltenyi MCD kit yielded consistently higher purity across a range of stromal contamination levels. Prior to purification, samples ranged from 20% to 95% human tissue composition and were consistently purified to greater than 93% purity after MCD. DNA from samples r