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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
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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
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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
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Proceedings, Part 2: Clinical Trials and Late-Breaking Abstracts
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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.
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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
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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
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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.
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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.
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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.
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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
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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
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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
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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.
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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
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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.
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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
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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
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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.
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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.
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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
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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
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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
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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.
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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
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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
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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.
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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
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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.
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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.
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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.
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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
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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).
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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.
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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
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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
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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),
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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
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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
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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
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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.
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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.
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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.
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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).
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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,
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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
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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.
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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
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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.
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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
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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
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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,
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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
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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
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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.
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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
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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,
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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
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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
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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.
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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,
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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.
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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.
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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
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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
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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.
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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
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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
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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
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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.
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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.
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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.
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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
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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
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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 inhibit