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ARVO 2016 Annual Meeting Abstracts 206 Eye Cancer Biology and Therapeutics Monday, May 02, 2016 8:30 AM–10:15 AM 608 Paper Session Program #/Board # Range: 1364–1369 Organizing Section: Anatomy and Pathology/Oncology Program Number: 1364 Presentation Time: 8:30 AM–8:45 AM Proteomic analysis of uveal melanoma (UM) secretome reveals novel biological insights and potential biomarkers Sarah E. Coupland1, 3, Sam Prendergast1, 3, Martina Angi3, Deborah Simpson2, Robert Beynon2, Helen Kalirai1, 3. 1Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom; 2Centre for Proteomics, University of Liverpool, Liverpool, United Kingdom; 3LOORG, University of Liverpool, Liverpool, United Kingdom. Purpose: Proteins secreted or shed from cancer cells, the “cancer secretome”, represent an important class of bioactive molecules thought to play a causal role in cancer progression. We investigated the UM secretome as a source of biomarkers of early metastatic disease and its capacity to provide novel insights into tumour biology. Methods: Secretomes of short-term UM cultures established from patient tumours with a high- (HR; n=10) or low- (LR; n=4) metastatic risk were analysed using nanoLC-MS/MS-based label-free quantitative proteomics. They were compared with the secretomes of normal cultured choroidal melanocytes (NCM; n=5). Protein identification was performed using Proteome Discover v3.1 and protein quantification was undertaken using Progenesis®v2.0. Bioinformatics analyses applied SecretomeP.v2.0, SignalP.v4.1, Exocarta.v5 and Ingenuity® Pathway Analysis (IPA). Results: Initial analysis produced a dataset of 1843 proteins, which were common to all three analysed subgroups (HR, LR, NCM). Of these, 758 proteins with ≥3 unique peptides were identified. These 758 proteins could be subdivided into: 25% classically/ non-classically secreted, 46% exosomal proteins, and 29% other. Taking only the 538 proteins into account that were secreted by classical, non-classical or exosomal mechanisms, IPA suggested that they have biological involvement in cell proliferation, growth and movement. Further, IPA highlighted top canonical signalling pathway involvement of these proteins, particularly associated with hepatic fibrosis/hepatic stellate cell activation and the mTORC1-S6K axis. Finally, CCN Family Member 3 and Neuropilin 2, both previously shown in cancer to promote metastasis, were highly upregulated in HR vs LR UM. Conclusions: UM secretome analysis identified cancer-associated proteins likely to represent important biomarkers of disease and/or play a functional role in disease progression. Exosomes are a likely mode of local and distal communication in UM. Commercial Relationships: Sarah E. Coupland, None; Sam Prendergast, None; Martina Angi, None; Deborah Simpson, None; Robert Beynon, None; Helen Kalirai, None Program Number: 1365 Presentation Time: 8:45 AM–9:00 AM Host Microenvironment Shapes Uveal Melanoma Tumor Properties Matthew W. McEwen1, Qing Zhang2, Bradley Gao1, Hua Yang2, Zachary Goldsmith1, Hans E. Grossniklaus2, Matthew W. Wilson1, 3, Vanessa M. Morales1, 4. 1Ophthalmology, University of Tennessee Health Science Center, Memphis, TN; 2Ophthalmology, Emory University, Atlanta, GA; 3Surgery, St. Jude Children’s Research Hospital, Memphis, TN; 4Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Purpose: Approximately 50% of patients diagnosed with uveal melanoma (UM) will develop liver metastases. Metastasis may be present at the time of initial diagnosis in the form of micrometastases. The pathways by which these UM micrometastases transform into clinically detectable metastases are not well understood. By comparing UM cells cultured in vitro with those recovered from an in vivo orthotopic xenograft, our study tested the hypothesis that tumormicroenvironment interactions in the liver affect UM cell properties. Methods: Athymic nude mice were inoculated intraocularly with Mel 270, a primary UM cell line known to metastasize. Livers and spleens were recovered and used to prepare ex vivo single cell suspensions. Mel 270 and its corresponding metastatic cell line OMM 2.5 were used as in vitro controls. Quantitative PCR (qPCR) was performed to determine relative gene expression among the tumor cells using HPRT1 controlled delta delta Ct analysis. Cell culture supernatants were examined using multiplex assay to quantify levels of secreted proteins from tumor cells. Results: qPCR analysis of genes associated with cytoskeletal signaling, angiogenesis, and cell death and survival revealed a liver-specific (compared to spleen) upregulation of pro-angiogenic molecules, including VEGFA (79.2-fold) and the transcription factor HIF1A (72.2-fold). Our work also revealed an in vivo, liver-specific blockade of the tumor suppressor TP53 (141.2-fold) in UM tumor cells by HDM2 (102.7-fold) and HDMX (96.0-fold) sequestration. Multiplex analysis of human secreted proteins revealed a high concentration of IFN-α2 (344.09 pg/mL) specific to the liver sample. VEGF concentration was elevated in both liver and spleen samples (377.17 pg/mL and 191.27 pg/mL, respectively). Conclusions: Our data show a host-dependent, liver-specific upregulation of several UM tumor genes and cytokines. This is consistent with our hypothesis that interaction with the host liver microenvironment plays a role in shaping UM tumor properties. Further study is needed to characterize the host tissue mediators. Commercial Relationships: Matthew W. McEwen, None; Qing Zhang, None; Bradley Gao, None; Hua Yang, None; Zachary Goldsmith, None; Hans E. Grossniklaus, None; Matthew W. Wilson, None; Vanessa M. Morales, None Support: Research to Prevent Blindness, SJCRH Endowment, West Cancer Center, NIH Medical Student Research Fellowship Program at UTHSC Program Number: 1366 Presentation Time: 9:00 AM–9:15 AM Deciphering the molecular basis of periocular infiltrative basal cell carcinoma John Bladen1, Michèle Beaconsfield2, Edel O’Toole1, Michael Philpott1. 1Centre for Cell Biology and Cutaneous Research, Blizard Institute, London, United Kingdom; 2Moorfields Eye Hospital, London, United Kingdom. Purpose: Basal cell carcinoma (BCC) is the commonest cancer worldwide and constitutes the vast majority of eyelid tumours. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Infiltrative BCC (iBCC) is a particularly aggressive subtype, which invades local tissue and adjacent structures in an uncompromising manner. Little is known about the genetics of iBCC and the differences that render it more aggressive compared to the more benign nodular (nBCC) subtype Methods: Fresh frozen tissue was collected from 20 BCC patients; 10 iBCC and 10 nBCC subtypes. Laser capture microdissection and nucleic acid extraction was performed. Whole exome sequencing of 20 BCC tumour and blood-matched controls were undertaken along with RNA sequencing of 3 iBCC and 3 nBCC tumour and stromamatched controls. Differential expression (DE) from normal eyelid was deemed significant if P<0.01, Log2FC >1 or <-1. Quantitative RT-PCR and protein immunohistochemistry was performed for validation. Results: For iBCC and nBCC, the average tumour mutational burden was 1533 and 2073, and UV signature was 88% and 85%, respectively. PTCH1 mutations were present in 80% of IBCC and 60% of nBCC. Novel iBCC driver included EPHA3 in 3 out of 10 patients. DE revealed 288 and 276 genes for iBCC and nBCC compared to normal, respectively. When both subtypes were compared, 128 genes were differentially expressed, with the majority upregulated in iBCC including LAMB3 and EPHB4. Shared genes included VCAN and GPC3. Immunohistochemistry confirmed Hedgehog (Hh) pathway expression, but to a much greater extent in iBCC including the surrounding non-tumour stromal tissue. Two key novel activated pathways were detected; axonal guidance and extracellular matrix (ECM) receptor interaction pathways. Conclusions: Despite a reduced mutational burden in iBCC, the presence of significant driver mutations may explain its aggressive nature. Furthermore, the hyper-expression of the Hh pathway compared to nBCC including surrounding non-tumour stromal tissue may aid its local migration. Discovery of novel axonal guidance and ECM receptor interaction pathways could also play a role in this behaviour. Regardless of trends, the extent of tumour heterogeneity demands personalised genetic mapping of the tumour to direct developing novel treatment modalities such as inhibitors of EPHA3, VCAN, Gli1/2 and EPHB4. Commercial Relationships: John Bladen; Michèle Beaconsfield, None; Edel O’Toole, None; Michael Philpott, None Support: Fight For Sight UK Program Number: 1367 Presentation Time: 9:15 AM–9:30 AM Successful treatment of recurrent ocular surface squamous neoplasia (OSSN) with topical cidofovir Matthew H. Ip1, 2, Robert George3, William Rawlinson1, 3, Minas T. Coroneo2, 1. 1Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia; 2Department of Ophthalmology, Prince of Wales Hospital, Sydney, NSW, Australia; 3South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, NSW, Australia. Purpose: Despite the success of topical medical treatments, particularly with Interferon alpha-2b in treatment of OSSN, there is a failure rate of ~2%. A previous study2 showed OSSN responds to cidofovir (CDV), although our data showed no herpes viruses present in OSSN1. We have previously demonstrated that 6.5% of OSSN specimens are HPV-positive1, and considered the possibility that patients unresponsive to interferon may be HPV-positive and respond to antiviral therapy with CDV. Methods: A single center retrospective observational case series was performed to evaluate the efficacy of topical CDV for treatment of recurrent OSSN in 7 eyes of 7 patients. Each patient had been previously diagnosed with OSSN utilizing slit-lamp examination confirmed with histopathology, and had failed other interventions including Interferon alpha-2b. Each patient was treated with 2.5mg/ml TDS topical CDV for a period of 4-6 weeks. Patients were then reviewed clinically with slit-lamp examination and photography to look for recurrence. Results: Topical CDV was effective in 6 out of 7 eyes treated, with improved clinical appearance. After 1-month of CDV treatment, each of the 6 eyes demonstrated a marked reduction in mass size. Furthermore, no residual scar tissue was visualized (Figure 1 and 2). The mean follow-up with no clinical relapse is currently 6.4 months, with a range of follow-up of 1-12 months. In 1/7 cases we detected high risk HPV in a biopsy specimen using a hybrid capture assay. A further 5/7 were negative for high risk HPV using PCR, and 1/7 remains untested. Conclusions: Topical CDV appears to be effective for treatmentrefractive OSSN in the short-medium term. The efficacy of CDV raises the possibility of a viral etiology of OSSN in cases resistant to treatment with Interferon Alfa-2b. 1. Woods M et al. Cornea 2013;54:8069-8078. 2. Sherman MD et al. Am J Ophthal 2002;134:432-3. Figure 1. The initial slit-lamp photograph of the right nasal conjunctiva of one patient. Figure 2. A comparative clinical photograph of the right nasal conjunctiva 12 months after biopsy and topical cidofovir administration for 6 weeks. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Commercial Relationships: Matthew H. Ip, None; Robert George, None; William Rawlinson, None; Minas T. Coroneo, None Program Number: 1368 Presentation Time: 9:30 AM–9:45 AM PDGF-PDGFR Signaling Sustain Angiogenesis in an Autocrine and Paracrine Fashion in Retinoblastoma Matthew W. Wilson1, 2, Zachary Goldsmith1, William Coppess1, Bradley Gao1, Matthew McEwen1, Andrew Irvine1, Rachel C. Brennan3, 1, Vanessa M. Morales1. 1Ophthalmology, Univ of Tennessee Health Sci Ctr, Memphis, TN; 2Surgery and Pathology, St Jude Children’s Research Hospital, Memphis, TN; 3Oncology, St Jude Children’s Research Hospital, Memphis, TN. Purpose: Purpose: The presence of vitreous seeding is considered a poor prognostic factor for ocular survival in Retinoblastoma (Rb) patients. External beam radiotherapy and chemotherapy have failed to increase ocular survival rates beyond 70% for eyes with vitreous seeding at the time of diagnosis, in part due to the presence of dormant tumor cells. Platelet derived growth factor is highly abundant in the vitreous microenvironment, especially in patients suffering from proliferative retinal disorders. We hypothesize reduction of the PDGF-PDGFR signaling may control vitreous seeds in Rb. Methods: Methods: We used the Rb cell lines Y79 and Weri-1 as they represent the metastatic and non-metastatic forms of the disease. Cells were cultured with or without PDGF-AB as primary stimulus and evaluated for the expression of angiogenesis-related genes, production of angiogenic factors, and morphological changes by qPCR, Multiplex assays and confocal microscopy. Next, we evaluated how PDGF regulates Rb tumor proliferation and angiogenesis via autocrine and paracrine signaling by modulation of the PDGFR-b by imatinib mesylate (IM) and a neutralization antibody against the PDGF-BB isoform. Results: Results: We found high expression of the PDGFA and PDGFB isoforms in Rb cells by qPCR analysis. Western blot, Multiplex and flow cytometry analyses revealed a reduction in the PDGF-AB/BB isoforms (p=0.04), FLT-3L (p=0.02), and VEGF (p=0.08) after concomitant incubation of IM and recombinant human PDGF-AB (rhPDGF-AB), when compared to untreated and rhPDGFAB stimulation. We observe striking differences in cellular migration both in 2D and 3D culture models when Rb cells were treated with IM. Additional studies utilizing the neutralizing antibody increased the magnitude of the angiogenic reduction. Conclusions: Conclusions: Our work suggests PDGF-PDGFR signaling sustain angiogenesis in an autocrine and paracrine fashion. These studies are a step in the pursuit of our goal of development of identifying targets of immunotherapy that will control the factors sustaining Rb. Commercial Relationships: Matthew W. Wilson, None; Zachary Goldsmith; William Coppess, None; Bradley Gao, None; Matthew McEwen, None; Andrew Irvine, None; Rachel C. Brennan, None; Vanessa M. Morales, None Support: Research to Prevent Blindness and St Jude Endowed Chair in Pediatric Oophthlamology Purpose: HER2 (ERBB2), a member of the epidermal growth factor family, is overexpressed in variety of malignancies including breast, ovarian, gastric, colorectal, pancreatic and endometrial cancers. Overexpression and gene amplification of HER2 is associated with aggressive malignancies accompanied by chemoresistance and poor prognosis. HER2 has been successfully targeted in breast cancer therapy by humanized anti-HER2 antibody trastuzumab (Herceptin) and an antibody drug conjugate trastuzumab-DM1 (Kadcyla). In this study, we tested the hypothesis that HER2 is expressed in retinoblastoma (RB), a childhood malignancy of the retina, and can be targeted by anti-HER2 antibody based therapeutics. Methods: To test the hypothesis that HER2 is expressed in retinoblastoma, we compared HER2 expression in four RB cell lines (Y79, WERI-RB27, RB143 and RB116) vs. breast cancer cell lines (BT474 and MDA-MB231), RB patient samples and breast cancer tumor arrays, as well as normal human ocular tissues by a variety of methods, including immunocytochemistry, flow cytometry, western immunoblot and RT-PCR. We utilized PCR primers that would detect all HER2 variants, as well as a variety of antibodies against HER2-specific epitopes. Once HER2 expression was confirmed, we tested the cytotoxic effect of trastuzumab and trastuzumab conjugates (T-vc-MMAE [DAR4 and DAR8]) on RB cells in vitro using an MTS assay. Results: HER2 expression was established in RB by flow cytometry, RT-PCR, immunohistochemistry of RB cell lines and an RB tumor tissue array. HER2 was not immunoreactive in normal ocular tissues. Western immunoblot analysis suggested a possible HER2 truncation in RB. This truncation spared the trastuzumab binding site and allowed us to test the cytotoxic effects of trastuzumab and its conjugates (T-vc-MMAE [DAR4 and DAR8]) on RB cells in vitro. Our data suggested that while trastuzumab itself was not cytotoxic in vitro, T-vc-MMAE was very effective in killing RB cells, where the DAR8 ADC (IC50=0.03-0.04 μM) was more potent than the DAR4 ADC (IC50=0.1-0.3 μM). Conclusions: For the first time, we have reported on the expression of HER2 in retinoblastoma and the potential for anti-HER2 based therapy. Our discovery of HER2 expression in RB may lead to innovative and targeted drug treatment options with a wider therapeutic index and fewer systemic side effects, designed to spare the eye and preserve vision in RB patients. Commercial Relationships: Gail M. Seigel, None; Sharad Sharma, None; Abigail Hackam, None; Dhavalkumar Shah, None Support: GMS is supported by the Cornell Center on the Microenvironment & Metastasis through Award Number U54CA143876 from the National Cancer Institute, as well as a grant from the SUNY Brain Network of Excellence. ASH is supported by the Karl Kirchgessner Foundation. Institutional support to Bascom Palmer Eye Institute was provided by a Research to Prevent Blindness Unrestricted Grant and an NEI Center Core Grant P30 EY014801. SS is supported by a Postdoctoral Fellowship grant from Roche Inc. to DKS. This work was in part supported by NIH grant GM114179 to DKS, and funding from the Center for Protein Therapeutics at the State University of New York at Buffalo. Program Number: 1369 Presentation Time: 9:45 AM–10:00 AM HER2: A novel therapeutic target for retinoblastoma Gail M. Seigel1, 2, Sharad Sharma3, Abigail Hackam4, Dhavalkumar Shah3, 2. 1Ctr for Hearing & Deafness, University at Buffalo, Buffalo, NY; 2SUNY Eye Institute, Buffalo, NY; 3 Pharmaceutical Sciences, University at Buffalo, Buffalo, NY; 4 Bascom Palmer Eye Institute, University of Miami, Miami, FL. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record.