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ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology 105 Retinoblastoma: Basic and clinical Sunday, May 03, 2015 8:30 AM–10:15 AM Exhibit Hall Poster Session Program #/Board # Range: 65–86/A0097–A0118 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Biochemistry/Molecular Biology, Clinical/ Epidemiologic Research, Genetics, Immunology/Microbiology, Physiology/Pharmacology Program Number: 65 Poster Board Number: A0097 Presentation Time: 8:30 AM–10:15 AM The cone-rod homeobox transcription factor (CRX) mRNA as a molecular marker in metastatic retinoblastoma Viviana E. Laurent1, Ana V. Torbidoni1, Claudia Sampor1, Daniela Ottaviani1, Mariano R. Gabri3, Jorge Rossi4, María T. García de Dávila2, Cristina Alonso1, Daniel F. Alonso3, Guillermo L. Chantada1. 1 Hematology-Oncology, Hospital de Pediatría Prof. Dr. J.P. Garrahan, Ciudad Autónoma de Buenos Aires, Argentina; 2Pathology Service, Hospital de Pediatría Prof. Dr. J.P. Garrahan, Ciudad Autónoma de buenos Aires, Argentina; 3Molecular Oncology Laboratory, Quilmes National University, Bernal, Argentina; 4Immunology Service, Hospital de Pediatría Prof. Dr. J.P. Garrahan, Ciudad Autónoma de buenos Aires, Argentina. Purpose: Disseminated retinoblastoma is still the major cause of mortality for this tumor worldwide. Research on the dissemination of this neoplasm has been hindered by the rarity of extraocular cases in developed countries. However, it would be of interest for developing countries. Using a prospective evaluation of a diagnostic clinical test, we evaluated the cone-rod homeobox transcription factor (CRX) as a new lineage-specific molecular marker for metastatic retinoblastoma and its usefulness as a tool for improving diagnostic accuracy and assessing the response to treatment and the patterns of disease dissemination in different clinical scenarios by the evaluation of minimal dissemination (MD) in extra-ocular sites. Methods: To validate CRX mRNA as a marker, we evaluated its expression in 17 retinoblastoma primary tumors, two cell lines, and 47 specimens from other malignancies as negative controls. Seventeen consecutive patients with metastatic retinoblastoma (9 at diagnosis, 8 at relapse) were included. CRX mRNA was evaluated by retrotranscription followed by real-time polymerase chain reaction (RT-qPCR) in bone marrow (BM), peripheral blood (PB), and cerebrospinal fluid (CSF) at diagnosis, after induction chemotherapy, and during follow-up after autologous BM transplantation. Results: With a sensitivity of 1 in 107 cells, CRX mRNA was expressed in all tumors and cell lines studied but it was negative in all control samples. BM metastatic cells showed expression of CRX in all nine children presenting with metastasis. After induction chemotherapy, no MD was evident in any of the eight responding children. In the CSF of children who had a metastatic relapse, CRX mRNA detection was positive in all 11 cases studied. MD in the CSF heralded a clinical relapse in three cases. No concomitant MD was evident in the BM in any case. Conclusions: CRX mRNA is a novel marker for retinoblastoma at extraocular sites. In patients with BM metastasis, there is quick, complete and sustained molecular response after induction chemotherapy. In all patients with secondary metastasis, CSF relapse occurs independently from the BM suggesting a sanctuary site. Commercial Relationships: Viviana E. Laurent, None; Ana V. Torbidoni, None; Claudia Sampor, None; Daniela Ottaviani, None; Mariano R. Gabri, None; Jorge Rossi, None; María T. García de Dávila, None; Cristina Alonso, None; Daniel F. Alonso, None; Guillermo L. Chantada, None Program Number: 66 Poster Board Number: A0098 Presentation Time: 8:30 AM–10:15 AM Adherence Capability of Cultivated Retinoblastoma Cells Narges Fazili6, Sahar Balagholi2, 1, Mozhgan Rezaeikanavi6, 3 , Somayeh Asadi4, 1, Seyed Bagher Hosseini5. 1Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, Tehran, Iran (the Islamic Republic of); 2Iran Blood Transfusion Organization, Tehran, Iran (the Islamic Republic of); 3Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran (the Islamic Republic of); 4K.N.Toosi of Technology, Tehran, Iran (the Islamic Republic of); 5Central Eye Bank of Iran, Tehran, Iran (the Islamic Republic of); 6Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran (the Islamic Republic of). Purpose: To investigate adherence capability of cultivated retinoblastoma (RB) tumorspheres with prolonged cultivation. Methods: RB cells from two Iranian patients were cultivated in DMEM supplemented with 15% FBS for four weeks in each passage. Fresh medium was added on a weekly basis and immunocytochemistry for synaptophysin was performed. All the experiments were done in duplicate. Cell attachment capability was studied during three consecutive passages. Results: A biphasic population of cultivated cells was observed during the first week in each passage; composed of RB tumorspheres and a single-cell suspension overlying a layer of fibroblastic cells that had adhered to the bottom of flask. Early adherence of RB tumorspheres to the bottom of flask, while surrounded by fibroblasts, was observed in the second week and increased by the fourth week (figure). Conclusions: Compared to previous data, this study demonstrated the adherence capability of RB tumorspheres to the underlying surface with prolonged cultivation; which after 4 weeks seems to be independent of the fibroblasts. Figure: Cultivated retinoblastoma (RB) cells. A-B: Note the presence of RB tumorspheres (circle 1), single-cell suspension (circle 2) in the first week C-F: Cultivated RB cells in the second week: the circled area shows adhered RB tumorspheres; merged FITC synaptophysine expression and DAPI in cultivated RB cells (C), adhered RB tumorsphere (D), DAPI of nucleus of RB and fibroblast cells (E), merged FITC synaptophysine expression and DAPI in cultivated RB cells (F). Commercial Relationships: Narges Fazili, None; Sahar Balagholi, None; Mozhgan Rezaeikanavi, None; Somayeh Asadi, None; Seyed Bagher Hosseini, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 67 Poster Board Number: A0099 Presentation Time: 8:30 AM–10:15 AM The Effects of Modulation of MMP-2 and MMP-9 in Angiogenesis and Invasive Potential in Retinoblastoma Anderson H. Webb1, Nabil Saleh1, Bradley T. Gao1, Ryan P. Lee1, Justin B. Lendermon1, Matthew W. Wilson1, Vanessa M. Morales1, 2 1 . Ophthalmology, University of Tennessee Health Science Center, Memphis, TN; 2Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Purpose: Retinoblastoma (Rb) is the most common primary intraocular tumor in children. Effective local treatment exists, but occasionally Rb can metastasize. Metastases are attributed to extraretina invasion of the ocular coats and the optic nerve. In this study we investigate modulation of Matrix Metalloproteinases (MMP), responsible for degradation of the extracellular matrix and invasion, as a potential adjuvant therapeutic target for Rb. Methods: Three different Rb cell lines, Rb-Y79, Rb-Weri and Rb355, were analyzed by gene expression, protein levels, and secretion of MMP-2 and MMP-9 by RT-PCR, Zymmography, and ELISA. Flow cytometry examined the levels of ICAM, VEGF, and CD31 as surrogates of adhesion, invasion, and angiogenesis. We evaluated the effect of pharmacological inhibition of MMP-2 (ARP100, Santa Cruz Biotechnology, Dallas, TX) and MMP-9 (AG-L-66085, Santa Cruz Biotechnology) by magnetic levitation (Nano3D Biosciences, Inc, Houston, TX) to determine their effects on angiogenesis and invasion. Results: Our results show the three different Rb cell lines express different levels of MMP-2 and MMP-9 mRNA. MMP-9 expression increased upon cell activation by using Phorbol 12-myristate 13-acetate (PMA) and was reduced upon use of the MMP-2 and -9 inhibitors. Magnetic levitation analysis showed reduction in Rb tumor masses in vitro by pharmacological inhibition of MMP-2 and MMP-9. Conclusions: Inhibition of MMP-2 and MMP-9, both markers of invasion, decreased expression of CD31 and VEGF, both markers of angiogenesis. MMP2 and MMP9 are potential candidates for targeted therapy. Commercial Relationships: Anderson H. Webb, None; Nabil Saleh, None; Bradley T. Gao, None; Ryan P. Lee, None; Justin B. Lendermon, None; Matthew W. Wilson, None; Vanessa M. Morales, None Program Number: 68 Poster Board Number: A0100 Presentation Time: 8:30 AM–10:15 AM HLA class I expression in cell lines derived from conjunctival melanoma and retinoblastoma using allele-specific monoclonal antibodies and flow cytometry Rind Smesseim1, Jinfeng Cao1, T H. Van Essen1, Arend Mulder1, Elsbeth van Zeeburg2, Bruce R. Ksander3, Martine J. Jager1. 1 Ophthalmology, Leiden University Medical Center, Leiden, Netherlands; 2The Rotterdam Ophthalmic Institute and the Rotterdam Eye Hospital, Rotterdam, Netherlands; 3Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA. Purpose: The expression of human leukocyte antigen (HLA) on tumor cells may influence the immunological recognition and response. Malignant cells often change their HLA class I antigens to escape immunosurveillance. To examine whether immunotherapy is possible in conjunctival melanoma and retinoblastoma we investigated the HLA class I surface expression by using allelespecific antibodies in flow cytometry. We furthermore analyzed the effect of interferon gamma (IFN-γ) stimulation on HLA expression. Methods: Three conjunctival melanoma (CRMM1, CRMM2 and CM2005.1) and three retinoblastoma (Rb116, Rb125 and Rb143) cell lines were HLA gene-typed at the Department for Immunohematology and Blood Transfusion (IHB), The Netherlands. Cells were grown in culture in the presence or absence of recombinant IFN-γ (100 and 200 U/ml) for 48 hours and prepared for flow cytometry. Based on the HLA-typing, HLA allele-specific monoclonal antibodies against class I were selected. Flow cytometry was performed and cellular surface HLA-expression measured. For Western blot analysis, a lysate was made to determine the expression of MHC class I. Results: We analyzed the HLA allele-specific expression on three conjunctival melanoma and three retinoblastoma cell lines by flow cytometry. In general, a lower expression of antigen specific and allele-specific HLA class I expression was observed in both types of malignancies. In one retinoblastoma cell line (Rb143) no class I expression was observed without IFN-γ stimulation but it was restored after incubation with IFN-γ. Two of the three conjunctival melanoma (CRMM1 and CRMM2) cell lines showed loss of expression of specific HLA class I alleles (respectively HLA-A2 and HLA-B44) which did not recover after IFN-γ stimulation. Conclusions: We were able to determine HLA expression on three new retinoblastoma cell lines and three conjunctival cell lines, and found a defect in expression of particular HLA specific alleles which were not restored by IFN-γ stimulation. This loss of antigen expression may help ocular tumors to escape the immune response and complicate the development of immunotherapy. Commercial Relationships: Rind Smesseim, None; Jinfeng Cao, None; T H. Van Essen, None; Arend Mulder, None; Elsbeth van Zeeburg, None; Bruce R. Ksander, None; Martine J. Jager, None Program Number: 69 Poster Board Number: A0101 Presentation Time: 8:30 AM–10:15 AM Gonadotropin releasing hormone receptor is expressed in retinoblastomas and a retinoblastoma cell line Sultan Aldrees, Pablo Zoroquiain, Mohammed F. Qutub, Sarah Alghamdi, Taylor Nayman, Miguel N. Burnier. Mcgill University, Montreal, QC, Canada. Purpose: Despite the fact that retinoblastoma treatment has dramatically increased the survival and vision preservation in these patients, it is still important to pursue new therapeutic targets to minimize the side-effects of current therapy. Gonadotropin releasing hormone (GnRH) has been shown to exert a direct antiproliferative effect on many types of reproductive tissue cancers, such as breast cancer, skin melanoma, and glioblastoma. The aim of this study is to describe the presence of GnRH receptor (GnRHR) in retinoblastoma in order to identify a new possible therapeutic target for this disease. Methods: Protein expression of GnRHR was studied by immunohistochemistry in 32 eyes with retinoblastoma and in the Y79 retinoblastoma cell line. Expression was scored according to intensity (1–3) and distribution (1–4), which were multiplied to generate an immunoreactive score (IRS). Low expression was considered an IRS score of 1 to 4, moderate 5 to 8, and high 9 to 12. GnRHR mRNA expression in Y79 cells were analyzed using reverse transcription polymerase chain reaction (RT-PCR). The Student’s t-test was used to compare GnRHIRS cases with or without each morphological high risk features. Results: GnRHR was expressed in all retinoblastoma cases and in the Y79 cell line. There was no expression in normal ocular structures. High, moderate, and low expression according to IRS score was evident in 16%, 36%, and 48% of cases. There were no differences in GnRH IRS with respect to uni- versus multifocal tumors, type of growth (mixed/endophytic), rosette formation, choroidal invasion, ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology extraocular extension or extension to the sclera, or optic nerve invasion. In Y79 cells, RT-PCR showed amplification of GnRHR mRNA. Conclusions: GnRHR is expressed in differing degrees in retinoblastomas, but did not correlate with prognostic factors of this particular tumor. Therefore, GnRHR may be a novel therapeutic target for the treatment of retinoblastoma. Further studies to analyze the response of the Y79 cell line to agonist and antagonist drugs are required to confirm the functionality of this receptor. Commercial Relationships: Sultan Aldrees, None; Pablo Zoroquiain, None; Mohammed F. Qutub, None; Sarah Alghamdi, None; Taylor Nayman, None; Miguel N. Burnier, None Program Number: 70 Poster Board Number: A0102 Presentation Time: 8:30 AM–10:15 AM Identification Of Differentially Expressed Protein Targets In HPV Infected Retinoblastoma Using 2D-DIGE Coupled Mass Spectrometry Approach Jasmine Naru1, 2, Ritu Aggarwal2, Ashok K. Mohanty3, Usha Singh4, Deepak Bansal5, Manoj K. Jena3, Surender Singh3, Nandita Kakkar6, Navneet Agnihotri1. 1Biochemistry, Panjab University, Chandigarh, India; 2Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India; 3Anima Biotechnology Center, National Dairy Research Institute, Karnal, India; 4 ophthalmology, Post Graduate Institute of Medical Education and Research, Chandigarh, India; 5Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India; 6histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India. Purpose: In India, population based cancer registries have reported retinoblastoma (RB) among the top five childhood cancers. There are reports on association of RB with Human Papilloma Virus (HPV) and abrogation of pRB function. But no reports are available on the proteomic profile of HPV positive and negative RB. We hypothesize that HPV may play a role in development of RB with several proteins differentially expressed in patients and controls that may act as potential candidates for therapy. Methods: Fresh RB tumor and normal retinal tissues (n=42each) were recruited. Screening of 21 different HPV genotypes was done using HPV genoarray kit. Proteomic analysis was performed on four samples each from HPV positive, HPV negative RB and controls. 2D-DIGE coupled MALDI- TOF/TOF mass spectrometry was employed. Dye swapping was done. Image analysis was done using Decyder 2D software. Differentially expressed spots were identified and picked. Using PCA, the two tumor groups could be delineated from normal tissue based on protein expression pattern. mRNA expression of the identified proteins was verified and some selected proteins were validated by western blot as well. Results: Disease was bilateral in 33% cases. Of the 39 eyes with nonfamilial RB, 25.6% tested positive for HR HPV16. Among the three groups, 102 protein spots were differentially regulated (p<0.05,±1.5 folds) constituted by 39 unique proteins determined by MALDI-TOF/ TOF. 12 proteins were up regulated in HPV positive cases vis-a-vis HPV negative. Patient group exhibited upregulation of 7 proteins compared to controls. Highly deregulated proteins were GFAP, RBP3, CRABP1, SAG and TF. Significant mRNA levels (p<0.05) of RBP3,GFAP,CRABP1,PDIA3,ATP5B,PITPNA were observed in RB compared to normal. Gene ontology revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). Pathway analysis identified to be the proteins involved in acute phase signalling in tumor(p=1.42-08). Whereas CTTNB1 and TP53 signalling pathways were more significantly regulated in HPV positive than HPV negative RB. Conclusions: The study provides a dynamic protein profile of retinoblastoma (HPV positive and negative) and highlights significantly relevant protein targets like GFAP, RBP3 and CRABP1. Their prospects of being used as potential candidates in therapy needs to be further explored. Commercial Relationships: Jasmine Naru, None; Ritu Aggarwal, None; Ashok K. Mohanty, None; Usha Singh, None; Deepak Bansal, None; Manoj K. Jena, None; Surender Singh, None; Nandita Kakkar, None; Navneet Agnihotri, None Program Number: 71 Poster Board Number: A0103 Presentation Time: 8:30 AM–10:15 AM Interferon-gamma is required for protecting against intraocular tumor growth after peripheral immunization but not for the generation of tumor-specific cytotoxic T-lymphocytes. Ann J. Ligocki, Jerry Y. Niederkorn. Ophthalmology, UT Southwestern Medical Center, Dallas, TX. Purpose: To determine the effect of interferon-gamma loss on ocular tumor growth after peripheral immunization. Methods: C57BL/6 or interferon-gamma knock-out mice (IFN-γ KO) were immunized with the syngeneic Ad5E1 tumor (adenovirus type 5 transformed mouse embryo cells) in the anterior chamber (AC), subcutaneously (SC), or in a protective model of SC immunization prior to an AC challenge. Ocular tumor growth was measured by percent AC occupied with tumor. Spleens were isolated from mice and tested for tumor-specific cytotoxic T-lymphocytes (CTLs) using a 51 Cr release assay. Results: AC immunization of tumor cells into C57BL/6 mice resulted in ocular tumor growth followed by necrotizing immune rejection of the tumor. However, when the same AC immunization was performed in IFN-γ KO mice, the tumor grew progressively in the eyes of all of the mice. SC immunization with the same tumor cells into both C57BL/6 and IFN-γ KO mice resulted in peripheral tumor rejection. Furthermore, both C57BL/6 and IFN-γ KO mice generated tumorspecific CTLs post SC immunization. In a protective immunization model, mice were immunized peripherally with tumor cells prior to an AC challenge. This SC immunization provided protection against ocular tumor growth in 79% of wild-type C57BL/6 mice (N=15/19). Conversely, 100% of the IFN-γ KO mice (N=18/18) developed progressive ocular tumors. Interestingly, both the C57BL/6 and IFN-γ KO mice still generated tumor-specific lysis CTL responses in the periphery, yet only the wild-type C57BL/6 mice rejected their intraocular tumors. Conclusions: IFN-γ is critical for immune rejection in this intraocular tumor model but is not required for the peripheral rejection of these tumors. Despite the generation of peripheral tumor-specific CTL responses in both C57BL/6 and IFN-γ KO mice, only an IFN-γ replete environment produces a protective immune response within the eye after a peripheral immunization. This suggests that IFN-γ is required for the protective CTLs to either enter or function within the eye. Commercial Relationships: Ann J. Ligocki, None; Jerry Y. Niederkorn, None Support: R01 EY05631-28A1 ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 72 Poster Board Number: A0104 Presentation Time: 8:30 AM–10:15 AM Effects of pretreatment with the radioprotector ortho-phosphoL-tyrosine (pTyr) on Rb+/- mice after radiation exposure – Implication for the treatment of retinoblastoma patients with radiotherapy Alexander Tschulakow1, Stephan Huber2, Monika Rittgarn1, HansPeter Rodemann3, Ulrich Schraermeyer1, 4, Sylvie Julien1, 4. 1Section of Experimental Vitreoretinal Surgery, Center for Ophthalmology, Tuebingen, Germany; 2Laboratory of Experimental Radiooncology, Division of Radiooncology, University of Tuebingen, Tuebingen, Germany; 3Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University Hospital, Tuebingen, Germany; 4STZ OcuTox, Preclinical Drug Assessment, Hechingen, Germany. Purpose: Retinoblastoma (Rb) is the most frequent ocular tumor in children and if let untreated, can cause death. Like the most head and neck tumors it is sensitive to radiotherapy (RT). However, the therapy has its risks like damage of healthy tissues recurrence and development of treatment-induced secondary tumors. The aim of this study is to investigate the ability of the radioprotector pTyr to prevent RT-induced secondary tumors and other side-effects observed after RT. Methods: B6;129-Rb1tm3Tyj/J mice having a mutation in one of the Rb -gen allele were used. Although these mice do not develop a Rb, this model was chosen because Rb-patients having a similar mutation have a higher risk of developing secondary tumors induced by RT. One group of mice was treated with intraperitoneal injections of pTyr 16 hours before each irradiation. Another group was only irradiated. Both groups were irradiated over a period of 3 weeks 3 times a week with a dosage of 5 Gy per exposure (Fig.1). All animals were investigated using SLO/OCT and histologically 1, 3, 6 and 9 months after irradiation (Ir) . Radiation-induced tumor induction as well as normal tissue radiation toxicity were evaluated as function of pTyrtreatment. Results: The first visible effect of the Ir was the graying of the hair coat in the area of Ir. This effect was reduced in the pTyr treated mice. The results of the OCT- analysis showed that 3 and 6 months after Ir the thickness of the retina of the mice was significantly lower in the untreated group (n=12) compared to the pTyr treated one (n=12) (p<0.05 3 months and p<0.001 6 months after Ir). The histological analysis of the retina showed a significant photoreceptor loss in the untreated group vs. the pTyr treated one (p<0.001 3 months and p<0.0001 6 months after Ir) . Conclusions: Our results show, that the application of pTyr before irradiation significantly reduces the negative effects of radiation on the hair coat and retina. The results of the analysis 9 months after Ir, the appearance of secondary tumors as well as the potential benefit of the pretreatment with pTyr are currently under investigation. fig. 1: (a) linear accelarator Linac-G (Phillips), (b) the mouse fixation units, (c) 6 Rb+/- mice fixed for radiation exposure, (d) scheme of the assembly of the experiment. Commercial Relationships: Alexander Tschulakow, None; Stephan Huber, None; Monika Rittgarn, None; Hans-Peter Rodemann, None; Ulrich Schraermeyer, None; Sylvie Julien, None Support: DKKS, DKS 2012.08 Program Number: 73 Poster Board Number: A0105 Presentation Time: 8:30 AM–10:15 AM Invasiveness and metastasis of retinoblastoma in an orthotopic zebrafish tumor model Xiaoyun Chen, Wei Xiao, Yizhi Liu. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China. Purpose: Retinoblastoma is a highly invasive malignant tumor that often invades the brain and metastasizes to distal organs through the blood stream. Invasiveness and metastasis of retinoblastoma can occur at the early stage of tumor development. However, an optimal preclinical model to study retinoblastoma invasiveness and metastasis in relation to drug treatment has not been developed. In this study, we developed an orthotopic zebrafish model in which retinoblastoma invasion and metastasis can be monitored at a single cell level. Methods: Human and mouse retinoblastoma cell lines were labeled with fluorescent DiI dye before injection. At 48 hours after fertilization, wild type and fli1: EGFP-transgenic zebrafish embryos which exhibit green fluorescent signals in blood vessels were anesthetized and injected about 100-200 retinoblastoma cells into the vitreous cavities using the Pneumatic PicoPump under the stereomicroscope. Zebrafish embryos were examined every 2 days under a fluorescent microscope to monitor tumor cell growth, invasion, metastasis, and the interactions between retinoblastoma cells and surrounding microvasculatures. Further, to prove this method can be used to evaluate the efficacy of new therapies, sunitinib was added directly to the aquaria water after tumor cells implantation to attain a final concentration of 1 μM, and zebrafish embryos were examined with a fluorescent microscopy after 4 days. Results: After 2 days implantation, dissemination of tumor cells from the primary sites could be detected using fluorescent microscopy. Total numbers of metastatic foci was progressively increased and reached the maximal level at day 6 after tumor implantation. Tumor cells could disseminate to the heads, the lateral healthy eyes and the tails of zebrafish. Additionally, the primary tumor masses could stably maintain in the vitreous cavity of zebrafish for 2 days after implantation, but progressively decreased after 4 days. We also found that most tumor cells formed clusters around the hyaloid vessels attached to lens at day 2 after tumor cell implantation. Finally, treatment with retinoblastoma-bearing zebrafish embryos with 1 μM of sunitinib could significantly attenuate retinoblastoma invasion and metastasis. Conclusions: Thus, this orthotopic retinoblastoma model in zebrafish offers a new and unique opportunity to study the early events of tumor invasion, metastasis and drug responses. Commercial Relationships: Xiaoyun Chen, None; Wei Xiao, None; Yizhi Liu, None Program Number: 74 Poster Board Number: A0106 Presentation Time: 8:30 AM–10:15 AM Kif14 overexpression accelerates tumor development in the TAgRB transgenic model of retinoblastoma Michael O’Hare1, 3, Shadmand Mehdi1, Timothy W. Corson1, 2. 1 Ophthalmology, Glick Eye Institute, Indianapolis, IN; 2Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN; 3Biomedical Science, University of Ulster, Coleraine, United Kingdom. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Purpose: The mitotic kinesin KIF14 is a molecular motor that plays a pivotal role in the final stages of cytokinesis. In most cases of retinoblastoma the KIF14 locus at 1q32.1 is gained as an important event after mutation of the RB1 gene. Moreover, KIF14 is overexpressed in retinoblastoma, strongly suggesting its role as an oncogene. Despite this, KIF14’s effects on retinoblastoma in vivo have not previously been analyzed. We aimed to determine Kif14’s role in promoting retinal tumor formation using a novel Kif14 overexpressing, TAg-RB retinoblastoma mouse model. Understanding the effects of Kif14 overexpression in vivo will allow for a greater understanding of the biology of post RB1 loss events and how they contribute to retinoblastoma progression. Methods: By crossing transgenic mice constitutively overexpressing Kif14 into the SV40 T-antigen expressing model of retinoblastoma (TAg-RB) we generated Kif14; TAg-RB double transgenic mice. The Micron III rodent imaging system was used to obtain fundus photographs as well as optical coherence tomography (OCT) images. Double transgenics and TAg-RB littermates were imaged in both eyes over a time-course to document tumor development. Results: Compared to the TAg-RB single transgenic mice, the Kif14; TAg-RB double transgenic mice showed greatly accelerated formation of tumor-like clusters of hyper-reflective cells in the inner nuclear layer of the retina from as early as 3-4 weeks of age, as visualized by OCT. Pale intraretinal tumors were first visible by funduscopy in the periphery of the retina by week 6 in the Kif14; TAg-RB mice compared to week 8 in the TAg-RB mice. Conclusions: The over-expression of the Kif14 oncogene in the TAg-RB model of retinoblastoma leads to accelerated onset of tumor formation, providing strong evidence that Kif14 promotes retinoblastoma formation in vivo. Further investigation will include quantitative immunohistochemical analysis of these mice to complement the OCT findings and further validate the importance of Kif14 for the genesis of retinoblastoma. Commercial Relationships: Michael O’Hare, None; Shadmand Mehdi, None; Timothy W. Corson, None Support: American Cancer Society Institutional Research Grant, Research to Prevent Blindness, NIH NCATS KL2TR001106 Program Number: 75 Poster Board Number: A0107 Presentation Time: 8:30 AM–10:15 AM Retinal Toxicity of Intravitreal Melphalan in Albino Rabbit Shai M. Bar-Sela1, 2, Shiri Zayit-Soudry3, 4, Amir Massarweh5, Anat Loewenstein1, 2, Ido Perlman5. 1Ophthalmology, Tel Aviv Medical Center, Tel Aviv, Israel; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3Ophthalmology, Rambam Medical Center, Haifa, Israel; 4Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Inst of Tech, Haifa, Israel; 5Physiology & Biophysics, Medicine, Technion-Israel Inst of Tech, Haifa, Israel. Purpose: Intravitreal melphalan injections at doses of 8-30mg have been successfully used for treating retinoblastoma with vitreous seeds, but there is insufficient data regarding their safety. This study was designed to evaluate the toxicity of intravitreal melphalan in a rabbit model. Methods: Eighteen albino rabbits were treated with a single intravitreal melphalan injection (0.1ml) to the right eye (experimental eye): group 1: 5mg, n=4; group 2: 15mg, n=4; group 3: 30mg, n=5; group 4: 60mg, n=5. The left eye of each rabbit was injected with 0.1ml saline (control eye). Indirect ophthalmoscopic examination, electroretinography (ERG) and visual evoked potentials (VEP) testing were performed at baseline and periodically during 4-week follow-up. After 4 weeks the retinas were prepared for histological examination and glial fibrillary acidic protein (GFAP) immunocytochemistry. Analysis of variance for repeated measures was used for statistical analysis of the electrophysiological parameters. Results: Indirect ophthalmoscopy after injections revealed sclerotic retinal vessels and retinal whitening in the experimental eyes of groups 2, 3 and 4, but not in rabbits of group 1, injected with the lowest melphalan dose. Mean (±SD) dark-adapted (DA) ERG b-wave Vmax ratios (experimental eye/control eye) in groups 1-4, measured at 4-weeks after injection were 1.12±0.08, 0.77±0.20, 0.42±0.11 and 0±0, respectively, and light adapted (LA) b-wave amplitude ratios were 1.19±0.18, 0.77±0.27, 0.43±0.24 and 0±0, respectively. Thus, increasing melphalan dosage significantly predicted reduced DA b-wave Vmax ratios (p<0.013) and reduced LA b-wave amplitude ratios (p<0.026), indicating dose-dependent functional retinal damage induced by melphalan. However, similar flash VEP responses were found in experimental and control eyes of all groups, suggesting no melphalan induced functional damage to the optic nerve. Morphological studies supported the ERG findings demonstrating retinal structural damage and GFAP expression in Müller cells, at a magnitude that paralleled the ERG deficit. Conclusions: Intravitreal melphalan dose of 5μg in rabbits, approximately equivalent to 10μg in human, appears to be safe to the retina. However, rabbit doses of 15μg and higher, roughly corresponding to human doses of 30μg and higher, are toxic, and their utilization for treating retinoblastoma should be executed with caution, particularly if visual potential exists. Commercial Relationships: Shai M. Bar-Sela, None; Shiri ZayitSoudry, None; Amir Massarweh, None; Anat Loewenstein, None; Ido Perlman, None Program Number: 76 Poster Board Number: A0108 Presentation Time: 8:30 AM–10:15 AM Ocular Toxicity of Intravitreal Melphalan for Retinoblastoma Stephen J. Smith1, Brian Smith2. 1Kellogg Eye Center, Univeristy of Michigan, Ann Arbor, MI; 2Hematology/Oncology, University of Rochester, Rochester, NY. Purpose: To describe the ocular side effects in patients receiving melphalan intravitreal injection therapy (IViT) for retinoblastoma. Methods: PUBMED (1946-present), SCOPUS (all years), Science Citation Index (1900 – present) and Conference Proceedings Citation Index - Science (1990 – present) electronic databases were searched to identify all published reports of therapeutic intravitreal injections for retinoblastoma in humans. Results: Eleven studies with original melphalan IViT ocular side effect data were included in this systematic review. In these combined reports, a total of 1311 intravitreal injections were given to 327 eyes of 317 patients. Melphalan IViT doses ranged from 8 to 50 micrograms. Ocular side effects occurred in 50 patients, with 37 patients experiencing side effects likely secondary to melphalan toxicity. The proportion of patients experiencing drug related ocular side effects following melphalan IViT regiments was 0.117 (37/317). Some patients experienced more than one side effect. Drug related side effects in patients receiving doses ranging from 8 to 40 micrograms included salt and pepper retinopathy, ERG reduction, iris atrophy, chorioretinal atrophy, and peripheral lens opacity. Four patients received a single dose of 50 micrograms of melphalan and developed retinal necrosis and gliosis, choroidal congestion, optic nerve atrophy, cataract, and retinal neovascularization. Conclusions: Ocular toxicity following melphalan IViT occurs in a dose dependent fashion, and can be severe. There appears to be significant variability in assessing and reporting ocular side effects, potentially underestimating drug related toxicity in these patients. Care must be taken in the dosing of intravitreal melphalan treatments to avoid potentially irreversible vision loss. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Commercial Relationships: Stephen J. Smith, None; Brian Smith, None Program Number: 77 Poster Board Number: A0109 Presentation Time: 8:30 AM–10:15 AM Toxic Effects of Melphalan on Retinal Pigment Epithelial Cells Sabine Aisenbrey1, 2, Ulrike Hagemann1, Merle Schrader1, 2, Kai Januschowski1, Sven Schnichels1, Daniela Suesskind1. 1Department of Ophthalmology, Eberhard Karls University Tuebingen, Tuebingen, Germany; 2University Eye Hospital, University of Oldenburg, Oldenburg, Germany. Purpose: The cytostatic drug Melphalan is widely used in the treatment of retinoblastoma. Special emphasis lies on superselective intraarterial and intravitreal chemotherapy with great success regarding tumor control. Recently, clinical evidence of retinal pigment epithelial (RPE) atrophy and vascular complications after intraarterial and intravitreal application of the drug have been reported in single cases. Facing this background we investigated cellular toxic effects of Melphalan on RPE in a cell culture model. Methods: ARPE19 cells were used after reaching 90% confluence. The effects of Melphalan (4, 10, 20, 50, 100, 166, 200 mg/ml) on cell morphology via phase contrast microscopy, proliferation using BrdU assay, cell viability via MTS assay, cell mass estimation using Crystal violet staining and apoptosis via Caspase 3/7 activity assay were examined in triplicate. Measurements were carried out after 24h of incubation time. Staurosporin and the Melphalan diluent were applied as positive and negative controls, respectively. Results: Morphologically increasing number and size of gaps were detectable in the cell layer with increasing Melphalan concentrations. In parallel proliferation of ARPE19 as well as their cell mass were decreasing. Cell viability was influenced by Melphalan concentrations of 50mg/ml and higher. After 24h apoptosis reached a maximum value with the 100mg/ml Melphalan concentration. Conclusions: In a cell culture model using ARPE19 cells we could observe a decrease in proliferative activity, cell amount, and cell viability of RPE cells as well as an increase in apoptosis after 24h Melphalan incubation suggesting a direct toxic effect of the chemotherapeutic remedy. A direct toxic effect of Melphalan in vivo after intraarterial or intravitreal application on the RPE may be probable and may explain the clinical and angiographic alterations. Additional cytostatic drugs currently used in retinoblastoma treatment have to be investigated in this context regarding tumor control versus toxicity. Commercial Relationships: Sabine Aisenbrey, None; Ulrike Hagemann, None; Merle Schrader, None; Kai Januschowski, None; Sven Schnichels, None; Daniela Suesskind, None Program Number: 78 Poster Board Number: A0110 Presentation Time: 8:30 AM–10:15 AM Diagnosing pathological prognostic factors in retinoblastoma: correlation between traditional microscopy and digital slides Christina Mastromonaco, Patrick T. Logan, Pablo Zoroquiain, Sarah Alghamdi, Matthew Balazsi, Miguel N. Burnier. Pathology, Henry C. Witelson Ocular Pathology Laboratoy, Montreal, QC, Canada. Purpose: Digital pathology is a tool that converts a microscope slide into a digital image using a scanner that can be viewed on a computer screen rather than on a microscope. Whole slide imaging (WSI) possess the unique feature of having a global view of the entire eye in order to visualize the relationships between the tumor and ocular structures. The aim of the present study was to determine the diagnostic accuracy, using WSI generated by a scanner, of high-risk prognostic factors and morphological characteristics of retinoblastomas. Methods: Forty-seven enucleated eyes with retinoblastoma from the Henry C. Witelson Ocular Pathology Laboratory, Montreal, Quebec, were stained with hematoxylin and eosin. Whole slide images at 40× magnification were reviewed by a pathologist using the Virtuoso image analyzer, and the following prognostic factors were evaluated: muticentricity, type of growth, choroidal, anterior chamber, and optic nerve invasion, rosette formation, necrosis, and Azzopardi effect. These results were compared with results obtained from the same pathologist after reviewing the slides in a random order using a regular microscope as the gold standard. McNemar’s test (MT), percentage of agreement (POA), and sensibility (S) and specificity (Sp) were evaluated between WSI and conventional microscopy. Results: There were no differences with respect to the determination of multicentricity, type of growth, rosette formation (Homer Wright, Flexner-Wintersteiner, and fleurettes), choroidal invasion, invasion of anterior chamber structures, extraocular extension, extension to the sclera (including vortex vessels), optic nerve invasion (head or prelaminar, laminar, or postlamellar invasion), necrosis, or Azzopardi effect between WSI analysis and light microscopy (MT, P = 1.0; POA = 100%; S = 100%; and Sp = 100%). Conclusions: To the best of our knowledge, this is the first report using digital pathology (WSI) to evaluate prognostic factors in eyes containing retinoblastomas. Using WSI, the pathologist was able to detect high-risk morphological features in retinoblastoma. WSI is an important tool now in particular for ophthalmic pathologists examining enucleation and exenteration specimens. WSI should be considered as a viable alternative to traditional microscopy in ocular pathology. Commercial Relationships: Christina Mastromonaco, None; Patrick T. Logan, None; Pablo Zoroquiain, None; Sarah Alghamdi, None; Matthew Balazsi, None; Miguel N. Burnier, None Program Number: 79 Poster Board Number: A0111 Presentation Time: 8:30 AM–10:15 AM PAR-1 and Maspin expression in Retinoblastoma and their correlation with histopathological prognostic features nadine marques1, Ana Beatriz T. Dias2, Sarah Alghamdi2, Cristina Fonseca3, Tânia Borges4, Miguel N. Burnier2. 1ophthalmology, hospital garcia de orta, Lisbon, Portugal; 2Ocular pathology, Mcgill, Montreal, QC, Canada; 3Ophthalmology, Centro hospitalar de Coimbra, Coimbra, Portugal; 4Ophthalmology, Centro hospitalar do Porto, Porto, Portugal. Purpose: Maspin is a tumor suppressor protein expressed in normal mammary and other epithelial cells and is reduced or absent in breast, ovarian carcinomas, and gliomas. Protease activated receptor 1 (PAR-1) is related to tumor growth and metastatic potential. Maspin expression, when co-expressed with PAR-1, can counteract its malignant potential, as the maspin gene is downstream of PAR-1 signaling. Our purpose was to evaluate, for the first time, maspin and PAR-1 expression in retinoblastomas and in normal retinas, and to correlate them with histopathological prognostic features. Methods: Maspin and PAR-1 expression were evaluated in 40 retinoblastoma eyes. Nine normal human eyes from the Eye Bank of Canada were used as controls. Positive controls were skin for maspin and pancreas for PAR-1. Maspin and PAR-1 immunostains were evaluated using a score considering extent of tumor/structure staining (0=none, 1=<50% and 2=>50%) and intensity (0=none, 1=intensity < positive controls, 2=intensity > positive controls). A total final score ranging between 0 and 4 was established (final score = proportion x intensity of staining). Invasive phenotype was considered when invasion of the optic nerve or choroid were present. Fisher exact and Student’s t-test analyses was performed to compare variables. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Results: A maspin score of 0 (neither nuclear nor cytoplasmic expression) correlated with invasive phenotype (P=0.03). No difference in invasive phenotype was found for isolated PAR-1 or PAR-1 without maspin expression in the same tumor. In normal eyes, no nuclear or cytoplasmic maspin staining in the retina was found. Both retinal plexiform layers showed PAR-1 expression in 6 of the same samples (4 with score 2 and 2 with score 4). No differences in PAR-1 score between normal retinas and retinoblastoma were found. Conclusions: Absence of maspin expression is associated with more aggressive biological behavior in this study. PAR-1 expression was found only in plexiform layers of normal retinas and does not differ according to retinoblastoma expression score. Maspin is not expressed in the normal retina. The identification of maspin suggests that it may be a novel therapeutic target for aggressive retinoblastomas. Commercial Relationships: nadine marques, None; Ana Beatriz T. Dias, None; Sarah Alghamdi, None; Cristina Fonseca, None; Tânia Borges, None; Miguel N. Burnier, None Program Number: 80 Poster Board Number: A0112 Presentation Time: 8:30 AM–10:15 AM Rates of Pineal Cysts Detected by MRI Associated with Successful Intravenous Chemotherapy Treatment of Bilateral Retinoblastoma Felix Y. Chau, Kai B. Kang, Michael P. Blair, Michael Shapiro. Ophthalmology & Visual Sciences, University of Illinois at Chicago UIC, Chicago, IL. Purpose: Retinoblastoma (RB) treatments include enucleation, radiotherapy, cryotherapy, laser photocoagulation and chemotherapy. Pinealoblastomas are known to potentially occur in germline RB, most often with bilateral RB (“trilateral RB”), and may regress to pineal cysts with systemic chemotherapy. The purpose of this study is to evaluate rates of pinealoblastomas and pineal cysts detected on magnetic resonance imaging (MRI) in bilateral RB patients treated with intravenous chemotherapy (vincristine, etoposide, carboplatin VEC) with focal consolidation or enucleation as primary therapy. Methods: Retrospective review of digital fundus images and medical records of RB patients who presented to the University of Illinois at Chicago, Illinois Eye and Ear Infirmary Retina Clinic from November 1, 2004 to November 1, 2014. Results: 34 patients received treatment over the study period. 18 patients had unilateral RB, and 16 had bilateral RB. The mean age at diagnosis was 16 months. No pinealoblastomas were detected by MRI in any RB patient. Pineal cysts detected by MRI occurred in none (0%) of the 18 unilateral patients and 2 of 16 (12.5%) bilateral patients. All bilateral RB patients received 6 cycles of VEC. One bilateral RB patient (International Classification of Retinoblastoma – ICRB Group B or C in both eyes) was diagnosed at age 12 months and developed pineal enhancement at age 18 months (within the last month of 6 rounds of VEC) and a pineal cyst at age 21 months, 3 months after completing VEC. The other bilateral RB patient (ICRB Group E OD [enucleated], Group C or D OS) was diagnosed at age 7 months and had a pineal cyst detected at age 26 months, 16 months after completing VEC. Conclusions: The rate of pinealoblastoma detected on MRI was 0% in bilateral RB patients receiving VEC. Pineal cysts were detected by MRI in 12.5% of bilateral RB patients receiving VEC. These findings may confirm the successful prevention of pinealoblastoma growth by systemic intravenous vincristine, etoposide, and carboplatin treatment in patients with bilateral retinoblastoma. Commercial Relationships: Felix Y. Chau, None; Kai B. Kang, None; Michael P. Blair, None; Michael Shapiro, None Support: Research to Prevent Blindness; K12 EY021475. Program Number: 81 Poster Board Number: A0113 Presentation Time: 8:30 AM–10:15 AM Identification of RB1 gene germline mutations in Mexican patients with sporadic unilateral retinoblastoma Dalia C. Guadarrama Vallejo1, Juan C. Zenteno1, 2, Arturo Flores Cuevas1. 1Genetics- retina, Instituto de Oftalmologia Conde de Valenciana, Mexico city, Mexico; 2Biochemistry, Faculty of Medicine, National Autonomous University of Mexico, Mexico, city, Mexico. Purpose: Purpose: to determinate the percentage of cases with sporadic unilateral retinoblastoma carrying RB1 gene germline mutations in a sample of Mexican patients. Methods: Methods: an observational, cross-sectional and descriptive study was performed; patients with sporadic (non-familial) unilateral retinoblastoma evaluated between March 2005 and December 2011 in a reference center in Mexico City were selected. Inclusion criteria were both genders, older than 12 months, and unilateral tumor. Patients with a family history of fibrosarcoma, lymphoma, leukemia, or melanoma were excluded. Patients developing contralateral retinoblastoma during the study were eliminated. Genomic DNA was obtained from peripheral blood leukocytes in each patient; PCR amplification and direct nucleotide sequencing of the 27 exons and exon/intron junctions of the RB1 gene was performed. Possible gross gene deletions or duplications were investigated by means of MLPA (Multiplex Ligation-Dependent Probe Analysis). In addition, promoter sequencing and DNA methylation analyses were performed. Potential pathogenicity of novel mutations was analyzed with PolyPhen software. Results: Results: Twenty (9 female and 11 male) Mexican patients with sporadic unilateral retinoblastoma were included; average age at diagnosis was 26.3 months. Germline mutations were identified in two patients (10%): a mutation in exon 17 predicting a nonsense mutation at residue 533 and a mutation in exon 20 predicting a p.R661W missense mutation. Parental DNA’s of these two cases were negative for the mutations, indicating de novo origin. No mutations in exonic or promoter regions or methylation of the RB1 promoter were demonstrated in the remaining 18 patients. Conclusions: Conclusion: This is the first study in Mexican population using a number of molecular diagnostic tests to identify RB1 germline defects in unilateral retinoblastoma. Our results contrast with figures from other populations showing up to 20% of RB1 mutations in sporadic unilateral retinoblastoma. Our results are of extreme importance for genetic counselling in this group of retinoblastoma patients and their families. Commercial Relationships: Dalia C. Guadarrama Vallejo, None; Juan C. Zenteno, None; Arturo Flores Cuevas, None Program Number: 82 Poster Board Number: A0114 Presentation Time: 8:30 AM–10:15 AM Retinoblastoma in South Africa – A 20-year retrospective study at two tertiary academic hospitals in Johannesburg Saadiah Goolam, Nicky D. Welsh, Ismail Mayet. Ophthalmology, University of Witwatersrand, Johannesburg, South Africa. Purpose: To characterise retinoblastoma in the South African population through a 20-year review of patient records at two tertiary academic hospitals in Johannesburg Methods: Retrospective clinical case series analysis of medical records of patients with retinoblastoma presenting to Charlotte Maxeke Johannesburg Academic Hospital and Chris Hani Baragwanath Academic Hospital between 01 January 1992 and 31 December 2011 Results: The total number of cases identified was 282, with 245 meeting the study inclusion criteria. Retinoblastoma comprised 6.9% ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology of total pediatric oncology presentations. 65.3% were unilateral, 34.3% bilateral and 0.4% trilateral. The overall male to female ratio was 1.08. Mean age at presentation overall was 32.6 months (median 28.0 months), unilateral 39.4 months (median 33.0 months) and bilateral 19.7 months (median 17.0 months). The mean delay to presentation overall was 7.0 months (median 4.0 months), unilateral 8.5 months (median 5.0 months) and bilateral 4.4 months (median 3.0 months). The most frequent presenting symptoms were leukocoria (37.1%) and proptosis (34.7%). Distribution of disease stage at presentation (International Retinoblastoma Staging System) was 1.6% with Stage 0, 24.1% with Stage I, 27.8% Stage II, 16.3% Stage III and 25.3% Stage IV. 26.5% of patients defaulted care. The fiveyear survival rate using the Kaplan-Meier survival curve was 57.7% in the overall study population, and according to disease stage at presentation: 95.3% - Stage I, 84.8% - Stage II, 49.7% - Stage III and 5.7% - Stage IV Conclusions: This study showed that delay in presentation of retinoblastoma cases remains a significant barrier to effective treatment in this African setting. Intervention to streamline referrals is indicated and may include outreach programs and education of referring hospitals Commercial Relationships: Saadiah Goolam, None; Nicky D. Welsh, None; Ismail Mayet, None Program Number: 83 Poster Board Number: A0115 Presentation Time: 8:30 AM–10:15 AM Development and Delivery of Candidate Retinoblastoma Therapeutics Eleanor M. Pritchard1, 2, Rachel Brennan5, Lyra Griffiths2, Elizabeth Stewart2, Cori Bradley2, Burgess B. Freeman3, William Caufield3, Michael A. Dyer2, 4, R K. Guy1. 1Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN; 2 Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN; 3Preclinical Pharmacokinetics Shared Resource,, St. Jude Children’s Research Hospital, Memphis, TN; 4Department of Ophthalmology, University of Tennessee Health Sciences Center, Memphis, TN; 5Oncology, St. Jude Children’s Research Hospital, Memphis, TN. Purpose: While mortality is low with aggressive multimodal therapy, partial or full loss of vision occurs in approximately 50% of patients with advanced bilateral retinoblastoma. There is an urgent need to develop targeted therapies that preserve vision and reduce late effects of current treatment modalities, which include facial malformations and increased incidence of secondary malignancies. Candidate therapeutics should ideally (1) completely clear disease to prevent progression and metastasis, (2) be well tolerated locally to preserve vision, and (3) act quickly to overcome rapid clearance from the eye. Phase I and II clinical trials for retinoblastoma are difficult due to the young age of the patient population and relative rarity of the disease. Therefore robust preclinical testing of new therapies is critical. Methods: To meet this need, we conducted library screening of approximately 300 compounds using focused libraries from St. Jude Children’s Research Hospital (SJCRH) chemical collection to determine in vitro potency in retinoblastoma cell lines (RB355, Weri, and Y79) and a normal human fibroblast control cell line (BJ). Further in vitro testing was conducted to prioritize “hits” (compounds active in retinoblastoma cells, but not BJ cells). For lead compounds, we determined speed of effect with washout studies and cidality with outgrowth studies. Synergy testing was performed to eliminate candidates with antagonistic interactions with current standard of care retinoblastoma drugs and prioritize any with strong agonism. We characterized pharmacokinetics (PK) to compare intraocular exposure for different routes of delivery (local and systemic) for high priority candidates. Results: Screening identified several candidates to advance to the preclinical testing phase, including histone deacetylase (HDAC) inhibitors and FDA-approved oncology drugs that might be repurposed for ocular use with local delivery methods. Consistent with treatment goals and the unique constraints of reaching an intraocular target, we prioritized HDAC inhibitors with rapid, cidal, potent and selective effects on retinoblastoma cells in vitro. Conclusions: In vitro and PK testing suggest local delivery of HDAC inhibitors represents a promising potential targeted therapy for retinoblastoma. Next steps will include toxicity and efficacy testing in preclinical animal models of retinoblastoma. Commercial Relationships: Eleanor M. Pritchard, None; Rachel Brennan, None; Lyra Griffiths, None; Elizabeth Stewart, None; Cori Bradley, None; Burgess B. Freeman, None; William Caufield, None; Michael A. Dyer, None; R K. Guy, None Support: Knights Templar Eye Foundation (KTEF) Pediatric Ophthalmology Career Starter Grant Program Number: 84 Poster Board Number: A0116 Presentation Time: 8:30 AM–10:15 AM Safety and efficacy of digoxin as a potential candidate agent for retinoblastoma treatment URSULA A. WINTER6, Emiliano Buitrago6, Hebe Mena1, Soledad Negrotto1, Maria J. del Sole2, Hakim Djaballah3, Juan O Croxatto4, Guillermo L. Chantada7, David H. Abramson5, Paula Schaiquevich6. 1 Experimental Thrombosis Laboratory, Institute of Experimental Medicine (IMEX), National Academy of Medicine-CONICET, Capital Federal, Argentina; 2Pharmacology Laboratory, CIVETANCONICET, Faculty of Veterinary, National University of the Centre of Buenos Aires, Tandil, Argentina; 3HTS Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY; 4Argentinean Ophthalmic Foundation Jorge Malbrán, Buenos Aires, Argentina; 5 Ophthalmic Oncology Service, Memorial Sloan-Kettering Cancer Center, New York, NY; 6Clinical Pharmacokinetic Unit, Hospital de Pediatria JP Garrahan, Buenos Aires, Argentina; 7Service of Hematology-Oncology, Hospital de Pediatria JP Garrahan, Buenos Aires, Argentina. Purpose: Despite recent advances of local routes for chemotherapy delivery there have been few agents incorporated in the chemotherapy armamentarium for retinoblastoma treatment with almost no new schedules for drug administration. This study assessed the anti-tumor and antiangiogenic effect of digoxin in vitro under conventional and protracted schedules and the ocular and systemic toxicity of repeated intravitreal injections in rabbits. Methods: Two retinoblastoma (Y79, WERI-RB1) and three endothelial cell types (HMEC, HUVEC, EPC) were exposed to increasing concentrations of digoxin after a single (1 day) or protracted (7 days) treatment fashion. Cytotoxicity was assessed with a vital dye and induction of apoptosis and the cell-cycle status were evaluated by flow cytometry. A cohort of 4 New Zealand rabbits (1.82.2 kg) received 4 bi-weekly doses of 1μg of intra-vitreal digoxin and the same volume (0.1 ml) of vehicle into the fellow eye. Animal controls included clinical, hematological and ocular evaluations (fundoscopy and electroretinograms). After 2 weeks of the last dose rabbits were euthanized and samples were obtained for retinal histology. Results: Digoxin was cytotoxic in retinoblastoma and endothelial cells after a single exposure. Single and protracted treatments of all five cell types did not show a significant difference in terms of the IC50 (p>0.05). Both treatment schedules with digoxin at the IC50 induced apoptosis and cell cycle arrest at G0. Retinal toxicity was ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology evident after the third intravitreal dose of digoxin with statistical changes of the ERG components against the control eye and considerable but local histologic damage of the retinas. Conclusions: Digoxin showed cytotoxic effects in retinoblastoma cell lines while exerting an antiangiogenic activity in vitro at similar concentrations. A protracted treatment was assessed in retinoblastoma cells without an advantage in terms of the dose for antitumor effect with respect to single dose. Despite promising antitumor and antiangiogenic activity in vitro four bi-weekly injections of digoxin lead to significant but local toxicity to the retina of rabbits. Thus, a counterbalance between efficacy and toxicity should be taken into account if translated into the clinics for retinoblastoma treatment. Commercial Relationships: URSULA A. WINTER, None; Emiliano Buitrago, None; Hebe Mena, None; Soledad Negrotto, None; Maria J. del Sole, None; Hakim Djaballah, None; Juan O Croxatto, None; Guillermo L. Chantada, None; David H. Abramson, None; Paula Schaiquevich, None Support: This work was supported by 2013 ARVO/Merck Collaborative Research Fellowship; Agencia Nacional de Promoción Científica-FONCYT (PICT Bicentenario, 2010-2271); Fund for Ophthalmic Knowledge (GLC, ACF), New York, NY, USA and Fundación Natalie D Flexer de Ayuda al Niño con Cáncer (GLC and ACF), Buenos Aires, Argentina. Program Number: 85 Poster Board Number: A0117 Presentation Time: 8:30 AM–10:15 AM Orbital Retinoblastoma: Enucleation vs. Ophthalmic Artery Chemosurgery for Advanced Intraocular Retinoblastoma Nicolas A. Yannuzzi1, Jasmine H. Francis1, Brian P. Marr1, Irina Belinsky1, Ira Dunkel1, Y. Pierre Gobin2, David H. Abramson1. 1 Ophthalmology, Memorial Sloan-Kettering Cancer Center, New York, NY; 2Interventional Radiology, Weill Cornell Medical College, New York, NY. Purpose: To determine the incidence of orbital recurrence following enucleation and ophthalmic artery chemosurgery (OAC) as primary treatments for advanced stage retinoblastoma. Methods: Single center retrospective study of 73 patients (74 eyes) of Reese-Ellsworth group V, or International Classification of Retinoblastoma group D or E primarily treated with enucleation and 76 patients (90 eyes) primarily treated with OAC. Endpoints analyzed were the development of orbital disease, incidence of metastasis, and death from metastatic retinoblastoma. Results: There were 6 orbital recurrences (incidence 8.1%) in the in the primary enucleation group and 1 orbital recurrence (incidence 1.1%) in the primary OAC group during median follow up times of 32.2 months (range 0.1-97.1) and 36.8 months (range 3.0-104.3) respectively. The 24-month Kaplan Meier estimate for orbital recurrence free survival was significantly worse for the enucleation group 92.6% (95% C.I. 83.2-96.8) than for the OAC group 98.5% (95% C.I. 89.9-99.7), Log-Rank p-value = 0.02. The enucleation group had 6 cases of metastatic disease and 2 deaths representing 8.2% and 2.7% of patients respectively. In the OAC group, there were 3 (3.9%) cases of metastatic disease and 0 deaths. Kaplan Meier analysis of metastasis free survival and overall survival yielded no statistically significant differences between the two treatment groups. Analysis of a large number of features of the two groups suggested no difference except more rubeotic eyes in the enucleated group (24%) than in the OAC group (6%). Conclusions: In this single institution retrospective study of advanced intraocular retinoblastoma there was significantly more orbital retinoblastoma in the primarily enucleated group. OAC for advanced intraocular retinoblastoma does not increase the chance of orbital recurrence or metastatic disease compared to primary enucleation. Commercial Relationships: Nicolas A. Yannuzzi, None; Jasmine H. Francis, None; Brian P. Marr, None; Irina Belinsky, None; Ira Dunkel, None; Y. Pierre Gobin, None; David H. Abramson, None Program Number: 86 Poster Board Number: A0118 Presentation Time: 8:30 AM–10:15 AM Rates and Features of Neovascularization Associated with Successful Intravenous Chemotherapy Treatment of Retinoblastoma Kai B. Kang, Michael Shapiro, Michael P. Blair, Felix Y. Chau. Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, Chicago, IL. Purpose: Retinoblastoma (RB) treatments include enucleation, radiotherapy, cryotherapy, laser photocoagulation and chemotherapy. Certain features of RB may be safely monitored without further treatment. The purpose of this study is to evaluate rates and describe features of retinal neovascularization associated with successful intravenous chemotherapy and focal consolidation of tumors with laser or cryotherapy. Methods: Retrospective review of digital fundus images and medical records of RB patients who presented to the University of Illinois at Chicago, Illinois Eye and Ear Infirmary Retina Clinic from November 1, 2004 to November 1, 2014. Only surviving eyes that received treatment for RB with a minimum length of follow up of one year were included. Results: 50 eyes of 34 patients received treatment over the study period. 18 patients had unilateral RB, and 16 had bilateral RB. The mean age at diagnosis was 16 months. Of the 50 eyes, 22 were enucleated upon diagnosis. Most (86%) of the enucleated eyes were classified as International Classification of Retinoblastoma (ICRB) Group E. Twenty-five eyes received intravenous chemotherapy consisting of six cycles of carboplatin, etoposide and vincristine as the primary treatment. Twenty-two (88%) eyes (with 2 in ICRB group A, 6 in Group B, 10 in Group C and 4 in Group D) showed no tumor progression over the study period. Neovascularization was identified in 3 eyes (14% of 22 eyes) in 3 patients. These eyes were monitored closely without further treatment. The three eyes showed no tumor growth over respective periods of 9.1 years, 3.2 years, and 1.2 years. All 3 were associated with calcified regression, and 2 with preretinal fibrosis and subretinal fluid (one with inferior detachment successfully treated with scleral buckle). Conclusions: The rate of retinal neovascularization in eyes with successful intravenous chemotherapy was approximately 14%. Neovascularization associated with retinoblastoma may be safely monitored without the need for further treatment if there are no signs of tumor progression. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology A) Fundus photos illustrating regressed tumor with calcified remnants after chemotherapy and laser with neovascularization and fibrosis from 2005 to 2009 Purpose: Melanin is an abundant endogenous chromophore in the iris, ciliary body and choroid of the eye, altered in many diseases such as macular degeneration and eye tumors. Here we attempt to evaluate melanin distribution in intact eyes using a novel photoacoustic imaging tool. Methods: Following St. Michael’s Hospital Research Ethics Board approval, we imaged whole eyes by photoacoustic imaging (MSOT inVision128, iThera Medical Inc., Germany), constructed with a Nd:YAG laser (1064/532 nm) with 8 ns pulse length and 10 Hz pulse frequency coupled to optical parameter oscillator. Five minipig eyes and three human eyes (Human Eye Biobank for Research) fixed in 10% formalin were embedded in 1.5% agar and placed in a clear plastic bag with distilled water in the imaging chamber. Images were taken in 300mm steps with wavelengths from 680-980 nm in 5 nm steps, and 4 averages per scan. Spectral unmixing was performed using a linear regression algorithm with spectra for deoxygenated and oxygenated hemoglobin and for melanin (Fig. 1A), providing images of entire eye sections. Known concentrations of synthetic melanin in 1.5% agar were used to determine the relationship between concentration of melanin and signal intensity. Results: In all human eyes, melanin was detected by photoacoustic imaging in the iris, ciliary body and choroid (Fig. 2A, melanin in green). In all Yucatan minipig eyes, in addition to the iris, ciliary body, and choroid, melanin was observed in optic nerve head (Fig. 2B, melanin in green). Histological examination of the eyes was used to confirm the location of melanin rich eye tissues. A linear relationship was found between the concentration of synthetic melanin and photoacoustic signal intensity (R2 = 0.99, Fig. 1B). Conclusions: Melanin distribution was reliably detected by photoacoustic imaging in whole intact human and minipig eyes. The linear relation between melanin and photoacoustic signal intensity supports the potential use for melanin quantification in eye specimens. Photoacoustic imaging may be useful for qualitative and quantitative assessment of the enucleated eye with uveal melanoma, including extraocular extension, and other eye diseases. B) Fundus photographs of the same eye illustrating continued neovascularization with fibrosis and adjacent calcified remnants without further tumor growth from 2011 to 2014 Commercial Relationships: Kai B. Kang, None; Michael Shapiro, None; Michael P. Blair, None; Felix Y. Chau, None 149 New challenges in anatomy Sunday, May 03, 2015 3:15 PM–5:00 PM 1AB Mile High Blrm Paper Session Program #/Board # Range: 896–901 Organizing Section: Anatomy and Pathology/Oncology Figure 1: Absorption spectra of chromophores used in spectral unmixing (A) and photoacoustic signal at known concentrations of synthetic melanin (B). Program Number: 896 Presentation Time: 3:15 PM–3:30 PM Melanin Distribution in Intact Human and Minipig Eyes Detected by Photoacoustic Imaging Shireen Khattak1, Neeru Gupta1, 2, Clinton Hupple3, Yeni H. Yucel1, 2 1 . Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, ON, Canada; 2Ophthalmology & Vision Sciences, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; 3iThera Medical GmbH, Munich, Germany. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology state in aniridic eyes due to elevated Wnt signaling, which may lead to AFS following surgery. Commercial Relationships: Yichen Wang, None; Yan Wang, None; Christopher Riemann, None; Melinda K. Duncan, None Support: NIH Grant EY015279 Figure 2: Photoacoustic images of human eye (A) and minipig eye (B) with melanin signal in green. Commercial Relationships: Shireen Khattak, None; Neeru Gupta, None; Clinton Hupple, iThera Medical (E); Yeni H. Yucel, None Support: Canadian Foundation for Innovation, Dorothy Pitts Fund, Nancy and Thor Eaton Fund, Henry Farrugia Fund Program Number: 897 Presentation Time: 3:30 PM–3:45 PM Molecular Mechanisms of Aniridia Fibrosis Syndrome (AFS) Yichen Wang1, Yan Wang1, Christopher Riemann2, Melinda K. Duncan1. 1Biological Sciences, University of Delaware, Newark, DE; 2 Cincinnati Eye Institute, Cincinnati, OH. Purpose: Congenital aniridia (CI) is defined as iris hypoplasia at birth, and often results from PAX6 mutations/deletions. Surgical interventions for common CI sequala such as cataract, glaucoma, and keratopathy may be complicated by AFS which can lead to devastating fibrotic complications. While little is known about the pathogenesis of AFS, previous studies showed that Pax6 negatively regulates Wnt signaling, the chronic upregulation of which can drive fibrosis. Thus, this work tests the hypothesis that haploinsufficiency of Pax6 leads to the upregulation of Wnt signaling, which may contribute to a pro-fibrotic state, resulting in AFS following surgeries. Methods: Immunofluorescence (IF) staining was used to characterize human AFS samples by detecting the expression of fibrotic markers. Penetrating central corneal wounding was performed in Pax6+/tm1Pgr and wildtype (WT) mice to study fibrotic responses at day 5/9 postsurgery, followed by IF staining to detect the expression of fibrotic markers. To study the signaling pathways, the activation state of Wnt and TGF-β signaling was measured by the levels of β-catenin and pSMAD3 respectively. Results: In human AFS samples, abundant cells expressing myofibroblast markers were found, confirming that AFS is a classic fibrotic disease. Further, both β-catenin and pSMAD3 was detected, indicating the activation of Wnt and TGF-β signaling. In unoperated Pax6+/tm1Pgr mice, elevated levels of β-catenin were observed in pockets of spontaneous fibrosis, suggesting the chronic elevation of Wnt signaling. After surgery, Pax6+/tm1Pgr mice displayed severe fibrosis at the injury site. They also developed distal fibrosis at the iris root, which was absent in WT mice. Higher levels of β-catenin and pSMAD3 were found in Pax6+/tm1Pgr mice at both the injury site and iris root compared to WT. Conclusions: AFS is a classic fibrotic disease. Chronic upregulation of Wnt signaling was confirmed in unoperated Pax6+/tm1Pgr mice. Compared to WT, Pax6 mutant mice exhibited increased fibrosis along with higher levels of β-catenin and pSMAD3 post-surgery. These data suggest that Pax6 haploinsufficiency leads to a pro-fibrotic Program Number: 898 Presentation Time: 3:45 PM–4:00 PM Analysis of the volumetric relationship among human ocular, orbital and visual cortical anatomy Michael P. Masters1, Emiliano Bruner2, Sarah Queer1, Sarah Traynor3, Jess Senjem3. 1Montana Tech, Butte, MT; 2Centro Nacional de Investigación sobre la Evolución Humana, Burgos, Spain; 3 University of Wisconsin, Madison, WI. Purpose: Recent research on the visual system has examined the volumetric relationship among the eye, orbit, and visual cortex in humans. Some studies have also suggested that light levels may drive eye size, which was hypothesized to in turn dictate orbital and visual cortical size, as a product of adapting or acclimating to differences in available daylight at disparate latitudes. However, further research is necessary in order to establish how these variables are related, and to what extent ocular volume influences orbital and visual cortical volume in humans. Methods: Relationships among these anatomical components were investigated using MRIs from a large sample of 83 individuals, which also included each subject’s height, age, sex, and uncorrected visual acuity scores. Frontal and occipital gyri volumes were calculated using two different cortical parcellation tools, Brain Parser 56 ROI and FreeSurfer 5.3.0, in order to provide a better understanding of how the eye and orbit vary in relation to the visual cortex, and in association with cerebral gyri of the frontal cortex that are not directly related to vision. Results: Results indicated that ocular and orbital volumes were weakly correlated, but that eye volume explains only 14.7% of the variance in orbital volume. Ocular and orbital volumes were also found to be equally, and in most cases, more highly correlated with five frontal lobe gyri than with occipital lobe gyri associated with V1, V2, and V3 of the visual cortex. Additionally, after accounting for age, sex, visual acuity, and body size differences, the relationships between eye and visual cortical volumes were no longer statistically significant for all variables using Brain Parser 56 ROI, and for all except the lingual gyrus using FreeSurfer 5.3.0. The relationship between orbital and visual cortical volumes remained significant for a number of occipital lobe gyri even after accounting for these cofactors, however it was again found to be equally, and often more highly correlated with the frontal lobe gyri than with occipital lobe gyri involved in visual processing. Conclusions: These results indicate that eye volume explains only a small amount of variation in orbital and visual cortical volume, and that the eye and orbit are generally more structurally associated with the frontal lobes than they are functionally associated with the visual cortex of the occipital lobes. Commercial Relationships: Michael P. Masters, None; Emiliano Bruner, None; Sarah Queer, None; Sarah Traynor, None; Jess Senjem, None Support: Montana INBRE - National Institute of General Medical Sciences of the National Institutes of Health, award number 8 P20 GM103474-12 ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 899 Presentation Time: 4:00 PM–4:15 PM Cell−ECM interactions during formation of the zebrafish hyaloid vasculature Andrea Hartsock, Victoria Arnold, Jeffrey Gross. Biological Sciences, University of Texas, Austin, Austin, TX. Purpose: Vasculature formation requires an orchestrated series of morphogenetic changes to generate an integrated vessel system. Previous work by our laboratory demonstrated there are three stages of hyaloid development in zebrafish: Stage I- arrival of hyaloid cells at the lens and formation of the hyaloid loop, Stage II- formation of a branched hyaloid network, and Stage III- refinement of the hyaloid network (Hartsock et al., 2014). The lens is not required for recruitment of hyaloid precursor cells, but is required for Stage II and III development and maturation. The lens is surrounded by the lens capsule, an ECM-rich basement membrane. It is not known how the ECM of the lens capsule contributes to hyaloid formation. Here, we test the hypothesis that distinct cell-ECM interactions play an integral role in building the hyaloid. Methods: Fixed sample and in vivo time lapse imaging of fli1a:GFP (a hyaloid marker) were performed in a variety of zebrafish lines possessing mutations in ECM components or their cellular interaction partners: lamining1 (lamc1), fibronectin1b (fn1b), integrin a5 (itga5), and integrin b1 (itgb1). All images were processed as maximum projections via ImageJ. Results: in vivo time-lapse imaging revealed that mutations in all cell-ECM components resulted in Stage II and III hyaloid defects. Unique defects were identified in each mutant. For example, Stage II hyaloid network branching was disrupted in lamc1 and fn1b mutants; fn1b mutants possessed clumps of vascular precursor cells on the lens that did not organize into mature vessels. itga5 mutants also possessed Stage II defects, with vessels that were reduced in branch number and thickness. Stage III, refinement of the vascular network appeared normal in itga5 and itgb1 mutants, but not in fn1b or lamc1 mutants. Conclusions: Analysis of hyaloid formation in cell-ECM component mutants revealed requirements for these proteins during hyaloid development. Mutations in ECM components (fn1b, lamc1) were more severe, likely affecting multiple cellular interacting partners. Conversely, hyaloid defects in mutants affecting the interacting partners (itga5, itgb1) were more limited, suggesting specific roles for these in distinct phases of hyaloid development. Further analyses of downstream regulators of these cell-ECM pathways will generate a comprehensive understanding of the cellular underpinnings of hyaloid morphogenesis during embryonic eye development. Commercial Relationships: Andrea Hartsock, None; Victoria Arnold, None; Jeffrey Gross, None Support: F32 EY023910 Program Number: 900 Presentation Time: 4:15 PM–4:30 PM Identification of a novel cause of ocular coloboma Sonya Widen1, Prajakta Desai2, Mika Asai-Coakwell2, Matthew Benson2, Curtis French2, Ordan J. Lehmann2, Andrew Waskiewicz1. 1 Biological Sciences, University of Alberta, Edmonton, AB, Canada; 2 Medical Genetics, University of Alberta, Edmonton, AB, Canada. Purpose: Ocular coloboma results from the incomplete fusion of the optic fissure and is a major cause of pediatric vision loss. We investigated a microphthalmia, anophthalmia and coloboma (MAC) cohort to advance understanding of these disorders’ genetic etiology. Methods: Exomic next generation sequencing (NGS) was performed in a large coloboma pedigree and the mutated gene was subsequently screened in 150 MAC DNA samples. Luciferase reporter assays, western blots and in silico ANOLEA modeling were used to investigate the pathogenicity of the identified mutations. Zebrafish morpholino (MO) inhibition was used to investigate gene function together with in situ hybridization (ISH), and analysis of transgenic GFP reporter lines. Results: NGS identified a BMP3 mutation in affected individuals of the MAC pedigree. The identified variant, A470P, alters a residue that is invariant across vertebrates. Four additional variants were identified in the larger cohort (A188D, K345N, S393F, F450Y), all of which were absent from controls. Consistent with these variants being disease causing, western immunoblots and luciferase reporter assays demonstrate significantly altered activity relative to wildtype BMP3. Supporting bmp3’s role in ocular development, antisense MO inhibition induces zebrafish coloboma and lens defects (85%, N=46). Furthermore, we show that bmp3 is expressed in a neural crest subpopulation (periocular mesenchyme, POM) with a critical role in eye development. Although POM migration to the eye in bmp3 morphants is unaffected, analysis of BMPRE:GFP (BMP Responsive Element) transgenics demonstrates retinal BMP signaling is profoundly altered (90%, N=10). Conclusions: We have identified a novel gene involved in MAC disorders, BMP3. While the precise regulation of ocular BMPs is required for optic fissure closure, BMP3’s role in eye development and disease is unstudied. Our research extends the role of BMPs in eye development and implicates BMP signaling in POM in this process. These data are compatible with a model in which POM bmp3 regulates retinal BMP signaling, identifying a mechanism for bmp3 in eye development and MAC disorders. Commercial Relationships: Sonya Widen, None; Prajakta Desai, None; Mika Asai-Coakwell, None; Matthew Benson, None; Curtis French, None; Ordan J. Lehmann, None; Andrew Waskiewicz, None Program Number: 901 Presentation Time: 4:30 PM–4:45 PM Hypothermic treatment induces the expression of cold sensing proteins CIRP and RBM3 in the retina, both in vitro and in vivo Alfredo Martinez1, Manuel Rey-Funes2, Daniela S. Contartese2, Verónica B. Dorfman3, Federico Rolón2, Anibal Sarotto2, Fabián Loidl2, 4, Ignacio Larrayoz1. 1Oncology, CIBIR, Logroño, Spain; 2 Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis”, University of Buenos Aires, Buenos Aires, Argentina; 3Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico (CEBBAD), Universidad Maimónides, Buenos Aires, Argentina; 4 Facultad de Medicina, Universidad Católica de Cuyo, San Juan, Argentina. Purpose: Hypothermia has been described as a very effective intervention to prevent or treat brain and retinal damage. Under cold conditions, there is a general reduction in protein expression, but there are 2 proteins whose expression is upregulated by hypothermia: CIRP and RBM3. Both are RNA-binding proteins that nowadays are considered cold sensors, thus providing a molecular mechanism of action for the advantages of hypothermia. These proteins have never been characterized in the retina and here we offer a preliminary description of their behaviour in this organ, both in vitro and in vivo. Methods: Retinal cell lines R28 (rat neural retina) and mRPE (monkey retinal pigment epithelium) were exposed to different temperatures in a time-dependent manner and the expression of RBM3 and CIRP was measured through quantitative real time PCR (qRT-PCR) and Western blotting. In addition, Sprague-Dawley albino rats of different ages (newborns and adults) were exposed to a cold environment (8 0C) for different periods of time. Retinas were either snap frozen for molecular analysis (qRT-PCR and ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Western blotting) or fixed in paraformaldehyde for histological and immunohistochemical analysis with specific antibodies against CIRP and RBM3. Data were analyzed with ANOVA tests. Results: In both cell lines, there was a time-dependent significant increase of RBM3 and CIRP expression after exposure to a cold environment. Maximum expression was reached after 6 h at 32 0C. Immunoreactivity (IR) for RBM3 and CIRP was negligible in retinas not exposed to hypothermia. On the other hand, retinas of both neonate and adult rats that had been exposed to hypothermia presented high levels of IR for both proteins with different colocalization patterns (Fig. 1). Conclusions: As happens in the central nervous system, cells in the retina can sense cold exposure through elevation of RBM3 and CIRP expression. These proteins are expressed in several cell types and can be responsible for the beneficial effects of hypothermia through the binding of specific molecules of mRNA. Figure 1. Retinas of newborn rats that were exposed to room temperature (CTL) or to hypothermia (HYP), stained with specific antibodies against RBM3 (green) and CIRP (red). Nuclear contrast was accomplished with DAPI (blue). Commercial Relationships: Alfredo Martinez, None; Manuel Rey-Funes, None; Daniela S. Contartese, None; Verónica B. Dorfman, None; Federico Rolón, None; Anibal Sarotto, None; Fabián Loidl, None; Ignacio Larrayoz, None Support: U.S. Department of Defense, Vision Research ProgramHypothesis Development Award (MR130239). 204 Tumors - Inside and around the eye, I Monday, May 04, 2015 8:30 AM–10:15 AM 1AB Mile High Blrm Paper Session Program #/Board # Range: 1287–1293 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Anatomy/Pathology Program Number: 1287 Presentation Time: 8:30 AM–8:45 AM Mitogen-activated protein kinase (MAPK) inhibitors in conjunctival melanoma cell lines Jinfeng Cao1, Robert M. Verdijk2, Aart G. Jochemsen3, Martine J. Jager1. 1Ophthalmology, Leiden University Medical Center, Leiden, Netherlands; 2Pathology, Erasmus Medical Center, Rotterdam, Netherlands; 3Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands. Purpose: Current therapies of conjunctival melanoma (CM) mainly focus on radical excision of the tumor, and are frequently supplemented with cryotherapy or local therapy. As activating mutations of BRAF and NRAS have been discovered in 47% of CMs, we studied the antitumor effect of mitogen-activated protein kinase (MAPK) inhibitors in CM cell lines. Methods: Sanger sequencing was used to detect the mutation status of three human CM cell lines (CRMM1, CRMM2 and CM2005.1). The MEK inhibitor MEK162 (ARRY-162) and BRAF inhibitors Vemurafenib (PLX4032) and Dabrafenib (GSK2118436) were chosen to treat CM cells. Cell viability was determined with WST-1 and in-cell western assay. The phosphorylation status of ERK protein was evaluated using western blot. Results: The BRAF T1799A mutation was identified in CRMM1 and CM2005.1, and the NRAS A182T mutation in CRMM2. The growth of all three CM cell lines was inhibited by MEK162 with a low concentration (IC50 < 45 nM). CRMM1 and CM2005.1 were sensitive to Vemurafenib (IC50 < 200 nM) and Dabrafenib (IC50 < 20 nM) in a dose-dependent manner, while CRMM2 was not. Consistent with the result of cell viability, a concentration-dependent decrease of ERK phosphorylation was detected in all three CM cell lines after MEK162 treatment, and in CRMM1 and CM2005.1 after Vemurafenib and Dabrafenib treatment. Paradoxically, the phosphorylated ERK of CRMM2 was activated by both BRAF inhibitors. Conclusions: Growth inhibition of CM cell lines can be achieved by the appropriate MEK or BRAF inhibitors. Our findings demonstrate that MAPK inhibitors might be potential therapeutic drugs against conjunctival melanoma and its metastasis. Commercial Relationships: Jinfeng Cao, None; Robert M. Verdijk, None; Aart G. Jochemsen, None; Martine J. Jager, None Support: China Scholarship Council Program Number: 1288 Presentation Time: 8:45 AM–9:00 AM Role of L1 Cell Adhesion Molecule in Adhesion-Mediated Proliferation and Chemoresistance of Retinoblastoma Dong Hyun Jo1, 2, Jin Hyoung Kim1, 4, Young Hoon Kim3, YoungLai Cho5, Young Suk Yu6, Jeong-Ki Min7, Jeong Hun Kim1, 2. 1Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea (the Republic of); 2Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Korea (the Republic of); 3Department of Pathology, College of Medicine, Seoul National University, Seoul, Korea (the Republic of); 4Tumor Microenvironment Research Center, Global Core Research Center, Seoul National University, Seoul, Korea (the Republic of); 5Center for Nanosafety Metrology, Korea Research Institute of Standards and Science, Daejeon, Korea (the Republic of); 6 Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Korea (the Republic of); 7Research Center for Integrated Cellulomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea (the Republic of). Purpose: Although retinoblastoma is the most common intraocular malignancy in children, there have been limited studies on which drives distinct proliferation patterns and responses to chemotherapy in retinoblastoma. In this study, we investigated the role of L1 cell adhesion molecule (L1CAM) in adhesion-mediated tumor behavior and chemoresistance of retinoblastoma. Methods: We evaluated the expression of L1CAM in retinoblastoma tissues from 30 patients by immunohistochemistry and in cell lines by Western blotting. To study the role of L1CAM in proliferation and chemoresistance in retinoblastoma, we utilized two retinoblastoma cell lines, Y79 and SNUOT-Rb1, with knockdown and overexpression of L1CAM, respectively. Then, the effects of L1CAM expression on proliferation, cell-cell adhesion, and chemoresistance of retinoblastoma cells were investigated. We injected naïve and stable cell lines into the vitreous cavity of BALB/c nude mice (n = ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology 6) to demonstrate the relation between L1CAM and in vivo tumor formation. Results: We observed varying degrees of L1CAM positivity in 86.6% (26/30) of retinoblastoma tissues. Interestingly, there was an inverse relation between the degree of Flexner-Wintersteiner rosette formation and that of L1CAM positivity. In vitro studies using stable cell lines demonstrated that L1CAM was associated with cell-cell adhesion and further adhesion-mediated proliferation of retinoblastoma cells. In addition, L1CAM was related with chemoresistance to carboplatin, one of the most widely utilized drug in the treatment of retinoblastoma. In line with in vitro results, in vivo tumor formation in rodent eyes was also more prominent with cell lines with higher expression of L1CAM. Conclusions: Our results show that L1CAM is expressed in retinoblastoma and plays an important role in adhesion-mediated proliferation and chemoresistance of tumor cells. We suggest that profound understanding of the roles of L1CAM may open up another therapeutic approach against retinoblastoma. Representative photographs of L1CAM expression in retinoblastoma tissues. Scale bar, 20 μm. Expression of L1CAM protein among different cells. HRMEC, human retinal microvascular endothelial cells; Rb1, SNUOT-Rb1 cells. Commercial Relationships: Dong Hyun Jo, None; Jin Hyoung Kim, None; Young Hoon Kim, None; Young-Lai Cho, None; Young Suk Yu, None; Jeong-Ki Min, None; Jeong Hun Kim, None Program Number: 1289 Presentation Time: 9:00 AM–9:15 AM Diagnosis of Vitreoretinal Lymphoma: The Use of Cytology, Gene Rearrangement, and IL-10/IL-6 Ratio Jacob Pe’er1, Shahar Frenkel1, Inna Kalickman2, Yoav Sherman3, Bela Maly3, Dina Ben Yehuda4, and Vivian Barak2 Departments of 1Ophthalmology, 2Oncology, 3Pathology, and 4 Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel . Jacob Pe’er. Ophthalmology, Hadassah-Hebrew Univ Med Ctr, Jerusalem, Israel. Purpose: Purpose: To compare three methods of diagnosis of suspected vitreoretinal lymphoma: cytology, IgH gene rearrangement, and IL-10/IL-6 ratio. . Methods: Methods: Diluted vitreous fluid obtained in diagnostic vitrectomy from patients with suspected vitreoretinal lymphoma were delivered immediately after the surgery to the cytopathology laboratory; to the hematology laboratory for PCR studies, and to the cancer-markers laboratory for ELISA test for IL-10/IL-6 ratio. . Results: Results: Forty-seven specimens were evaluated using the three methods: 21 of them (44.7%) were positive for lymphoma in cytology examination, 13 (27.6%) were positive in Ig-H gene rearrangement evaluation, and 25 (53.2%) were positive by IL10/IL-6 ratio that was higher than 1.0. Nine of 21 specimens (42.8%) with positive cytology were positive also for Ig-H gene rearrangement and 18 of the 21 (85.7%) were positive also for IL-10/ IL-6 ratio. Nine specimens were positive for lymphoma via all three methods. Seven specimens in which the IL-10/IL-6 ratio was positive for lymphoma were negative in cytology, 4 of them developed CNS lymphoma, and 3 that were positive for Ig-H gene rearrangement were negative in cytology. Conclusions: Conclusions: In our series, the examination of the IL10/IL-6 ratio was much more sensitive and much more comparable to cytology than examination for Ig-H gene rearrangement which showed low sensitivity. Commercial Relationships: Jacob Pe’er, None Program Number: 1290 Presentation Time: 9:15 AM–9:30 AM The Classification of Vitreous Seeds in Retinoblastoma: Response to Intravitreal Melphalan Jasmine H. Francis1, 2, David H. Abramson1, 2, Marie-Claire Gaillard3, Brian P. Marr1, 2, Maya Beck-Popovic4, Francis L. Munier3. 1 Ophthalmic Oncology, Memorial Sloan Kettering Cancer Center, New York, NY; 2Weill-Cornel Medical Center, New York, NY; 3JulesGonin Eye Hospital, Lausanne, Switzerland; 4University Hospital CHUV, Lausanne, Switzerland. Purpose: Vitreous seeds in retinoblastoma have clinical heterogeneity and we have previously proposed a classification to distinguish between them. This study evaluates the clinical characteristics of the three vitreous seed classes: dust (class 1), spheres (class 2) and clouds (class 3) and their responses to intravitreal melphalan. Methods: Bi-institutional cohort study of 87 patient eyes that received 475 intravitreal injections of melphalan (median dose 30mg) given weekly, a median of 5 times (range 1-12). At presentation, the vitreous seeds were classified into three groups: dust, spheres and clouds. Indirect ophthalmoscopy, fundus photography, ultrasonography and ultrasonic biomicroscopy were used to evaluate ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology clinical response to weekly intravitreal melphalan injections and time to regression of vitreous seeds. Kaplan-Meier estimates of time to regression, ocular, patient and event-free survival were calculated and Log-rank test of curve comparison. Results: There was a significant difference in time to regression for the three seed classes (p<0.0001): the median time to regression was 0.6 months, 1.7 months and 7.7 months for dust, spheres and clouds, respectively. Dust received significantly fewer injections, a lower median and cumulative dose of melphalan; while clouds received significantly more injections, a higher median and cumulative dose of melphalan. Kaplan-Meier estimates for two-year ocular, patient, and event-free survival (related to target seeds) was not significantly different between the three seed groups: overall, they were 90.4% (95% confidence interval (CI) 79.7-95.6), 100%, 98.5% (95%CI 9099.7), respectively. Conclusions: The three vitreous seed classes have a significantly different time to regression in response to intravitreal melphalan. The vitreous seed classification can help to predict time to regression, number, median dose and cumulative dose of intravitreal melphalan injections required. Commercial Relationships: Jasmine H. Francis, None; David H. Abramson, None; Marie-Claire Gaillard, None; Brian P. Marr, None; Maya Beck-Popovic, None; Francis L. Munier, None Support: The Fund for Ophthalmic Knowledge and Perry’s Promise Fund Program Number: 1291 Presentation Time: 9:30 AM–9:45 AM Evaluating the in vivo efficacy of a novel first in class drug for the treatment of primary uveal melanoma Patrick T. Logan, Sultan Aldrees, Mohammed F. Qutub, Natalia Vila, Vasco Bravo-Filho, Miguel N. Burnier. Henry C Witelson Lab, Ocular Pathology, McGill University, Montreal, QC, Canada. Purpose: The standard for treating uveal melanoma is plaque radiotherapy; however, tumors that are too large or close to the optic nerve cannot be treated using this method. Moreover, as radiotherapy is a non-specific treatment, it can cause serious side-effects, such as retinopathy, cataract, and glaucoma. Thus, there is a need for a specific uveal melanoma treatment with limited side-effects. Herein, we tested the efficacy of a nanoparticle with a conjugated photoactive dye (collectively, AU-011) in a uveal melanoma rabbit model. Methods: One million 92.1 uveal melanoma cells were injected into the suprachoroidal space of twenty albino New Zealand rabbits. Tumor growth was assessed weekly by ultrasound and fundoscopy. After the presence of a tumor was confirmed clinically, treatment commenced. Treatment consisted of intravitreal injection of AU-011; 20 hours after injection, tumors were lasered using an ophthalmic laser (690 nm, 50 J/cm2) to activate the photoactive dye. This treatment was repeated weekly for 3 weeks, and animals were sacrificed 1 week after the last treatment. Post-mortem, tumors were examined on gross pathology and by histopathology. Results: Twelve of the 20 animals developed intraocular tumors. Two animals with tumors died unexpectedly due to cyclosporine complications and did not receive treatment; these animals acted as controls. For the other 10 treated animals, a noticeable tumor response was observed, which was characterized by three elements: 1) induction of extensive tumor necrosis; 2) change in the growth pattern (“sleeve-like pattern”); and 3) sparing of the adjacent retina. On ultrasound, tumor growth cessation or shrinkage was generally observed. In 4 of these treated animals, histopathology revealed that no tumor was present (complete response); instead, the intraocular masses noted by ultrasound and fundus were comprised of only inflammatory cells and fibrosis. Histopathology of all treated animals showed widespread necrosis (>80% in most cases) that was not seen in controls or in previous animal models (<20% necrosis). Conclusions: In this pilot study, we show that AU-011 is efficacious for treating uveal melanoma tumors in an orthotopic xenograft rabbit model. In addition to the widespread necrosis noted in treated tumors, several animals experienced complete responses. Future studies investigating different AU-011 dosing regimens are currently underway. Commercial Relationships: Patrick T. Logan, Aura Biosciences (C); Sultan Aldrees, None; Mohammed F. Qutub, None; Natalia Vila, None; Vasco Bravo-Filho, None; Miguel N. Burnier, Aura Biosciences (C) Program Number: 1292 Presentation Time: 9:45 AM–10:00 AM Intravitreal Aflibercept as Rescue Therapy for Post-Radiation Cystoid Macular Edema Mohammed A. Khan1, 2, Arman Mashayekhi1, Jerry A. Shields1, Carol L. Shields1. 1Ocular Oncology Service, Wills Eye Hospital, Philadelphia, PA; 2Retina Service, Wills Eye Hospital, Philadelphia, PA. Purpose: To investigate the safety and efficacy of intravitreal aflibercept as rescue therapy for persistent post-radiation cystoid macular edema (CME) following prior treatment with intravitreal bevacizumab. Methods: Retrospective, observational, consecutive case series of patients who received intravitreal aflibercept (2mg/0.05mL) for persistent post-radiation CME by the Oncology Service, Wills Eye Hospital (Philadelphia, PA). Primary outcomes were change in central macular thickness (CMT) by optical coherence tomography (OCT) and visual acuity after initiation of intravitreal aflibercept therapy. Results: Five eyes of five patients with persistent CME following plaque radiotherapy for choroidal malignant melanoma were included. Mean tumor basal diameter was 10.4 mm (range 6 - 16 mm) and mean tumor thickness of 3.56 mm (range 2.2 – 4.7 mm). Mean radiation dose to the macula was 6158 cGy (mean rate to macula 65.5 cGy/hour). OCT evident CME occurred at a mean of 22.8 months (range 8 – 43 months) post plaque radiotherapy. All patients were prior treated with intravitreal bevacizumab (mean 11.6 injections per patient, range 6 – 22 injections). Following treatment with three monthly doses of intravitreal aflibercept, logMar visual acuity improved from mean 0.47 (standard deviation 0.08, Snellen equivalent 20/60) to mean 0.30 (standard deviation 0.24, Snellen equivalent 20/40) (p=0.19). CMT reduced significantly from mean 478.8 microns (standard deviation 123.3) to mean 292.4 microns (standard deviation 46.7)(p=0.01). No patient experienced worsening of CMT or visual acuity while treated with intravitreal aflibercept. No alternative therapies were necessary. No patient experienced an adverse event. At mean final follow-up of 4.2 months (range 3-8 months), mean CMT and logMar visual acuity remained stable at 294.6 microns and 0.35 (Snellen equivalent 20/44), respectively. Conclusions: Intravitreal aflibercept can be an effective rescue therapy for persistent post-radiation CME in patients with poor response to bevacizumab, with reduction in central macular thickness and improvement in visual acuity. Commercial Relationships: Mohammed A. Khan, None; Arman Mashayekhi, None; Jerry A. Shields, None; Carol L. Shields, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 1293 Presentation Time: 10:00 AM–10:15 AM Second primary cancers in uveal melanoma survivors and the role of radiotherapy: a long-term population-based study Inês Laíns1, 2, Carla Bartosch3, Vera Mondim4, Deeba Husain1, Joan W. Miller1. 1Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA; 2Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 3Instituto Português de Oncologia do Porto, Porto, Portugal; 4Centro Hospitalar de Lisboa Central, Lisboa, Portugal. Purpose: The long term survival in uveal melanoma (UM) patients is about 50% and these patients may be at higher risk of developing second primary cancers (SPC). In other malignancies, the risk of SPC has been linked with radiotherapy (RT). Despite this knowledge, no population-based studies have been done on UM survivors after radiation became standard of care. The aim of this study was to characterize the long-term SPC risk in subjects previously diagnosed with UM and to determine if RT is an independent risk factor. Methods: This is a retrospective cohort study. Using the Surveillance, Epidemiology and End Results (SEER) 9 database from the United States of America, we identified patients with a diagnosis of UM as their first malignancy between 1973 to 2011. The second cancer events were defined as those diagnosed at least two months after the UM. Poisson regression was used to model standardized incidence ratios (SIR) and excess absolute risks (EAR) of SPC, compared with a reference SEER population matched for sex, 5-year age group, race, site and calendar year. Multivariate Cox regression model was used to evaluate the effect of RT in SPC risk. Results: Of the 3,736 UM patients identified, 15.3% developed a SPC during a median follow-up of 139 months (range: 12-463 months). This represented a 10% higher risk compared to the general reference population, mainly due to a significantly increased risk of salivary gland tumors (SIR=4.27, p<0.05, EAR=1.22), skin melanomas (SIR=2.94, p<0.05, EAR=10.18) and kidney tumors (SIR=2.05, p<0.05, EAR=3.67). The occurrence of second UM was also increased (SIR=16.95, p<0.05, EAR=3.26), which may represent a recurrence of melanoma or true second primary malignancies. RT was performed in 38.3% of the patients. The multivariate analysis revealed that this treatment was not an independent risk factor for SPC (HR=1.27, 95%CI: 0.97-1.65, p=0.08), after adjusting for age at diagnosis, sex, race and surgical treatment. Conclusions: Uveal melanoma survivors presented a 10% higher risk of SPC as compared to the general population. RT does not seem to be an independent risk factor for second primary cancers. Commercial Relationships: Inês Laíns, None; Carla Bartosch, None; Vera Mondim, None; Deeba Husain, None; Joan W. Miller, None 229 Tumors - Retinoblastoma Monday, May 04, 2015 11:00 AM–12:45 PM 1AB Mile High Blrm Paper Session Program #/Board # Range: 1660–1665 Organizing Section: Anatomy and Pathology/Oncology Program Number: 1660 Presentation Time: 11:00 AM–11:15 AM Generation of in vitro intraocular tumor models for pre-clinical drug screening using nanotechnology Justin B. Lendermon1, Nabil Saleh1, Anderson H. Webb1, Bradley T. Gao1, Ryan P. Lee1, Matthew W. Wilson1, Jena J. Steinle1, Vanessa M. Morales1, 2. 1Ophthalmology, University of Tennessee Health Science Center, Memphis, TN; 2Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Purpose: Three dimensional (3D) tumor cell cultures more closely resemble tumors in vivo, thus providing a better model for pre-clinical drug screening. This technology, Nano3D (Nano3D Biosciences, Inc., Houston, TX), was recently developed by Souza and colleagues to study glioblastomas. We adopted Souza’s techniques to generate 3D cell cultures of retinoblastoma and uveal melanoma for pre-clinical drug screening. Methods: We used two retinoblastoma cell lines, Y79 and Weri, and three uveal melanoma cell lines, OMM1, 92.1 and Mel 270. First, we determined the minimum number of tumor cells to obtain significant data, maximizing their potential. Cells were co-cultured with nanoshuttle particles, made of iron oxide, to allow the formation of tumor spheroids upon contact with magnet in the dock station with capacity to record, at 37°C. Cells were dissociated and analyzed for cell viability and different surface markers. Then, we generated, in vitro, an extracellular matrix-like microenvironment to analyze tumor migration potential and disruption of extracellular matrix, in a similar fashion to the classical “scratch wound assay”. Third, we optimized tumor cell number and media volume to analyze cell growth by spheroid size. Results: Comparison of tumor growth for functional analyses in flat 2D surfaces versus the 3D nanotechnology system revealed significant differences. First, we found a more homogeneous phenotype in 2D flat surfaces than in the 3D. Second, we observed slight differences in adhesion molecules, specifically in UM, as they are adherent in 2D surfaces and not in the 3D. Conclusions: The use of 3D cell culture is a powerful tool with potential applications for preclinical drug screening in intraocular tumors. Commercial Relationships: Justin B. Lendermon, None; Nabil Saleh, None; Anderson H. Webb, None; Bradley T. Gao, None; Ryan P. Lee, None; Matthew W. Wilson, None; Jena J. Steinle, None; Vanessa M. Morales, None Program Number: 1661 Presentation Time: 11:15 AM–11:30 AM Discovery of novel cell cycle regulatory and signal transduction modules driving Retinoblastoma using a correlative multi-omics approach Arkasubhra Ghosh1, 2, Ashwin C. Mallipatna1, Nilanjan Guha3, Deepak S A3, Syed Lateef3, Vishnu Babu1, Seetaramanjaneyulu Gundimeda3, Arunkumar Padmanabhan3, Rohit Shetty1, 2. 1GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India; 2Singapore Eye Research Institute, Singapore, Singapore; 3 Agilent Technologies, Bangalore, India. Purpose: Applying a multi-omics methodology to molecular analysis of primary intraocular retinoblastoma tumor samples covering global ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology transcriptomic and metabolomics assays for elucidating functional pathways driving this cancer. Methods: All samples were collected after informed written consent and approval of Institutional Ethics Committee. We obtained total RNA from tumors of 9 patients (5 male, 4 female; age range 4-30 months) who underwent enucleation of the affected eyes. Additional tumor, aqueous humor, vitreous humor and tear samples were also collected from the same patients for metabolomics analyses. Control retina, vitreous and aqueous humor was extracted from pediatric donor eyes. Global gene expression and miRNA microarrays were performed simultaneously with the tumor RNA, followed by pathway analysis. Metabolites were extracted using monophasic solvent extraction of aqueous and vitreous humor samples followed by analysis on Accurate Mass QTOF mass spectrometer on reverse phase C18 and HILIC columns. Results: Differential expression analysis carried out using moderated t-test with Benjamini Hochberg multiple testing correction revealed 1404 significantly regulated genes (p≤0.005 and fold change≥10) as compared to normal pediatric retina. Analysis of miRNA arrays revealed 18 previously unreported, deregulated miRNAs (p≤0.005 and fold change≥10). Using these data sets in concert, pathway analysis of the miRNAs and their possible target differentially expressed genes revealed novel networks. The results show that cell cycle, mTOR, PI3-AKT, retinal function and Rap1 signaling modules are most significantly deregulated. We further validated 20 genes selected from these deregulated pathways by quantitative PCR. IHC staining of 33 independent patient tumor cohort further validated E2F and Rap1 up regulation. Analysis of the differentially expressed metabolites in vitreous and aqueous humor revealed enrichment of several additional pathways. Conclusions: The study illustrates an integrated method of discovering new biological insights that are made accessible by correlating data from different large scale techniques in the same patient sample, thereby reducing intra-cohort bias. In particular, E2F, Rap1, CDK and cyclin dependent signaling pathways were up regulated while retinal function and visual cycle related pathways were down regulated in Rb patients. Commercial Relationships: Arkasubhra Ghosh, None; Ashwin C. Mallipatna, None; Nilanjan Guha, None; Deepak S A, None; Syed Lateef, None; Vishnu Babu, None; Seetaramanjaneyulu Gundimeda, None; Arunkumar Padmanabhan, None; Rohit Shetty, None Program Number: 1662 Presentation Time: 11:30 AM–11:45 AM Targeting Notch signaling as a novel therapy for Retinoblastoma Laura Asnaghi1, Arushi Tripathy1, Charles Eberhart1, 2. 1 Neuropathology, Johns Hopkins University, Baltimore, MD; 2 Ophthalmology, Johns Hopkins University, Baltimore, MD. Purpose: Retinoblastoma, a malignant tumor of the retina, is the most common intraocular cancer of childhood. Our goal was to study the role of Notch signaling in promoting growth and proliferation of retinoblastoma cells to determine if inhibiting this pathway might represent a novel approach to treatment. Methods: WERI Rb1 and Y79 retinoblastoma lines were used as model. Expression of Notch pathway components was measured using qPCR and Western blot. Downregulation of the Jag2 ligand was achieved using short hairpin RNA (shRNA). Cell growth, proliferation, and invasion of these cell lines, upon downregulation of Jag2, were determined respectively using the colorimetric CCK8 (Cell Counting kit8) assay, bromodeoxyuridine incorporation and transwell invasion assays. Results: We found that Notch pathway components, including Jag1-2 ligands, Notch receptors and Hes1, Hey1-2 target genes, were expressed at various levels in WERI Rb1 and Y79 retinoblastoma lines. Notch ligand Jag2 RNA and protein were more highly expressed as compared to Jag1 in the retinoblastoma lines. Notch1 receptor was found to be more abundantly expressed than Notch2 and Notch3 receptors in these lines. The cleaved, active form of Notch1 was detected under standard culture conditions, further supporting a potential role for the pathway in retinoblastoma. Because Jag2 was highly expressed, we performed loss-of-function studies using shRNA. Downregulation of Jag2 significantly suppressed cell growth and proliferation by 40 to 50% in both lines, as determined respectively by CCK8 and bromodeoxyuridine assays, and partially reduced invasion, as found by transwell invasion assay. Conclusions: Our findings indicate that Notch pathway components are expressed in WERI Rb1 and Y79 retinoblastoma lines, with the presence of cleaved Notch1 receptor and downstream targets further supporting pathway activity. Jag2 ligand appeared to be highly expressed and promoted retinoblastoma growth. These data suggest that Notch inhibitors represent a new potential therapeutic strategy in retinoblastoma. Commercial Relationships: Laura Asnaghi, None; Arushi Tripathy, None; Charles Eberhart, None Support: Knights Templar Eye Foundation Grant for Career Development to Laura Asnaghi Program Number: 1663 Presentation Time: 11:45 AM–12:00 PM Aqueous seeding: fall of the ultimate intraocular retinoblastoma sanctuary by a new in situ chemotherapy technique Francis L. Munier1, Marie-Claire Gaillard1, Sarah Decembrini1, Maja Beck-Popovic2. 1Ophthalmology Department, Jules-Gonin Eye Hospital, Lausanne, Switzerland; 2Pediatric Hematology Oncology Unit, University Hospital CHUV, Lausanne, Switzerland. Purpose: The presence of primary or secondary aqueous seeding in retinoblastoma (rb) represents a universal indication for enucleation. All previous attempts at conservative treatment have been associated with 100% failure rate. Here we present a novel technique of in situ chemotherapy specifically developped to eradicate aqueous seeding. Methods: Retrospective review of two patients presenting with primary (patient#1) and secondary (patient# 2) aqueous seeding respectively. Combined injections of melphalan were given in the vitreous (200 μg/mL) and in both posterior (PC) and anterior (AC) chambers (15 μg/mL). The intra-cameral procedure consisted of 5 successive steps: 1) long needle passage (34G) across the peripheral clear cornea, 2) aqueous aspiration of of both AC and PC volumes, 3) melphalan injection of 1/3 of the paracentesis volume in AC, 4) transiridal injection into the PC of the last 2/3 and AC retrofilling, 5) triple freeze and thaw cryo-application at the entry site. Per-operative intravenous acetazolamide is administred to suppress aqueous secretion. Results: Patient#1 was diagnosed with unilateral group E anterior diffuse rb at 11 years of age. Following first line intra-arterial chemotherapy, she received 10 intravitreal and 12 intra-cameral injections. Complete regression was achieved with an event-free follow-up of 27 months. The patient is binocular with visual acuity of 20/20 OU. Patient#2 was diagnosed with bilateral rb, group D OD and group E OS at 6 months of age. Following first line systemic chemotherapy and intra-arterial injections in OS, he developed secondary anterior seeding in OD at 25 months of age. He received 4 intravitreal and 5 intra-cameral injections. Complete regression was obtained with an event-free follow-up of 7 months since the last injection. The patient has good fixation without nystagmus. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Conclusions: This is the first study to report globe salvage in case of anterior chamber seeding using a new intra-cameral injection technique. Moreover, both eyes retained normal visual function including binocularity in patient#1. We would like to stress that this approach is safe and efficient provided that 1) careful 35mHz ultrasonic biomicroscopy have excluded invasion of the ciliary body, iris and Schlemm’s canal, 2) the injection is performed in the PC with retofilling of the AC, 3) pharmacologic suppression of aqueous secretion is observed. Commercial Relationships: Francis L. Munier, None; MarieClaire Gaillard, None; Sarah Decembrini, None; Maja BeckPopovic, None Program Number: 1664 Presentation Time: 12:00 PM–12:15 PM Intra-arterial chemotherapy rescue for recurrent or persistent subretinal seeds in retinoblastoma: Tumor control and globe salvage in 30 eyes Prashanth G. Iyer2, Emil A. Say1, Murat Hasanreisoglu1, Sara Lally1, Pascal Jabbour3, Carol L. Shields1. 1Ocular Oncology Service, Wills Eye Hospital, Philadelphia, PA; 2Sydney Kimmel Medical College, Philadelphia, PA; 3Thomas Jefferson University Hospital, Philadelphia, PA. Purpose: To describe the efficacy of intra-arterial chemotherapy (IAC) for control of persistent or recurrent subretinal seeds in retinoblastoma. Methods: In this retrospective non-comparative interventional case series, there were thirty eyes in 29 patients with retinoblastoma who have failed previous treatment with systemic chemotherapy, plaque radiation, or focal therapy and persistent or recurrent subretinal seeds. Superselective ophthalmic artery chemotherapy infusion under fluoroscopic guidance with melphalan (3, 5, or 7.5 mg) and additional topotecan (1 mg) and/or carboplatin (30 mg) was administered as necessary. The main outcome measures were tumor control and globe salvage. Results: The mean patient age was 19 months. Secondary IAC was given to patients with recurrent or persistent subretinal seeds who failed previous treatment regimens (n=30 eyes). Each eye received a mean of 3 IAC per eye (median, 2; range, 1-7). After IAC with a mean follow-up of 14 months, 27 of the 30 eyes (90%) had subretinal seed regression after completion of IAC cycles. Longterm control with global salvage was maintained in 19 eyes (63%), with enucleation being required for recurrent subretinal seeds in 3 eyes (27%) and for recurrent subretinal and vitreous seeds in 3 eyes (27%). Conclusions: IAC is effective as a secondary agent in the management of subretinal seeds in retinoblastoma achieving complete regression of subretinal seeds in 90% of eyes. Commercial Relationships: Prashanth G. Iyer, None; Emil A. Say, None; Murat Hasanreisoglu, None; Sara Lally, None; Pascal Jabbour, None; Carol L. Shields, None Support: Eye Tumor Research Foundation Program Number: 1665 Presentation Time: 12:15 PM–12:30 PM Management of retinal detachment after first-line intra-arterial chemotherapy for advanced retinoblastoma Jelena Potic1, 2, Jean-Antoine C. Pournaras1, Marie-Claire Gaillard1, Thomas Wolfensberger1, Maya Beck-Popovic3, Francis L. Munier1. 1 Jules-Gonin Eye Hospital, Lausanne, Switzerland; 2Univ Eye Hosp, Clinical Center of Serbia, Belgrade, Serbia; 3Paediatric Oncology and Haematology, CHUV, Lausanne, Switzerland. Purpose: First line intra-arterial chemotherapy was proposed recently as a new conservative treatment for retinoblastoma patients. The aim of this retrospective study is to analyze the occurrence of rhegmatogenous retinal detachment in patients with advanced retinoblastoma treated with first line intra-arterial chemotherapy and their surgical outcome. Methods: All consecutive patients with advanced retinoblastoma treated with first line intra-arterial chemotherapy at the Jules-Gonin Eye Hospital (November 2008 to October 2014) were included. Indications for retinal detachment surgery were either new or persistent detachment. Scleral buckle surgery with anterior chamber ponction was performed without drainage of the sub-retinal fluid. Results: 30 eyes from 30 patients responded to the inclusion criteria, including 3 eyes from bilaterally affected patients: Group C (n=1), Group D (n=23), Group D/E (n=2), Group E (n=4). Average age at the first intra-arterial injection was 28.7 months. Cumulative injected dose of Melphalan was 11.44 mg, with a mean number of 2.7 injections. Before treatment, 11 patients (37%) had retinal detachment at presentation, and 19 (63%) patients had none. After chemotherapy, 16/19 (84.2%) patients developed retinal detachment (1 localized, 6 subtotal, 9 total). All the 11 patients presenting retinal detachment at the first examination regressed spontaneously after treatment (100%). Mean time between the intra-arterial chemotherapy and beginning of retinal detachment was 41 days. Average observation period before surgery was 4.1 months. In 10 patients, scleral buckle was indicated, but only 9 underwent surgery. In 1 patient with shallow retinal detachment, it was decided to continue with clinical follow-up only. Regression of retinal detachment was seen in 8/9 cases (89%), with complete reapplication in 6/9 patients (67%). One patient showed no change (11%). Conclusions: First line intra-arterial chemotherapy for advanced retinoblastoma provides good tumor control, but in this series secondary retinal detachment occurred in 16/30 cases (50%). Retinal reapplication was obtained with scleral buckling in more than two thirds of them. The early treatment of this complication is important for salvage of the globe and visual function. Commercial Relationships: Jelena Potic, None; Jean-Antoine C. Pournaras, None; Marie-Claire Gaillard, None; Thomas Wolfensberger, None; Maya Beck-Popovic, None; Francis L. Munier, None 275 Myopia Monday, May 04, 2015 3:45 PM–5:30 PM Exhibit Hall Poster Session Program #/Board # Range: 2148–2182/B0001–B0035 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Clinical/Epidemiologic Research Program Number: 2148 Poster Board Number: B0001 Presentation Time: 3:45 PM–5:30 PM The effect of combination of white and monochromatic light on eye growth of normal chicks Rachel Ka-man Chun1, Danyang Wang1, 2, King Kit Li1, Thomas C Lam1, Quan Liu2, Chi Ho To1, 2. 1Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hong Kong, Hong Kong; 2State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China. Purpose: To examine the effect of combination of white and monochromatic light on eye growth of normal chicks Methods: White Leghorn chicks aged 4 days (n = 8 in each group, three groups in total) were raised in a cabinet of 80 x 80 x 125 cm ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology under three different lighting. They were white (585 nm), white (585 nm) and red (630 nm), white (585 nm) and blue (450 nm). The average luminances in these three environments were around 260 lux. The ratio of those light combination was 50:50. The ocular parameters and refractive errors were measured before and after 14 days of exposure. The ocular parameters were measured by high frequency A-scan ultrasonography while the refractive errors were examined by streak retinoscopy. Percentage changes in the ocular parameters and refractive errors relative to the baseline were calculated and compared among different groups by two-way ANOVA and Fisher’s least significant difference (LSD) post hoc test. Results: After 14 days of exposure, the percentage increase in anterior chamber depth (ACD), lens thickness, vitreous chamber depth (VCD), retinal thickness and axial length were found to be significantly greater in chicks raised under white and red light when compared with those under white light only (p < 0.001). Besides, more myopic shift was shown in the chicks under white and red light (p < 0.05). However, the choroid did not demonstrate the thinning as expected but significant thickening was found (mean choroidal thickness ± SEM; white vs. white and red; 264.3 ± 4.43 mm vs. 280.8 ± 5.18 mm). Chicks raised in white and blue light had a smaller increase in ACD, lens thickness, VCD and axial length when compared with chicks under white light only (p< 0.001). Significant thinning of the retina was found (percentage change of the retinal thickness ± SEM; white vs. white and blue; -6.1 ± 1.2% vs -11.6 ± 0.7%, p = 0.001). Changes in refractive errors and choroidal thickness were comparable to the chicks under white light only. Conclusions: Combination of the white and monochromatic light provided two peak wavelengths across the spectrum. It significantly affected the eye growth of normal chicks. Greater elongation of eyeball was found in the chicks under white and red light than those under white light only whereas ocular growth was slower in the chicks under white and blue light. Monochromatic light seems to play a role in modulating the ocular growth in chicks. Commercial Relationships: Rachel Ka-man Chun, None; Danyang Wang, None; King Kit Li, None; Thomas C Lam, None; Quan Liu, None; Chi Ho To, None Support: This work was supported by research grants GU986, GU839, GUA32 and GYK89 from The Hong Kong Polytechnic University and the Henry G. Leong Endowed Professorship in Elderly Vision Health. Program Number: 2149 Poster Board Number: B0002 Presentation Time: 3:45 PM–5:30 PM Effects of 430nm monochromatic light on defocus-induced myopia in guinea pigs Yi-Feng Qian1, rui liu2, jinhui dai2. 1Department of Ophthalmology, First Affiliated Hospital of Soochow University, Suzhou, China; 2 Department of Opthalmology, Eye & ENT Hospital of Fudan University, Shanghai, China. Purpose: To investigate the effects of 430nm monochromatic light on defocus-induced myopia in guinea pigs. Methods: Eighteen 2-week-old pigmented guinea pigs were randomly assigned to two groups based on the mode of illumination: short-wavelength light (SL) for 8 weeks and broad-band white light (BL) for 8 weeks. All animals of the two groups were worn -5D lenses on right eye. Biometric and refractive measurements were then performed every 2 weeks. The illuminative parameters of all groups were identical and the light quantum number was 3×10-4μmolcm-2s-1. Results: After the beginning of the experiment, the right eyes of the two groups decreased in refraction. At the end of the experiment, relative myopia of the right eye was about 2.72D in the SL and about 3.03D in the BL when compared with the fellow eye. But a relative hyperopia, about 1.2D, was induced in the SL compared with the BL group in the end. From 4 to 8 week, there was significant difference in radius of corneal curvature between the two eyes of the SL group. But, there was no significant difference in corneal curvature between the two eyes of the BL group. At the end of the experiment, there was significant difference in radius of corneal curvature between the right eyes of the two groups. The difference was not significant in vitreous length of right eye between the two groups from beginning to the end of experiment. There was no significant difference in vitreous length between the two eyes of the SL group in the end. But finally, significant difference existed in vitreous length between the two eyes of the BL group. There were no significant inter-group or intra-group differences in length of anterior segment and Lens thickness. Conclusions: 430nm monochromatic light could interfere with the development of defocus-induced myopia in guinea pigs. The effect of the monochromatic light may be achieved by influencing the developments of vitreous chamber and corneal curvature. The recognition of defocus under the monochromatic light may be achieved by the function from only one type of cone. Commercial Relationships: Yi-Feng Qian, None; rui liu, None; jinhui dai, None Support: This work was supported by Grants 81400429, 81100689,81271040 from the National Natural Science Foundation of China Program Number: 2150 Poster Board Number: B0003 Presentation Time: 3:45 PM–5:30 PM Antagonistic effects of atropine and timolol on the color and luminance emmetropization mechanisms Laura A. Goldberg, Frances J. Rucker. New England College of Optometry, Boston, MA. Purpose: The role of the autonomic nervous system in the color and luminance emmetropization mechanisms is unknown. This study analyzed the response to the non-selective, parasympathetic antagonist, atropine, and the sympatholytic, beta-adrenergic antagonist, timolol, in chicks subjected to illumination conditions that selectively stimulate the color and luminance emmetropization mechanisms. Methods: Chicks were binocularly exposed eight hours each day, for four days, to one of three illumination conditions: 2Hz sinusoidal luminance flicker (LUM), 2Hz sinusoidal color flicker (B/Y), or steady light. Mean illuminance was 680 lux. Eyes received daily injections of either 20μl atropine (18nmol) (N=8), 2 drops of 0.5% timolol (N=8), 20μl phosphate-buffered saline (N=8), or no injection (N=8). Measurements of the axial dimensions of ocular components and refraction were performed using A-scan ultrasonography, photorefraction and a Hardinger Refractometer. In each illumination condition, the saline effect was subtracted from the drug effect [Drug (ΔX-ΔN) – Saline (ΔX-ΔN)]. Results: LUM flicker demonstrated opposite effects on eye growth and refraction with atropine and timolol treatment. Atropine caused a reduction in eye growth (-0.08 ± 0.02 mm, p=0.01) and a reduction in vitreous chamber depth (-0.10 ± 0.02 mm, p=0.004), evoking a hyperopic shift in refraction (3.40 ± 1.77 D), despite an antagonistic increase in lens thickness (0.14 ± 0.05 mm, p=0.004). In contrast, timolol elicited a myopic shift in refraction (-4.07 ± 0.92 D, p=0.001), due to an increase in eye length (0.045 ± 0.030 mm). Color flicker induced choroidal compensation for eye growth, preventing refractive shifts with atropine and timolol. With atropine, hyperopia was not observed, because a reduction in eye length (-0.05 ± 0.02 mm, p=0.01) was compensated for by choroidal thinning (-0.05 ± 0.02 mm, p=0.03). With timolol, myopia did not occur because a reduction ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology in eye length (-0.05 ± 0.018 mm, p=0.02) was also compensated for by choroidal thinning (-0.052 ± 0.015 mm, p=0.01). Conclusions: The opposing growth and refractive effects of atropine and timolol with luminance flicker, and the compensatory choroidal compensation with color flicker, suggest a precise balancing mechanism between the parasympathetic and sympathetic nervous system, and the visual environment, in achieving emmetropization. Commercial Relationships: Laura A. Goldberg, None; Frances J. Rucker, None Support: NEI T35 Student Research Fellowship and Beta Sigma Kappa Student Research Grant Program Number: 2151 Poster Board Number: B0004 Presentation Time: 3:45 PM–5:30 PM Antagonistic Effect of Ciliary and Superior Cervical Ganglion Sections on the Color and Luminance Emmetropization Mechanisms Frances J. Rucker1, Falk Schroedl2. 1Biomedical Science, New England Coll of Optometry, Boston, MA; 2Paracelsus Medical University, Salzburg, Austria. Purpose: Longitudinal chromatic aberration produces changes in retinal color and luminance contrast that guides emmetropization. An eye exposed to luminance contrast becomes hyperopic, like an atropine treated eye, an eye exposed to color contrast becomes more myopic. This experiment investigates the role of the autonomic nervous system in the control of emmetropization. Methods: One to two week old, white leghorn chicks, underwent unilateral lesion of the ciliary ganglion (CGX; N=16) or superior cervical ganglion (SCGX; N=16). Animals were allowed to recover for one week, and were then placed in cages illuminated with sinusoidally modulated light (2 Hz: 80% contrast) that changed in luminance (LUM) contrast or COLOR (red to green) contrast (mean illumination 680 lux). Animals were kept in these illumination conditions for three days (9am-5pm), and otherwise in the dark. Changes in ocular components after the recovery period, and after exposure to the illuminants, were measured with OCT (Lenstar) and refraction with a Hartinger Refractometer. Changes in the lesioned eye were compared with the unlesioned fellow eye. Results: After recovery: CGX produced an eye with relative hyperopia (2.01 ± 0.63D; p=0.006) and thinning of the anterior chamber (25 ± 11 mm; p=0.037). SCGX produced an enlarged eye (114 ± 26 mm; p<0.001) with longer vitreous chamber depth (154 ± 22 mm; p<0.001). With subsequent exposure to flicker: With CGX, LUM prevented significant eye growth (47 ± 30 mm) but the choroid thinned slightly (-17 ± 17 mm; p = 0.03) increasing vitreous depth (79 ± 17 mm; p < 0.01) without refractive shift (-1.1 ± 1.45 D; p = 0.44). The lens thickened (36 ± 8 mm; p = 0.002) and anterior chamber thinned (-41 ± 12 mm; p = 0.009). COLOR increased eye growth (101 ± 34 mm; p<0.05) but the choroid thickened (41 ± 19 mm), preventing significant vitreal (59 ± 33 mm) and refractive change (0.38 ± 1.0 D). With SCGX, LUM prevented vitreal growth (-13 ± 19 mm), and the choroid thickened slightly (16 ± 6 mm; p = 0.03), without refractive shift (0.3 ± 0.6 D). The lens thinned (32 ± 9 mm). COLOR increased vitreal growth (40 ± 14 mm; p=0.02) without refractive shift (0.8 ± 1.0 D). Conclusions: The results indicate common neural pathways for CGX and LUM flicker, slowing growth and mostly affecting the anterior eye, and for SCGX and color flicker, increasing growth and affecting the posterior eye. Commercial Relationships: Frances J. Rucker, None; Falk Schroedl, None Support: Research Promotion Fund of the Paracelsus University S13/05/007-SCH: F. Rucker and F. Schroedl. Neural pathways for Emmetropization Program Number: 2152 Poster Board Number: B0005 Presentation Time: 3:45 PM–5:30 PM Scotopic and Photopic Lighting Prevents Lens-induced Myopia in Mice Erica Landis1, Han na Park2, Megan Prunty2, 3, Curran Sidhu2, P M. Iuvone2, 4, Machelle T. Pardue2, 3. 1Neuroscience, Emory University, Atlanta, GA; 2Ophthalmology, Emory University, Atlanta, GA; 3Rehab Center for Excellence, Atlanta VA, Atlanta, GA; 4 Pharmacology, Emory University, Atlanta, GA. Purpose: The goal of this study was to determine the effects of different ambient illumination levels on dopaminergic signaling in the retina and on the susceptibility of the mouse eye to lens-induced myopia. Previous studies have shown photopic lighting to protect against myopia in both controlled animal studies and correlational human population studies. Photopic lighting was tested as well as scotopic lighting since rods may be needed for emmetropization (Park et al IOVS 2014). Methods: Male C57BL/6J mice were exposed to photopic (15,000 lux, n=38), mesopic (50 lux, n=36), or scotopic (0.005 lux, n=38) lighting during the light phase of a 12:12 hr light cycle, starting at postnatal day 23 (P23). At P28, half the mice received head-mounted monocular lens defocus (-10D). Retinas were enucleated at P36 and analyzed for dopamine and DOPAC levels via HPLC. At each time point, the refractive error, corneal curvature, and ocular parameters of the mice were measured. The DOPAC/dopamine ratios were calculated as a measure of dopamine turnover. Results: After two weeks of exposure to photopic, mesopic, or scotopic light there was no myopic shift (OD minus OS) in the refractive development of the control animals. Mice with lens defocus under mesopic light had significantly larger myopic shifts (normalized to P28, -4.741±0.608; p<0.005) by P34 compared to mice exposed to photopic (-2.604±0.544) or scotopic (-1.807±0.608). Additionally, in a subset of mice, the difference in DOPAC/dopamine ratio between the lens defocused and opposite eyes was significantly increased in mice exposed to scotopic light levels (0.022±0.007); decreased in mice exposed to mesopic light levels (-0.020±0.003; p<0.001); and showed no significant change in mice exposed to photopic light (0.002 ±0.003). No significant differences were found in corneal curvatures or axial lengths. Conclusions: While photopic and scotopic light levels were protective against lens induced myopia with increases in dopamine turnover, mesopic light levels increased the development of lensinduced myopia with a decrease in dopamine turnover. This implies that high and low intensities of light may prevent myopia, while intermediate intensities, similar to indoor lighting, promote myopia. Commercial Relationships: Erica Landis, None; Han na Park, None; Megan Prunty, None; Curran Sidhu, None; P M. Iuvone, None; Machelle T. Pardue, None Support: NEI Grant EY016435, NEI Grant EY004864, NEI Core Grant P30EY006360, Research to Prevent Blindness ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 2153 Poster Board Number: B0006 Presentation Time: 3:45 PM–5:30 PM Retinal-specific Dopamine Knock-out Mice are Myopic Michael A. Bergen3, 4, Han na Park1, Ranjay Chakraborty1, 4, Erica G. Landis5, 4, Curran Sidhu1, P M. Iuvone1, 2, Machelle T. Pardue4, 1. 1 Ophthalmology, Emory University School of Medicine, Atlanta, GA; 2 Pharmacology, Emory University School of Medicine, Atlanta, GA; 3 Biology, Emory University, Atlanta, GA; 4Rehab R&D Center of Excellence, Atlanta VA Medical Center, Decatur, GA; 5Neuroscience, Emory University, Atlanta, GA. Purpose: Dopamine has been implicated as a stop signal for refractive eye growth based on pharmacological studies in chickens, mammals, and primates. More recently, dopamine receptor knockout mice have been used to elucidate dopaminergic mechanisms of refractive development (Huang et al. IOVS 2014). In this study, a Cre-mediated, retinal-specific tyrosine hydroxylase knockout (KO) mouse was studied to determine the effect of eliminating retinal dopamine on refractive development and susceptibility to form deprivation (FD) myopia. Methods: KO mice were on a C57BL/6J background and were homozygous for both the Chx10 Cre-recombinase and floxed tyrosine hydroxylase alleles. Mice were randomly assigned to two groups, one undergoing normal refractive development and the other undergoing FD. Refractive development of KO mice and age-matched C57BL/6J wild-type (WT) mice was measured every 2 weeks from post-natal day 28 (P28) to P112. Under the FD paradigm, mice received a headmounted diffuser goggle at P28 over their right eye (OD) and were measured weekly until P77. Measurements of refractive error, corneal curvature, and ocular biometrics were obtained using an automated photorefractor, a keratometer, and a spectral-domain optical coherence tomography system, respectively. Results: During normal refractive development, KO mice were significantly more myopic (at P70, KO 2.74 ± 2.18 D, n=19; WT 7.08 ± 1.47 D, n=9; p < 0.01) and had significantly shorter axial lengths (at P56, KO 3.18 ± 0.01 mm; WT 3.23 ± 0.04 mm; p < 0.05) than their WT counterparts. KO mice also had significantly steeper corneas than WT mice (at P56, KO 1.42 ± 0.03 mm; WT 1.44 ± .03 mm; p < 0.05). Both WT and KO form-deprived mice showed similar magnitudes of myopic shift (difference of right and left eyes) (at P42, KO -2.29 ± 3.59 D, n=8; WT -3.85 ± 1.35 D; n=7). Conclusions: Our results support the hypothesis that dopamine is a stop signal for refractive eye growth. KO mice showed greater variability in FD myopic shifts than WT mice, which may indicate varying levels of retinal dopamine depletion in this model. Future work will correlate dopamine and DOPAC levels with refractive error and ocular parameters to comprehensively examine how dopamine concentration affects refractive development Commercial Relationships: Michael A. Bergen, None; Han na Park, None; Ranjay Chakraborty, None; Erica G. Landis, None; Curran Sidhu, None; P M. Iuvone, None; Machelle T. Pardue, None Support: NEI Grant EY016435, NIH Core grant P30EY006360, Research to Prevent Blindness Grant Program Number: 2154 Poster Board Number: B0007 Presentation Time: 3:45 PM–5:30 PM Apomorphine attenuates form-deprivation myopia (FDM) by a dopamine D2R-independent mechanism Xiangtian Zhou, Furong Huang, Jiangfan Chen, Jia Qu. School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China. Purpose: The dopamine agonist apomorphine (APO) can profoundly attenuate form-deprivation myopia (FDM) development in animals. Here, we determine whether APO acts at dopamine D2 receptors (D2R) to exert its effect on myopia development using D2R knockout (KO) mice. Methods: Wild-type (WT) littermates and D2R KO were subjected to FDM at postnatal days 28-56. Both groups were intraperitoneally injected daily with either APO (5 mg/kg/day) or vehicle for 4 weeks (starting from postnatal day 28). Their body weight, refraction, corneal radius of curvature and ocular axial components were measured at the end of 4-week treatment. Results: Consistent with our recent report, D2R KO attenuated FDM development compared to WT littermates. As expected, APO treatment attenuated myopia development compared with vehicle treatment in WT mice. Importantly, APO treatment in D2R KO mice further attenuated myopia development compared with the vehicle treatment in D2R KO mice. In parallel with refractory changes, D2R KO alone or APO alone also attenuated FDM-induced elongation of vitreous chamber depth and axial length compared to their corresponding controls. Moreover, combined treatment of D2R KO an APO treatment attenuated FDM-induced elongation of vitreous chamber depth and axial length compared to the D2R KO treated with vehicle. Conclusions: The inhibition of APO on FDM development was still effective in absence of D2Rs, suggesting that APO attenuates myopia development by a D2R-independent mechanism. Commercial Relationships: Xiangtian Zhou, None; Furong Huang, None; Jiangfan Chen, None; Jia Qu, None Support: 973 project:2001CB504602 Program Number: 2155 Poster Board Number: B0008 Presentation Time: 3:45 PM–5:30 PM Effectiveness of Low-dose Atropine on the Marmoset Eye Alexandra Benavente-Perez1, Ann Nour1, Eric R. Ritchey2, David Troilo1. 1Biological Sciences, SUNY College of Optometry, New York, NY; 2Johnson & Johnson Vision Care, Inc, Jacksonville, FL. Purpose: Low dose atropine (0.5%, 0.1% and 0.01%) has been shown to reduce myopia progression in humans by up to 24.5% (0.01%) to 40% (0.5%). The purpose of this study was to develop reliable measures to evaluate the effects of low doses of atropine on marmoset eyes for studies of myopia control. Methods: Nine age-matched young adult marmosets were divided into three treatment groups of low-dose atropine (0.1%, 0.01% or 0.005%). Subjects received one drop of atropine in one eye. The contralateral eye served as control. Pupil diameter (PD) under fixed background illumination (254lux) and pupillary light reflex (PLR) to ophthalmoscope illumination were monitored at baseline and after atropine every 5mins for the first 30mins, every hour for the first 9hrs, and daily for 7 days. The accommodative response to 1D of imposed hyperopic defocus was measured with a Shin-Nippon autorefractor in one marmoset from each group for 5 days following the atropine drop. Results: The interocular PD difference, normalized to baseline, reached maximum at 4hrs (0.1%), 3hrs (0.01%) and 2hrs (0.005%) after instillation (+1.96±0.26mm,+1.46±0.28mm and +1.45±0.49mm respectively). PDs returned to baseline levels 6 (0.1%) or 7 days (0.01% and 0.005%) after instillation. The PLR was blocked 15 to 25mins after 0.1% instillation and recovered after 2 days, but was always present for the 0.01% and 0.005% dosages. Accommodation was reduced 3hrs after instillation of 0.1% and recovered after 4 days (baseline accommodation: 1.11±0.56D; 3hrs post: 0.05±1.08D, p=0.03; 4days post: 1.45±1.20D, p>0.05). Lower doses of atropine did not block the accommodative response (p>0.05). Conclusions: A single drop of atropine had measurable effects on pupil function lasting several days and showed a dose response. Only ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology the highest dose tested affected accommodation. While the effects of extended treatment have yet to be examined, these results will help guide further investigation of the effects of low-dose atropine for myopia control using the marmoset model. Commercial Relationships: Alexandra Benavente-Perez, 2Johnson & Johnson Vision Care, Inc (F); Ann Nour, Johnson & Johnson Vision Care, Inc (F); Eric R. Ritchey, Johnson & Johnson Vision Care, Inc (E); David Troilo, Johnson & Johnson Vision Care, Inc (C), Johnson & Johnson Vision Care, Inc (F) Support: Johnson & Johnson Vision Care, Inc Program Number: 2156 Poster Board Number: B0009 Presentation Time: 3:45 PM–5:30 PM The control effects of Fenofibrate on the axial length elongation in lens-induced myopia chicken model Panfeng Wang1, 2, Thomas C Lam2, Chi Ho To2. 1Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; 2 Laboratory of Experimental Optometry,Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hong Kong, Hong Kong. Purpose: Apolipoprotein A1 (ApoA1), the major component of high density lipoprotein, was suggested to be down-regulated in the retina of lens-induced myopia (LIM) animal model. The growth of axial length was reduced in LIM chicken possibly through an up-regulation of the ApoA1. Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is the first line therapy to regulate lipid metabolism by increasing the synthesis of ApoA1. The current study investigated the efficacy of fenofibrate on the growth of eye axial in lens-induced myopia chicken model. Methods: At 4-day old, male chicken was divided into different groups after gender determination by PCR method. In the LIM group, negative powered lenses (-10D) were worn on the right eyes and the left eyes were kept untreated as controls. In the treatment groups, 20uM, 100uM, 200uM fenofibrate delivered at an injection volume of 10ul, were injected into the bottom of the vitreous chamber of the right eye, and vehicle solutions were injected into the left eyes as control on day 5. Then, lenses (-10D) were worn on both eyes of treated chicken. An extra group of chicken without lens received 200uM fenofibrate intravitreal injection at the right eyes, and the left eyes were kept untreated as normal control. A high-frequency A-scan ultrasound system was used to measure the ocular parameters of all the chicken before the treatment and on day 8. Chicken with body weight growth abnormality or vitreous hemorrhage were ruled out. The changes of ocular parameters among different treated eyes were statistically analyzed by t-test. Results: Fenofibrate dose dependently suppressed the growth of ocular axial length, and statistical difference was recorded at the concentration of 200uM. On day 8, LIM chicken (n=15) treated by fenofibrate (200uM) had shorter axial length than LIM chicken (n=13) by 32.7% (P= 5.15078E-07). The fenofibrate showed no effect on the growth of ocular axial length of normal eyes. There was no different between fenofibrate treated eyes and LIM eyes at the choroid recovery changes after the negative lenses were taken off on day 8. Conclusions: 200uM of Fenofibrate is protective against LIM development. The results not only demonstrate the therapeutic effects of fenofibrate on myopia, but they also support the possible role of PPARα-dependent mechanism in the development of myopia. Commercial Relationships: Panfeng Wang, None; Thomas C Lam, None; Chi Ho To, None Support: XJ2012061 Program Number: 2157 Poster Board Number: B0010 Presentation Time: 3:45 PM–5:30 PM Effect of oral administration of nicotinic acid on ocular growth of lens-induced myopic chicks Hu XIAO, Panfeng Wang, King Kit Li, Rachel Ka-man Chun, Thomas C Lam, Chi Ho To. School of Optometry, The Hong Kong Polytechnic University, Hong Kong, Hong Kong. Purpose: To explore the effect of oral administration of nicotinic acid on eye growth of normal and myopic chicks. The nicotinic acid is the normal drug used in human to raising the HDL in blood. The Apolipoprotein A1 is the potential in protect myopic eye growth in previous studies. Methods: White Leghorn chicks aged at 4 days (n = 48 in total) were randomly allocated into 4 groups. Chicks in group A and B were orally administered a single dose of nicotinic acid daily (150mg/ ml, 1ml per chick) while the chicks in group C and D were received saline orally as control (1ml per chick). The oral administration last for 11 consecutive days. After 7 days of oral administration, -10D lenses were attached to both eyes of the chicks in group A and C while the chicks in group B and D worn plano lenses for 4 days. The refractive errors and ocular dimension components were examined using streak retinoscopy and high resolution A-scan ultrasonography before and after 11 days of the oral administration respectively. The changes of refractive errors and vitreous chamber depth between 1 days and 11 days of oral administration were gained. T-test was used to analysis the difference. Results: After 4 days of lens wear, chicks with -10D lenses became significantly more myopic than the chicks with plano lenses (A and B group: P= 0.0274; C and D group: P= 0.0013; t-test). In the groups with -10D lenses (group A and C), the changes in vitreous chamber depth (VCD) in chicks treated with nicotinic acid (mean ± SEM; 0.568 ± 0.146mm) were significantly less than that of the salinetreated chicks (0.778 ± 0.197 mm, p = 0.007, t-test). The change in refractive errors in chicks treated with nicotinic acid (mean ± SEM; -8.35 ± 1.11D) were significantly less than that of the saline-treated chicks (-10.81± 0.75D, p = 2.20E-06, t-test). There was no significant difference in VCD between the groups wearing plano lenses (group B: nicotinic acid vs. group D: saline; 0.398 ± 0.202mm, vs. 0.514± 0.151mm, p = 0.125, t-test). But there was significant difference in refractive errors between the groups wearing plano lenses (group B: nicotinic acid vs. group D: saline; -3.25 ± 0.30D, vs. -4.54 ± 0.35D, p = 2.20E-09, t-test). Conclusions: The nicotinic acid intake could retard the elongation of VCD in lens-induced myopic chicks. Its effect on the normal ocular growth is however not apparent. Commercial Relationships: Hu XIAO, None; Panfeng Wang, None; King Kit Li, None; Rachel Ka-man Chun, None; Thomas C Lam, None; Chi Ho To, None Support: PolyU research grants: GYK89, GU986; RGC GRF: BQ29N Program Number: 2158 Poster Board Number: B0011 Presentation Time: 3:45 PM–5:30 PM Form-deprived highly myopic chick eyes have lower than normal corneal stiffness than emmetropic eyes Byung Soo Kang1, Li Ke Wang2, Yong-Ping Zheng2, Chea-su Kee1. 1 School of Optometry, The Hong Kong Polytechnic University, Hong Kong, Hong Kong; 2Interdisciplinary Division of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong, Hong Kong. Purpose: This study aimed to determine whether corneal stiffness differed between normal and highly myopic eyes in the chick model of myopia ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Methods: Starting from day 5 post-hatching, the right eyes of 21 chicks (Gallus Gallus Domesticus) were covered with a translucent occluder for 7 days to induce form-deprived myopia. At the end of the treatment period, spherical equivalent (SE) refractive error was measured under anesthesia (1.5% Isoflurane) by Hartinger coincidence refractometry, and in-situ corneal stiffness (CS) was measured using a custom-made air-jet optical coherent tomography (OCT) system. Specifically, triplicate CS measurements were obtained, with corneal deformation (in mm) assessed in response to 5 cycles of increasing/decreasing air pressure (in N). A custom algorithm calculated the slope of CS load-deformation curve (N/mm) and the correlation between these parameters Results: Compared to fellow untreated eyes, form-deprived eyes developed significant myopia (mean±SEM: SE= -20.65 ± 1.41D vs. -1.22 ± 0.26D; paired t-test, p<0.001) and exhibited reduced corneal stiffness (mean±SEM: CS= 0.0203 ± 0.0008mm vs. 0.0239 ± 0.0009mm; paired t-test, p<0.05). When data from both eyes were used for correlation analyses, both SE (r=+0.462, p<0.002) and J45 astigmatic component (r=+0.351, p<0.05) were moderately correlated with corneal stiffness. Expressing the corneal stiffness as percentage of interocular difference in CS [100 %*( treated eye – fellow eye) / fellow eye], we found that in the 17 birds that showed a reduction in corneal stiffness in the treated eyes (range= -0.35% ~ -46.65%), eleven (64.7 %) of them had at least 15% reduction in corneal stiffness (mean= -24.46%, one-sample t-test, T= -4.57, p<0.01) Conclusions: Form deprivation induced high myopia and reduced corneal stiffness. The correlation between SE and CS indicates that the changes occurring in the biomechanical properties of the cornea may be quantitatively related to those occurring in the sclera Commercial Relationships: Byung Soo Kang, None; Li Ke Wang, None; Yong-Ping Zheng, None; Chea-su Kee, None Program Number: 2159 Poster Board Number: B0012 Presentation Time: 3:45 PM–5:30 PM Regional Differences in Gene Expression with Imposed Defocus in Chick RPE Yan Zhang, Emily Eng, Christine F. Wildsoet. School of Optometry, Univ of California, Berkeley, Berkeley, CA. Purpose: We previously reported bidirectional gene regulation of members of the Bone Morphogenetic Protein (BMP) family (2, 4, & 7) in chick retinal pigment epithelium (RPE) in response to as little as 2 h of imposed optical defocus. This study investigated whether there were also regional differences in the effects of imposed defocus on the expression of these genes in chick RPE. Methods: 19-day old White-Leghorn chicks wore monocular +10 or -10 D lenses for 2 h. At the end of the treatment period, RPE was isolated and divided into 3 circular zones using punches of 3 and 6 mm radius: central 3 mm zone (T3 or F3; T: treated eyes, F: fellow controls), middle 3-6 mm zone (T6 or F6), and peripheral 6-9 mm circular zone (T9 or F9). RPE RNA was purified and reverse transcribed to cDNA. qPCR was performed to examine the gene expression of BMP2, 4, and 7. Expression levels were compared between lens treated and fellow control eyes. Paired Student’s t test was used for statistical analysis. Results: The +10 D lens treatment induced up-regulation of both BMP2 and BMP4 gene expression in the central and middle zones (T3 & T6 regions compared to F3 & F6). For BMP2, gene expression was highly up-regulated, by 129- and 28-fold (T3/F3, n = 3, p = 0.09; T6/F6, n = 4, p = 0.05). The equivalent values for BMP4 gene expression were 13- and 10-fold (T3/F3, n = 3, p < 0.05; T6/F6, n = 4, p < 0.05). BMP7 expression was 3-fold up-regulated for the T3/ F3 comparison only. The most peripheral region (T9/F9) did not show differential gene expression between treated and fellows for any of the genes. As expected, the -10 D lens induced down- instead of up-regulation; significant changes were recorded for both BMP2 and BMP4, for both T3/F3 and T6/F6 comparisons. BMP2 was down-regulated by 32- and 12-fold for T3/F3 and T6/F6 comparison, respectively (n = 4), but not for the T9/F9 comparison. Likewise, BMP4 gene expression was down-regulated 12- and 3.6-fold in central (T3/F3) and middle (T6/F6) regions respectively. BMP7 gene expression did not change for any of these three regions. Conclusions: This study provides evidence of regional variations in the response of chick RPE to the same defocus, i.e. as imposed by single vision lenses, generally decreasing with increasing eccentricity. The significant up- or down-regulation of BMP gene expression in central RPE points to a critical role of the central retina/RPE in early stage of eye growth regulation. Commercial Relationships: Yan Zhang, None; Emily Eng, None; Christine F. Wildsoet, None Support: NIH grants R01EY012392 (CFW), K08EY023609 (YZ), K12EY017269 (YZ), T35EY007139 (EE) Program Number: 2160 Poster Board Number: B0013 Presentation Time: 3:45 PM–5:30 PM Retinal profile asymmetries in myopes and emmetropes Christopher A. Clark, Ann E. Elsner, Bryan Haggerty, Joel A. Papay. School of Optometry, University of Indiana, Bloomington, IN. Purpose: Previous work has shown that peripheral refractive error asymmetries exist between different locations in the retina and those asymmetries are greater in myopes than emmetropes. This could be due to a number of factors including physical restriction from the optic nerve and differences in optical alignment between the optical/ visual axis. The purpose of this study is to investigate the source of asymmetry. Methods: Fifty-six subjects (refractive error +1.50 to -11.15) had a battery of tests performed including axial length, corneal topography, anterior chamber depth, peripheral refraction, peripheral partial coherence interferometry, and SD OCT for retinal thickness. Turning point location (TPL) was classified as the retinal location in degrees where the retinal profile was at a minimum. Angle alpha was measured as the distance from the apex of the corneal topography to the visual axis. Statistics including repeated measures ANOVA were performed with SPSS (IBM, Endicott, NY.) Results: The TPL was greatest in high myopes with an average displacement of three degrees temporal from the fovea compared to approximately zero degrees for emmetropes (P = 0.045.) No correlation was found in this study between angle alpha and either the TPL or retinal profile asymmetry along the horizontal axis. Asymmetry did increase with refractive error (P = 0.008.) Visual inspection of the SD OCT images showed greater optic nerve head tilt in subjects with higher asymmetries regardless of refractive error. Conclusions: Retinal profile asymmetries increase with refractive error which is consistent with previously reported data. These asymmetries appear to be largely due to physical constriction by the optic nerve rather than to do optical effects such as angle alpha. Commercial Relationships: Christopher A. Clark, None; Ann E. Elsner, None; Bryan Haggerty, None; Joel A. Papay, None Support: NIH Grant K23EY022064 Program Number: 2161 Poster Board Number: B0014 Presentation Time: 3:45 PM–5:30 PM Effects of 6-hydroxydopamine on refractive development and form-deprivation myopia in C57BL/6 mice Shi-Jun Weng, Xiao-Hua Wu, Yun-Yun LI, Kang-Wei Qian, Xiong-Li Yang, Yong-Mei Zhong. Institute of Neurobiology, Fudan University, Shanghai, China. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Purpose: In various species, reduced retinal dopamine (DA) levels are thought to mediate the development of myopia. However, recent evidence shows that in the C57BL/6 mouse, retinal DA levels are unaltered when form-deprivation myopia is developed. Here, to further explore the role of retinal DA in mouse eye growth, we examined whether refractive development could be disturbed by destroying retinal DA pathway in this mouse strain. Methods: 6-hydroxydopamine (6-OHDA, 50 μg) was intravitreally applied to the right eye using a micro-injector, and the left eye serves as the control. Refractive errors were measured using an automated eccentric infrared photorefractor, in animals raised in normal visual environment, or in those with the injected eye wearing an occluder for 4 weeks to induce form-deprivation myopia. The levels of retinal DA and its primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were assessed by HPLC analysis. Results: Administration of 6-OHDA significantly reduced retinal DA levels by 40-80%, and the effect lasted for at least 31 days. With normal visual experience, the 6-OHDA-injected eyes became markedly myopic relative to their fellow eyes (~6D of interocular difference). Furthermore, in injected eyes, form-deprivation did not induce further myopic shifts, nor did it cause further reduction in retinal DA and DOPAC levels. Conclusions: An intact retinal dopaminergic system, including healthy dopaminergic amacrine cells and complete retinal DA stores, is essential for both normal refractive development and the generation of form-deprivation myopia in the C57BL/6 mouse. In this mouse strain, refractive development could be interfered by reducing retinal DA levels dramatically, even though the generation of formdeprivation myopia is not associated with retinal DA levels. Commercial Relationships: Shi-Jun Weng, None; Xiao-Hua Wu, None; Yun-Yun LI, None; Kang-Wei Qian, None; Xiong-Li Yang, None; Yong-Mei Zhong, None Support: Ministry of Science and Technology of China (2011CB504602); the National Natural Science Foundation of China (31171055, 31121061, 31100796); ARVO/Pfizer Collaborative Research Fellowship to Shi-Jun Weng Program Number: 2162 Poster Board Number: B0015 Presentation Time: 3:45 PM–5:30 PM Identification of apolipoprotein A-I as a novel retinoic acid binding protein Jody A. Summers1, Angelica Harper1, Hanke Van-Der-Wel2, Marcela Hermann3, Christopher M. West2. 1Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK; 2 Biochemistry, University of Oklahoma Health Science Center, Oklahoma City, OK; 3Medical Biochemistry, Medical University of Vienna, Vienna, Austria. Purpose: All-trans-retinoic acid (atRA) may be an important molecular signal in the postnatal control of eye size. Retinoids are closely associated with atRA binding proteins, which are important in regulating their transport, metabolism and biological activity. We and others (Mertz and Wallman, Exp. Eye Res. 2000) have previously identified a protein with an apparent Mr of 27 kD (p27) that represents the major secreted atRA binding protein in chick choroids. The goal of the current study was to identify p27 as we hypothesize that p27 may play a role in the regulation of atRA activity during visually guided ocular growth. Methods: atRA binding proteins were initially identified from conditioned medium of chick ocular tissues by photoaffinity 3H-atRA labeling, SDS-PAGE and autoradiography. 3H-atRA binding proteins were purified using a combination of anion exchange (Q-sepharose), gel filtration (Superdex 200) columns and SDS-PAGE. Unlabeled samples were processed in parallel and peak fractions of unlabeled protein, corresponding to the elution position and mass of 3H-atRAlabelled protein were identified by mass spectrometry. The identify of p27 was confirmed using immunoprecipitation of 3H-atRA-labelled p27 from conditioned medium using anti-chick apolipoprotein A-I antibodies. Results: Following photoaffinity labeling of choroid and sclera conditioned medium, radiolabeled proteins migrating at 60 kD and 27 kD were detected by autoradiography. These proteins coeluted from Q-sepharose and Superdex 200. Mass spectrometry analyses identified the 60 kD protein as serum albumin and the 27 kD protein as apolipoprotein A-I. Following immunoprecipitation of 3H-atRA labeled proteins from sclera conditioned medium with anti-chick apolipoprotein A-I, a single 27 kD band was detected on autoradiograms. Conclusions: Apolipoprotein A-I is the 27 kD 3H-atRA-binding protein present in chick choroid and sclera conditioned medium. The expression of this protein may play a role in the regulation of atRA signaling in the choroid and sclera in postnatal ocular growth. Commercial Relationships: Jody A. Summers, None; Angelica Harper, None; Hanke Van-Der-Wel, None; Marcela Hermann, None; Christopher M. West, None Support: NIH Grant EY09391 Program Number: 2163 Poster Board Number: B0016 Presentation Time: 3:45 PM–5:30 PM Transcriptome Analysis Indicates Adaptive Responses to Physiological Stress in Recovery from FDM Loretta Giummarra1, Nina Riddell1, Nathan Hall2, 3, Melanie Murphy1, Sheila G. Crewther1. 1Psychological Science, La Trobe University, Melbourne, VIC, Australia; 2Life Sciences Computation Centre (LSCC), Victorial Life Sciences Computation Centre (VLSCI), Melbourne, VIC, Australia; 3La Trobe University, Melbourne, VIC, Australia. Purpose: Form deprivation myopia (FDM) is associated with dramatic increases in ocular volume, axial length, thinning of the retina and choroid and hyperosmotic stress. Thus this study aimed to assess the associated gene pathway changes using high throughput RNA-sequencing and comprehensive bioinformatic analysis. Given our previous ultrastructural and elemental microanalyses it was hypothesized that profile changes would involve energy metabolism, ionic solute changes and evidence of oxidative stress Methods: Twelve male hatchling chicks were monocularly occluded from days 4-11 after which chicks were given T=0hr, T=6hr or T=24hr of normal vision. Four chicks were used as aged-matched unoccluded controls. Biometrics were measured prior to tissue collection. RNA was isolated from choroid/retina/RPE tissue and prepared for sequencing on the Illumina HiSeq™ 1500. Raw reads were mapped onto the chicken genome and counts determined for each gene. Differential expression analysis was undertaken with voom/EdgeR with an FDR of 0.05. Gene Set Enrichment Analysis (GSEA) software was used to determine whether a priori defined set of genes were significantly altered (FDR<0.25) during the induction and recovery of FDM. Curated gene sets were obtained from BioCarta, KEGG, and the Pathway Interaction Database and the Reactome database. Results: FD Chicks were ~20D myopic. Refractive normalization began with removal of occlusion. GSEA analysis revealed an overall suppression in genes associated with metabolism and ion homeostasis at T=0hr. GSEA during the recovery period revealed an increase in expression of genes associated with glucose metabolism, potassium transport and hypoxia. These changes were positively correlated with reduction in refraction. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Conclusions: Increased axial growth during FD is accompanied by suppression of gene pathways associated with retinal metabolism and refractive normalization. Removal of FD and reintroduction of the normal visual environment with constantly changing luminance levels requires upregulation of metabolic pathways and normalization of ion distribution profiles across the eye. These results confirm our previous work and build our understanding of the importance of osmoadaptive pathways that use energy metabolism, ion transport, to reduce hypoxia and restore osmotic homeostasis. Commercial Relationships: Loretta Giummarra, None; Nina Riddell, None; Nathan Hall, None; Melanie Murphy, None; Sheila G. Crewther, None Program Number: 2164 Poster Board Number: B0017 Presentation Time: 3:45 PM–5:30 PM RNAseq gene expression analysis highlights the correlation of changes in metabolic and structural pathways with axial elongation during refractive compensation. Nina Riddell1, Loretta Giummarra1, Nathan Hall4, 2, Melanie Murphy1, David P. Crewther3, Sheila G. Crewther1. 1Psychological Science, La Trobe University, Bundoora, VIC, Australia; 2La Trobe University, Melbourne, VIC, Australia; 3Swinburne University, Melbourne, VIC, Australia; 4Life Sciences Computation Centre (LSCC), VLSCI, Melbourne, VIC, Australia. Purpose: High throughput transcriptome studies in animal models of refractive error have primarily analysed data at the single-gene level. Results from these studies are disparate and a comprehensive framework for understanding the biological cascades underlying ocular growth regulation remains elusive. Thus, this study aimed to identify characteristic biological features of refractive compensation to myopic and hyperopic defocus in chick by correlating axial length across lens-groups during defocus induction with expression of genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Methods: Chicks were raised with ±10D lenses, or no lens. Following biometric measurements at 1, 2, and 3 days, 3-4 chicks per lens-group were euthanized and RNA extracted from the retina/RPE/ choroid. Libraries were sequenced on the Illumina HiSeq1500, raw reads mapped to the chick genome, and counts determined for each gene. Counts/million were imported into GSEA and expression of KEGG pathways correlated with axial length phenotype across lensgroups at each time-point (FDR cut-off <.25). Results: Refractive and axial length change was rapid during the first day of defocus for both lens-types and slower over subsequent days (particularly for plus lenses). Consistent with the initially rapid change in ocular morphology, expression of structural pathways including focal adhesion, tight junction, and vascular smooth muscle contraction was positively correlated with axial length at 1 day. Fatty acid metabolic and signalling pathways were also correlated with axial length at this time. Although no structural pathways were identified following 2 and 3 days of lens-wear when morphological changes had slowed, metabolic pathways (such as oxidative phosphorylation) were implicated at both time-points. Conclusions: This study is the first to correlate ocular axial length changes with pathway enrichment across a period of refractive compensation to lenses. Results suggest that changes in structural pathway expression are linked to periods of rapid axial growth change. Perturbed metabolism was characteristic of all stages of compensation, with implication of oxidative phosphorylation and related pathways suggesting that growth changes elicit a shift in energy homeostasis that may alter redox state and vulnerability to later development of ocular pathologies. Commercial Relationships: Nina Riddell, None; Loretta Giummarra, None; Nathan Hall, None; Melanie Murphy, None; David P. Crewther, None; Sheila G. Crewther, None Program Number: 2165 Poster Board Number: B0018 Presentation Time: 3:45 PM–5:30 PM Anti-Diuretic Hormone in the Regulation of Ocular Volume in Compensation to Defocus Melanie Murphy1, Loretta Giummarra1, Nina Riddell1, David P. Crewther2, Vinh Nguyen1, Sheila G. Crewther1. 1Psychological Science, La Trobe University, Melbourne, VIC, Australia; 2Centre for Human Psychopharmacology, Swinburne University of Technology, Melbourne, VIC, Australia. Purpose: The hormone Arginine Vasopressin (AVP) is a vasoconstrictor and anti-diuretic that is commonly associated with stress. Our previous results show that AVP causes a myopic shift in refractive compensation (RC) to +10D defocus (ARVO, 2013). Further, environmental stress in the form of asymmetric flicker impacts ocular growth (Crewther et al, 2006). Thus the current experiment aimed to investigate whether AVP plays a role in RC to defocus, and whether flicker affects this process. Methods: Experiment 1: RNA was extracted from the retina/RPE/ choroid of chicks with + or -10D, or no defocus on days 5-7 posthatching (n = 3 per lens group, per day) and prepared for sequencing on the Illumina HiSeq1500. Raw reads were mapped onto the chick genome and counts determined for each gene. Counts per million were imported into Pathway studio and GSEA conducted using the Mann-Whitney U-test algorithm (p<.05). Experiment 2: Chicks (n=360) were raised from day 5-9, with or without asymmetric flicker in the 12 hr day cycle, with + or -10D defocus (or non lens), following intravitreal injection of 5ml of either PBS, AVP or the AVP receptor antagonist ([des-Gly9-β-Mercapto-β, β cyclopentamethylenepropionyl1, O-Et-Tyr2,Val4,Arg8]Vasopressin) (in PBS) into the experimental eye. Fellow eyes were injected with PBS. Retinoscopy and A-scan ultrasonography was performed on day 9. Tissue was collected and prepared for immunohistochemistry to examine AQP-4 and Kir4.1 expression. Results: Experiment 1: RNAseq revealed sign-dependent changes in AVP-related pathways over 3 days of rearing with defocus. Experiment 2: Flicker alone induced a myopic shift in both lens conditions. Flicker+AVP reduced hyperopia, axial elongation and anterior chamber depth in +10D lenses. Flicker+AVP antagonist reduced RC and ocular growth to -10D lenses. Immunohistochemistry showed altered AQP-4 and Kir4.1 staining across flicker conditions. Conclusions: Results indicate that changes in AVP-related gene expression occur concomitantly with changes in ocular volume during the induction of RC. Further, AVP and its antagonist also differentially interfered with the typical pattern of compensation to lenswear. Physiological stress induced by flickering light further influenced this. These results implicate stress-induced changes in the rate of transretinal fluid movement in the development of refractive error. Commercial Relationships: Melanie Murphy, None; Loretta Giummarra, None; Nina Riddell, None; David P. Crewther, None; Vinh Nguyen, None; Sheila G. Crewther, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 2166 Poster Board Number: B0019 Presentation Time: 3:45 PM–5:30 PM Hyperosmotic Stress and Osmo-Gene Adaptation During Early Induction of Refractive Errors. Sheila G. Crewther1, Nina Riddell1, Alan Marshall1, Loretta Giummarra1, Melanie Murphy1, Melinda J. Goodyear1, David P. Crewther2. 1Psychological Science, La Trobe University, Melbourne, VIC, Australia; 2CHP, Swinburne University of Technology, Melbourne, VIC, Australia. Purpose: Why is myopia a common risk factor for most sight threatening disorders? Our earlier biometric, ultrastructural and elemental analyses of the chick form deprivation model have provided evidence of severe physiological, oxidative and hyperosmotic stress. More recently prolonged hyperosmotic stress has been shown to lead to chronic inflammation in a number of diseases (Brocker etal 2013). We hypothesized that perturbation of axial growth during induction of refractive errors would also be accompanied by hyperosmosis and osmoadaptative gene changes, that should be demonstratable with elemental microanalysis (EDX) and RNA seq respectively. Methods: Chicks were raised with ±10D lenses, or no lens. Following biometric measurements at 1, 2, and 3 days, 8 chicks per lens group were euthanized. RNA was extracted from the retina/RPE/ choroid of 4. Four were used for scanning electron-microscopy and EDX. Libraries were sequenced on the Illumina HiSeq1500. Counts per million were imported into GSEA and expression of KEGG and Reactome pathways during myopia/hyperopia induction compared to age-matched no lens chicks (FDR cut-off <.25). Results: Refractive compensation (RC) to -10D defocus continued for 72hrs whereas RC to +10D was in near completion after 24hours. EDX shows sodium and chloride ion distributions were greatly upregulated in outer retina by -10D over the 72hrs but only at the retino-vitreal border in +10D at 72hrs. Potassium profiles in RC to +10D remained upregulated across the retina for 72 hrs with concurrent up-regulation of reactome potassium channel pathways at 72hrs in RNAseq data. Consistent with altered osmotic and oxidative stress, implicated pathways during refractive compensation included those related to synthesis of small molecule osmolytes, structural remodelling, inflammation, and metabolism. Conclusions: The EDX results demonstrate that RC to optical defocus is accompanied by hyperosmotic shifts in ion distribution profiles across the entire posterior eye, while concurrent changes in gene expression profiles were seen in metabolic and ion solute processes. These pathways have previously been associated with osmoadaptation and more severe disease states such as ARM and diabetes. The findings suggest the need for further experimental considerations of hyperosmotic changes as risk factors for severe visual impairments and for development of therapeutics. Commercial Relationships: Sheila G. Crewther, None; Nina Riddell, None; Alan Marshall, None; Loretta Giummarra, None; Melanie Murphy, None; Melinda J. Goodyear, None; David P. Crewther, None Program Number: 2167 Poster Board Number: B0020 Presentation Time: 3:45 PM–5:30 PM Differences in the sensitivity to myopia-inducing stimuli of young guinea pigs sourced from different colonies Mariana Garcia2, David Hammond1, Christine F. Wildsoet2. 1Deakin University, Geelong, VIC, Australia; 2Vision Science, University of California, Berkeley, Berkeley, CA. Purpose: To characterize the responses of guinea pigs sourced from different breeding colonies to myopia-inducing stimuli using negative lens and form deprivation paradigms. Methods: English Short Hair guinea pig breeders were obtained from a commercial vendor (Elm Hill Labs, Chelmsford, MA – designated “Elm Hill” guinea pigs) and from a University-based breeding colony (University of Auckland, NZ – designated “NZ” guinea pigs). Elm Hill guinea pig pups were fitted with either negative lenses (-10, -5, or 0 D) or diffusers at 10 days of age. NZ guinea pigs were fitted with diffusers at 7 days of age. Both sets of animals were treated for 4 weeks. Ocular axial lengths were measured twice a week using high frequency A-scan ultrasonography, cycloplegic refractions were measured on treatment days 0, 14, and 28, and behavioral visual acuity measured on treatment day 28. Results: Elm Hill guinea pigs fitted with lenses exhibited minimal interocular differences in axial length and refractive error after 28 days of treatment; likewise, form deprivation (FD) failed to significantly affect the rate of ocular elongation or to induce a myopic shift in refractive error. Overall changes in interocular difference in axial length (treated minus control) were 0.03±0.1 mm for -10 D lenses, -0.20±0.43 mm for -5D lenses, -0.01±0.21 mm for 0 D lenses, and 0.07±0.16 mm for FD. Conversely, the NZ guinea pigs exhibited a 0.17±0.12 mm increase in interocular differences in axial length after 28 days of FD treatment. Conclusions: A systematic study of the ocular growth responses of young guinea pigs to myopia-inducing stimuli revealed significant strain-related differences. These results point to genetically determined differences in the sensitivity of emmetropization mechanisms to visual manipulation, even within the same breed. Finally, this works suggests that research groups wishing to work with a guinea pig myopia model should carefully consider the source of their animals. Commercial Relationships: Mariana Garcia, None; David Hammond, None; Christine F. Wildsoet, None Support: EY012392 Program Number: 2168 Poster Board Number: B0021 Presentation Time: 3:45 PM–5:30 PM Compensatory eye growth responded to the imposed defocus is influenced by spatial content in chick Man Pan Chin, Zhe Chuang Li, Allen Ming Yan Cheong, Ho Lung Henry Chan. School of Optometry, The Hong Kong Polytechnic University, Hong Kong, Hong Kong. Purpose: Emmetopization is a visually guided eye growth, and the compensatory eye growth is depending on the imposed defocus. This study hypothesized if different spatial contents can affect the compensatory eye growth responded to imposed defocus. Methods: The right eyes of White Leghorn chicks from 10 to 12 days old were glued with a cone-shaped lens system using Velco. Animals were divided into six groups (n=8 to 12) for various levels of defocus magnitude, including plano, -15D and -25D (lenses were attached at the proximal end of the cone), with two different spatial stimulus patterns at the other end of the cone: 1) high spatial frequency: 0.88 cycle/deg (0.4mm white/black checkers) with 100% of contrast, and 2) a lower spatial frequency: 0.28 cycle/deg (1.25mm white/black checkers) with 100% of contrast. Axial ocular dimensions, including anterior chamber depth, lens thickness, vitreous chamber depth (VCD) and axial length, were obtained using A-scan ultrasound. Measurement was carried out prior to fitting the lens system and on the fourth day after treatment. Analysis of variance (ANOVA) was used for statistical analysis. Results: After 4 days of wearing the cones, the VCD of the right eye increased with the imposed defocuses. Under the same magnitude of defocus, chick eyes with low spatial stimulus had consistently longer VCD than those with high spatial stimulus (p<0.05). Both defocus and spatial stimulus showed significant main effect on VCD ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology percentage change (ANOVA, p<0.005). Significant group differences of VCD percentage change were observed in Group -15D (low vs high, 10.4±2.2 vs 7.25±3.35, p<0.05) and -25D (9.53±3.82 vs 4.93±4.79, p<0.05), but not in plano group. Similar trend was also observed in axial length, but neither in anterior chamber depth nor lens thickness. Conclusions: Our results confirm our hypothesis that spatial content in emmetropization can affect the compensatory eye growth responded to same magnitude of imposed defocus. Effects of different optical defocus on compensatory eye growth significantly interacted with the spatial frequency of the visual stimulus. Further studies are important to understand the mechanism of defocus and spatial interaction in emmetropization. Commercial Relationships: Man Pan Chin, None; Zhe Chuang Li, None; Allen Ming Yan Cheong, None; Ho Lung Henry Chan, None Support: General Research Funds (PolyU5605/13M), Health and Medical Research Fund (01121876) and PolyU Internal Grants (G-YBBS, G-UA2E, Z-0GF, G-YM70) Program Number: 2169 Poster Board Number: B0022 Presentation Time: 3:45 PM–5:30 PM Effects of the relative strength of the more positive-powered component in dual focus lenses on emmetropization in macaques Baskar Arumugam1, 2, Li-Fang Hung1, 2, Chi-ho To3, Brien A. Holden2, Earl L. Smith1, 2. 1College of Optometry, University of Houston, Houston, TX; 2Vision CRC, Sydney, NSW, Australia; 3Hong Kong Polytechnic University, Kowloon, Hong Kong. Purpose: Dual focus lenses that impose relative myopic defocus over a large part of the visual field can slow myopia progression in children. Our aim was to determine how the relative surface area devoted to the more positive-powered lens component influenced the ability of dual focus lenses to alter refractive development. Methods: Beginning at 3 weeks of age, infant rhesus monkeys were reared with Fresnel lenses that had central 2mm zones of zero power and concentric annular zones that had alternating powers of +3.0D or 0D. The relative spatial widths of the annular zones were varied from 1:1 (i.e., equal widths) to 1:4.5 (+3D:0D) between treatment groups (n≥6 per group). The monkeys wore the treatment lenses over both eyes continuously until 151±4.2 days. Comparison data were obtained from monkeys reared with full field +3D lenses over both eyes (FF+3D, n=6) and from 34 control monkeys reared with unrestricted vision. Refractive status, corneal power and axial dimensions were assessed every 2 weeks throughout the lens rearing period. Results: All of the dual focus lens designs produced relative hyperopia. At the end of the treatment period, the median refractive errors for the monkeys reared with dual focus lenses that had width ratios of 1:1, 1:2, 1:3 and 1:4.5 were +5.25D, +5.19D, +4.31D, and +4.28D, respectively, which were similar to the refractive errors exhibited by animals reared with FF+3D lenses (+4.63D; p=0.22 to 0.94), but significantly more hyperopic than those found in agematched control monkeys (+2.50 D; p=0.0002 to 0.004). The average vitreous chamber depths for the dual-lens-reared animals were also not significantly different from those found in FF +3D lens-reared monkeys (OD:+3D/pl 1:1; 9.31±0.34mm, 1:2; 9.44±0.60mm, 1:3; 9.74±0.38mm, 1:4.5; 9.55±0.25mm vs 9.58±0.32mm, respectively, p=0.18 to 0.87). In addition, there were no significant differences in either the median refractive errors (p=0.08 to 1.0) or the average vitreous chamber depths (p=0.06 to 0.71) between the dual focus lens groups. Conclusions: The results demonstrate that even when the more positive-powered zones make up only about 1/5th of a dual-focus lens’ surface area, refractive development is dominated by relative myopic defocus. Overall, the results emphasize that myopic defocus distributed across the visual field evokes strong signals that can slow eye growth in primates. Commercial Relationships: Baskar Arumugam, None; Li-Fang Hung, None; Chi-ho To, Inventor (P); Brien A. Holden, Zeiss (P); Earl L. Smith, Zeiss (P) Support: National Eye Institutes EY03611 and EY07551 and funds from Vision CRC, Sydney, Australia Program Number: 2170 Poster Board Number: B0023 Presentation Time: 3:45 PM–5:30 PM Two-zone bifocal lenses with peripheral negative additions control lens-induced hyperopia in young chicks Huamao Miao1, Christine F. Wildsoet2. 1Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China; 2Center for Eye Disease & Development, School of Optometry, University of California Berkeley, Berkeley, CA. Purpose: It has been proven that bifocal lenses designed with relative positive additions can slow ocular elongation and thus myopia progression. This study addressed a related question of whether similar designed lenses with relative negative additions can control hyperopia using young chicks as an animal model. Methods: To induce hyperopia, chickens wore monocular +5 diopter (D) single vision lenses (SVLs) from 7 days of age for 3 days; the lenses were then switched for either +10 D SVLs or 2-zone concentric bifocal lenses (BFLs), which were worn for 5 days. BFLs had a central zone power of +10 D and one of 3 peripheral zone powers (plano, +5 or +8 D, corresponding to additions of -10, -5 or -2 D, respectively). For all BFL designs, both 2.5 and 4.5 mm central zone diameters (CZDs) were tested. Central refractive errors and ocular axial parameters were measured using static retinoscopy and high frequency A-scan ultrasonography. Results: At the last time point, the control group (i.e., wearing +10 D SVLs) was most hyperopic (+9.37 D), with the group wearing 2.5 mm CZD BFLs with the highest (-10 D) addition being least hyperopic (+4.28 D). For both the 2.5 and 4.5 mm CZDs, there were trends towards decreasing induced hyperopia with increasing negative add power, with this dose effect being significant for 2.5 mm CZD lenses. Induced changes in vitreous chamber depth and optical axial length (relative shortening) as well as choroid thickening followed trends consistent with induced refractive errors. Conclusions: Our study explored the possible application of BFLs as a treatment to control hyperopia. Our data provide evidence that 2-zone concentric BFLs incorporating peripheral negative additions could restrain lens-induced hyperopia progression in young chicks, and treatment effects increasing with both add power and larger peripheral zones. Further studies are warranted to examine whether mammals and primates show similar beneficial effects. Commercial Relationships: Huamao Miao, None; Christine F. Wildsoet, None Support: NIH/NEI Grants R01 EY012392 (CFW) & exchange program fund for doctoral student, Graduate Medical Education, Fudan University (HMM) ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 2171 Poster Board Number: B0024 Presentation Time: 3:45 PM–5:30 PM Evaluation on the changes of 90 degree visual field defects accompanied peripheral retinal degenerative lesions in high myopic eye——the roles in following-up and prophylaxis in retinal detachment Yining Shi1, Yanming Chen2, 3, Ji Liu3. 1Department of Ophthalmology, Shaanxi Provincial People’s Hospital, Xian, China; 2Department of Ophthalmology, China Medical University, Shenyang, China; 3 Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT. Purpose: To observe the relationship between peripheral retinal degenerative changes in high myopic eyes and 90 degree visual fields defects, and the influence with the refractive and aging factors. We performed a retrospective, observational clinical study to provide a safer prophylactic treatment of retinal detachment (RD). Methods: 360 cases of high myopia were tested by LVP, OCTOPUS 101 perimeter. and compared with the 161 fellow eyes of high myopia with RD, 118 eyes of high myopia with RD, 41 fellow eyes of low myopia with RD, 54 eyes of low myopia with RD, and 108 normal eyes.High risk RD eyes were treated by defined equatorial pan-retinal photocoagulation, at meantime detected the visual field changes and posterior vitreous detachment(PVD) developing, posttreated complications. Results: The 90 degree mean light sensitivity (MS) of high myopia eyes was (21.34±5.40)dB (Fig. 1). The MS in high myopia was declined with aging and severity of myopia, the linear regression formula was R=32.981-0.161ages+0.468 diopters.There was definite co-relationship with 2 kinds of visual defect and the degenerations: 1. The peripheral absolute defects, related with lattice degeneration; 2. The peripheral MS declining, with white-without-pressure. Following 32 to 74 months after the defined equatorial pan-retinal photocoagulation,there were complete PVD formation observed under split lamp with 90°Volk pre-set lens (Fig. 2), and there were no significant changes between the MSs pre- and post treatment. Conclusions: There are linear relations with MS and aging, myopic degree. The MS may indicate the peripheral degenerative lesions and its degrees, played a roles in following-up and prophylaxis in retinal detachment.The defined equatorial pan-retinal photocoagulation is a safer, effective intervene for prophylaxis of retinal detachment and forming iatrogenous PVD. Commercial Relationships: Yining Shi, None; Yanming Chen, None; Ji Liu, None Program Number: 2172 Poster Board Number: B0025 Presentation Time: 3:45 PM–5:30 PM Identification of Elements of the BMP2 Signaling Pathway in Cultured Chick Scleral Fibroblast Emilia A. Zin, Yan Zhang, Christine F. Wildsoet. University of California, Berkeley, Berkeley, CA. Purpose: Previous studies have indicated the role of Bone Morphogenetic Protein 2 (BMP2) in eye growth regulation. This study investigated the role of BMP2 in scleral remodeling using a chick scleral fibroblast culture model to look for evidence of BMP2 signaling. Methods: Primary chick scleral fibroblast (CSF) were cultured on 24-well plates or chamber slides in DMEM/F-12 medium with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. CSF total RNA was collected and purified using RNeasy Mini Kit and then subjected to cDNA synthesis and real-time PCR semi-quantification. Gene expression was examined for BMP2, BMP receptors (BMPR1A, 1B, -2), SMAD1, -5, and -9, and the localization of relevant proteins, BMP2, BMPR1A, BMPR2, SMAD1, -4, and -5, phosphorylated SMAD1 (p-SMAD1), and p-SMAD1/5 in CSF and 293T cell lines was investigated using immunocytochemistry. Protein expression was also validated with Simon automated western blot. Results: Cultured CSF showed detectible expression of BMP2, BMP receptors, and SMAD 1, 5, 9 genes and immunohistochemistry confirmed the expression of BMPR1A, BMPR2, SMAD1, -4, and -5 proteins in CSF as well as 293T cells. Western blot analyses confirmed expression of the p-SMAD1/5 protein in both CSF and 293T cells, while SMAD1 and p-SMAD1 proteins were only detected in 293T cells. Conclusions: That cultured chick scleral fibroblasts express many of the components of the BMP2 signaling pathway, at both gene and protein levels, points to their likely involvement in scleral remodeling and ocular growth. These results provide a foundation for future in vivo studies into the role of BMP2 in ocular (scleral) growth regulation. Commercial Relationships: Emilia A. Zin, None; Yan Zhang, None; Christine F. Wildsoet, None Support: NIH grant R01EY012392, NIH grant K08EY023609, NIH grant K12EY017269 ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 2173 Poster Board Number: B0026 Presentation Time: 3:45 PM–5:30 PM Growth and completion of emmetropization in the normally developing chick eye Zheng Shao1, 2, Marsha Kisilak1, Elizabeth L. Irving2, Melanie C. Campbell1, 2. 1Physics and Astronomy, University of Waterloo, Waterloo, ON, Canada; 2School of Optometry and Vision Science, University of Waterloo, Waterloo, ON, Canada. Purpose: Normal emmetropization has long been assumed to result in zero refractive error, but recently this has been questioned. In normally growing chick eyes, we are interested in objectively determining when emmetropization is complete. We are also interested in whether growth during and following normal emmetropization differs. Methods: From literature values of chick eye parameters, functions were fit to MOR (mean ocular refraction or spherical equivalent) and optical axial length (OAL; anterior cornea to anterior retina) vs. age. Dioptric length (K’) and eye power (F) were derived up to day 75 using our previously reported method to calculate eye power. Pupil size data were also used to calculate the angular and linear retinal blurs (EB and LRB) due to defocus. Results: Eye power and K’ decrease exponentially with age at slightly different rates until power and K’ reach almost equal values about day 35. Subsequently, power and K’ decrease almost identically with age. This gives an initial rapid exponential decrease in MOR, which reaches a relatively stable value of 1.0 D of hyperopia beyond day 35. The completion of emmetropization is defined as the first time point beyond which MOR remains relatively stable, estimated as between 30 and 35 days. EB and LRB decrease almost exponentially until day 35. After emmetropization is complete, MOR changes little, EB remains almost constant while LRB increases slowly from about day 45 to the end of available measurements on day 75, in agreement with predictions of an almost uniformly expanding eye model. The radius of the blur on the retina is larger than cone spacing prior to completion of emmetropization, and approaches cone spacing as emmetropization is completed. Conclusions: Concurrent variations in eye power and length combine to produce the smaller, more rapid changes in MOR during emmetropization. The time point at which emmetropization is complete can be defined as the first time point after which MOR and angular retinal blur are stable. Emmetropization appears to be driven by an active reduction of EB to a value close to cone resolution. After emmetropization is complete, the subsequent change in retinal blur is consistent with a slow, almost uniform ocular expansion. However, after the age when normal emmetropization is complete, an emmetropization response to additional imposed defocus blur has been observed. Commercial Relationships: Zheng Shao, None; Marsha Kisilak, None; Elizabeth L. Irving, None; Melanie C. Campbell, None Support: NSERC Canada 35321 Program Number: 2174 Poster Board Number: B0027 Presentation Time: 3:45 PM–5:30 PM Adaptive optics measurements of cone density in chick eyes during lens-induced myopia Marsha Kisilak, Laura Emptage, Ian Andrews, Melanie C. Campbell. University of Waterloo, Waterloo, ON, Canada. Purpose: In vivo measurements of cones in the chick eye, an animal model of myopia, are desirable as a marker of retinal changes during axial length increases. In vivo images allow longitudinal measurements of the angular cone spacing in the growing chick eye and during lens-induced myopia. We can then compare measured densities with models of retinal changes during eye growth and myopia development. Methods: Four Ross Ross chicks were acquired on the day of hatching. Axial length was measured using A scan ultrasound and aberrations and defocus were measured in a custom built HartmannShack aberrometer. Eyes were imaged in an adaptive optics corrected scanning laser ophthalmoscope modified for small animal use (2.5 mm diameter pupil). After this, the right eyes were goggled with -15D lenses. Measurements were repeated on days 7 and 14. All measurements were taken close to the optical axis and the anatomical position of the area centralis. Angular cone densities were measured directly. Linear cone spacings on the retina were calculated from published schematic eye models modified for measured eye lengths and cone packing properties were assessed. Paired t-tests were performed to compare between days and between treated and control eyes. Results: By day 14 goggled eyes were on average 15D myopic. Cones were successfully imaged on all days. The angular density of cones was not significantly different between control and goggled eyes (p > 0.2) on any day. As seen in previous control birds, angular density was not significantly different between days 0 and 7 (p = 0.1) in control eyes, after which it significantly increased (p < 0.02). Goggled eyes showed no significant change in angular density with growth. The calculated linear distance between cones increased significantly from 6.4 microns on day 0 to 7.0 microns on day 14 in control eyes and did not differ from goggled values of 6.2 microns on day 0 (before goggling) and 8.3 microns on day 14. On average, cones were 38% hexagonally packed across all days and for both control and treated eyes. Conclusions: Average cone spacings in control eyes on day 14 were within 10% of some literature values. Results for control eyes, showing initial uniform expansion followed by either cone migration or optic pole elongation are consistent with our previous data. Eyes with lens-induced myopia expand uniformly relative to control eyes. Commercial Relationships: Marsha Kisilak, None; Laura Emptage, None; Ian Andrews, None; Melanie C. Campbell, None Support: NSERC Canada 35321 Program Number: 2175 Poster Board Number: B0028 Presentation Time: 3:45 PM–5:30 PM Peripheral Wavefront Aberration in Myopia with and without Orthokeratology Lenses Young Sik Yoo1, Kyung-Sun Na1, Choun-Ki Joo1, Geunyoung Yoon2. 1 The Catholic University of Korea, Seoul, Korea (the Republic of); 2 University of Rochester, Rochester, NY. Purpose: Peripheral refractive error degrades the quality of retinal images and has been hypothesized as a potential factor to stimulate the development of refractive error. Various contact lens designs based on the hypothesis have shown the efficacy of controlling myopia progression. The aim of the study was to evaluate the impact of orthokeratology lens (OK lens) on the peripheral wavefront aberration in myopic eyes. Methods: We conducted a cross-sectional study to evaluate the effect of OK lens on the peripheral aberration profile of myopic subjects in adolescents. Study subjects were divided into two groups; one was OK lens group and the other was myopic patients who did not experience the OK lens correction. A custom-developed Shack-Hartmann aberrometer was used to measure ocular wavefront aberrations along different horizontal retinal eccentricities with ten degree step across the central 30 degrees of visual field. The study subjects maintain their natural foveal fixation while the aberrometer is rotated around the eye for the off-axis measurements. Wavefront ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology refraction for each retinal eccentricity was quantified over 4 and 6 mm pupils. Results: The mean refractive error was -1.92D ± 0.83 in the OK lens group and -5.84D ± 2.47 in the naked eye group, respectively. Hyperopic defocus at the peripheral visual field in the myopic eyes increases with increasing amounts of forveal refractive error. These effects varied with the degree of the corrected power after a treatment of OK lens. Significant difference (p < 0.05) in the value of defocus was found at the peripheral retinal eccentricity in the OK lens group compared to the naked eye group. In the analysis of high order aberration, values of on-axis and off-axis showed a different tendency along with the symmetry of each aberration. Conclusions: OK lens treatment is found to be effective in reducing the degree of peripheral hyperopic defocus in myopic eyes. Commercial Relationships: Young Sik Yoo, None; Kyung-Sun Na, None; Choun-Ki Joo, None; Geunyoung Yoon, None Program Number: 2176 Poster Board Number: B0029 Presentation Time: 3:45 PM–5:30 PM Hyperoxic Myopia: A Prospective Study of Twelve Divers with Six Hours of Exposure to 1.35 ATM PO2 for Five Consecutive Days Jonathan W. Brugger1, 2, Anita Gupta1, Barbara Shykoff2, John Florian2. 1Ophthalmology, New York Eye & Ear, New York, NY; 2 Navy Experimental Diving Unit, Panama City Beach, FL. Purpose: Hyperoxic myopia is a phenomenon associated with prolonged exposure to an increased partial pressure of oxygen (PO2) resulting in a myopic shift of refractive error. This has been described in patients undergoing hyperbaric oxygen therapy and in divers exposed to high PO2. The mechanism of action for hyperoxic myopia is not understood. This prospective study collected ocular data in healthy divers exposed to 1.35 ATM PO2 at the Navy Experimental Diving Unit to better characterize hyperoxic myopia PO2 thresholds and the mechanism of action. Methods: Twelve healthy healthy U.S. Navy Divers participated in five consecutive days of exposure to 100% Oxygen via surfacedsupplied, open-circuit MK20 breathing apparatuses at the bottom of a 15-foot pool (PO2 of 1.35 ATM) for 6 hours. Prior to diving, and three days after the last dive, subjects had an ocular examination consisting of visual acuity (VA), autorefraction, intraocular pressure (IOP), biometry, and corneal topography. Before and after every dive, subjects had VA, and autorefraction. IOP was measured on the first, third, and fifth day. Results: Two of the twelve divers had subjective symptoms of blurry vision 2-3 days after the last dive. The first diver had a myopic shift of -0.50 diopters OS via autorefraction and VA change from 20/16 to 20/20-2. The other diver had a myopic shift of approximately -0.25 diopters OU via autorefraction with a VA shift from 20/30-1 to 20/100 OD and 20/20-1 to 20/40 OS. Both subjects had no significant changes in IOP, topography, and biometry measurements and both had spontaneous resolution of their myopia over two to three weeks with no residual symptoms. Conclusions: Two healthy divers exposed to an increased PO2 (1.35ATM for 30 hours in 5 days) developed symptomatic myopia with no changes in corneal topography and biometry (axial length, lens thickness, aqueous depth). With no appreciable changes in eye structure, a change in refractive index of the lenticular crystalline lens is likely responsible for the myopic shift. Hyperoxic myopia is a risk for those conducting intense diving with a PO2 between 1.3-1.6 ATM and warrants additional studies to better define risk factors, recovery time, mechanism of action, and PO2 thresholds. Commercial Relationships: Jonathan W. Brugger, None; Anita Gupta, None; Barbara Shykoff, None; John Florian, None Program Number: 2177 Poster Board Number: B0030 Presentation Time: 3:45 PM–5:30 PM Malondialdehyde in high myopia. Amparo Navea1, Francisco Bosch-Morell2, 1, Salvador Mérida Donoso2. 1Oftalmología Médica, FISABIO, Valencia, Spain; 2Instituto de Ciencias Biomédicas, Universidad CEU Cardenal Herrera, Valencia, Spain. Purpose: Malonyldialdehyde (MDA), a secondary product of lipid peroxidation is widely used as an indicator of lipid peroxidation. Lipids and lipid-soluble compounds are essential constituents of the cells and tissues that comprise the eye. Simultaneously, lipids are also crucial targets of the attack by reactive oxygen species such as oxygen free radicals. The role of lipid peroxidation, a process under which oxidants such as free radicals attack lipids containing carboncarbon double bond(s), especially polyunsaturated fatty acids, has been described in several ocular pathologies in the past decades. The aim of this work is to establish, if any, the relationship between myopia and oxidative damage in subretinal fluid of myopic patients with retinal detachment. Methods: Protein content and MDA was evaluated in subretinal fluid of 71 myopic and no myopic patients with retinal detachment. Samples were collected in three different groups attending to myopia degree of the subjects: group 1, non-myopic, group 2, low myopia (patients with less than 6 dioptries) and group 3, high myopia (patients with more than 6 dioptries). Results: Similar data were obtained for groups 1 and 2 (group 1: 0,20 ± 0,09 mM MDA and 9,24 ± 4,54 mg protein/ml; group 2: 0,22 ± 0,06 mM MDA and 9,26 ± 4,29 mg protein/ml). However high myopia patients displayed statistically significant higher values (p<0,001) of both components: MDA (0,39 ± 0,10 mM) and proteins (17,47 ± 4,55 mg/ml). One of the most remarkable result was the high positive correlation obtained (r=0,87) when representing individual data pairs of MDA and myopia degree of myopic patients. Conclusions: These results ratify the direct contribution of oxidative stress in retinal detachment. They also suggest that myopia may play a role (qualitative and quantitative), that deteriorate the natural course of ocular diseases that involve oxidative stress. Commercial Relationships: Amparo Navea, None; Francisco Bosch-Morell, None; Salvador Mérida Donoso, None Support: CEU-SANTANDER PRCEU-UCH 13/17 Program Number: 2178 Poster Board Number: B0031 Presentation Time: 3:45 PM–5:30 PM Collagen crosslinking using genipin diminishes cyclic softening of tree shrew sclera during lens-induced myopia development Alexander Levy2, Sarah M. Baldivia2, Rafael Grytz1. 1Ophthalmology, University of Alabama at Birmingham, Birmingham, AL; 2 Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL. Purpose: To assess the effect of exogenous crosslinking using genipin on the cyclic softening response of the remodeling tree shrew sclera during monocular -5 diopter (D) lens wear. Methods: Cyclic tensile tests were performed on 2-mm wide scleral strips, first at physiological loads (50 cycles, 0-3.3 g, 30 sec/cycle) and subsequently after 10 minutes rest at supra-physiological loads (50 cycles, 0-33.3 g, 60 sec/cycle) conditions. Two scleral strips were obtained from each eye of two juvenile tree shrews exposed to 4 days of monocular -5 D lens wear to induce axial elongation and myopia. The scleral strips of the control eye were mechanically tested immediately after enucleation or after 24 hours incubation at 37°C in PBS. The scleral strips of the lens treated eye were tested after 24 hours incubation in PBS or PBS supplemented with genipin at a low cytotoxicity concentration (1mM). Cyclic softening was defined as ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology the incremental strain increase from one to the next cycle. This value was averaged over both animals and cycles 5 to 50. Cycles that led to tissue failure were excluded. Results: At both loading conditions (physiological / supraphysiological loads), the average incremental strain increase (% per cycle) was nearly identical in the fresh (0.03 /0.2) and PBS incubated tissue of the control eye (0.03/0.26). The cyclic softening was approximately four times higher in the sclera of the myopic eye (0.14/0.81) and two orders of magnitude lower after genipin crosslinking (0.001/0.004). Conclusions: Results indicate that cyclic tensile loading leads to continued softening of the juvenile tree shrew sclera. The softening rate increases during lens-induced myopia and is diminished after genipin crosslinking. This finding suggests that axial elongation in myopia may be due to a remodeling mechanism that increases the cyclic softening response of the sclera, which can be inhibited by scleral crosslinking using genipin. Cyclic tensile tests at supra-physiological loads of three scleral strips of one animal showing the increased cyclic softening leading to tissue failure in the treated sclera versus the control and the diminished cyclic softening after genipin crosslinking. Commercial Relationships: Alexander Levy, None; Sarah M. Baldivia, None; Rafael Grytz, None Support: NIH grant EY003909 (P30); EyeSight Foundation of Alabama; Research to Prevent Blindness Program Number: 2179 Poster Board Number: B0032 Presentation Time: 3:45 PM–5:30 PM Immunolesioning of glucagonergic amacrine cells in the chick retina Diane Nava1, 2, Tatiana Lupashina2, Bhavna Antony1, Li Zhang3, Michael D. Abramoff3, Christine F. Wildsoet1, 2. 1Vision Science Graduate Group, UC Berkeley, Berkeley, CA; 2Center for Eye Disease and Development, Berkeley, CA; 3Department of Electrical and Computer Engineering, University of Iowa, Iowa City, IA. Purpose: Neurotoxins have been used in myopia research to ablate inner retinal cells in order to study their contributions to eye growth and refractive error regulation, although those used in past studies have been relatively unselective. The purpose of this study is to investigate immunolesioning as a potential tool to selectively ablate glucagon amacrine cells (GACs) in the chick and to compare its selectivity to previous methods. Methods: A Saporin immunotoxin conjugated to a primary antiglucagon antibody was injected intravitreally in the left eyes of 7-day old chicks as a 10uL solution in one of 5 concentrations (0.125, 0.25, 0.5, 0.75 or 1 uM). TUNEL staining (Roche) was used to determine the distribution of apoptotic cells and immunohistochemistry on vertical sections used to assess changes in the glucagonergic cell population. Optical coherence tomography imaging was used to investigate changes in the retina and choroid, and flash electroretinograms (ERGs) were recorded to assess changes in retinal function 4, 6 and 9 days after injection. Results: The maximum loss of GACs was seen with the 1 uM concentration and for concentrations lower than 1 uM, the central retina seems to be more affected than peripheral retina, where GAC immunoreactivity in the IPL/INL boundary was more apparent than in the center. The 1uM concentration significantly attenuated the photopic negative response of the flash ERG at both 4 and 7 days (p=0.0236 & p=0.0393), with no significant effect on b-wave and a-wave amplitudes. The peak of the flash ERG at approximately 200 ms was also significantly attenuated at 4 days after injection (p=0.03), but not at later time points. With the 1 uM concentration, total retinal thickness was not significantly reduced in injected eyes at any time point, while choroidal thickness underlying the area centralis was significantly increased compared to values for the contralateral eyes at both 6 and 9 days post injection (p=0.0475 & p=0.04097 respectively). Conclusions: The above immunotoxin conjugate, injected intravitreally, appears to more selectively lesion GACs in the chick retina than previously tested neurotoxins, as evidenced by histological as well as retinal structural and functional data, and thus represents a suitable tool for investigating the role of GACs in eye growth regulation. The finding of choroidal thickening in lesioned eyes is a novel finding that warrants further investigation. Commercial Relationships: Diane Nava, None; Tatiana Lupashina, None; Bhavna Antony, None; Li Zhang, None; Michael D. Abramoff, University of Iowa (P); Christine F. Wildsoet, None Support: NIGMS 3R25GM090110-04S1 Program Number: 2180 Poster Board Number: B0033 Presentation Time: 3:45 PM–5:30 PM Ciliary Muscle Cell Changes During Guinea Pig Emmetropization Andrew D. Pucker1, Ashely R. Carpenter2, Hugh J. Morris1, Andrew J. Fischer3, Kirk M. McHugh2, Donald O. Mutti1. 1Optometry, Ohio State University, Columbus, OH; 2Center for Molecular and Human Genetics, Nationwide Children’s Hospital, Columbus, OH; 3 Department of Neuroscience, Ohio State University, Columbus, OH. Purpose: To establish normal morphological parameters and to characterize ciliary muscle (CM) cell changes with age during guinea pig emmetropization. Methods: Three pigmented guinea pig eyes were collected at three different ages (n = 9 eyes). Mean refractive error was determined with retinoscopy by two trained examiners. Eyes were then enucleated, hemisected, and fixed with paraformaldehyde. Temporal eye segments were then embedded in OCT compound and 30 mm serial sections were collected; the two most temporal slides of each eye were then labeled with anti-α-smooth muscle actin antibodies (smooth muscle) and Draq5 nuclear stain. Sections were then visualized with a fluorescent microscope (Leica Microsystems) and analyzed with Stereo Investigator (MBF Bioscience) to determine the mean CM cross-sectional area, nuclei number, and cell crosssectional area. Results: Guinea pigs displayed emmetropization as refractive error decreased from +9.08 ± 2.75 D at 1-day-old to +2.91 ± 0.88 D at 90-days-old. The mean CM length and CM cross-sectional area both ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology significantly increased with age, from 0.42 ± 0.035 mm to 0.90 ± 0.095 mm (p = 0.003) and from 0.037 ± 0.005 mm2 to 0.057 ± 0.015 mm2 (p = 0.011), respectively. The mean cross-sectional area covered by each CM cell did not change (p = 0.82), which is consistent with the marginal increase in the mean CM cell number from 72.0 ± 7.4 cells to 105.5 ± 19.2 cells per section (p = 0.059; all by JonckheereTerpstra test). Conclusions: Guinea pig CM undergoes morphologic changes during development in the first 90 days of life characterized by significant increases in cross-sectional area and length while the mean area occupied by each cell does not significantly change. These data suggest that the CM grows via cell proliferation rather than through hypertrophy. These normative data will be useful when contrasted with potential CM changes during myopia induction experiments. Commercial Relationships: Andrew D. Pucker, None; Ashely R. Carpenter, None; Hugh J. Morris, None; Andrew J. Fischer, None; Kirk M. McHugh, None; Donald O. Mutti, None Support: NIH/NEI: K08EY023264 Program Number: 2181 Poster Board Number: B0034 Presentation Time: 3:45 PM–5:30 PM Eye size and shape in newborn children and its relation to axial length and refraction at three years Laurence S. Lim1, 2, Sharon Chua2, Pei Ting T. [email protected], Shirong Cai2, Yap Seng Chong2, Kenneth Kwek3, Marielle Fortier3, Cheryl Ngo2, Anqi Qiu2, Seang-Mei Saw2. 1Ophthalmology, Singapore National Eye Center, Singapore, Singapore; 2National University of Singapore, Singapore, Singapore; 3KK Women’s and Children’s Hospital, Singapore, Singapore. Purpose: Eye shape has been postulated to be a risk factor for refractive error. The purpose of this study is to determine if eye shape and size at birth are associated with refractive error and eye size 3 years later. Methods: A subset of 173 full-term newborn infants from the Growing Up in Singapore Towards healthy Outcomes (GUSTO) birth cohort underwent magnetic resonance imaging (MRI) to measure the axial length (AL), height, width, volume and surface area of the internal eye at birth. Eye shape was assessed by an index of oblateness, calculated as 1–(AL/width) or 1–(AL/height). Oblate eyes had oblateness >+0.01, spherical eyes had oblateness between +0.01 and -0.01, and prolate eyes had oblateness <-0.01. Cycloplegic autorefraction and optical biometry (IOLMaster) were performed 3 years later. Results: In total, 346 eyes of 173 children were analysed. The majority were male (94 children, 54%) and of Malay (43%) or Chinese (43%) origin. Most eyes were prolate at birth. At three years, the mean AL was 21.74±0.68mm (range 19.77-23.84), representing a mean increase from birth of 4.47±0.94mm (1.71–7.20). The mean spherical equivalent refraction(SER) was 0.91±0.80D (-2.40 to +3.47) and only a small proportion of eyes was myopic (8 eyes, 3.6%). After multivariate adjustment, eyes with longer AL at birth had smaller increases in AL at 3 years (p<0.001). Eyes with larger baseline volumes and surface areas had smaller increases in AL at 3 years (p<0.001 for both). Eyes which were more oblate at birth had greater increases in AL at 3 years (p<0.001). Using width to calculate oblateness, prolate eyes had smaller increases in AL at 3 years compared to oblate eyes (p<0.001), and, using height, prolate and spherical eyes had smaller increases in AL at 3 years compared to oblate eyes (p<0.001 for both). There were no significant associations between eye size and shape at birth and SER, corneal curvature or myopia at 3 years. Conclusions: Eyes that are longer, larger and have prolate or spherical shapes at birth exhibit smaller increases in AL over the first 3 years of life. Eye size and shape at birth influence subsequent eye growth but not the development of refractive error, suggesting adequate compensatory mechanisms to maintain emmetropia for at least the first 3 years of life. Commercial Relationships: Laurence S. Lim, None; Sharon Chua, None; Pei Ting T. [email protected], None; Shirong Cai, None; Yap Seng Chong, None; Kenneth Kwek, None; Marielle Fortier, None; Cheryl Ngo, None; Anqi Qiu, None; Seang-Mei Saw, None Support: This work is supported by the Translational Clinical Research (TCR) Flagship Program on Developmental Pathways to Metabolic Disease funded by the National Research Foundation (NRF) and administered by the National Medical Research Council (NMRC), Singapore- NMRC/TCR/004-NUS/2008. Additional funding is provided by the Young Investigator Award at the National University of Singapore (NUSYIA FY10 P07) and Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2012-T2-2-130). Program Number: 2182 Poster Board Number: B0035 Presentation Time: 3:45 PM–5:30 PM Normative ocular biometric values for the adult mouse, rat, rabbit, dog, pig, non-human primate, and human Joshua S. Eaton1, Andrea D. Rodrigues2, Craig B. Struble3, Sara M. Thomasy4, Christopher J. Murphy4, 1. 1Ocular Services on Demand, Madison, WI; 2Non-Clinical Development - Toxicology, Allergan, Irvine, CA; 3Covance Laboratories, Madison, WI; 4Department of Surgical and Radiological Sciences, University of California - Davis, Davis, CA. Purpose: Confidence in knowledge of biometric dimensions of the ocular chambers and structures is essential in pharmacokinetic/ dynamic modeling and preclinical development of therapeutic compounds and devices. Furthermore, knowledge of comparative biometric values between animal models and humans can impact dosing methods, concentration delivered, and techniques employed in nonclinical studies. Consideration of comparative differences also improves accuracy in prediction of human safety risk. Our objective was to summarize available normative data for the human and six common animal models - mouse, rat, rabbit, dog, pig, and non-human primate (NHP). Methods: References citing normative ocular biometric values in all seven species were collected by searching online publication databases, textbooks, and journals available to the authors. Biometric parameters researched included: axial globe length (AGL), anterior chamber depth (ACD), axial lens diameter (ALD), vitreous chamber depth (VCD), and vitreous chamber volume (VCV). Available data were assembled and evaluated. For scarce or unavailable values, additional data was generated to establish normative values. All data are presented as mean ± SD. Results: Reported values were obtained using both in vivo and ex vivo techniques as well as calculation and/or estimation. Parameters with values of higher intraspecies variability (>25% coefficient of variation) included: ACD in the mouse (0.50 ± 0.17); ALD in the rat and rabbit (3.55 ± 1.08 and 6.18 ± 1.88 mm, respectively); VCD in the mouse and NHP (0.61 ± 0.16 and 8.72 ± 3.58 mm, respectively); and VCV in the mouse, rat, dog, and NHP (0.01 ± 0.01, 0.03 ± 0.02, 2.63 ± 0.81, and 2.20 ± 0.89 ml, respectively). Relative to other laboratory species, fewer values were reported for AGL in the rat; ACD in the rat, rabbit, pig, and NHP; VCD in the rabbit, pig, and NHP; and VCV in the rat, dog, and NHP. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Conclusions: Reliable normative ocular biometric values are critical for use of animal models in eye research and preclinical ocular drug and device development. This review summarizes published data, highlighting parameters of greater variability and identifying data gaps for species commonly used in the development of new therapeutics. Commercial Relationships: Joshua S. Eaton, None; Andrea D. Rodrigues, None; Craig B. Struble, None; Sara M. Thomasy, None; Christopher J. Murphy, None 302 Glial cell pathology in blinding disease - Minisymposium Tuesday, May 05, 2015 8:30 AM–10:15 AM 1AB Mile High Blrm Minisymposium Program #/Board # Range: 2561–2567 Organizing Section: Anatomy and Pathology/Oncology Program Number: 2561 Presentation Time: 8:30 AM–8:45 AM Why Glial Cells Matter to Retinal Function? Vijay P. Sarthy. Northwestern University, Chicago, IL. Presentation Description: Retinal health and function are supported by several types of glial cells: Müller cells, astrocytes, microglia and oligodendrocytes. This presentation will focus on our current knowledge of the structural, nutritional and trophic roles of retinal glia. It will describe novel roles for Müller cells in retinal regeneration following injury and in the cone-visual cycle, and potential roles of astrocytes in retinal ganglion cell function. Commercial Relationships: Vijay P. Sarthy, None Support: EY019325 Program Number: 2562 Presentation Time: 8:45 AM–9:00 AM Retinal glial cells in development and aging Tailoi Chan-Ling. University of Sydney, Sydney, NSW, Australia. Presentation Description: The retina is an unistratified avascular neuroepithelium early in human embryonic development. Retinal glia including Muller cells and astrocytes play pivotal roles in the normal development of the retina. This presentation will review the complex interactions between astrocytes, pericytes, blood vessels and neurons and how these complex interactions regulate retinal vascularization during development and disease. Changes in astrocytes including the decrease in the ratio of astrocytes servicing neurons likely affect the ability of astrocytes to service the functions of neurons with aging and thus contribute to disease pathogenesis. Commercial Relationships: Tailoi Chan-Ling, None Support: National Health and Medical Research Council of Australia. Rebecca Cooper Medical Research Foundation Program Number: 2563 Presentation Time: 9:00 AM–9:15 AM Glial cell pathology in glaucoma Yeni H. Yucel. 1University ofToronto, Toronto, ON, Canada; 2Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, ON, Canada. Presentation Description: In addition to neuron loss, glial cell pathology is implicated in glaucoma damage. Glial cell pathology in optic nerve and central visual pathways will be reviewed, in addition to relevant potential strategies to prevent vision loss in glaucoma. Commercial Relationships: Yeni H. Yucel, None Support: Canadian Institutes of Health Reseacrh, Canada Foudation for Innovation, Glaucoma Research Society of Canada, Program Number: 2564 Presentation Time: 9:15 AM–9:30 AM Role of retinal microglia in retinal degeneration Wai T. Wong. NEI, Bethesda, MD. Presentation Description: Microglia, the resident immune cell in the retina, are prominently involved in the pathology of retinal degenerations. However, whether microglia contribute or are simply reactive to photoreceptor degeneration has been unclear. This presentation describes how retinal microglia are altered during the course of retinal degeneration and the mechanisms by which they contribute to the progression of degeneration. Commercial Relationships: Wai T. Wong, None Support: NEI Intramural Research Program Program Number: 2565 Presentation Time: 9:30 AM–9:45 AM Astrocyte and Muller cell pathology in diabetic retinopathy Elia J. Duh. Johns Hopkins School of Medicine, Baltimore, MD. Presentation Description: Diabetic retinopathy (DR) is one of the leading causes of blindness in industrialized countries. Long regarded as a microvascular disease, it is increasingly evident that other retinal cell types are involved in the pathogenesis of DR. There is increasing awareness of the potential role of glial cells - Muller cells and astrocytes. Emerging evidence supports the concept that glial elements may contribute significantly to the progression of DR. This talk will discuss current knowledge of pathologic changes in astrocytes and Muller cells in diabetic retinopathy as well as their possible role in progression of the disease. Commercial Relationships: Elia J. Duh, None Support: NIH Grant EY022383, EY022683 Program Number: 2566 Presentation Time: 9:45 AM–10:00 AM Lessons from glial cell tumors of the eye Charles Eberhart. 1Wilmer Eye Institute, Baltimore, MD; 2Pathology, Johns Hopkins University, Baltimore, MD. Presentation Description: Ocular glia can proliferate and form both neoplastic and reactive mass lesions. The most common ocular gliomas occur in the optic nerve, and are generally low grade pilocytic astrocytoma. Many of these are associated with neurofibromatosis type 1 (NF1), and activation of the BRAF/MEK and AKT/mTOR pathways has been documented in NF1 deficient lesions. It has also recently been shown that small duplications of the BRAF gene leading to a constitutively active fusion protein are common in sporadic optic nerve gliomas not associated with NF1 mutation. The activation of BRAF in both syndromic and sporadic tumors leads to induction of p16 and oncogene induced senescence, which most likely explains the growth arrest and even regression often seen clinically in optic nerve low grade gliomas. Unfortunately, directly targeting mutant BRAF can cause paradoxical tumor growth, thus more sophisticated pharmacological approaches will be necessary to block this molecular arm in aggressive optic nerve gliomas. The mTOR pathway is also active in the majority of both NF1 associated and sporadic optic nerve gliomas, and clinical trials are underway in children. Finally, the Notch pathway can drive ocular glioma formation in mice, and is active in many human tumors. Intraocular gliomas are rare, and include retinal astrocytic neoplasms in tuberous sclerosis patients similar to giant cell astrocytomas seen in the brain. A number of “reactive retinal astrocytic tumors” have also been reported, and while their biology is not entirely clear, genetic studies suggest that they are not clonal neoplasms. The clinical, pathological and molecular features of the above tumors will be discussed. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Commercial Relationships: Charles Eberhart, None Program Number: 2567 Presentation Time: 10:00 AM–10:15 AM Harnessing glial cells to understand and treat diseases of the retina Deniz Dalkara. Institut de la Vision INSERM, Paris, France. Presentation Description: Müller glial cells are the principal glial cells of the retina. They provide architectural support and form the limits of the neurosensory retina at the outer and inner limiting membranes. They are involved in virtually every aspect of retinal homeostasis and maintenance. They are thus a main player in both acquired and inherited retinal disease and a target for therapeutic intervention. Müller glia respond to retinal disease and injury in ways that can be protective or detrimental to retinal function. Here, I will speak about viral tools to direct gene expression to Müller cells. Such viral tools allow us to both investigate Müller glial response to retinal disease and harness glial cells in gene therapeutic intervention to slow down or reverse retinal disease. Commercial Relationships: Deniz Dalkara, INSERM (P) Support: Association Francaise contre les Myopathies (AFM); Grant number: 14853, LABEX LIFESENSES [reference ANR-10LABX-65] were identified as multiple, small, amorphic calcified material within the dermis that was positive for calcium with Von Kossa stain. The spherical calcified deposits were surrounded by foreign body giant cells and lymphoplasmocytic chronic inflammation (image 1). On the other hand, the biopsy findings in elderly patients (3 cases) were characterized by a single, large, well-demarcated amorphous calcified material that also stained positive with Von Kossa, and was surrounded by fibrous tissue without chronic inflammation or foreign body reaction (image 2) Conclusions: SCN of the eyelid is a rare condition, but should be considered in any patient presenting with a painless white to yellowish colored nodule of the ocular adnexa. Clinician should be aware that this entity could be associated particularly in elderly patients, with systemic conditions. We have found 2 different histopathological patterns according to patients’ age that are associated with SCN of the eyelid and have not been described yet. 345 Tumors - Inside and around the eye, II Tuesday, May 05, 2015 11:00 AM–12:45 PM Exhibit Hall Poster Session Program #/Board # Range: 3407–3435/C0218–C0246 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Cornea, Eye Movements/Strabismus/ Amblyopia/Neuro-Ophthalmology, Immunology/Microbiology, Multidisciplinary Ophthalmic Imaging, Physiology/Pharmacology Program Number: 3407 Poster Board Number: C0218 Presentation Time: 11:00 AM–12:45 PM Subepidermal calcified nodule of the eyelid Saeed AlWadani2, Charles Eberhart3, Hind ALKatan1, Maria J. Suarez B3, Jonathan Kass3. 1KKESH, Riyadh, Saudi Arabia; 2 Ophthalmology department, King Saud University, Riyadh, Saudi Arabia; 3johns Hopkins Hospital, Baltimore, MD. Purpose: Subepidermal calcified nodule of the eyelid (SCN) is considered one of the types of Calcinosis cutis.The purpose of this study was to study the clinical features and histopathological findings in patient diagnosis with SCN of the eyelid Methods: There were 13 patients identified who had been diagnosed with SCN of the eyelid. A chart review was conducted on all patients to identify any salient past medical history, trauma or concurrent systemic disease. Histopathological analysis was performed for each case using hematoxylin-eosin stains. In selected cases, Von Kossa stain was also used to identify and characterize the nature of calcium deposition, as well as immunostains including CD3, CD20 for chronic inflammation and CD68 to recognize granulomatous inflammation. Results: We have found 13 cases of SCN diagnosis in our ophthalmic pathology archive. Clinical information is summarized in table 1.Two of these patients presented with systemic disease association including hypertension and gout. The other 11 patients were otherwise healthy and most of them presented as white, firm, nodules. All cases from our review received surgical excision of the lesion as treatment under local anesthsia. Microscopically, we described 2 different histopathological patterns according to the age of patients. In younger patients (10 cases, histopathological features ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology pattern and it may be confused with other tumors, thus in order to make the diagnosis of this adenoma, resection is needed along with histopathologic analysis and the identification of specific immunohistochemical markers. Commercial Relationships: Laura Andrea Torrado, None; Juan Carlos Serna-Ojeda, None; Enrique Ariza-Camacho, None; Alberto Collado- Solorzano, None; Blanca C. Flores- Sanchez, None; Abelardo Rodriguez- Reyes, None; Emiliano Fulda-Graue, None Commercial Relationships: Saeed AlWadani, None; Charles Eberhart, None; Hind ALKatan, None; Maria J. Suarez B, None; Jonathan Kass, None Support: Non Program Number: 3408 Poster Board Number: C0219 Presentation Time: 11:00 AM–12:45 PM Adenoma of the nonpigmented ciliary body and iris epithelium in Mexican mestizo patients Laura Andrea Torrado1, Juan Carlos Serna-Ojeda1, Enrique ArizaCamacho1, Alberto Collado- Solorzano1, Blanca C. Flores- Sanchez2, Abelardo Rodriguez- Reyes2, Emiliano Fulda-Graue1. 1Instituto de Oftalmologia, Mexico, Mexico; 2APEC, Mexico, Mexico. Purpose: Describe the clinical characteristics, ultrasound findings, and histopathologic and immunohistochemical results from mexican mestizo patients with adenoma of the nonpigmented ciliary body and iris epithelium Methods: Retrospective analysis of the interventional case series of four patients with final diagnosis of adenoma of the nonpigmented ciliary body and iris epithelium. Results: Median age at presentation was 50 years (range 15 - 75 years). Half of the patients presented with decreased visual acuity and the other half with changes in iris pigmentation. Ultrasound revealed a solid mass of homogeneous density and high internal reflectivity and the biomicroscopy showed the anatomical origin. In all the patients an excisional biopsy with partial lamellar sclerouvectomy was performed for accurate diagnosis. One patient required a 23-gauge vitrectomy at the same surgical time because of retinal detachment. Histopathology reported sheets of cells with clear cytoplasm surrounded by basement membrane and tubular differentiation. Inmunohitochemistry was positive por vimentin, S-100, epithelial membrane antigen (EMA) and neuron specific enolase (NSE). Conclusions: The adenoma of the nonpigmented ciliary body is a benign rare tumor with few reports in literature, being this the first latin american case series of this entity. Because of its origin, the clinical presentation is varied without an established Program Number: 3409 Poster Board Number: C0220 Presentation Time: 11:00 AM–12:45 PM Topographic distribution of ocular vascular lesions: a 20-year study Tânia Borges1, 2, Taylor Nayman2, Ana Beatriz T. Dias2, Sultan Aldrees2, Beatriz Nugent da Cunha2, Miguel N. Burnier2. 1Centro Hospitalar do Porto, Porto, Portugal; 2Henry C. Witelson Ocular Pathology Laboratory, Montreal, QC, Canada. Purpose: The clinical diagnosis of ocular vascular lesions is challenging for ophthalmologists due to similarities between the different categories of these lesions and between them and other nonvascular lesions. The purpose of this study is to describe the clinical and pathological characteristics and the frequency of different ocular vascular lesions in order to assist with differential diagnoses. Methods: We reviewed 15,512 cases diagnosed at the Henry C. Witelson Ocular Pathology Laboratory at McGill University during a 20-year period. Clinical and pathological data of all ocular vascular lesions diagnosed during this period were retrieved. Descriptive analysis of various lesions based on site (eyelid, conjunctiva, and orbit), frequency, gender, and age was performed. Results: Of the 15,512 specimens reviewed, 115 (0.74%) cases were ocular vascular lesions. Female patients represented approximately half the study population (55.17%). Most patients presented in the 41–60 year age group (42.61%). The most common site of involvement was the eyelid (52.17%), followed by the orbit (27.83%) and conjunctiva (20%). The most common eyelid lesion was capillary hemangioma (36.67%). However, the most common orbit and conjunctiva lesions were cavernous hemangioma (81.25%) and vascular hamartoma (52.17%), respectively. Four malignant lesions were found: Kaposi’s sarcoma (n = 2) and epithelioid hemangioendothelioma (n = 2). Three of these lesions were in the eyelid and one case was conjunctival. One case was misdiagnosed clinically as a benign lesion. Of the 101 cases with clinical diagnoses, 28 cases (27.72%) were misdiagnosed clinically as non-vascular lesions. Encountered lesions include: cavernous hemangioma (38.26%), vascular hamartoma (25.22%), capillary hemangioma (22.61%), lymphangioma (6.96%), vascular ectasia with thrombosis (1.74%), epithelioid hemangioma (0.87%), and intravascular papillary endothelial hyperplasia (0.87%). Conclusions: The location of ocular vascular lesions is an important feature for differential diagnoses. There was a lack of a correlation between clinical and histopathological diagnosis in one third of cases. The eyelid is the most common site of ocular vascular lesions. Capillary hemangioma is the most common eyelid vascular lesion; however, cavernous hemangioma is the most frequent lesion of all studied sites. Malignant ocular vascular lesions are exceedingly rare. Commercial Relationships: Tânia Borges, None; Taylor Nayman, None; Ana Beatriz T. Dias, None; Sultan Aldrees, None; Beatriz Nugent da Cunha, None; Miguel N. Burnier, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 3410 Poster Board Number: C0221 Presentation Time: 11:00 AM–12:45 PM The Occurrence of Lens Tumors in Humans and Other Species and a Perceived Opportunity for Further Study Krishna R. Surapaneni1, Paul O. Phelps1, Bradley Thuro1, Richard R. Dubielzig2, Daniel M. Albert1. 1Department of Ophthalmology and Visual Sciences, University of Wisconsin – Madison, Madison, WI; 2 Department of Pathobiological Sciences, University of Wisconsin – Madison, Madison, WI. Purpose: The purpose of this census was to determine in which species and under what conditions lens tumor occurs using human and veterinary pathological material available to us. Methods: Material in two major archived collections at the University of Wisconsin medical and veterinary schools were studied for occurrence of lens tumors. A database of material from 1920 to 2014 was included, which was approximately 17500 human eye pathology cases, and 45000 veterinary cases. In addition, cases presented in every major eye pathology society (VerhoeffZimmerman, Eastern Ophthalmic Pathology Society, and Armed Forces Institute of Pathology Alumni Society) were reviewed. Finally, a careful search of the literature was carried out using variations of the terms “Lens, Crystalline”[Mesh]) AND “Neoplasms”[Majr]. Results: The database search revealed that lens tumors occurred under natural conditions in cats and rabbits. Five percent of feline neoplasms (257/5048) in the veterinary school database were designated a Feline Ocular Post-traumatic Sarcoma (FOPTS), a tumor demonstrated to be of lens epithelial origin. Three similar tumors seen in chronically disease rabbit eyes are suspected because or remarkable similarity to FOPTS. There are three reports of these FOPTS-like tumors rabbits in the literature. All cat and rabbit sarcomas had a history of either ocular trauma or protracted uveitis. Literature search also revealed cases where lens tumors were induced in zebrafish, rainbow trout, hamsters, and mice, by carcinogenic agents (methylcholanthrene, thioacetamide), oncogenic viruses (SV40, HPV-16), and genetic manipulation (bumper, p53). No evidence of lens tumors was found in human pathologic material or extensive literature search. Conclusions: Following lens capsule rupture a malignant lens tumor can occur in other species, but not in humans. We hypothesize that a genetic mechanism exists which prevents lens tumors in man. We have begun a search for candidate genes in other species involved in tumor formation for subsequent comparison in humans. Commercial Relationships: Krishna R. Surapaneni, None; Paul O. Phelps, None; Bradley Thuro, None; Richard R. Dubielzig, None; Daniel M. Albert, None Support: Department of Ophthalmology and Visual Sciences CORE Grant (UW – Madison), and Financial Support from Ocular Services on Demand (OSOD) Program Number: 3411 Poster Board Number: C0222 Presentation Time: 11:00 AM–12:45 PM Clinical relevance of EMT in eyelid sebaceous gland carcinoma: an immunohistochemical and molecular study Mansi Bhardwaj1, Seema Sen1, Anjana Sharma2, Neelam Pushker3, Seema Kashyap1, Mandeep S. Bajaj3, Kunzang Chosdol4, Sameer Bakhshi5. 1Ocular Pathology, Dr.R.P.Centre for Ophthalmic Sciences, All India Institue of Medical Sciences, New Delhi, India; 2Ocular microbiology, Dr.R.P.Centre, AIIMS, New Delhi, India; 3Ophthalmology, Dr.R.P.Centre, AIIMS, New Delhi, India; 4 Biochemistry, AIIMS, New Delhi, India; 5Medical Oncology, IRCH, AIIMS, New Delhi, India. Purpose: Sebaceous gland carcinoma (SGC) is the most common malignant eyelid tumor with a high rate of incidence in Asia. Invasion and metastasis in several carcinomas is promoted by ‘Epithelial Mesenchymal Transition’ (EMT) where epithelial cells acquire a mesenchymal phenotype.It is mediated by transcription factors like Slug,ZEB1 & ZEB2 which causes loss of cell-cell adhesion molecule E-cadherin.The present study was designed to determine the status of EMT markers Slug,ZEB1,ZEB2 and E-cadherin and to correlate with high risk features of eyelid SGC. Methods: Prospective analysis of 30 eyelid SGC patients was undertaken. Clinicopathological features were noted and H&E stained sections were reviewed to confirm the diagnosis.AJCC staging (2009) was done and patients were followed up for 37 months(Mean: 22.5±9.08).Immunohistochemistry(IHC) was performed using antibodies against ZEB1,ZEB2,Slug and E-cadherin. mRNA analysis by quantitative Real Time PCR was performed in all the tumour samples and adjoining normal skin tissues.Results were correlated with high risk features and follow up data.Statistical analysis was performed using Fisher Exact and Spearman’s Rank Correlation Tests. Results: Mean age of patients was 58.9±13.2yrs (male:female ratio1.1:1).IHC and mRNA analysis revealed ZEB2,ZEB1 and Slug overexpression in 67%(20/30),40%(12/30) and 10%(3/30) cases respectively.E-cadherin loss was seen in 63%(19/30) cases. IHC expression of all 4 EMT markers significantly correlated with mRNA levels.ZEB2 overexpression was significantly associated with high risk features like poor histological differentiation (P=0.017) & pagetoid spread (P=0.023),whereas E-cadherin loss was significantly associated with large tumour size(P=0.023) & pagetoid spread (P=0.018).Both ZEB2 overexpression and E-cadherin loss were present in 88%(7/8) cases with recurrence.ZEB2 overexpression was also seen in 75%(6/8) cases with lymph node metastasis.There was a significant inverse correlation between ZEB2 and E-cadherin expression (P=0.038).Other EMT markers ZEB1 and Slug did not correlate with any clinicopathological high risk features. Conclusions: EMT is an important phenomenon in eyelid SGC which could be responsible for its aggressive behaviour.Both ZEB2 and E-cadherin are important biomarkers for detection of high risk SGC cases.Further validation on a larger series of cases,with longer follow up is however necessary. Commercial Relationships: Mansi Bhardwaj, None; Seema Sen, None; Anjana Sharma, None; Neelam Pushker, None; Seema Kashyap, None; Mandeep S. Bajaj, None; Kunzang Chosdol, None; Sameer Bakhshi, None Program Number: 3412 Poster Board Number: C0223 Presentation Time: 11:00 AM–12:45 PM Ocular Ultrastructural Features of Gaucher Disease Mones S. Abu-Asab, Christopher Ardeljan, Chi-Chao Chan. Lab of Immunol/Sect of Immunopath, National Eye Institute, Bethesda, MD. Purpose: Gaucher disease is a lysosomal storage disorder resulting from mutations in the enzyme glucocerebrosidase (aka b-glucosidase). It leads to the massive accumulation of glucocerebroside (glucosylceramide) within phagocytic cells throughout the body, particularly white blood cells such as macrophages. Lysosomes turn into Gaucher bodies, and cells with Gaucher bodies turn into Gaucher cells. In addition to histology, we have undertaken an ultrastructural approach to examine a case of Gaucher disease in order to uncover the state of affected ocular tissues and their cellular organelles. Currently, there is no adequate ultrastructural survey of Gaucher in the eye. Methods: A postmortem ultrastructural examination and immunohistochemistry was performed on ciliary body (OD & OS), retina (OD & OS), choroid (OD & OS), sclera (OD & OS), corneal ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology limbus (OS), and cornea (OD). The patient was a 61 year old woman with type I Gaucher since the age of 14. The tissues were prepared for transmission electron microscopy. Results: By light microscopy, Gaucher body inclusions (GBIs) were seen in ciliary body, choroid, sclera, and some infiltration in the retina. There were lipid-laden foamy histiocytes throughout the choroid, many CD68+ macrophages, and a few of CD45RO+ T cells, CD3+ T cells, and CD20+ B cells. EM showed GBIs present in ciliary pigmentary epithelium, while ciliary non-pigmentary epithelium appeared vacuolated probably due to the remains of storage vacuoles. Axons of the ciliary body had abnormal and degenerate myelin sheaths and numerous membranous myelin inclusions; and Schwann cells contained cholesterol inclusions. Choroid was full of Gaucher cells filled with GBIs. GBIs were found mainly along the vessels in sclera. In the retina, some neuronal cells contained fine homogenous granular materials that had replaced normal cytoplasmic organelles mainly in the GCL and INL. No GBIs were seen in the cornea, corneal limbus, neural retina, or RPE. In the cornea, there were melanosome laden macrophages, but no GBIs. Conclusions: Gaucher disease affected the ocular tissues and GBIs were evident in the ciliary body, choroid, and sclera. Furthermore, there were other pathological transformations such as degenerate axons in ciliary body, large inclusions in retinal ganglion cell layer, melanosome-laden macrophages in the cornea, and condensed Bruch’s membrane with loss of elastin fibrils. Commercial Relationships: Mones S. Abu-Asab, None; Christopher Ardeljan, None; Chi-Chao Chan, None Program Number: 3413 Poster Board Number: C0224 Presentation Time: 11:00 AM–12:45 PM An immunohistochemical study for the tumorigenesis of feline ocular post-traumatic sarcoma Hiroki Takahashi1, Shunichiro Ueda1, Jun Matsubayashi2, Leandro B. Teixeira3, Richard R. Dubielzig3, Hiroshi Goto1. 1Ophthalmology, Tokyo Medical University, Shinjyuku-ku, Japan; 2Anatomic Pathology, Tokyo Medical University, Tokyo, Japan; 3Pathological Sciences, University of Wisconsin, Madison, WI. Purpose: Feline ocular post-traumatic sarcomas (FOPTS) are malignant intraocular neoplasms that are frequently associated with a history of ocular trauma and chronic inflammation. However, the tumorigenesis of FOPTS is unknown. In this study, we evaluated the possible association between the epithelial mesenchymal transition (EMT) of lens epithelial cells (LEC) and the tumorigenesis of FOPTS. Methods: The database of the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) was searched and records of FOPTS spindle cell variant were examined. FOPTS spindle cell variant were divided into 4 categories: Spindle cell metaplasia (No tumor but LECs have proliferated), Early FOPTS (LECs were extending beyond the lens capsule), Full FOPTS (Fully developed tumors which have spread entirely around the eye), and Extensive FOPTS (Fully developed tumors extend beyond the sclera). 17 cases from each category were examined immunohistochemical phenotypes. Immunohistochemistry were performed to detect expression of Pancytokeratin (P-CK: AE1/AE3), Vimentin, α-SMA, S100A4, and Ki-67. We also compared the number of P-CK and α-SMA positive case of the inside and outside the lens capsule. Results: In all cases, tumor cells were positive for Vimentin and S100A4. The number of P-CK positive cases gradually decreased with tumor progression. On the other hand, the number of α-SMA positive cases gradually increased with tumor progression. Ki-67 labeling index also gradually increased with tumor progression. In cases of early FOPTS intracapsular LECs were more frequently P-CK positive than extracapsular LECs and extracapsular LECs were more frequently α-SMA positive than intracapsular LECs. However, there was no significant difference between the number of P-CK and α-SMA positive cells of the inside and outside the lens capsule in Full FOPTS. Conclusions: In FOPTS, EMT phenomenon was enhanced with the category progression. Our immunohistochemical findings were suggested that lens epithelial cells might associate with tumorigenesis of FOPTS. Commercial Relationships: Hiroki Takahashi, None; Shunichiro Ueda, None; Jun Matsubayashi, None; Leandro B. Teixeira, None; Richard R. Dubielzig, None; Hiroshi Goto, None Program Number: 3414 Poster Board Number: C0225 Presentation Time: 11:00 AM–12:45 PM A case series of canine pleomorphic iridociliary adenocarcinomas Gillian C. Shaw, Leandro B. Teixeira, Richard R. Dubielzig. COPLOW & Pathobiological Sciences, University of Wisconsin, Madison, WI. Purpose: To describe a case series of pleomorphic iridociliary adenocarcinomas in dogs. Methods: The COPLOW database was mined for cases of canine pleomorphic iridociliary adenocarcinomas. Cases were reviewed and described, and additional clinical data were collected and summarized. Immunohistochemical staining was performed on a subset of cases. Results: There are 22 cases of canine pleomorphic iridociliary adenocarcinomas in the COPLOW collection representing 1.5% of all canine iridociliary epithelial tumors. The average age at enucleation was 10.9 years and there were 13 males and 8 females. There were 7 Labrador retrievers, 4 golden retrievers, 2 shih tzus, 3 mixed breeds and a single dog from the following breeds: border collie, Shetland sheepdog, boxer, vizsla, Maltese, dachshund. Elevated intraocular pressure, uveitis, corneal disease and hyphema were common presenting complaints. Seven cases (31.8%) had a history of chemical ciliary body ablation with an intravitreal gentamycin injection and three (13.6%) had known or suspected ocular trauma. All had a history of ocular abnormalities for months to years. Histologically, the tumors are composed of cuboidal to polygonal cells forming cords, trabeculae and sheets with variably prominent PAS-positive basement membranes and exhibit extensive invasion of intraocular structures and/or sclera. Of the tumors stained immunohistochemically, the neoplastic cells stain positively for vimentin and variably positive for pancytokeratin. Conclusions: These canine neoplasms represent the most malignant of tumors arising from the anterior uvea and share histologic and immunohistochemical features with human pleomorphic adenocarcinomas of the ciliary body. The development of these tumors in canine globes is associated with a history of chemical ciliary body ablation, trauma and long standing ocular disease. Commercial Relationships: Gillian C. Shaw, None; Leandro B. Teixeira, None; Richard R. Dubielzig, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 3415 Poster Board Number: C0226 Presentation Time: 11:00 AM–12:45 PM Mantle Cell Lymphoma of the Ocular Adnexa Roxana Saucedo Urdapilleta2, Abelardo A. Rodriguez-Reyes2, Hector A. Rodriguez-Martinez1, Carmen Lome-Maldonado3, Ivette Hernandez-Ayuso2, Rosario Gulias-Cañizo2, Dolores Ríos y VallesValles2. 1Experimental Medicine, Hospital General de México, Mexico city, Mexico; 2Ophthalmic Pathology, Asociación para Evitar la Ceguera en México I.A.P. Dr. Luis Sánchez Bulnes, Mexico city, Mexico; 3Pathology, Instituto Nacional de Ciencias Médicas y Nutrición, Salvador Zubirán, Mexico city, Mexico. Purpose: Mantle cell lymphoma (MCL) is a rare type of lymphoma. Reports of MCL in the ocular adnexa have been published in only some studies with a limited number of cases. We reviewed the frequency of MCL of the ocular adnexal region in a large Mexican referral center. Methods: Retrospective review of clinical records, histopathology and immunohistochemistry of medical cases (all genders and ages) diagnose with MCL of ocular adnexal from 1958-2013. a. Files from the Ophthalmic Pathology Service from the “Asociación para Evitar la Ceguera en México I.A.P. Dr. Luis Sánchez Bulnes”. b. Cases with diagnosis of Mantle cell lymphoma. c. Clinical data recorded for each patient (included year of diagnosis, gender, age, symptoms and clinical findings). d. Evaluation of clinical pictures, ocular ultrasound, computed tomography and magnetic resonance imaging. e. Followup. Results: Twelve patients with MCL of the ocular adnexa were identified comprising 8% (12/155) of all lymphomas in the ocular region. There were 6 male patients and 6 female patients with an age range from 32 to 79 years old (median 66 years). Forty two percent had bilateral involvement. The orbit (83%) and the lacrimal gland (42%) were the most commonly affected sites. MCL of the adnexal region was the first manifestation of systemic disease. Fifty percent presented in stage II, one stage IV. Microscopically all the cases had a diffuse architectural pattern and expressed CD20 and cycline D1. Conclusions: Twelve of one hundred and fifty five patients were diagnosed with MCL. Commonly ocular adnexa MCL presents in elderly males, however our patients did no present predominance of gender. The orbit and lacrimal gland were frequently involved. Less than 50% of our patients had bilateral orbital involvement. In the current series the end clinical stage was not as common as other series in the literature. Inmmunohistochemistry is mandatory to establish the definitive diagnosis. Commercial Relationships: Roxana Saucedo Urdapilleta, None; Abelardo A. Rodriguez-Reyes, None; Hector A. RodriguezMartinez, None; Carmen Lome-Maldonado, None; Ivette Hernandez-Ayuso, None; Rosario Gulias-Cañizo, None; Dolores Ríos y Valles-Valles, None Program Number: 3416 Poster Board Number: C0227 Presentation Time: 11:00 AM–12:45 PM Lymphoproliferative Disease of the Ocular Adnexa: Clinical Features and Subtypes Nitika Arora1, Catherine E. Cuite2. 1Internal Medicine, University of Illinois College of medicine, Peoria, IL; 2Oculoplastics, Illinois Eye Center, Peoria, IL. Purpose: To study the clinical features of patients presenting with ocular adnexal Lymphoproliferative disease (OALD) and their relationship with different histologic subtypes. Methods: Data was collected retrospectively for 36 cases of OALD that were biopsied by one surgeon between 2000 and 2014. All patients were classified according to the World Health Organization modification of the Revised European American Classification. Descriptive statistics were calculated for demographics, histologic subtype of tumor, clinical stage at presentation, tumor location, presenting symptoms and tumor related mortality. Tumor location was reported as orbital, lacrimal or conjunctival. Presenting symptoms included swelling, double vision, prominent globe and tearing. Relationships between the histologic subtype and other variables were explored using two sample t test and Fisher’s exact test. Results: The mean age of the patients was 68.6 ± 13.4 years, and 47% were males. Fifty percent of patients had mucosa associated lymphoid tissue (MALT) followed by follicular type in 27.8%. The rest were small cell, large cell and mantle cell lymphomas. On comparing MALT and follicular type, there was no significant difference in the age of the presentation, gender, location of the tumor or clinical features of presentation (p>0.05 for all). Eighty percent of follicular lymphomas are secondary as compared to 11% in case of follicular (p=0.023). 69.4% of all the tumors were primary. Conclusions: The diagnosis of orbital lymphomas is challenging because these present with few specific features. According to our study, clinical presentation, age and gender are not significant determinants of histologic subtype. Although majority of tumors were primary, follicular Lymphoma is more likely to be a secondary tumor. Follicular Lymphoma may have increased tumor-related death but our small cohort could not detect statistical significance of this finding. Commercial Relationships: Nitika Arora, None; Catherine E. Cuite, None Program Number: 3417 Poster Board Number: C0228 Presentation Time: 11:00 AM–12:45 PM Incidence of retinal detachment in primary vitreoretinal lymphoma (VRL) Irina Belinsky1, Song Eun Lee2, William M. Schiff3, Brian P. Marr1. 1 Ophthalmic Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY; 2Ophthalmology, Harkness Eye Institute, Columbia University, New York, NY; 3Ophthalmology, Manhattan Eye Ear and Throat Hospital, New York, NY. Purpose: Primary vitreoretinal lymphoma (VRL) is rare but potentially fatal, presenting a diagnostic and therapeutic challenge by masquerading as infectious or inflammatory conditions. We have observed an increased incidence of retinal detachment in our cohort of patients with VRL. To characterize this observation further, we studied this population with attention to demographics, clinical course, and treatment. Methods: Retrospective review of 26 consecutive cases at a large Ocular Oncology Center. Results: Between 2006 and 2014, a total of 26 patients (44 eyes) were identified with primary vitreoretinal lymphoma, of whom 81% were female and 19% male, 92% were Caucasian, and the mean age at presentation was 62 years. The mean follow up period was 32 months. 81% (n=21) had biopsy proven VRL and 29% (n=5) were diagnosed clinically. 69% (n=18) had bilateral disease and 31% (n=8) unilateral. 27% (n=7) of patients had a history of retinal detachment ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology (RD), 86% (n=6) unilateral and 14% (n=1) bilateral and one patient developed a recurrent RD in one eye, totaling 9 retinal detachments in this cohort. 33% (n=3) of RD’s occurred prior to the diagnosis of VRL, 56% (n=5) of RD’s occurred at presentation, and 11% (n=1) occurred during the course of the disease. 89% (n=8) received some form of surgical treatment, with no recurrences except one to date. 58% (n=15) of patients had associated primary central nervous system lymphoma (PCNSL) and 12% (n=3) died as a result of their lymphoma. Conclusions: We observe a high incidence of retinal detachment in patients with primary vitreoretinal lymphoma. The etiology of this association is unclear. Retinal detachments may be caused by some effect of lymphoma on the vitreous, the retina, or the interface. Furthermore, these patients receive numerous diagnostic and therapeutic interventions. Further studies with more patients and longer follow up are needed. We propose that this observation is clinically relevant and warrants careful surveillance for retinal detachment during follow up and treatment as well as patient counseling. Commercial Relationships: Irina Belinsky, None; Song Eun Lee, None; William M. Schiff, None; Brian P. Marr, None Program Number: 3418 Poster Board Number: C0229 Presentation Time: 11:00 AM–12:45 PM A large series of blind painful eyes: potential causes and associated histopathological features Cristina Fonseca1, 2, Norah Alsaif2, Mohammed F. Qutub2, Silvin Bakalian2, Vasco Bravo-Filho2, Miguel N. Burnier2. 1Opthalmology, Centro Hospitalar e Universitario de Coimbra, Coimbra, Portugal; 2 Henry C. Witelson Ocular Pathology Laboratory, Montreal, QC, Canada. Purpose: A blind painful eye is the end stage of several ocular conditions, including inflammatory, vascular, and glaucomatous changes. The aim of this project is to describe the morphological findings of blind painful eyes and to evaluate the most frequent histopathological features of this condition. Methods: One hundred and seventy-two cases of enucleated or eviscerated blind painful eyes were reviewed from the Henry C. Witelson Ocular Pathology Laboratory, McGill University over a 21 year period (1993–2014). A review of the histopathological features to establish criteria for the diagnosis of blind painful eyes was performed. The underlying causes of the blind painful eyes were determined clinically and histopathologically. Results: Of the 172 blind painful eyes, 95 (55.2%) were eviscerated and 77 (44.7%) were enucleated. Phthisical eyes were diagnosed by the presence of disorganization with osseous metaplasia and calcification in shrunken eyes (<16 mm in diameter), and represented 18% of all cases. Mean patient age was 55.6 years (1–88) with equal sex distribution. Fifty-four histopathological features were identified, the most common being retinal gliosis (70.9%), chronic keratitis (69.1%), and non-granulomatous chronic uveitis (50%). Other findings included total retinal detachment (34.3%), subretinal hemorrhage (32.5%), anterior synechia (29.6%), optic nerve atrophy (25.5%), subepithelial pannus (25%), bone metaplasia (16.8%), and rubeosis iridis (8.1%), among others. The histopathological changes were further classified according to clinicopathologic features: over 90% of cases had findings consistent with retinal vascular diseases, retinal detachment (83%), uveitis (79%), keratitis (72%), glaucomatous changes (37%), cataract (21%), and corneal decompensation (18%). Conclusions: To the best of our knowledge, this is the first study of blind painful eyes describing the histopathological features and the underlying ocular disease. The clinical criteria for the diagnosis of a blind painful eye includes retinal detachment and gliosis, disorganization of ocular structures, and glaucomatous atrophic changes. A wide spectrum of ocular conditions can lead to a blind painful eye. Pathological evaluation of blind painful eyes may help ophthalmologists formulate an accurate clinicopathological correlation of the baseline ocular disease. Commercial Relationships: Cristina Fonseca, None; Norah Alsaif, None; Mohammed F. Qutub, None; Silvin Bakalian, None; Vasco Bravo-Filho, None; Miguel N. Burnier, None Program Number: 3419 Poster Board Number: C0230 Presentation Time: 11:00 AM–12:45 PM Genetic alterations in conjunctival melanoma and relation to clinical outcome Nihal Kenawy1, Sarah E. Coupland1, Bertil E. Damato2, Heinrich Heimann3, Sarah L. Lake1. 1University of Liverpool, Liverpool, United Kingdom; 2Ocular Oncology Service, University of California, San Fransisco, CA; 3Liverpool Ocular Oncology Centre, Royal Liverpool University Hospital, Liverpool, United Kingdom. Purpose: Despite improved understanding of the molecular changes in conjunctival melanoma (CoM), the underlying aetiology of this tumor remains unclear. In this study, we determined gene copy number variations (CNV) in CoM aiming to identify disease-specific genetic biomarkers to facilitate prognostication, as has been achieved in uveal melanoma. Methods: Ninety two patients with primary CoM seen between 2005 and 2014 were recruited in eight international ocular oncology centres. Clinical and histological data were collected. DNA was extracted from paraffin-embedded samples. Fifty-nine samples yielded sufficient DNA for Affymetrix 6.0 Single Nucleotide Polymorphism (SNP) microarray testing. SNP data were analysed by Partek Genomic suite. Patients who developed regional lymph nodes and/or systemic metastases were compared to a sex, age-matched cohort of low risk clinical and pathological criteria for dissemination and with similar follow up duration. Results: Over 40% of the samples showed amplifications of histone gene cluster (6p22.2), ETV1 (7p21.2), FOXQ1 (6p25.3 - 6p25.2) and SOX4 (6p22.3) and deletions in ASNS (7q21.3), CHEK2P2 (15q11.1), RET (10q11.21) and BAGE (21p11.1). CNVs in the two groups of metastatic CoM and the low risk comparative group were consistent. None of the chromosomes identified with gains or losses showed loss of heterozygosity or isodisomy. Based on the current median follow-up time of 2.5 years, no statistically significant correlation has been detected between any of the genetic alterations and features of poor outcome, i.e. caruncular involvement, epithelioid cell type or metastatic spread. Conclusions: This is the largest cohort of CoM samples analysed by SNP genotyping to date and describes CNVs not previously reported. Longer follow-up is required to facilitate our understanding of CoM and identification of patients at high metastatic risk. Commercial Relationships: Nihal Kenawy, None; Sarah E. Coupland, None; Bertil E. Damato, None; Heinrich Heimann, None; Sarah L. Lake, None Support: Eye Tumour Research Fund A0722/CF Program Number: 3420 Poster Board Number: C0231 Presentation Time: 11:00 AM–12:45 PM HSP90 expression is a useful tool for the differential diagnosis of ocular surface squamous neoplasia Ana Beatriz T. Dias, Pablo Zoroquiain, Dominique F. souza, Dana Faingold, Patrick T. Logan, Miguel N. Burnier. McGill University, Montreal, QC, Canada. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Purpose: HSP90 is a chaperone protein that stabilizes and activates client proteins involved in malignant transformation. HSP90 is overexpressed in many malignant tumors, including squamous cell carcinomas of the head and neck. The aim of this study is to evaluate HSP90 expression in ocular surface squamous neoplasia (OSSN) and to assess its diagnostic value for the different lesions of the OSSN spectrum. Methods: Seventy conjunctival squamous lesions, including 19 papillomas (Pa), 22 conjunctival intraepithelial neoplasia (CIN) I, 11 CIN II, 6 CIN III, and 12 squamous carcinoma (sqCA), were evaluated using automated immunohistochemical staining against HSP-90. As controls, 15 normal conjunctiva specimens were used. The staining was scored by evaluating staining intensity (0–3) and extent (0–4) and these values were multiplied to generate an immunoreactive score (IRS; 0–12). This process was performed for nuclear (nIRS) and cytoplasmic (cIRS) HSP90 staining. Also, intraepithelium staining was evaluated as a percentage of total thickness (PTE). Results: All OSSN and 90% of controls were positive for HSP90; however, mean benign and malignant OSSN IRS were significantly higher than normal conjunctiva (P<0.0001). With respect to OSSN, cIRS had a higher HSP90 IRS in high grade compared to low grade lesions (Pa and CIN I; P<0.001). nIRS was significantly different between high grade lesions (CIN II vs CIN III-sqCA; P=0.0162). A cIRS >6 correlated with a high grade lesion (sensitivity [S] 58.3%, specificity [Sp] 97.4%), while a cIRS <4 correlated with a low grade lesion (S 43.6%, Sp 100%). An nIRS >6 in high grade lesions correlated with CIN III-sqCA (S 55.6%, Sp 81.8%), while an nIRS <4 correlated with CINII (S 45.5%, Sp 94.4%). PTE staining of <73% differentiated between CIN III and sqCA with an S of 91.7% and Sp of 100% for sqCA. nIRS, cIRS, and cIRS + nIRS did not correlate with the depth of infiltration or sqCA differentiation degree. Conclusions: To the best of our knowledge, this is the first study using HSP90 as a marker to differentiate between benign, premalignant, and malignant lesions of the conjunctiva. The expression of HSP90 is particularly useful to differentiate low from high grade CIN and invasive sqCA of the conjunctiva. HSP90 may play an important role in stabilizing and activating client proteins involved in this malignant transformation. Commercial Relationships: Ana Beatriz T. Dias, None; Pablo Zoroquiain, None; Dominique F. souza, None; Dana Faingold, None; Patrick T. Logan, None; Miguel N. Burnier, None Program Number: 3421 Poster Board Number: C0232 Presentation Time: 11:00 AM–12:45 PM Expression of nestin in conjunctival melanoma Marie-Sophie Hanet, Henning Thomasen, Henrike Westekemper, Klaus-Peter Steuhl, Daniel Meller. Ophthalmology, University of Duisburg-Essen, Essen, Germany. Purpose: Conjunctival melanoma is a rare malignant tumor, of which the biology remains largely unknown. Nestin, an intermediate filament protein described as neural stem cell marker, has been reported in cutaneous melanocytic tumors with potential implication for their diagnosis and staging. We analyzed the immunological properties of conjunctival melanomas and hypothesized that nestin could also have clinical interest as a biomarker in conjunctival melanomas. Methods: Samples of tumoral tissue from five patients with a primary conjunctival melanoma and four cell lines (UKE29, UKE17, CR1 and CR2) derived from conjunctival melanoma were analyzed. Ten samples of limbal (n=5) or fornical (n=5) conjunctiva obtained from healthy subjects were used as controls. Expression of nestin and other pluripotency markers (Sox-2, Oct-4, Nanog, c-Myc, ABCG2, p63, and c-KIT) was examined using real-time polymerase chain reaction. Expression of nestin was additionally examined using immunohistochemical staining. Results: Expression of nestin was significantly higher in melanoma tissue than in controls (p<0.008 vs. limbal and p<0.02 vs. fornical conjunctiva), which was consistent with the results of immunological staining. The expression of other markers of pluripotency was not statistically different between melanomas and normal tissues from either limbal or fornical conjunctiva. In all cell lines the expression of nestin was inferior to the melanoma tissues. The expression of other markers of pluripotency was generally lower in cell lines compared to melanoma tissues, except for ABCG2 in UKE29 and p63 in UKE17. Compared to control tissues, nestin expression was higher in the cell line UKE17 than in limbal controls, and higher than both limbal and fornical controls in the cell line UKE29. The expression of other pluripotency markers was inferior in cell lines compared to all controls, apart from c-Myc in the cell line CR1 and ABCG2 in UKE 29. Conclusions: Nestin is significantly overexpressed in conjunctival melanoma, whereas the expression shows a decrease in cell lines consistent with the general pattern of expression of other pluripotency markers in those cell lines. This observation supports the hypothesis that nestin can potentially serve as diagnostic adjunct in conjunctival melanoma. Commercial Relationships: Marie-Sophie Hanet, None; Henning Thomasen, None; Henrike Westekemper, None; Klaus-Peter Steuhl, None; Daniel Meller, None Program Number: 3422 Poster Board Number: C0233 Presentation Time: 11:00 AM–12:45 PM Clinicopathologic Characterization of Amyloid Deposition in Ocular Surface and Adnexa Maria J. Suarez B1, Roxana Rivera-Michlig2, Fausto Rodriguez1. 1 Ophthalmic Pathology, Johns Hopkins University, Baltimore, MD; 2 Ophthalmology, Wilmer Eye Institute/Johns Hopkins, Baltimore, MD. Purpose: To describe the histopathological features of amyloid deposition seen in the ocular surface and/or adnexa biopsy specimens and further characterize the type of amyloid with proteomic analysis. Methods: This is a retrospective study in which the medical records from patients that were diagnosed with primary and secondary ocular and orbital amyloid deposition at our institution were retrieved between 1991-2014. The demographic data, clinical findings and pathology reports were also reviewed. Mass spectrometry-based proteomic analysis was performed in one case using formalin-fixed paraffin-embedded tissue. Results: The study included 9 patients (5 females, 4 males). The mean age was 59.1 years (range 39 – 88 years). Eight cases presented as unilateral lesions in otherwise healthy patients and one case was bilateral, in a patient with a previous history of multiple myeloma confirmed by electrophoresis. Four cases involved the conjunctiva, three cases with lesions in the eyelid and two cases presented as orbital masses, one of them with ptosis. Congo red stain was positive in eight cases; one case was unequivocal but moderately positive for Thioflavine T. Proteomic analysis performed in one of the orbital masses demonstrated lambda light chain-derived peptides (but not kappa). Systematic clinical evaluation in this patient was performed, and no evidence of a systemic plasma cell dyscrasia was identified. Conclusions: Our study describes the clinicopathologic features of amyloid deposition in the ocular surface and adnexa in patients with no evidence of predisposing disease, as well as secondary amyloid orbital deposition seen in one patient with a preexisting plasma cell ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology dyscrasia (multiple myeloma). Proteomic analysis may be of value in biopsies from these patients and deserves further study. Commercial Relationships: Maria J. Suarez B, None; Roxana Rivera-Michlig, None; Fausto Rodriguez, None Program Number: 3423 Poster Board Number: C0234 Presentation Time: 11:00 AM–12:45 PM Secreted Ly6/urokinase-type plasminogen activator receptor related protein-1 (Slurp1) expression by strain, age, gender and ocular surface health Sudha Swamynathan, Emili E. Delp, Stephen A. Harvey, Leela Raju, Shivalingappa K. Swamynathan. Ophthalmology, University of Pittsburgh, Pittsburgh, PA. Purpose: Previously, we demonstrated that Slurp1 serves an immunomodulatory function in the ocular surface. Although Slurp1 transcripts are the 11th most abundant in the mouse cornea, it is not known if this high expression is reflected at the protein level. Also, several tear proteomic studies have not identified SLURP1, resulting in ambiguity about the baseline SLURP1 concentration in human tears. Here, we examine the mouse Slurp1 expression in different strains, age-groups, genders, and ocular surface health conditions, and quantify SLURP1 expression in human tears collected from healthy or inflamed ocular surfaces. Methods: Early embryonic (E) and postnatal (PN) mouse corneal Slurp1 expression was quantified using QPCR. The influence of mouse genetic background, age, and gender on Slurp1 expression was examined by QPCR and immunofluorescent staining in Balb/C, FVBN, C57Bl/6, and DBA/2J strains at PN10, PN20 and PN56. Human tears were collected from 34 adults (18-80 years) with absorbent wicks using an IRB-approved protocol, and the SLURP1 levels quantified by ELISA. Results: Slurp1 expression, undetectable in E13, E16, and PN1 mouse corneas and barely detectable in PN10, increased rapidly in post-eyelid opening stages levelling off around PN20. Slurp1 expression did not differ significantly between different strains, or genders. Human tear SLURP1 levels did not vary significantly between genders and age groups. However, tears from inflamed human ocular surface contained significantly decreased amounts of SLURP1 (0.35 ng/100ng tear protein) compared with those from healthy individuals (0.68 ng/100ng tear protein). Conclusions: These data provide the first detailed description of the influence of age, gender, and genetic background on Slurp1 expression in the mouse cornea, establish the baseline for SLURP1 concentration in human tears, and demonstrate that the tears from inflamed ocular surface contain decreased amounts of SLURP1. Commercial Relationships: Sudha Swamynathan, University of Pittsburgh (P); Emili E. Delp, None; Stephen A. Harvey, None; Leela Raju, None; Shivalingappa K. Swamynathan, University of Pittsburgh (P) Support: NIH Grant R01EY022898 Program Number: 3424 Poster Board Number: C0235 Presentation Time: 11:00 AM–12:45 PM Orbital Invasion From Ocular Surface Squamous Neoplasia Benjamin P. Erickson1, Anat Galor2, Thomas Johnson1, Wendy W. Lee1, Carol L. Karp2. 1Oculoplastics, Bascom Palmer Eye Institute, Miami, FL; 2Cornea, Bascom Palmer Eye Institute, Maim, FL. Purpose: Orbital invasion from ocular surface squamous neoplasia (OSSN) is a rare and potentially devastating complication. The majority of studies arise from Sub-Saharan Africa and the Middle East, where HIV and poor access to care are significant factors. The North American literature primarily consists of isolated reports. We present our experience with 7 cases of OSSN invading the orbit. Methods: Retrospective review of 500 consecutive patients with biopsy-confirmed OSSN and radiologic and/or histologic evidence of orbital invasion. Results: Seven patients with orbital extension of OSSN were identified. Average age was 59.7±15.8 years old; 5 patients were male and 2 female. 5/7 were black and the remainder Hispanic (1/7) or non-Hispanic Caucasian (1/7). 4/7 were current or former smokers. 3/7 patients (age 48±10 years old) were HIV positive. None had a known history of hematogenous malignancy, head & neck radiation, immunosuppressive medication use, skin cancer, or HPV. Five of the lesions were primary, while the remaining 2 represented recurrence of previously treated OSSN. Lesions occupied 6.8±5.0 clock hours, with 5 of 7 cases involving the perilimbal conjunctiva. The one case with multiple recurrences had only a single clock hour of superficial involvement. Orbital invasion was clinically detected in the form of significant motility restriction in 4/7 patients, diplopia in 2/7, profound vision loss (light projection or worse) in 2/7, and epiphora in 2/7. Only the patient with multiple recurrences lacked clear external stigmata of invasion, which was identified by computed tomography (CT). Scans revealed involvement of the extraconal fat in 6/7, intraconal fat in 2/7, lacrimal drainage system in 2/7, and optic nerve in 1/7. Four of the 7 patients underwent total, and 2/7 lid sparing, exenteration; the remaining patient (age 88) refused surgery and died of unrelated causes. 2/7 underwent negative sentinel node biopsy. One patient each was treated with adjuvant chemotherapy and radiation. A single patient had disease specific mortality. Conclusions: OSSN is statistically a disease of older Caucasian men with history of sun exposure, but the majority of patients in our study were black with delayed primary diagnosis and/or underlying immunosuppression. Orbital invasion appears rare in cases of appropriately treated but recurrent OSSN, but associated external findings can be minimal and close surveillance is essential. Commercial Relationships: Benjamin P. Erickson, None; Anat Galor, None; Thomas Johnson, None; Wendy W. Lee, None; Carol L. Karp, None Program Number: 3425 Poster Board Number: C0236 Presentation Time: 11:00 AM–12:45 PM Fibrosis, Gene Expression, and Orbital Inflammatory Disease Stephen R. Planck1, 2, Dongseok Choi1, Christina A. Harrington4, David J. Wilson1, Hans E. Grossniklaus3, Patrick Stauffer1, James T. Rosenbaum1, 2. 1Casey Eye Institute, Oregon Health & Science Univ, Portland, OR; 2Devers Eye Institute, Legacy Health System, Portland, OR; 3Ophthalmology, Emory University, Atlanta, GA; 4Integrated Genomics Laboratory, Oregon Health & Science Univ, Portland, OR. Purpose: Fibrosis is an important association with inflammation that affects the orbital tissue. To clarify the pathogenesis of fibrosis in orbital diseases, we analyzed the gene expression in orbital biopsies and compared our results to those reported for idiopathic pulmonary fibrosis. Methods: We collected 153 biopsies including 68 from the lacrimal gland and 85 from orbital fat. Diagnoses included healthy controls (n=27), nonspecific orbital inflammation (NSOI) (n=64), thyroid eye disease (TED) (n=33), sarcoidosis (n=20), and granulomatosis with polyangiitis (GPA) (n=9). Fibrosis was scored on a zero to three scale by two expert, ophthalmic pathologists. Gene expression was quantified using Affymetrix U133 plus 2.0 microarray. Results: Within orbital fat, fibrosis was greatest among subjects with GPA (2.75±0.46) and significantly increased in tissue from subjects with GPA, NSOI, or sarcoid (p<0.01), but not for TED, compared to controls (1.13±0.69). For the lacrimal gland, the average fibrosis score among healthy controls (1.36±0.48) did not differ statistically from any of the 4 disease groups. 73 probe sets identified transcripts ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology (~54 genes) correlating with fibrosis in orbital fat (false discovery rate < 0.05 and fold-difference >1.5). Transcripts with increased expression included fibronectin, lumican, thrombospondin, and collagen types I and VIII. Many of these transcripts were also increased in pulmonary fibrosis. Conclusions: A pathologist’s recognition of fibrosis in orbital tissue correlates well with increased expression of transcripts considered essential in fibrosis. Furthermore, overlapping genes have been detected in pulmonary fibrosis. The study supports the accuracy of histological scoring of fibrosis and conversely, the results help to validate gene expression analysis from formalin-fixed tissue as an accurate methodology to understand pathophysiology. Commercial Relationships: Stephen R. Planck, None; Dongseok Choi, None; Christina A. Harrington, None; David J. Wilson, None; Hans E. Grossniklaus, None; Patrick Stauffer, None; James T. Rosenbaum, Genentech (C) Support: NIH Grants EY020249, EY010572 and RR024140; Research to Prevent Blindness Program Number: 3426 Poster Board Number: C0237 Presentation Time: 11:00 AM–12:45 PM A Novel Murine Model of Orbital Inflammation N. Grace Lee1, Lindsay L. Wong2, Dhanesh Amarnani2, Suzanne K. Freitag1, Diane Bielenberg3, Patricia A. D’Amore2, Leo A. Kim1, 2 1 . Ophthalmology, Harvard Medical School - MEEI, Boston, MA; 2 Schepens Eye Research Institute, Boston, MA; 3Boston Children’s Hospital, Boston, MA. Purpose: Orbital inflammation from Graves’ disease or nonspecific orbital inflammation can cause devastating compressive optic neuropathy. Current therapeutic options which primarily consist of steroids and decompression surgery may not be sufficient to resolve the optic neuropathy. Progress in this area is hindered by the lack of animal models of acute orbital inflammation. We have developed an animal protocol to induce inflammation in the murine orbit with the use of a skin sensitizer, oxazolone. Methods: Eight-week-old female BALB/c mice were sensitized with a topical application of 2% oxazolone solution in olive oil/acetone (2:1 vol/vol) to the shaved abdomen. Five days after sensitization (day 0), the right eye was challenged with a 10uL orbital injection of 2% oxazolone solution. The left eye, serving as a control, received a sham injection of the vehicle alone (olive oil/acetone). Mice then underwent daily magnetic resonance imaging (MRI) before being euthanized at various time points. Their exenterated orbits were examined histologically. Results: Twenty-four hours following the orbital challenge, mice were observed to demonstrate mild proptosis and dermatitis. MRI on day 1 confirmed the findings of exophthalmos as well as retrobulbar inflammation and periorbital edema. On day 3, there was relative reduction of edema and proptosis compared to day 1. Histopathologic examination of the mouse orbit from day 1 to day 5 corroborated the MRI findings of periocular and intraocular inflammation consisting of neutrophils with a transition to chronic inflammation with lymphocytes by day 3 and early fibrosis on day 5. Conclusions: We present a novel animal model of orbital inflammation utilizing a type 4 hypersensitivity reaction. This model should allow a better understanding of why the orbit is preferentially affected in certain systemic disorders and may provide a way to evaluate potential alternatives to steroid and surgical treatment. A. Sub-Tenon’s injection of 2% oxazolone in the right orbit and vehicle in the left on Day 0. B. Dermatitis of the right upper and lower eyelids, right facial edema, and right orbital inflammation in contrast to the normal-appearing left side on Day 1. C. MRI reveals asymmetric retrobulbar and facial edema right greater than left on day 1. D. Exenterated right orbit on day 1 shows retrobulbar edema and an aggregate of neutrophils, suggesting acute inflammatory response. Commercial Relationships: N. Grace Lee, None; Lindsay L. Wong, None; Dhanesh Amarnani, None; Suzanne K. Freitag, None; Diane Bielenberg, None; Patricia A. D’Amore, AGTC (C), Eleven Biotherapeutics (S); Leo A. Kim, None Support: American Thyroid Association Grant, Lions Eye Foundation Research Award Program Number: 3427 Poster Board Number: C0238 Presentation Time: 11:00 AM–12:45 PM Differences in the expression of immunohistochemical markers in solid and fibrosing basal cell carcinoma Viola Graham1, 2, Ute Kaiser1, 2, Frank G. Holz1, Martina C. Herwig1, 2 , Karin U. Loeffler1, 2. 1Department of Ophthalmology, University of Bonn, Bonn, Germany; 2Department of Ophthalmology, Division of Ophthalmic Pathology, University of Bonn, Bonn, Germany. Purpose: Basal cell carcinoma (BCC) is the most frequent cancer of the ocular adnexae. Semimalignant, it can be subdivided into a solid type and a more aggressive and invasive fibrosing type. In this study, different molecules related to infiltrative growth were investigated to further help our understanding of these different growth patterns. Methods: 20 formalin-fixed paraffin-embedded specimens of basal cell carcinoma of the eyelid were investigated and subdivided into two groups. Group I consisted of 10 solid BCC, group II of 10 fibrosing BCC (mean age 71 versus 75,1 yrs). There were 4 male and 6 female patients in both groups. Immunohistochemistry was performed for matrix metalloproteinases (MMP) 1, 2 and 9 (Santa Cruz), Ki67 (Dako), p53 (Dako) and FHOD1 (Novusbio). Immunoreactivity was evaluated by light microscopy and graded semiquantitatively by two readers using a score from 0 to 3. Statistical analysis was performed with SPSS (IBM SPSS Statistics 20; Armonk, NY). The probability for the alpha error was set to p < 0.05. Results: As could be expected, MMP 1, 2 and 9 were present in a membrane-bound fashion, whereas Ki67 and p53 were located in the nucleus. MMP1, MMP2 and p53 showed a stronger expression ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology in fibrosing BCC compared to solid BCC. This was only statistically significant for MMP1 (p=0,024) with a mean staining reaction of 0,95 for solid BCC and 1,7 for fibrosing BCC. For MMP2 (p=0,105) and p53 (p=0,233), there was only a trend for a more intense staining in fibrosing BCC (MMP2: 2,45; p53: 1,9) compared to solid BCC ( MMP2: 2,05; p53:1,4). MMP9 and FHOD1 did not as yet yield reliable results. Regarding Ki67, there was no significant difference between both groups. Conclusions: Molecules associated with infiltrative growth such as MMP1 showed a higher staining intensity in fibrosing BCC and may explain - among other factors - their invasive behaviour. Adressing those as therapeutic target might perhaps help in developing new adjuvant treatment options. To confirm our observations, studies with a larger number of basal cell carcinomas are warranted and may also address this topic on a genetic level. Commercial Relationships: Viola Graham, None; Ute Kaiser, None; Frank G. Holz, None; Martina C. Herwig, None; Karin U. Loeffler, None Program Number: 3428 Poster Board Number: C0239 Presentation Time: 11:00 AM–12:45 PM Macrophages in basal cell carcinoma Karin U. Loeffler1, Ute Kaiser1, Frank G. Holz2, Martina C. Herwig1. 1 Division of Ophthalmic Pathology, University Eye Department, Bonn, Germany; 2Ophthalmology, University of Bonn, Bonn, Germany. Purpose: Basal cell carcinoma (BCC) is a locally invasive tumor which can be subdivided in a fairly benign solid variant and a more aggressive fibrosing type. Since in oncology there is increasing evidence for the importance of macrophages (Ms) interacting between tumor cells and their surroundings, thereby affecting not only the infiltrative potential but also the patients’ prognosis, we wanted to compare these two BCC variants regarding the presence and localisation of tumor-associated Ms. Methods: We studied 15 specimens of solid BCC (BI) and 15 of fibrosing BCC (BII). Mean age in both groups was similar (73 vs.75 years). Fibrosing BCC was located almost exclusively in the lower eyelid while solid BCC was located more diversely around the eye. H&E sections were evaluated for the presence of associated inflammation (score + to +++). Immunohistochemistry was performed with antibodies against CD68 (Dako;1:100), CD163 (EnzoLifeSciences;1:100) and Ki67 (Dako;1:100). Positive reacting cells were visualized either using AEC or immunofluorescence and counted in a standard fashion and/or by means of Automatic Nuclei Counter plug-in for ImageJ. Evaluation was performed by at least 2 different readers, and IBM SPSS 20 was used for statistical analysis. Results: In all specimens, an inflammatory component was observed which - as judged by H&E - was significantly more pronounced in BII compared to BI (p<0.001). Ms were also more frequent in BII (p<0,05), however, no significant difference in M subtype was found between both groups. In both BI and BII, Ms were much more frequent in the surrounding stroma compared to the tumor itself (p<0.001). Ms within the tumor were more frequent in BII compared to BI (p<0,05), and in both groups those Ms always belonged to the M1(CD68+CD163-) category. Ki67 index was similar in both groups. Conclusions: Fibrosing BCC, more likely to be located at the lower eyelid, is associated with a significantly more intense inflammatory reaction in the surrounding tissue. From our data, however, there was no significant difference in the subtype of Ms when comparing both groups. Thus, in BCC, macrophage polarization does not seem to be particularly relevant regarding infiltrative growth, and tumorassociated chemokines attracting/stimulating inflammatory cells other than Ms might also be important in tissue destruction and thereby invasive potential. It remains unclear whether localisation of the tumor plays a major role in this. Commercial Relationships: Karin U. Loeffler, None; Ute Kaiser, None; Frank G. Holz, None; Martina C. Herwig, None Program Number: 3429 Poster Board Number: C0240 Presentation Time: 11:00 AM–12:45 PM Clinical and histopathological characteristics of periocular basal cell carcinoma in a low UV geographic region Beatriz Nugent da Cunha1, Dominique F. de Souza2, Nadine Marques2, Patrick T. Logan2, Jordan Discepola2. 1Federal University of São Paulo, São Paulo, Brazil; 2Ophthalmology, McGill University, Montreal, QC, Canada. Purpose: Basal cell carcinoma (BCC) accounts for more than 90% of all eyelid malignancies. Risk factors for BCC include age, ultraviolet light [UV] exposure, genetic background, and fair skin. The objective of this study is to investigate the frequency of periocular BCC subtypes, in particular sclerosing type, in a low UV exposure area (Quebec, Canada) and to compare these results to published data from a high UV exposure area (Australian Mohs Nationwide Database). Methods: A total of 387 excised periocular BCCs from January 1998 to June 2014 from the Henry C. Witelson Ocular Pathology Laboratory, Montreal, Quebec were evaluated. Demographic data were retrieved, including gender, age, tumor localization, prior clinical diagnosis, histological subtype, and ulceration. For comparisons with a high UV geographic location, we obtained published data (ocular location and histopathological subtype) from the Australian Mohs Database, which includes 1,295 periocular BCC cases. Results: The average age of our patients was 67.6 ± 15.3 years (range: 8–101). Tumors were more frequent in women (57.1%). Clinical misdiagnosis occurred in 15.2% of the cases, 70% of which were diagnosed as benign lesions. BCC location frequency in descending order was the lower eyelid (58%), upper eyelid (12%), and medial canthus (8.5%). Histopathological subtypes identified were as follows: nodular (79.6%), sclerosing type (7.5%), squamous differentiation (6.5%), sebaceous differentiation (2.8%), and mixed types (3.6%). Twenty tumors were ulcerated (5.2%). The Australian Mohs Database published the following results: 615 (47.5%) BCCs were located in the lower eyelid; 627 (48.3%) in the medial canthus; and 51 (3.9%) in the upper eyelid. The most common subtype in this geographic region was nodular (39.5%) followed by sclerosing type in 34.8% of cases. Conclusions: There are significant differences in periocular BCC location and subtype between low and high UV geographic locations. In Quebec, the most common ocular location is the lower eyelid, whereas in Australia it is the medial canthus. Sclerosing type was significantly higher in Australia compared to Quebec. The higher UV exposure in Australia relative to Quebec is the likely factor explaining the much higher frequency of this most aggressive type of periocular BCC in the Australian population. Commercial Relationships: Beatriz Nugent da Cunha, None; Dominique F. de Souza, None; Nadine Marques, None; Patrick T. Logan, None; Jordan Discepola, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 3430 Poster Board Number: C0241 Presentation Time: 11:00 AM–12:45 PM Multispecialty approach to management of sebaceous carcinoma of the eyelids Seanna R. Grob1, 2, N. Grace Lee1, 3, Francis Creighton4, Frederick Jakobiec1, 5, Kevin Emerick4, Suzanne K. Freitag3, 1. 1Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA; 2Ophthalmology, Harvard Medical School, Boston, MA; 3Oculoplastics and Reconstructive Surgery, Massachusetts Eye and Ear Infirmary, Boston, MA; 4Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, MA; 5Ophthalmic Pathology, Massachusetts Eye and Ear Infirmary, Boston, MA. Purpose: To report a multispecialty approach to the management of periocular sebaceous carcinoma and the clinical outcomes of patients who have been treated with this approach. Methods: Retrospective review of 6 patients with periocular sebaceous carcinoma managed by the Oculoplastics Service at Massachusetts Eye and Ear Infirmary (MEEI) was conducted. Data collected included duration of symptoms, previous ocular diagnoses, eyelids affected, method of excision and reconstruction, results of sentinel lymph node biopsy and metastatic work up, recurrence rate, and duration of follow up. Results: Six patients were identified (mean 70.5 years, 4 female). All were of a Caucasian background. Four patients had a family history of any cancer; only one patient had a prior history of skin cancer – basal cell carcinoma. Prior to evaluation at MEEI, four patients were diagnosed with blepharitis. The mean duration of symptoms prior to biopsy proven diagnosis and evaluation at MEEI was 28.5 months. The location of the sebaceous carcinoma was most commonly in the upper lids (4 patients). After diagnosis was confirmed by histopathologic analysis, all patients were evaluated by the head and neck oncology service at MEEI for discussion of sentinel lymph node biopsy, medical oncology for staging and metastatic work up, and radiation oncology for possible adjuvant therapy. An average of 2.2 total sentinel lymph nodes were removed per case. The parotid and cervical level 2 sentinel lymph node basins were the most common sites (each 50% of cases). Surgical margins were evaluated by frozen section and confirmed with rapid permanent section within 24 hours. All patients had negative lymph node biopsies and metastatic work ups on initial presentation. Only one patient has had recurrence since initiation of care at MEEI, but this patient had extensive eyelid and ocular surface disease on initial presentation. Conclusions: The optimal management of sebaceous carcinoma of the ocular surface and adnexa requires an integrated multidisciplinary approach involving an ophthalmic plastic surgeon, a head and neck oncologist, a medical and radiation oncologist, experienced pathologists and radiologists, and at times, an anterior segment surgeon specializing in ocular surface tumors. Commercial Relationships: Seanna R. Grob, None; N. Grace Lee, None; Francis Creighton, None; Frederick Jakobiec, None; Kevin Emerick, None; Suzanne K. Freitag, None Program Number: 3431 Poster Board Number: C0242 Presentation Time: 11:00 AM–12:45 PM Visual Outcomes in Non-Neurofibromatosis-Related Optic Pathway Gliomas Michael J. Wan, Gena H. Heidary. Ophthalmology, Boston Children’s Hospital and Harvard University, Boston, MA. Purpose: Optic pathway gliomas (OPGs) are low-grade tumors of childhood that have a high survival rate but can cause significant visual impairment. While commonly associated with Neurofibromatosis Type 1 (NF1), non-NF1-related OPGs have been reported to cause more visual disability, but long-term data are limited. The purpose of this study was to report the long-term visual outcomes of a cohort of pediatric patients with non-NF1-related OPGs. Methods: This was a retrospective cohort study at a tertiary care pediatric hospital and cancer institute. The study included all patients with non-NF1-related OPGs seen between 1995-2013. The clinical features at presentation, tumor location, type of treatment, evidence of progression during treatment or recurrence after treatment, and the results of all visual assessments were recorded. The primary outcome was visual acuity at final follow-up. Results: There were a total of 61 patients with non-NF1-related OPGs. The most common presenting features were vision loss (26%), nystagmus (26%), neurological findings (23%), strabismus (13%) and headache (12%). The median age of onset was 2.6 years old and 52 cases (85%) presented with bilateral involvement. Fifty-four patients (89%) received treatment; the most common treatment was chemotherapy and surgery (44%), followed by chemotherapy alone (39%) and surgery alone (5%). Fifteen patients (28%) showed progression during treatment requiring a change in therapy and 29 patients (54%) had evidence of recurrence after completion of initial therapy. Of the 54 patients who received treatment, 18 (33%) did not have evidence of progression or recurrence. The median follow-up interval was 5.1 years after presentation. In the worse eye at final follow-up, 18 patients (30%) were ≥ 20/30, 9 patients (15%) were between 20/40-20/80, and 34 patients (56%) were ≤ 20/100. In the better eye at final follow-up, 35 patients (57%) were ≥ 20/30, 11 patients (18%) were between 20/40-20/80, and 15 patients (25%) were ≤ 20/100. Conclusions: In this cohort of pediatric patients with non-NF1related OPGs, the long-term visual outcomes were poor, with severe visual deficits in over half of the patients at last follow-up. Even with treatment, the majority of patients had evidence of either progression or recurrence. Frequent visual assessments for patients with nonNF1-related OPSs, both during treatment and afterward, are crucial to minimize long-term visual impairment. Commercial Relationships: Michael J. Wan, None; Gena H. Heidary, None Program Number: 3432 Poster Board Number: C0243 Presentation Time: 11:00 AM–12:45 PM The influence of race and gender on the risk of hair loss secondary to methotrexate. Sarah Escott1, Julia Malalis1, Yosuke Harada1, David Mai2, Debra A. Goldstein1. 1Ophthalmology, Northwestern University, Chicago, IL; 2 University of Illinois, Peoria, IL. Purpose: To determine if there is a difference in terms of selfreported race and gender in the incidence of hair loss with methotrexate (MTX). Methods: A retrospective review of all patients at a University based practice seen between August 2012 and September 2014 with a history of MTX treatment for uveitis was performed. Data collected included self-reported race, diagnosis, age at start of MTX therapy, self-reported hair loss, and both dose and duration of MTX at time of hair loss. To qualify for inclusion patients had a minimum of 6 months follow up after reported hair loss and cessation of MTX therapy. Results: Complete records of 97 uveitis patients who met the above criteria were reviewed. There were 25 males and 72 females. Of these, 28 patients self-reported African American race, 60 reported Caucasian, and the remaining 9 reported Asian, Hispanic, or Arabic ethnicity. Hair loss was reported in 9.2% of patients. Average age at the time of hair loss was 38.6 years (range 9-59 years). The incidence of hair loss was similar in women (10%) and men (8%); ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology but was twice as common in African American (AA) patients (14%) compared with Caucasian patients (6.6%). All AA patients reporting hair loss were female, although there were only 3 AA males included in the analysis. Hair loss developed on weekly doses of 10-25mg MTX after an average exposure time of 24.8 months (range 7 months – 8 years). Reversal of hair loss occurred in all cases following cessation of treatment. Conclusions: These findings suggest hair loss is twice as common in African Americans compared to Caucasians exposed to MTX. There was no effect of gender on risk of hair loss. Alopecia may develop on any dose of the drug, and is reversible on cessation of therapy. This information may be useful to clinicians when considering this medication for treatment, and in counseling patients on risk profile. Commercial Relationships: Sarah Escott, None; Julia Malalis, None; Yosuke Harada, None; David Mai, None; Debra A. Goldstein, None Program Number: 3433 Poster Board Number: C0244 Presentation Time: 11:00 AM–12:45 PM The effect of tumor formation and treatment on retinal dysfunction in mouse models of Neurofibromatosis type 1 (NF1)associated optic glioma Joseph Toonen, Aparna Kaul, Scott Gianino, David Gutmann. Neurology, Washington University in St. Louis, Saint Louis, MO. Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome, affecting 1 in 2,500 people worldwide. Approximately 15-20% of children with NF1 develop low-grade gliomas of the optic pathway (OPG), leading to visual imparment in 30-50% of affected children. While bi-allelic inactivation of the NF1 gene is the most common cause of OPG, a small group of patients with OPGs have additional genetic alterations resulting in greater tumor volume and proliferation. Our aim was to determine the impact of optic glioma formation on retinal dysfunction utilizing genetically engineered mouse (GEM) models. Additionally, we tested the outcome of tumor treatment on retinal function. Methods: Mice used in the study were Nf1 flox/mut; GFAP-Cre (FMC) Nf1flox/mut; f-BRAF; GFAP-Cre mice (FMBC) and Nf1flox/mut; Pten flox/ wt ; GFAP-Cre (FMPC) mice. Mice were perfused at 3 months of age and processed for paraffin embedding. Immunohistochemistry was performed on paraffin sections using antibodies Brn3a and SMI-32. TUNEL staining was also performed. 3 month old FMC mice were treated for 4 weeks with PD901, BKM120, or vehicle controls via oral gavage (n=6). Results: FMC, FMBC, and FMPC retinae had a 6-fold (±5.6% –6.9%) increase in RGC death and a 31-45% reduction in RGCs compared to controls. While all of the Nf1-OPG mouse strains had decreased RNFL thickness, FMPC mice had 3-fold decreased RNFL thickness in comparison with 2-fold reductions observed in FMC and FMBC mice. We found an inverse correlation (R2 = 0.803) between RGC death and RNFL thickness among all 3 mouse models. Following treatment with either BKM120 or PD901 which each reduced FMC-OPG volume to wild-type levels, we found 1.9-fold and 4.9 -fold decreases in TUNEL+ cells in BKM120-treated and PD901-treated mice, respectively. Similarly, BKM120-treated and PD901-treated mice retained 40% (± 14%-57%) and 50% (± 34%60%) more RGCs than controls. Lastly, RNFL thickness following BKM120 and PD901 treatment was increased by 1.6-fold and 2.3fold, respectively, compared to vehicle-treated FMC mice. Conclusions: The results from this study show that a larger, more proliferative OPG leads to increased retinal dysfunction. Likewise, using biological targets that inhibit tumor growth reversed these effects. These findings have implications for assessing visual outcome in children with NF1-OPG. Commercial Relationships: Joseph Toonen, None; Aparna Kaul, None; Scott Gianino, None; David Gutmann, None Support: NIH Grant NS007205 Program Number: 3434 Poster Board Number: C0245 Presentation Time: 11:00 AM–12:45 PM The utility and technique for infraorbital and supraorbital nerve biopsies Valerie Chen, Hee Joon Kim, Brent Hayek, Hans E. Grossniklaus, Ted H. Wojno. Ophthalmology, Emory University, Atlanta, GA. Purpose: Enlargement of the infraorbital and supraorbital nerves can most commonly indicate perineural invasion of a malignancy or benign conditions such as idiopathic orbital pseudotumor. The purpose of this study is to review the role of supraorbital and infraorbital nerve biopsies in patients presenting with radiographic enlargement of these nerves and to elucidate the surgical technique involved in obtaining these biopsies. Methods: A 5-year chart review (2009-2014) was performed at The Emory Clinics. Patients with radiographic confirmation of enlarged supraorbital and/or infraorbital nerves that underwent a biopsy were included in the review. Charts were reviewed for the following data: patient demographics and history, clinical symptoms and findings, radiographic findings, surgical method, and treatment. Results: A total of 6 patients met the inclusion criteria. Five patients (83%) were female and 1 (17%) was male with the average age being 72.3, ranging from 36-90 years. Five of the 6 patients had a history of a cutaneous malignancy. All 6 patients presented with either diplopia and/or dysesthesias on the affected side. Clinical examination confirmed decreased V1 and/or V2 sensation for 5 of the 6 patients. Imaging revealed enlargement of V1, V2, and/or V3 for all of the patients. Supraorbital nerve biopsies were performed for 2 patients via a sub-brow incision onto the superior orbital rim with reflection of the periosteum that revealed the nerve. One confirmed squamous cell carcinoma and one confirmed mucoepidermoid carcinoma. The remaining 4 patients underwent infraorbital nerve biopsies via a transconjunctival fornix-based orbitotomy with subperiosteal dissection along the orbital floor followed by unroofing of the infraorbital canal. The biopsy confirmed squamous cell carcinoma for the 3 patients with a history of cutaneous squamous cell carcinoma. One patient confirmed idiopathic orbital inflammation. Five of the 6 patients had initiation of treatment in less than a month. One patient was lost to follow-up. Conclusions: For patients presenting with enlarged supraorbital and/ or infraorbital nerves, biopsies of these nerves can rapidly confirm the underlying condition that can facilitate early treatment. A subbrow approach offers a direct access to the supraorbital nerve while a transconjunctival fornix-based anterior orbitotomy with unroofing of the infraorbital canal allow access to the infraorbital nerve. Commercial Relationships: Valerie Chen, None; Hee Joon Kim, None; Brent Hayek, None; Hans E. Grossniklaus, None; Ted H. Wojno, None Program Number: 3435 Poster Board Number: C0246 Presentation Time: 11:00 AM–12:45 PM Prevalence of intraocular tumors detected by ultrasonography in eyes with opaque media Saranya C. Balasubramaniam, Sophie J. Bakri. Mayo Clinic, Richmond, VA. Purpose: To study the prevalence of intraocular tumors detected by screening ultrasonography in eyes with opaque media. Methods: A retrospective review of ultrasounds done in 119 eyes that had opaque media and a diagnosis of blindness or phthisis ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology between January 1, 1994 and December 31, 2013. Patients were excluded if ultrasound was ordered in eyes with clear media or when ultrasound was used for acute etiologies of vision loss including endophthalmitis, retinal detachment, or acute vitreous hemorrhage. Data were extracted on visual acuity, intraocular pressure, presence or absence of ocular pain, etiology of opaque media, number of ultrasounds received during study time period, and ultrasound findings. Follow up was defined as the time range for which an eye was followed from initial documentation of opaque media to last visit with opaque media. In addition, ultrasounds obtained for screening prior to evisceration or enucleation was noted along with pathology findings. Results: A total of 173 ultrasounds corresponding to 119 eyes were reviewed. No intraocular tumors were detected. Mean age of patients was 59 years. Visual acuity was hand motions or worse in 89 eyes (74.8%), elevated intraocular pressure was found in 23 eyes (19.3%) and ocular pain was noted in 30 eyes (25.4%). 50 eyes had less than twelve months of follow up. The remaining 69 eyes with opaque media (58%) had at least one year follow up. The mean follow was 65 months (median 56 months; range 12-129). Of these, 2 eyes (2.9%) had an annual ultrasound, 43 eyes (62%), had an ultrasound done every 13 months- 5 years, and 19 eyes (27.5%) had an ultrasound every 61 months to 10 years. In addition, 16 eyes with opaque media for at least 6 years only received a screening ultrasonography at presentation (11 eyes had 6-8 years follow up; 5 eyes had more than 8 years of follow up). 6 eyes had screening ultrasonography prior to evisceration or enucleation with pathology clear of intraocular tumors. Conclusions: No prior studies have been done to examine the utility of interval screening ultrasonography in blind eyes with opaque media. In this series of eyes with opaque media, no intraocular tumors were detected by screening ultrasonography. Further long term study is necessary to determine the utility of consecutive ultrasonography in eyes with opaque media and the appropriate interval of screening ultrasonography. Commercial Relationships: Saranya C. Balasubramaniam, None; Sophie J. Bakri, None Support: Research to Prevent Blindness 427 Tumors - Uveal melanoma Wednesday, May 06, 2015 11:00 AM–12:45 PM 1AB Mile High Blrm Paper Session Program #/Board # Range: 4331–4337 Organizing Section: Anatomy and Pathology/Oncology Program Number: 4331 Presentation Time: 11:00 AM–11:15 AM HIF-1α upregulates ANGPTL4 to promote angiogenesis in Uveal Melanoma Ke Hu1, 2, Savalan Babapoor-Farrokhran1, Murilo W. Rodrigues1, Brooks Puchner1, Monika Deshpande1, Laura Asnaghi1, Charles Eberhart1, Silvia Montaner1, Akrit Sodhi1. 1Wilmer Eye Institute, Johns Hopkins School of Medicine., Baltimore, MD; 2The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. Purpose: The transcriptional response promoted by hypoxiainducible factor (HIF)-1α has been associated with angiogenesis and metastatic spread in uveal melanoma (UM). Expression of one HIF-1 target gene, vascular endothelial growth factor (VEGF) correlates with tumor vascularity in UM, as well as other tumors. However, treatment of patients with therapies targeting VEGF has not proven sufficient alone to prevent UM growth or spread. Here we set out to evaluate the potential role of a novel HIF-regulated gene product, angiopoietin-like 4 (ANGPTL4), in the promotion of angiogenesis in UM. Methods: UM cell lines were examined for expression of HIF1α, VEGF, and ANGPTL4. Expression of HIF-1α, VEGF, and ANGPTL4 were further assessed by immunohistochemical analysis in UM tissue and quantitated using a UM tissue array. Expression of ANGPTL4 was also examined in vitreous biopsies from patients with uveal melanoma. The role of ANGPTL4 in angiogenesis in UM was assessed using endothelial cell (EC) tubule formation (TF) assays. Inhibition of HIF-1α, VEGF, and ANGPTL4 was performed using RNAi. Results: Expression of HIF-1α was detected in all UM cell lines tested. Hypoxic stabilization of HIF-1α resulted in the promotion of TF in treated ECs in vitro; this affect was inhibited by blocking HIF-1α translation, but only partially inhibited by blocking VEGF expression, implicating additional HIF-regulated genes in the promotion of angiogenesis in UM. We demonstrate that HIF-1α stabilization results in ANGPTL4 mRNA and protein expression in UM cell lines. These results were corroborated in tissue samples from patients with UM. Using a UM tissue array, we further observed expression of HIF-1α in a majority of tumors in the array. Expression of either ANGPTL4 or VEGF was detected in almost all (99%) of the tumors in the UM tissue array. ANGPTL4 expression was increased more than 50 fold in vitreous biopsies from UM patients at levels that were sufficient to promote angiogenesis in vitro. RNAi targeting ANGPTL4 inhibited the promotion of TF induced by UM cell lines; this effect was additive to RNAi targeting VEGF. Conclusions: We propose that therapies targeting both VEGF and ANGPTL4 will be necessary to effectively inhibit tumor-induced angiogenesis in patients with UM. Commercial Relationships: Ke Hu, None; Savalan BabapoorFarrokhran, None; Murilo W. Rodrigues, None; Brooks Puchner, None; Monika Deshpande, None; Laura Asnaghi, None; Charles Eberhart, None; Silvia Montaner, None; Akrit Sodhi, None Support: The K08 grant: K08EY021189, The RPB Career Development Award: 109622 Program Number: 4332 Presentation Time: 11:15 AM–11:30 AM Modulation of Cytoskeletal Associated Proteins Regulates Cell Death and Survival in Uveal Melanoma Matthew W. Wilson1, 2, Ryan P. Lee1, Sumana R. Chintalapudi1, Bradley T. Gao1, Justin B. Lendermon1, Nabil Saleh1, Anderson H. Webb1, Hans E. Grossniklaus4, Vanessa M. Morales1, 3. 1Ophthal/ Hamilton Eye Int, Univ of Tennessee Health Sci Ctr, Memphis, TN; 2Surgery, St Jude Children’s Research Hospital, Memphis, TN; 3Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN; 4Emory Eye Center, Eye Univeristy, Atlanta, GA. Purpose: Purpose: Approximately 50% of Uveal Melanoma (UM) patients will be diagnosed with liver metastases within 5-years of diagnosis. Micro-metastases are present in the liver as early as 2-years before diagnosis of the intraocular malignancy. Recent work from our laboratory suggests that inhibiting the upregulation of cytoskeletal signaling could offer potential therapeutic targets for metastasis control. In this study we elucidate the mechanism(s) involved. Methods: Methods: We performed gene transcription, flow cytometry as well as cellular and molecular biology analyses after pharmacological modulation of paxillin Y118 using the paxillin inhibitor 6-B345TTQ. The effects of paxillin inhibition (pY118) were measured on pAKT, Protein Kinase C-delta (PKC-δ), pro-apoptotic ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology molecules expression, actin polymerization and cellular proliferation in UM cell lines and immunohistochemical (IHC) analyses in normal and with UM. We performed cutting edge Nano3D (Nano 3D Biosciences Inc, Houston, TX) technology to investigate changes in cellular migration and proliferation after paxillin inhibition. Results: Results: We observed an increase in the alpha-4 subunit of VLA-4 and paxillin both in the UM cell lines by flow cytometry analysis (p<0.05) and in UM eyes compared to normal eyes by IHC. Targeting of paxillin through inhibition of pY118 reduced cellular proliferation in primary, not metastatic, UM. Mechanistically, we observed a significant reduction in pAKT signaling in metastatic UM (p<0.05) by Western blot analysis and phospho-PKC-δ in the metastatic UM cell lines by flow cytometry. IHC analysis in human eyes revealed differences in PKC-δ expression in normal and UM eyes. Conclusions: Conclusions: Our in vitro results identified paxillin as a potential therapeutic target to arrest UM cellular proliferation and migration. Inhibition of paxillin preferentially affected primary UM cells compared to metastatic cell lines through pAKT and PKC-δ and may represent a potential targeted therapy for micrometastases. Commercial Relationships: Matthew W. Wilson, None; Ryan P. Lee, None; Sumana R. Chintalapudi, None; Bradley T. Gao, None; Justin B. Lendermon, None; Nabil Saleh, None; Anderson H. Webb, None; Hans E. Grossniklaus, None; Vanessa M. Morales, None Support: Unrestricted Grant from Research to Prevent Blindness, Inc. New York, New York Program Number: 4333 Presentation Time: 11:30 AM–11:45 AM FNAB of Uveal Melanoma : Outcomes and Complications Arun D. Singh1, Carlos A. Medina Mendez2, Nakul Singh2, Mary E. Aronow3, Hassan A. Aziz1, Charles V. Biscotti4. 1Ophthalmic Oncology, Cole Eye Institute, Cleveland, OH; 2Case Western Reserve University, Cleveland, OH; 3WIlmer Eye Institute, Baltimore, MD; 4 Anatomy PAthology, Cleveland Clinic, Cleveland, OH. Purpose: To report outcomes and complications of diagnostic fineneedle aspiration biopsy (FNAB) of uveal melanoma. Methods: Prospective interventional series of 150 consecutive patients with uveal melanoma treated at the Cleveland Clinic Cole Eye Institute between May 2009 and August 2012. The FNAB approach (trans corneal [TCO], trans scleral [TSC], and trans vitreal [TSV] were primarily determined by the location of the tumor. The FNAB was performed using 25 Gauge needle using previously published technique. All aspirated material was flushed into in Cytolyt® solution for ThinPrep® processing. The diagnosis of uveal melanoma was based upon characteristic cellular features. The cytological reporting was divided into 4 conventional categories: 1. Unsatisfactory for interpretation, 2. Negative for melanoma, 3. Atypical cells (not diagnostic or consistent with melanoma) and 4. Positive for melanoma. Patients were evaluated 1-4 weeks postoperatively, then every 3 months for the first year, followed by every 6 months thereafter. Data were analyzed using STATA version 11. Results: FNAB was obtained via TCO (8), TSC (71), and TVT (64) approach and impression smear in 7 cases. Diagnostic yield was 92% (Positive for melanoma 122, Atypical cells : consistent with melanoma 9). False negative results were oserved in 8% (Unsatisfactory 7, Negative for melanoma 3, Atypical cells : not diagnostic 2). Diagnostic yield was significantly correlated to biopsy approach (TCO 100%, TSC 96%, 86%; p = 0.029, Fisher’s exact test) and tumor size (basal diameter >5.0 mm; height >2.5 mm). Visual acuity (<20/40 and > 20/400) at baseline and at 3 months was recorded in 68%,13% and 57%, 22% respectively). Visually significant complications such as persistent hemorrhage (sub retinal hemorrhage or vitreous) requiring surgical intervention (1%) and rhegmatogenous retinal detachment (1%) were rare. Endophthalmitis, hypotony, and detectable needle tract seeding were not observed. Conclusions: FNAB for uveal melanoma with 25-gauge needle is a safe procedure that can yield diagnostic samples in more than 90% of cases. Possibility of negative diagnostic FNAB yield should be considered when counseling patients with small tumors. Commercial Relationships: Arun D. Singh, None; Carlos A. Medina Mendez, None; Nakul Singh, None; Mary E. Aronow, None; Hassan A. Aziz, None; Charles V. Biscotti, None Support: Falk Trust; Supported in part by the Cole Eye Institute, Research to Prevent Blindness Unrestricted Grant. Program Number: 4334 Presentation Time: 11:45 AM–12:00 PM Tumor diameter contributes prognostic information that enhances the accuracy of gene expression profile molecular classification in uveal melanoma Scott Walter1, Daniel L. Chao2, Joyce C. Schiffman1, William J. Feuer1, J. William Harbour1, 3. 1Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, FL; 2 Department of Ophthalmology, UCSF School of Medicine, San Francisco, CA; 3Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL. Purpose: Molecular classification using gene expression profiling (GEP) is currently the most accurate single predictor of mortality from uveal melanoma. The purpose of this study was to determine whether clinical or cytopathologic features may provide independent prognostic value in assessing mortality risk. Methods: 336 consecutive cases of uveal melanoma were analyzed using Kaplan-Meier survival analysis and Cox regression. The primary outcome measure was 5-year all cause mortality. Dependent variables included GEP molecular classification (class 1 vs. 2), patient age and sex, tumor thickness and basal diameter, ciliary body involvement and cell type. Results: After controlling for GEP molecular classification, no clinicopathologic features provided independent prognostic information except basal tumor diameter (p=0.002, Cox regression). For both class 1 and class 2 tumors, the best dichotomous cutoff for basal tumor diameter was <12mm (small) vs. ≥12mm (large). The risk of mortality relative to small class 1 tumors was 8.6 for large class 1 tumors, 9.1 for small class 2 tumors, and 78.1 for large class 2 tumors (all p<0.05). Similar results were demonstrated for melanoma-specific mortality. The model-based predicted 5-year survival was 98% for small class 1 tumors, 85-87% for large class 1 and small class 2 tumors, and 16% for large class 2 tumors. Conclusions: For both class 1 and class 2 uveal melanomas, basal tumor diameter was the only clinicopathologic factor that provided additional prognostic information that enhanced the accuracy of GEP molecular classification. Basal tumor diameter, in combination with GEP, may be a useful metric for risk stratification, especially for tumors less than 12mm in diameter. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology However, because receiving both Class 1 and Class 2 test results are possible in the setting of a non-melanoma malignancy as we have demonstrated, we recommend that cytopathology and/or other melanoma-specific testing, (e.g. fluorescence in-situ hybradization for monosomy 3, GNAQ mutation analysis) also be performed. DecisionDx-UM GEP testing alone should be interpreted with caution in choroidal lesions which are not melanomas. Commercial Relationships: Michael A. Klufas, None; Sujit Itty, None; Colin A. McCannel, None; Ben J. Glasgow, None; Christian Moreno, None; Tara A. McCannel, None Figure 1: Reverse Kaplan-Meier plot of all cause mortality over time for patients with class 1 (A) and class 2 (B) uveal melanoma by gene expression profiling. Results are stratified according to basal tumor diameter <12mm (blue line) or ≥12mm (green line). Vertical steps represent mortality events. Vertical notches represent patients alive at last follow-up. Commercial Relationships: Scott Walter, None; Daniel L. Chao, None; Joyce C. Schiffman, None; William J. Feuer, None; J. William Harbour, Castle Biosciences (C), Castle Biosciences (P) Support: R01 CA125970, Melanoma Research Alliance, Melanoma Research Foundation, and Dr. Mark J. Daily Fund (to JWH), and NIH Core Grant P30EY014801, Research to Prevent Blindness Unrestricted Grant, and Department of Defense Grant #W81XWH-09-1-0675 (to Bascom Palmer Eye Institute). Funding organizations had no role in the design or conduct of this research. Program Number: 4335 Presentation Time: 12:00 PM–12:15 PM Variable Results for Uveal Melanoma Specific Gene Expression Profile Prognostic Test in Choroidal Metastasis Michael A. Klufas, Sujit Itty, Colin A. McCannel, Ben J. Glasgow, Christian Moreno, Tara A. McCannel. Ophthalmology, Jules Stein Eye Institute/UCLA, Los Angeles, CA. Purpose: DecisionDx-UM (Castle Bioscience, Phoenix, AZ) gene expression profiling (GEP) of uveal melanoma tissue obtained by fine needle aspiration biopsy is a frequently used prognostic test for the identification of patients at high risk for uveal melanoma metastasis. Benign melanocytic lesions are believed to provide a “Class 1”, or low-risk for metastasis designation. However, cytopathology is not typically obtained to confirm the actual lesion diagnosis. At some centers, performing GEP testing on indeterminate choroidal lesions may be used to direct a decision for treatment. We report our experience with uveal melanoma specific GEP testing on a series of choroidal metastatic tumors confirmed by cytopathology. Methods: Retrospective review of all cytopathology and DecisionDx-UM GEP reports between 2012 to 2014 from intraoperative fine needle aspiration biopsy (FNAB) of choroidal tumors undergoing brachytherapy at our center. Patient medical records including ancillary testing (fundus photography, fluorescein angiography, B-scan ultrasonography) were also reviewed. Results: Four patients (four eyes) were identified to have cytopathology consistent with a non-melanoma primary. All patients presented with a unilateral, single choroidal lesion. 50% of the patients were male. Mean patient age was 74.75 years (median 72.5 years, range 67-87 years). Two patients (50%) had a history of treated systemic cancer with no previous evidence of metastatic disease (one prostate, one lung). For the two patients with no history of systemic cancer, further systemic work-up revealed primary lung cancer in both cases. A GEP result was provided for each patient, three were Class 1A, one was Class 2. Conclusions: DecisionDx-UM GEP may be a helpful test to provide molecular prognostication in patients with uveal melanoma. Program Number: 4336 Presentation Time: 12:15 PM–12:30 PM Correlation of Iris Color with Gene Expression Profile in 146 consecutive Fine Needle Biopsies of Uveal Melanoma Peter G. Hovland1, Marc Zafferani2. 1Ophthalmology, Colorado Retina Associates, Denver, CO; 2College of Osteopathic Medicine, Rocky Vista University, Parker, CO. Purpose: It has been previously demonstrated that light iris color is a risk factor for the development of uveal melanoma. This study is estimates the correlation of iris color to gene expression profile (GEP), which is an independent measure of likelihood of metastasis. Methods: A retropective chart review of a consecutive series of patients treated for uveal melanoma was performed. Patients were selected for study if they had been treated for uveal melanoma with either brachytherapy or enucleation, and if they had GEP classification (Castle BioSciences) into Class 1A, Class1B, or Class 2. Iris color classification was derived from drivers license information as administered by the Department of Motor Vehicles. Iris color was classified into one of four groups: Brown, Blue, Hazel, or Green. Complete data was available for 146 patients. Differences in GEP distributions in the separate classes of iris color was analyzed with students t-test and chi-squared analysis. Results: The population of patients was predominantly Caucasian (98%). The incidence of iris colors in the 146 patients with uveal melanoma was: Blue 66/146 (45%), Hazel 26/146 (18%), Green 14/146 (10%), Brown 40/146 (27%). Blue iris color had the following percentage distribution of GEP results: Class 1A 58%, Class 1B 14%, Class 2 29%. Brown iris color had the following percentage distribution of GEP results: Class 1A 35%, Class 1B 33%, Class 2 33%. Differences in the distributions of GEP class in different iris colors was found to be not statistically significant. Conclusions: This study is consistent with previous studies which show light iris color is a risk factor for uveal melanoma in Caucasians in that light iris color is predominant in this patient group, with brown iris color represented in a minority of 25%. The separate classes of iris color, however, do not appear to be associated with significant differences in GEP result. This study supports the hypothesis that iris color does not confer clinically significant information regarding risk of developing metastatic disease in patients with uveal melanoma. The limitations of this study are it’s relatively small size, and the dependence on the unknown relaibility of iris color determination as present in the database of the department of motor vehicles. Commercial Relationships: Peter G. Hovland, None; Marc Zafferani, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 4337 Presentation Time: 12:30 PM–12:45 PM Novel combinatorial treatment option for metastatic uveal melanoma Shahar Frenkel1, Dudi Shneor1, 2, Alik Honigman2, Jacob Pe’er1. 1 Ophthalmology, Hadassah-Hebrew Univ Med Ctr, Mevaseret Zion, Israel; 2Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel. Purpose: To date, chemotherapy for metastatic uveal melanoma (mUM) is limited to dacarbazine (DTIC) and fotemustine. We tested the effect of the common chemotherapeutic drug doxorubicin (DOX) on cell mortality in order to expand the chemotherapeutic arsenal for mUM. Methods: We examined the effect of both DITC and DOX in five different uveal melanoma cell lines – originating from metastases (OMM1, OMM2.3 and OMM2.5) and from primary tumors (92.1 and MEL270) and performed dose response tests using both drugs. Based on our previous results, we hypothesized that combining DOX and knockdown of CREB will increase cellular death. To test our hypothesis, we infected cells with replicative competent retroviruses (RCR) expressing shRNA against CREB to create a continuous infective knockdown of CREB. Results: Both chemotherapeutic drugs induced cell death in a dose dependent manner. Knockdown of CREB in these cells increased the effect of DOX on cell mortality. Conclusions: Treatment with DOX is at least as efficient and in some cases even more efficient than DTIC in inducing UM cell mortality in vitro. Moreover, the ability of combining CREB knockdown and DOX treatment to achieve the same amount of cell death in lower concentrations of DOX may result in fewer side effects from DOX. This combination is a possible new treatment for metastatic uveal melanoma. Commercial Relationships: Shahar Frenkel, None; Dudi Shneor, None; Alik Honigman, None; Jacob Pe’er, None Support: Israel Science Foundation (ISF) for physician-researcher 444 Orbit, whole globe, components Wednesday, May 06, 2015 11:00 AM–12:45 PM Exhibit Hall Poster Session Program #/Board # Range: 4719–4732/D0164–D0177 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Clinical/Epidemiologic Research, Eye Movements/Strabismus/Amblyopia/Neuro-Ophthalmology, Glaucoma Program Number: 4719 Poster Board Number: D0164 Presentation Time: 11:00 AM–12:45 PM Isolation Of Several Choroidal Cell Types And Retinal Pigmented Epithelial Cells From A Single Rabbit Eye Stephanie Proulx2, 1, Rémi Parenteau Bareil2, Olivier RochetteDrouin2, Solange Landreville2, 1. 1Ophthalmology, Laval University, Quebec, QC, Canada; 2Centre de recherche du Centre hospitalier universitaire (CHU) de Québec, axe médecine régénératrice, Quebec, QC, Canada. Purpose: Melanocytes, fibroblasts and endothelial vascular (EV) cells from the choroid as well as retinal pigment epithelial (RPE) cells are required to generate functional tissue-engineered choroidal substitutes. The purpose of this study was to develop an isolation protocol that enables the establishment of pure cultures of choroidal and RPE cells. Methods: Enucleated rabbit eyes were cut in a half just below the ora serrata. The retina and the optic nerve were then carefully removed. A 20 min dispase treatment (2.5%) and/or gentle pipetting were used to separate small sheets of RPE that were then put in culture. After a complete removal of the RPE, the choroid was peeled out of the sclera and incubated overnight in collagenase H (0,125U). The resulting cell suspension was centrifuged and incubated 10 min in trypsin-EDTA (0,05%-0,01%). The cell suspension was then filtered with a cell strainer. Dynabeads and anti-CD31 antibody were used to sort choroidal EV cells from the cell suspension. The residual mixture was equally plated in two selective growth media: a non-restrictive medium allowing fibroblast proliferation and a melanocyte growth medium containing G418 selection agent for eradication of contaminating fibroblasts. Cells were seeded onto glass coverslips and purity was assessed by phase contrast microscopy and immunofluorescence (K8/18, CD31, HMB45, vimentin). Results: The method described herein allowed for the isolation of four ocular cell types. RPE cells expressing K8/18 were successfully isolated. EV cells positive for CD31 were expended for at least three passages without any loss of phenotype. Pure cultures of HMB45positive melanocytes were obtained after a two-week culture period in G418 selective medium. Finally, cultures of fibroblasts positive for vimentin and negative for the other markers were established after the third passage. At low passages (P1), less than 1% of melanocytes were present in the fibroblast cultures. Melanocyte contamination was absent at higher passages (P3 and over). Conclusions: Our results show the feasibility of obtaining pure cultures of four ocular cell types required to develop functional tissue-engineered choroidal substitutes. These substitutes may be used as allogeneic sub-retinal grafts in preclinical studies for the treatment of retinal diseases. Commercial Relationships: Stephanie Proulx, None; Rémi Parenteau Bareil, None; Olivier Rochette-Drouin, None; Solange Landreville, None Support: Foundation Fighting Blindness, FRQ-S, CFI Program Number: 4720 Poster Board Number: D0165 Presentation Time: 11:00 AM–12:45 PM Choroidal mast cells in retinal pathology: a potential target for intervention Francine F. Behar-Cohen1, 2, elodie bousquet2, Min Zhao2, Brigitte Goldenberg2, Lorena Vieira2, Marie-Christine Naud2, Ciara Bergin1, Yvonne De Kozak2. 1Jules-Gonin Eye Hospital, Lausanne, Paris, France; 2INSERM U1138, Centre de Recherche des Coreliers, Paris, France. Purpose: We previously showed that mast cells play a role in the initiation of acute ocular inflammation but the exact consequences of mast cell degranulation on ocular pathology have not been fully characterized. The aim of this study was to analyse in depth the kinetics of events following local and acute ocular mast cells degranulation. Methods: Adult female Lewis rats (6–8 weeks old, Janvier, Le Genest-Saint-Isle, France) were used in this study. Animals were housed in a 12-hrs light and 12-hrs dark cycle and fed water and dried ration ad libitum. Experimental procedures were submitted and approved by the ethic committee of Paris Descartes University (number: Ce5/2012/122). We induced mast cells depletion of their inflammatory mediators by the local unilateral sub-conjunctival injection of 48/80 drug. Clinical examination (slit-lamp, SD-OCT) were preformed and animals were sacrificed at different time points for pathology and cytokines analysis. Results: Initial degranulation of mast cells was observed in the choroid 15 min after the injection of 48/80, with degranulation continuing to increase up to 3-6 hrs after the injection. The signs ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology of anterior segment clinical inflammation paralleled mast cell degranulation. Posterior segment imaging using optical coherence tomography showed increased choroidal thickness and serous retinal detachments, which were confirmed by histological analysis. The infiltration of polymorphonuclear cells was associated with increased ocular media levels of TNF-a at 1 and 3 hrs, followed by CXCL1, IL6, IL-5, CCL-2 at 6 and 24 hrs, and IL-1b at 24 hrs. Analysis of levels of VEGF and IL-18 at all time intervals showed an opposite evolution of VEGF as compared to IL-18 levels. Conclusions: These findings suggest that the local degranulation of ocular mast cells provokes acute ocular inflammation, increased choroidal vascular permeability and serous retinal detachments. The involvement of mast cells in retinal diseases, particularly when associated with serous detachments, should be investigated and the pharmacological inhibition of mast cells degranulation considered as a potential intervention target. Commercial Relationships: Francine F. Behar-Cohen, None; elodie bousquet, None; Min Zhao, None; Brigitte Goldenberg, None; Lorena Vieira, None; Marie-Christine Naud, None; Ciara Bergin, None; Yvonne De Kozak, None Program Number: 4721 Poster Board Number: D0166 Presentation Time: 11:00 AM–12:45 PM N-cadherin regulates morphogenesis and secretion of the ciliary body in the mouse eye Yi Zhou1, 2, Christopher P. Tanzie1, 2, Ting Xie1, 2. 1Xie Lab, Stowers Inst for Medical Research, Kansas City, MO; 2Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS. Purpose: High intraocular pressure (IOP) is often a risk factor for glaucoma. The ciliary body (CB) is an apically adherent epithelial structure responsible for aqueous humor secretion and thus IOP modulation. However, the regulation of its development and secretion remains poorly understood. This study reveals an important role of N-cadherin in the regulation of CB morphogenesis and a surprising role in the regulation of CB secretion. Methods: Trp1-cre was used to conditionally delete N-cadherin (Ncad) in both CB epithelial layers. Mutant phenotypes were characterized by standard procedures, including immunohistochemistry, mRNA in-situ hybridization, BrdU labeling, TUNEL labeling and Western blotting. Additionally, coimmunoprecipitation was performed to verify protein interactions. To further support our hypothesis, injection of Par3 shRNA lentiviruses into developing CBs was utilized to demonstrate the role of the cell polarity in CB morphogenesis. Results: Ncad mutant CBs show defective morphogenesis. Those mutant CBs maintain normal expression of the CB markers, Msx1, Otx1, Pax6 and Notch2, suggesting that N-cadherin is dispensable for CB specification. Our BrdU labeling results show that cell proliferation is drastically reduced in the Ncad mutant CBs. In addition, the cellular localization of the Par3/Par6/aPKC polarity complex is compromised in the Ncad mutant CBs, and shRNAmediated knockdown of Par3 in the developing CB also disrupts its morphogenesis, suggesting that one of the N-cadherin functions in the regulation of CB morphogenesis is to maintain the epithelial polarity. Finally, secretion of Collagen IX is also reduced in the Ncad mutant CBs. Our co-IP results show that N-cadherin physically interacts with gap junction protein Connexin43, which is essential for aqueous humor secretion, indicating that N-cadherin regulates CB secretion by modulating the function of Connexin43. Conclusions: Our findings in this study have revealed two important roles of N-cadherin in the CB: regulation of CB morphogenesis and secretion. N-cadherin regulates CB morphogenesis at least in part by stabilizing the epithelial polarity and maintaining cell proliferation. N-cadherin also regulates aqueous humor secretion by directly modulating the function of the gap junction protein Connexin43. Therefore, this study has provided important insights into how CB morphogenesis and secretion are regulated at the molecular level. Commercial Relationships: Yi Zhou, None; Christopher P. Tanzie, None; Ting Xie, None Program Number: 4722 Poster Board Number: D0167 Presentation Time: 11:00 AM–12:45 PM COMPARISON OF BRUCH MEMBRAN OPENING BETWEEN HEALTHY SUBJECTS, GLAUCOMATOUS EYES ET CENTRAL VEIN OCCLUSION (CRVO) EYES Arnaud GEORGE. Ophtalmologie, Clinique Sainte Jeanne D’arc, Saint-Brieuc, France. Purpose: To determine if there is any correlation between Bruch Membran Opening Area and CRVO, and if a small BMO area is a risk factor for CRVO. To our knowledge, this is the first study using the Heidelberg Spectralis’ new algorithm. Methods: We compare the Bruch Membran Opening Area, as measured by the new algorithm of the Spectralis (R) Optical Coherence Tomography in 20 patients with Central Retinal Vein Occlusion, their fellow eyes, 20 glaucomatous eyes and 20 normal eyes, comparable in age, gender and sex. Results: The first results show no correlation between BMO area and CRVO, as hypothesized by Hayreh. Conclusions: In this small study, we found no correlation between BMO and CRVO. We need further studies and larger samples to confirm these results. BMO area sample BMO area sample ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Commercial Relationships: Arnaud GEORGE, None Program Number: 4723 Poster Board Number: D0168 Presentation Time: 11:00 AM–12:45 PM Age related changes in the Korean pediatric human orbit on CT Choi Woo seok, Hee-Bae Ahn, Sang wook Jin, Woo Jin Jung, Yoon Hyung Kwon, Won Yeol Ryu, Hyun Wook Shin. Ophthalmology, DongA medical center, Busan, Korea (the Republic of). Purpose: The purpose of this study was to determine the relationships among orbital dimensions, globe diameter and age. Methods: A retrospective analysis of 136 CT scans of subjects with no orbital or globe disease was performed and 10 lengh measurements and 3 angle measurement of various aspects of the orbit was obtained. Results: 136 CT scans of 136 korean subjects without identifiable globe and orbital disease were included in this study. 29 subjects ≥ 17 years of age were considered mature adults and grouped together, while the remaining 107 subjects were grouped according to age. All orbital length measurements except globe protrusion increased most rapidly over the first 12 to 24 months. Vertical and horizontal orbital length was reached to 97-98% of their adults value at age 8 years and highly correlated to linear orbital measurement. Central orbital axis angle and intraorbital angle in neonate group were found to be greater than their adults group and decreased rapidly over the first 12 months. Orbital protrusion continued to increase through the neonate until adulthood. Conclusions: The growth of the korean human orbit increased most rapidly over the first 12 to 24 months and reached to 96-98% of their adults value at age 8 years. With this attempt to define normal agerelated measured orbital value and orbital changes, this is helpful to treat the pediatric orbital disease and abnormalies. Commercial Relationships: Choi Woo seok, None; Hee-Bae Ahn, None; Sang wook Jin, None; Woo Jin Jung, None; Yoon Hyung Kwon, None; Won Yeol Ryu, None; Hyun Wook Shin, None Program Number: 4724 Poster Board Number: D0169 Presentation Time: 11:00 AM–12:45 PM Changes in Angle of Optic Nerve and Angle of Ocular Orbit with Increasing Age in Japanese Children Hideyuki Tsukitome1, Yoshikazu Hatsukawa2, Tomoko Morimitsu2, Teiji Yagasaki3, Mineo Kondo1. 1Department of Ophthalmology, Mie University School of Medicine, Tsu, Japan; 2Osaka Medical Center and Research Institute for Maternal and Child Health, Sakai, Japan; 3 Yagasaki Eye Clinic, Ichinomiya-shi, Japan. Purpose: To study the changes in the opening angle of the optic nerve and angle of the ocular orbit with increasing age in normal Japanese children. Methods: We studied 147 normal children whose ages ranged from 6 months to 18 years who had undergone computed tomography(CT) as a diagnostic procedure. Measurements were done on the axial CT images that included the entire optic nerves of both eyes. The opening angle of the optic nerve was defined as the angle formed by the intersection of a line running through the left optic nerve and a vertical line passing through the center of the nose. The opening angle of the orbit was defined as the angle formed by the intersection of a line running tangentially along the deep lateral wall of left orbit and a vertical line passing through the center of the nose. The relationships between the age and these opening angles were analyzed by regression analysis. Results: The correlation between the age and the opening angle of the optic nerve was not significant. The opening angle of the orbit decreased relatively rapidly until about 2-3 years of age, and then it stabilized. The decrease in the opening angle of the orbit with increasing age was significant(P<0.001). The relationship between these two parameters was best fit by a logarithmic regression curve. Conclusions: Because the Opening angle of the orbit decreased significantly with increasing age, this factor must be considered when diagnosing and treating strabismus in children. Commercial Relationships: Hideyuki Tsukitome, None; Yoshikazu Hatsukawa, None; Tomoko Morimitsu, None; Teiji Yagasaki, None; Mineo Kondo, None Program Number: 4725 Poster Board Number: D0170 Presentation Time: 11:00 AM–12:45 PM Gene expression profiling of orbital adipose tissue in thyroid orbitopathy Jwu Jin Khong1, 2, Lynn Wang3, Gordon Smyth3, Alan A. McNab2, Thomas Hardy2, Dinesh Selva4, Shiwani Sharma6, Kathryn P. Burdon5, Ebeling Peter1, 7, Jamie E. Craig6. 1Department of Medicine, University of Melbourne, Melbourne, VIC, Australia; 2 Orbital, Plastics and Lacrimal Unit, The Royal Victorian Eye and Ear Hospital, Melbourne, VIC, Australia; 3Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; 4Ophthalmology and Visual Sciences, South Australian Institute of Ophthalmology, Adelaide, SA, Australia; 5Menzies Institute for Medical Research, Hobart, TAS, Australia; 6Department of Ophthalmology, Flinders University, Adelaide, SA, Australia; 7 Department of Medicine, Monash University, Melbourne, VIC, Australia. Purpose: Thyroid orbitopathy(TO) results from autoimmunity against orbital tissue. The underlying molecular mechanisms remain incompletely understood. We aim to determine differentially expressed genes that may be involved in stimulating orbital inflammation and fat expansion in TO using microarray gene expression profiling in a case control study. Methods: Human orbital adipose samples were obtained during orbital decompression and upper lid surgeries in patients with TO. Patients with TO were subclassified as active(n=12) or inactive(n=21). Normal controls(n=21) were patients without autoimmune thyroid disease with adipose tissue harvested from corresponding anatomical locations at the time of unrelated orbital and lid surgeries. RNA was extracted then hybridized to Illumina humanHT12 v4 microarray. Expression signals were analyzed using R. Top differentially expressed genes were compared between active and inactive TO, and between active TO and normal controls ranked by fold change, level of expression and adjusted false discovery rate<0.05. Top ranked and some lower ranked genes of interest were validated by real time PCR in additional 8 active, 13 inactive TO and 11 normal controls. Gene set enrichment analysis(GSEA) was performed. Molecular pathways were analysed using DAVID bioinformatics. Results: 721 probes representing 626 annotated genes were differerentially expressed in active compared to inactive TO. Defensins(DEFA1, DEFA1B, DEFA3) were overexpressed by 3.05-4.14 fold. TIMD4 was over-expressed by 4.3 fold. CD247, CD3A, CAMP, SLAM family member 6, GZMA, NKG7 were upregulated by 1.75-1.95 fold. Several markers for adipogenesis were overexpressed by 1.91-2.6 fold including SCD, FADS and ELOVL6. Increased expression of FADS1 and SCD were confirmed in active TO versus normal controls. GSEA revealed a significant proportion of genes upregulated in gene sets tyrosine kinase protein CSK pathway, cytotoxic T lymphocyte, T cell apoptosis, glycolysis, IL-12, T helper, T cell receptor activation, caspase cascade, TOB1, cell to cell adhesion signaling and strathmin ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology pathways. Molecular pathway analysis correlated well with GSEA analysis. Conclusions: Active TO is marked by up-regulation of genes involved in cellular, innate immune and inflammatory response and enhanced orbital adipogenesis.TIMD4, DEFA1, DEFA1B and DEFA3 may be involved in stimulating immune mediated orbital inflammation. Commercial Relationships: Jwu Jin Khong, None; Lynn Wang, None; Gordon Smyth, None; Alan A. McNab, None; Thomas Hardy, None; Dinesh Selva, None; Shiwani Sharma, None; Kathryn P. Burdon, None; Ebeling Peter, None; Jamie E. Craig, None Support: Ophthalmic Research Institute of Australia new investigator grant Program Number: 4726 Poster Board Number: D0171 Presentation Time: 11:00 AM–12:45 PM Serum Total IgG and IgG4 Levels in Thyroid Eye Disease Aileen Sy, Rona Silkiss. Ophthalmology, California Pacific Medical Center, San Francisco, CA. Purpose: IgG4-related disease (IgG4-RD) has recently been recognized as an etiology of previously described cases of orbital inflammatory disease or pseudotumor. The most common orbital inflammatory disease is thyroid eye disease (TED). The authors sought to evaluate total IgG and IgG4 levels in a TED population to determine if a subset of this population demonstrated latent IgG4-RD. Additionally, the relationship between total IgG, IgG4 and TSI levels was evaluated. Methods: TED patients evaluated in the practice of one author between December 2013 and July 2014 were reviewed. Patients with total and IgG subclass and thyroid function tests were included in the study. Results: Of 19 patients studied, three were found to have elevated serum IgG4 levels by current criteria. All three patients presented with proptosis, extraocular muscle enlargement on MRI and elevated TSI, but no other systemic findings. One patient had elevated IgG4 and IgG4: total IgG ratio, positive by IgG4-RD criteria. The IgG4 levels of the remaining two patients were not positive by IgG4-RD criteria, but were positive by IgG4: total IgG ratio. In all patients, elevated IgG4 levels were not reflected in the total IgG levels. Additionally, elevated TSI levels were not reflected in levels of total IgG or any particular subclass of IgG. Conclusions: We measured the serum IgG4 and IgG4: total IgG levels in patients with TED. Measurement of these levels in TED patients specifically has not been previously reported. Total IgG levels were not an adequate proxy for either IgG4 or TSI elevation. The results of this study suggest there may be a subpopulation of TED patients with elevated IgG4 without other systemic findings to suggest a type of IgG4-RD. This subpopulation of TED may be responsive to rituximab selectively. Further immunologic evaluation of TED patients may provide insight into the pathogenesis of the disease and aid in the selection of novel biologic therapy. Commercial Relationships: Aileen Sy, None; Rona Silkiss, None Program Number: 4727 Poster Board Number: D0172 Presentation Time: 11:00 AM–12:45 PM Galanin receptor detection in the human eye: first results Falk Schroedl1, 2, Alexandra Kaser-Eichberger1, Andrea Trost1, Barbara Bogner1, Christian Runge1, Karolina Motloch1, Daniela Bruckner1, Clemens Strohmaier1, Barbara Kofler3, Herbert A. Reitsamer1. 1Ophthalmology and Optometry, Paracelsus Medical University Salzburg, Salzburg, Austria; 2Anatomy, Paracelsus Medical University Salzburg, Salzburg, Austria; 3Dept. of Pediatrics, Laura-Bassi Centre of Expertise, THERAPEP, Paraelsus Medical University Salzburg, Salzburg, Austria. Purpose: The neuropeptide galanin (GAL) is widely distributed within intrinsic and extrinsic sources supplying the eye. It is involved in regulation of the vascular tone, thus important for ocular homeostasis. Since the presence/distribution of its receptors is unknown, we here screen for the presence of the various GAL receptors in the human eye. Methods: Meeting the Helsinki-Declaration, human eyes (n=6; 45 -83 years of age, of both sex, post mortem time 1019 hrs) were obtained from the cornea bank and prepared for immunohistochemistry against GAL receptors 1 to 3 (GALR1GALR3). Over-expressing cell assays served as positive controls and confocal laser-scanning microscopy was used for documentation. Results: In the cornea, GALR1-GALR3 were detected in basal layers of the epithelium, stroma, endothelium, as well as in adjacent conjunctiva. In the iris, GALR1-GALR3 were detected in iris sphincter, dilator and iris vessels. In the ciliary body, GALR1GALR3 were detected in ciliary muscle, with highest signal for GALR3, and in ciliary body epithelium (GALR1 >>GALR3> GALR2), while ciliary body vessels were positive for GALR3 only. In the retina, GALR1 was present in fibers of the IPL/NFL, many cells of the INL and only few cells of the ONL. GALR3 and GALR2 were present in few neurons of the INL, while GALR2 was also found surrounding retinal vessels. In the choroid, GALR1-3 were detectable in nerve fibers surrounding vessels and in intrinsic choroidal neurons. Conclusions: This is the first report of the various GALRs in the human eye. While the presence of GALR in cornea is enigmatic, the detection of GALR1-3 in ocular vessels (iris, choroid) highlights the role of GAL in vessel dynamics. High GALR3 presence in ciliary body vessels might indicate importance for aqueous humor production, whereas retinal GALR distribution might contribute to signal transduction. Commercial Relationships: Falk Schroedl, None; Alexandra Kaser-Eichberger, None; Andrea Trost, None; Barbara Bogner, None; Christian Runge, None; Karolina Motloch, None; Daniela Bruckner, None; Clemens Strohmaier, None; Barbara Kofler, None; Herbert A. Reitsamer, None Support: Research Promotion Fund of the Paracelsus Medical University PMU-FFF (E-11713/068-SRO), The Fuchs Foundation, Adele Rabensteiner Foundation, Lotte Schwarz Endowment Program Number: 4728 Poster Board Number: D0173 Presentation Time: 11:00 AM–12:45 PM Optical clearing of the human eye using the See Deep Brain (SeeDB) technique Antonio Bergua1, Bettina Hohberger1, Winfried Neuhuber2. 1 Department of Ophthalmology, University of Erlangen-Nuremberg, Erlangen, Germany; 2Department of Anatomy, University of Erlangen-Nuremberg, Erlangen, Germany. Purpose: Previous studies showed clearing of the brain using a water-based optical clearing method denominated See Deep Brain (SeeDB). However, although the eye belongs to the central nervous ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology system, this histological technique was not used until now. Applying this new clearing technique could open new possibilities for the histological visualization of the normal anatomy or pathological changes of the eye maintaining original architecture. We applied this optical clearing method for visualization of intraocular structures. Methods: Four eyes of cornea-donors (2 male, 2 female: 73 to 84 years) obtained from the Cornea Bank of the Department of Ophthalmology, were used. After enucleation and extraction of the corneoscleral button, bulbi were fixed with 4% paraformaldehyde in PBS and treated with increasing concentrations of aqueous fructose solution with 0.5% α-thioglycerol. After the optical clearing procedure, transscleral microphotographs of the choroid were taken using confocal microscopy. Results: Complete transparency of the sclera was obtained in the SeeDB treated enucleated human eyes. Transscleral visualization of the choroid is possible. The transparency of the choroid is only partial, because the SeeDB method does not act on melanocytes of the uvea. Microscopical observation and photographic documentation of vessels and other choroidal tissues is also allowed without additional processing of the specimens. Conclusions: The SeeDB method allows visualization of intraocular structures in fixed enucleated human eyes through a completely translucent sclera. This innovative imaging technique could facilitate comprehensive qualitative and quantitative studies of the whole human eye, preserving its 3D relations. Of special interest, supra- and intrachoroidal ganglionic plexus could be visualized transsclerally. Also choroidal vessels and melanocytes are now accessible for direct visualization without opening the fixed, enucleated eyes. Finally, clinical-pathological correlations of some intraocular diseases – e.g. choroidal or retinal tumors will be possible in intact eyes. Commercial Relationships: Antonio Bergua, None; Bettina Hohberger, None; Winfried Neuhuber, None Program Number: 4729 Poster Board Number: D0174 Presentation Time: 11:00 AM–12:45 PM Surgical Considerations in Vascularized Composite Allotransplantation of the Eye Nikisha Richards4, Edward Davidson1, Eric Wang3, Jenny Y. Yu4, Juan Fernandez-Miranda2, Kia M. Washington1. 1Plastic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA; 2 Neurosurgery, University of Pittsburgh Medical Center, Pittsburgh, PA; 3Otolaryngology, University of Pittsburgh Medical Center, Pittsburgh, PA; 4Ophthalmology, University of Pittsburgh Medical Center, Pittsburgh, PA. Purpose: The concept of eye transplantation is not entirely novel. In 1885, Chibret unsuccessfully transplanted a rabbit eye into a blind girl and in 1977, the National Eye Institute called for a limited and thoughtful laboratory effort in eye transplantation. Methods for reconstruction or replacement of a non-functioning eye have to date traditionally been limited to prostheses with the limiting factors of whole eye transplantation including: failure by ganglion cell axon survival, insuring adequate circulation of blood to the transplanted eye and immune rejection of foreign tissue. We propose the surgical considerations in patient selection, implantation and surgical procedure in this prospective experimental model. Methods: The critical technique prerequisite for successful transplantation surgery was the development of techniques for suturing blood vessels. Vascular anastomosis techniques have evolved tremendously most recently with vascularized composite allotransplantation (VCA). Anastomosis of donor and recipient ophthalmic arteries is well within the capabilities of a microsurgeon. Through cadaveric studies, we propose the anatomic considerations for the use of the angular or internal maxillary artery (IMAX) as ideal recipient vessels, a solution to the challenge of an anastomosis to the ophthalmic artery due to limited exposure and need to escape the zone of injury or pathology. Results: Given the larger caliber of the IMAX, it is preferred over the angular artery in some instances. We have successfully duplicated a surgical approach to the IMAX for recipient grafting: bicoronal incision followed by temporalis muscle turndown and extended lateral orbitotomy. Conclusions: Given the highly specialized function of the eye and its unique anatomical components, VCA of the eye is an appealing novel method for restoration, replacement and reconstruction of the non-functioning eye. With the advent of image guidance and evolution of endoscopic techniques, this is certainly within the realm of practicality and this trial helps to bring eye transplantation closer to clinical reality. Commercial Relationships: Nikisha Richards, None; Edward Davidson, None; Eric Wang, None; Jenny Y. Yu, None; Juan Fernandez-Miranda, None; Kia M. Washington, None Program Number: 4730 Poster Board Number: D0175 Presentation Time: 11:00 AM–12:45 PM Suture fixation for Monocanalicular stenting Milap Mehta1, 2, Nitasha Gupta2. 1Eye and Vision, Northshore University, Glenview, IL; 2Surgery, Division of Ophthalmology, University of Chicago, Chicago, IL. Purpose: Epiphora may result from multiple etiologies including increased lacrimation, decreased tear outflow, and eyelid malposition. Monocanalicular stenting using the mini-Monoka has been used to treat punctal stenosis but often results in premature stent extrusion or migration. We sought to test a simple technique to reduce stent extrusion using an externalized fixation suture.Our study was a retrospective chart review of 3 patients undergoing a modified surgical technique. Methods: Our exclusion criteria included patients with canalicular stenosis, nasolacrimal duct obstruction, dry eyes or significant ectropion as a cause for epiphora. Probing and irrigation were performed on each patient to demonstrate canalicular and nasolacrimal duct patency. Tear break-up time and fluoroscein staining were used to evaluate for dry eyes. Three patients (4 puncta) fulfilled our exclusion criteria and demonstrated only punctal stenosis. One patient had bilateral punctal stenosis. Our cohort included 2 male and 1 female patients with an average age of 52 years. Each patient underwent punctal dilation, limited punctoplasty with a single vertical blade incision using a # 11 blade, insertion of mini-Monoka® stenting, and suture fixation. A double-armed 6-0 chromic suture or double-armed 6-0 vicryl suture was first looped around the stent lip. Each arm was then passed from the punctal lumen through the eyelid skin and externalized. The ends of the sutures were tied 3 millimeters from the eyelid margin to prevent corneal irritation. The sutures were allowed to reabsorb and the patients were followed at 1 week, 1 month and 3 month intervals. Our end points were extrusion or migration of the stent prior to surgical removal. Each stent was removed after 3 months. Results: There were no cases of premature migration or extrusion. All 3 patients underwent uncomplicated stent removal after 3 months. One punctum (25%) developed a conjunctival pyogenic granuloma which was treated conservatively. All 3 patients (100%) developed symptomatic relief and all 4 puncta (100%) maintained anatomic patency at 3 month follow-up with a repeat probe and irrigation. Conclusions: The mini-Monoka® stent is an excellent option for the treatment of epiphora from punctal stenosis. Suture fixation with a double-armed 6-0 chromic or 6-0 vicryl suture reduces the risk of stent migration and extrusion with limited complications. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Commercial Relationships: Milap Mehta, None; Nitasha Gupta, None Program Number: 4731 Poster Board Number: D0176 Presentation Time: 11:00 AM–12:45 PM Immunopathology of conjunctival biopsies in patients with punctal stenosis Amit Reddy1, Meredith Baker1, Amanda Maltry1, Nasreen A. Syed1, 2, Richard C. Allen1, 3. 1Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, IA; 2Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA; 3Otolaryngology - Head and Neck Surgery, University of Iowa Hospitals and Clinics, Iowa City, IA. Purpose: Numerous etiologic processes and pharmacologic agents have been associated with punctal stenosis, yet until two recent papers examining punctal histopathology in patients with punctal stenosis, little was known about the underlying pathologic processes. These two papers reported that nearly all punctal specimens displayed signs of chronic inflammation. The purpose of this study is to expand upon these previous findings and examine the utility of conjunctival biopsy in patients with punctal stenosis without known risk factors. Methods: A retrospective chart review was performed of patients who presented to the University of Iowa Hospital and Clinics between August 2009 and October 2014 with idiopathic epiphora, were clinically diagnosed with punctal stenosis, and underwent conjunctival biopsy for histopathologic and direct immunofluorescent (DIF) examination. Patients with known systemic or pharmacologic etiologies were excluded. Results: 12 patients met inclusion criteria. Conjunctival biopsies from all 12 patients – 18 specimens – underwent histological examination. All 18 specimens showed a stromal lymphocytic infiltrate. Conjunctival biopsies from nine of the 12 patients – 12 specimens – were also evaluated by DIF. Three patients (33.3%) had depositions of shaggy and/or duplicative fibrinogen along the basement membrane zone. Conclusions: Conjunctival specimens of all patients displayed stromal lymphocytic infiltrate, confirming the previously reported findings in punctoplasty specimens. While these previous studies were performed on punctal specimens, conjunctival tissue tends to be easier to access and provides greater amounts of tissue. That conjunctival biopsies displayed similar findings to those in the previous punctal specimens suggests that conjunctival biopsies may be useful in studying these patients. Three of nine patients showed abnormal fibrinogen morphologies that are consistent with lichen planus (LP). None of these patients had signs or symptoms of LP elsewhere in the body. These cases suggest that isolated LP may be a common and underdiagnosed cause of punctal stenosis in patients without other risk factors. Thus, patients meeting these criteria may benefit from prompt histopathologic and DIF examination. Commercial Relationships: Amit Reddy, None; Meredith Baker, None; Amanda Maltry, None; Nasreen A. Syed, None; Richard C. Allen, None Program Number: 4732 Poster Board Number: D0177 Presentation Time: 11:00 AM–12:45 PM Feline Neovascular Vitreoretinopathy: A Histological Study of a Newly Described Cause of Feline Glaucoma Richard R. Dubielzig1, Billie Beckwith-Cohen1, Alison Hoffman2. 1 Pathobiol Sciences, Univ of Wisconsin-Madison, Madison, WI; 2Eye Care for Animals, Pasadina, CA. Purpose: To characterize the histopathology of twenty-one feline globes enucleated because of intractable glaucoma. Methods: The submission forms of twenty-one feline globes diagnosed with neovascular vitreoretinopathy were examined retrospectively. Case histories and follow-up information were reviewed when available. Globes were examined grossly and histologically. Histological sections were stained with hematoxylin and eosin and selected special and immunohistochemical stains were used in individual cases. Results: The median±SD age of cats diagnosed with feline neovascular vitreoretinopathy was 6±14 months. Eighteen cats were domestic, 2 were Persian and one was a British shorthair. All cats (21/21) had clinical or histological signs of glaucoma and buphthalmos. Intraocular pressure measurements were available for twelve cats and had a median±SD of 47±11mmHg. Histologically in 21/21 globes the peripheral retina was avascular and gliotic and there were epiretinal vascular profiles extending from the peripapillary retina into the vitreous. Other common histological abnormalities included: anterior segment dysgenesis (20/21), retinal detachment (20/21), lymphoplasmacytic anterior uveitis (18/21), preiridal fibrovascular membranes (21/21), peripheral anterior synechia (19/21), and ectropion uvea in 16/18 eyes where the pupillary margin was sampled. Keratitis was present in 14/21 globes, likely secondary to exposure. The status of the fellow eye was known in seventeen cats: the fellow eye was variably affected in ten. Physical examination results were available for thirteen cats and were otherwise unremarkable. Conclusions: Feline neovascular vitreoretinopathy is a rare disease of kittens and young cats. Either one or both eyes may be involved and cats often present with multiple ocular abnormalities. Etiology remains unknown. Commercial Relationships: Richard R. Dubielzig, None; Billie Beckwith-Cohen, None; Alison Hoffman, None 505 Tumors - Melanomas Thursday, May 07, 2015 8:30 AM–10:15 AM Exhibit Hall Poster Session Program #/Board # Range: 5307–5349/A0156–A0198 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Biochemistry/Molecular Biology, Genetics, Physiology/Pharmacology Program Number: 5307 Poster Board Number: A0156 Presentation Time: 8:30 AM–10:15 AM A novel classification of canine uveal melanoma: the importance of melanocytoid-type of uveal melanoma in dogs Miguel N. Burnier1, Silvin Bakalian1, Erin Mayo Goldberg2, Eduardo Perlmann3, Paulo S M. Barros3, Nancy E. Mayo4. 1Ophthalmology, McGill University, Montreal, QC, Canada; 2Henry C. Witelson Ocular Pathology Laboratory, McGill University, Montreal, QC, Canada; 3Surgery/School of Vet Medicine, University of Sao Paulo, Sao Paulo, Brazil; 4McGill University, Montreal, QC, Canada. Purpose: Canine uveal melanoma is a pigmented uveal tumor that rarely metastasizes. They are erroneously referred in the literature as melanocytomas, which is a benign melanocytic tumor. In contrast to human melanoma, the histopathological features of canine uveal melanomas exhibit classical and melanocytoma-type cells that display malignant features. The aim of this study is to describe these intraocular melanocytic lesions in dogs, propose a new classification, and compare these features with human uveal melanoma. Methods: Sixty-seven enucleated canine eyes with the clinical presentation of an intraocular pigmented tumor were evaluated. Three cases were excluded from the study (one squamous cell carcinoma and two melanocytomas). In total, 64 malignant melanomas were ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology classified into two groups (melanocytoid or classic) according to the presence or absence of large melanocytes with abundant cytoplasm and medium sized nuclei (M cells), respectively. Histopathological characteristics were compared between the two groups using Chisquare and t-tests. Multivariate discriminant analysis was conducted to identify features that discriminated the two groups. Results: Among the 64 tumors, 28 were classic and 36 were melanocytoid type. Classic melanomas were larger than melanocytoid type (18.0 mm vs. 13.6 mm; P=0.0033). Four variables (tumor size, presence of histiocytes, degree of pigmentation, and mitotic activity) were identified as discriminating between these two types, with an accuracy of 68% for classic and 100% for melanocytoid. Melanocytoid-type tumors had a smaller average size, abundant histiocytes, high degree of pigmentation, and the majority low mitotic activity (0–1 mitoses). Conclusions: To the best of our knowledge, this is the largest series of canine uveal melanomas. Canine uveal melanoma should be further classified into classic and melanoytoid-type. The melanocytoid-type melanomas possess fewer malignant features and are characterized by the presence of M cells. This classification suggests that melanocytoid-type tumors have a better prognosis than the classic type. This study reclassifies previous tumors that were called “melanocytomas” as malignant “melanocytoid-type” tumors. Thus, the term melanocytoma used ubiquitously is a misnomer in dogs as it is suggestive of a benign lesion when these lesions have an infiltrative pattern. Commercial Relationships: Miguel N. Burnier, None; Silvin Bakalian, None; Erin Mayo Goldberg, None; Eduardo Perlmann, None; Paulo S M. Barros, None; Nancy E. Mayo, None Program Number: 5308 Poster Board Number: A0157 Presentation Time: 8:30 AM–10:15 AM Inferring an evolutionary tree of uveal melanoma Nakul Singh1, Arun D. Singh3, Winston Hide2. 1School of Medicine, Case Western Reserve University, Solon, OH; 2Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom; 3 Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH. Purpose: Lack of repeated access to pathologic samples over time in a given patient limits study of tumor evolution. Herein, we apply tools developed within the field of evolutionary biology for the study of evolution of species to the genomes of primary uveal melanoma. Methods: Primary uveal melanoma genomic DNA was assayed on the Illumina Human660WQuad v1.0 DNA Analysis Bead Chip. Raw signal intensity data were quantile normalized, which were then used to estimate copy number aberration with the Genome Alteration Print algorithm. Distance between samples was calculated as the Manhattan distance between the copy number profiles of the tumors. From the distance matrix, a phylogenetic network (evolutionary relationship inference) was estimated using the SplitsTree package. Each copy number segment was tested for an association with tumor clade by means of a Fisher’s exact test, using a Benjamini-Hochberg corrected p value of 0.05. Results: Of the 57 tumors, 1(1.7%) was discarded because of a failed assay, and 8 (13.8%) revealed to be mixtures of several cell populations that could not be resolved by the GAP algorithm. Three clades of tumor, each following a distinct evolutionary path, were identified. The clades contained 29 (59.2%), 16 (32.7%), and 3 (6.1%) samples each, and were labeled Clade A, B and C, respectively. These clades were associated with metastatic status (p value 0.04 by Fisher’s exact test). From a normal diploid melanocyte, a few tumors (Clade C) lose a large portion of chromosome 6q. A few tumors within this group subsequently lose almost all of chromosome arm 1p. Clade C tumors do not develop any mutations on 8q. In an alternate path, the vast majority of tumors (Clade A and Clade B) gain a copy of the telomeric half of 8q. A majority of these tumors (Clade A) then subsequently lose a copy of chromosome 3, as well as the rest of 8q. The other tumors (Clade B) gain copies of 6p, as well as regions on 11p and 22q. Conclusions: Applying an evolutionary framework to uveal melanoma genome reveals that there are distinct subtypes of uveal melanoma, and these subtypes resemble each other at the beginning of their development, but diverge soon thereafter. Our data also suggests that there is little overlap in the subtypes of uveal melanoma after divergence that are not likely to crossover or transform from one major clade to another major clade. Commercial Relationships: Nakul Singh, None; Arun D. Singh, None; Winston Hide, None Program Number: 5309 Poster Board Number: A0158 Presentation Time: 8:30 AM–10:15 AM Generation of Luc2-eGFP-UM Cell Lines for Pre-Clinical Studies on Metastatic Uveal Melanoma Ryan P. Lee1, Michelle Sims2, Bradley T. Gao1, Hans E. Grossniklaus3, Lawrence M. Pfeffer2, Matthew W. Wilson1, Vanessa M. Morales1, 4. 1Ophthalmology, University of Tennessee Health Science Center, Memphis, TN; 2Pathology, University of Tennessee Health Science Center, Memphis, TN; 3Ophthalmology, Emory University, Atlanta, GA; 4Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Purpose: Metastatic Uveal Melanoma (UM) has no available treatment. UM is the most common primary intraocular malignancy in adults. Almost half of cases metastasize to the liver, resulting in a very poor 2-year survival, but little is known about how UM invades new tissue. Of particular interest is the ability of UM to remain dormant as sub-clinical micro-metastasis. The goal of our study was to generate UM cell lines transduced with Luciferase bioluminescent protein (Luc2) and a fluorescent reporter gene (eGFP) to investigate in vivo (1) how UM migrates to, seeds, and invades the liver and (2) the kinetics of the transition from micro-metastasis to detectable metastasis. Methods: The UM cell lines, Mel 270, 92.1, and OMM1, were transduced with a lentiviral construct (pLenti-UBC-Luc2-EGFP). Transduced cell lines were screened using IVIS® Xenogen (Caliper Life Sciences, MA) for bioluminescence and confocal microscopy for fluorescence. Validation of the transduced cell lines was performed using flow cytometry to analyze (a) cell surface expression of ICAM-1 (CD54), ICAM-2 (CD102), VCAM-1 (CD106), ALCAM (CD166), EpCAM (CD326), and VLA-4 (CD49d), (b) cell death by Annexin V and Propidium Iodide (PI), and (c) proliferative capacity by Ki-67. Cell line authentication was performed by Genetica DNA Laboratories, Inc. (Cincinnati, OH) by comparison to published material. Results: All three UM cell lines exhibited significant bioluminescence and green fluorescence in vitro 48 hours after transduction, which was sustained through time, evidencing stable transduction. Comparing transduced to non-transduced control cells, we found no difference in (a) the cohort of surface molecules, (b) the live/dead cell ratio by Annexin V and PI, and (c) proliferative capacity by Ki-67. Authentication analysis showed cell line properties were consistent with published characterization data. Conclusions: We have generated a valuable tool for the in vivo study of metastatic UM, allowing for extremely sensitive tracking of cancer cells in animal models. Using this non-invasive tool, we are currently performing in vivo analyses of the kinetics of UM metastasis. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Commercial Relationships: Ryan P. Lee, None; Michelle Sims, None; Bradley T. Gao, None; Hans E. Grossniklaus, None; Lawrence M. Pfeffer, None; Matthew W. Wilson, None; Vanessa M. Morales, None Support: Fight for Sight - Summer Fellowship Program Number: 5310 Poster Board Number: A0159 Presentation Time: 8:30 AM–10:15 AM Phenotypic heterogeneity of uveal melanoma cell lines Michael J. Young1, Natalia Vila2, Vasco Bravo2, Petr Y. Baranov1, Burke Lieppman1, Alexis Palazzolo1, Miguel N. Burnier2. 1Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, MA; 2 Ophthalmology, McGill University, Montreal, QC, Canada. Purpose: Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with 2,500 new cases diagnosed every year in United States. The “cancer stem cell” paradigm has been explored in skin melanoma and has led to better understanding of disease manifestation, progression and metastasis. The expression of stem cell markers (ABCB5, CD133, Sox2, Pax6, nestin) by small subpopulation of uveal melanoma cells in vitro and in vivo has also been previously described, although the possibility of using this “stem” subpopulation for drug discovery has not been addressed. The goal of this study was to identify new targets for drug therapy, which would be focused on suppressing proliferative potential of dedifferentiated melanoma cells. Methods: Immunocytochemistry, flow cytometry and RT-PCRT analysis was performed for 3 adherent UM cell lines: MKT-BR, OCM1, 92.1. The expression of stemness (Oct4, Nanog, Sox2, Klf4, cMyc, NMyc, LMyc, Lin28, hTERT), proliferative (FGFR, HGFR, Ki67, EpoR), eye field and melanocyte (Lhx2, Mitf, Nestin, TRP1, TRPM1), known skin melanoma markers (ABCA5, ABCB5, ABCG2) as well as several cell surface markers, expressed through neural crest (CD73, CD38, PSA-NCAM, PTK7, A2B5, CD133, HLA-ABC). For proliferation assays we have plated cells at 10,000 cells/well in 96 well plate, cultured them for 24 hours before drug treatment and assessed the population change at 48 hours after stimulation using CyQuant. The dose-response curves (7-point dilution) were calculated for several small molecules and growth factors. Results: We have observed the uniform expression of early development markers (Sox2, Klf4), proliferative marker Ki67 and melanoma markers (ABCA5, ABCB5, ABCG2) within population. We have also confirmed the expression of growth factor receptors. Cultured cell lines (92.1, MKT-BR and OCM1) contain a small subpopulation of Lhx2, CD38 (2%, 3%, 8%), CD24 (2%, 11%, 5%), PSA-NCAM (2%, 3%, 4%) and CD133 (15%, 6% and 3%, respectively) – positive cells. Proliferation studies showed the cytostatic effect of Iodoacetic acid, Rapamycin, PI3K inhibitor, Akt inhibitor, DAPT, AMPK inhibitor and Valproic acid. Wnt-3a, EGF and FGF stimulated the proliferation. Conclusions: We have identified several immature markers that are heterogeneously expressed in 3 uveal melanoma cell lines. This identified subpopulation may be considered as a target for drug therapy. Commercial Relationships: Michael J. Young, None; Natalia Vila, None; Vasco Bravo, None; Petr Y. Baranov, None; Burke Lieppman, None; Alexis Palazzolo, None; Miguel N. Burnier, None Program Number: 5311 Poster Board Number: A0160 Presentation Time: 8:30 AM–10:15 AM Identification and proteomic analysis of exosomes derived from human uveal melanoma cultures Manuel Bande1, Maria Santiago-Varela1, Maria Jose BlancoTeijeiro1, Carmela Capeans1, Antonio Pineiro1, Maria Pardo2. 1Ocular oncology Unit. Ophthalmology. Complexo Hospitalario Santiago de Compostela, Santiago de Compostela, Spain; 2Grupo obesidomica, Laboratorio de Endocrinologia molecular, Complexo Hospitalario Santiago de Compostela (SERGAS), Santiago de Compostela, Spain. Purpose: Exosomes are particles derived from cell membranes with sizes ranging between 20-120 nm. They are also attributed a role of intercellular messengers since the interior contains a variety of proteins and microRNAs. This fact makes them object of study as oncogenic biomarkers with prognostic value. The objective was to determine whether uveal melanoma cells (UM) release exosomes and to analyze their proteome. Methods: The secretome from a primary human UM cell culture established previously by our group (UM-A), and from a spontaneous cell line arisen from this culture, characterized by more invasive capability, were collected. Exosomes were isolated (ExoQuick-TC) and analyzed by 2-dimensional Differential In Gel Electrophoresis (DIGE) to quantitatively compare the differences in the protein content of exosomes from both cultures. Significant protein differences among exosomes (SameSpots, TOTALLAB) were identified by mass spectrometry analysis (MALDI-TOF/TOF). Results: 118 significant differences (p<0.001) between the two samples of exosomes were detected. From 67 proteins identified, proteins such as the mitogen activated protein kinase 3 (ERK1), and transcription regulators such as zinc finger protein 224 or LAMTOR3, were elevated in exosomes secreted by the more invasive cell line; angiogenesis inhibitors such as PEGF were found in the less invasive primary culture. Conclusions: We show for the first time the identification and characterization of exosomes released by UM cells with different invasion potential. Our results indicate that the proteomic exosome profiles vary depending on the aggressiveness of the cell line. Certain proteins from UM exosomes could be considered as potential biomarkers or treatment targets in the future. Commercial Relationships: Manuel Bande, None; Maria Santiago-Varela, None; Maria Jose Blanco-Teijeiro, None; Carmela Capeans, None; Antonio Pineiro, None; Maria Pardo, None Support: Grant Instituto de Salud Carlos III PI11/00972 Program Number: 5312 Poster Board Number: A0161 Presentation Time: 8:30 AM–10:15 AM Epigenetic Drugs Inhibit Uveal Melanoma Cell Proliferation and Cell Cycle Progression Weiwei Chen1, Jiao Wang1, Dan-Ning Hu2, Dongsheng Yan1. 1school of opthalmology and optometry, Wenzhou medical university, Wenzhou, China; 2The New York Eye and Ear Infirmary, New York, NY. Purpose: Emerging evidence indicates that epigenetic drugs, such as DNA hypomethylating agents and histone deacetylase (HDAC) inhibitors have substantial efficacy in treating some cancers. Their effects on uveal melanoma, however, are largely unknown. To deal with this question, we determined the effects of four epigenetic drugs on uveal melanoma cell proliferation and apoptosis. The drugs used include two hypomethylating agents and two HDAC inhibitors. Methods: The uveal melanoma cell lines M23 and SP6.5 were cultured and treated, respectively, with 5-azacytidine and decitabine, two hypomethylating agents approved by the FDA for the treatment ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology of myelodysplastic syndrome, SAHA and romidepsin, two HDAC inhibitors approved by the FDA for cancer therapy in treating T-cell lymphomas. The MTS assay measured uveal melanoma cell proliferation. Flow cytometry analyzed cell cycle progression. Caspase 3/7 assays examined cell apoptosis. Results: Uveal melanoma cell proliferation was dramatically inhibited after treatment with each of these four epigenetic drugs. 5-azacytidine, SAHA and romidepsin induced G1-arrest, while decitabine blocked the G2 phase. All of these epigenetic drugs did had no obvious effects on cell apoptosis. Conclusions: Our results imply that these epigenetic drugs may be developed as potential agents in uveal melanoma treatment. Commercial Relationships: Weiwei Chen, None; Jiao Wang, None; Dan-Ning Hu, None; Dongsheng Yan, None Support: National Natural Science Foundation of China(81077682 & 81272286) Program Number: 5313 Poster Board Number: A0162 Presentation Time: 8:30 AM–10:15 AM Oncogenic GNAQ orchestrates multiple signaling pathways via ARF6 Dallas S. Shi1, 3, Jae Hyuk Yoo2, Weiquan Zhu4, Dean Li1, 2. 1Human Genetics, University of Utah, Salt Lake City, UT; 2Oncological Sciences, University of Utah, Salt Lake City, UT; 3MD/PhD Program, University of Utah, Salt Lake City, UT; 4Molecular Medicine, University of Utah, Salt Lake City, UT. Purpose: Activating mutations in GNAQ and GNA11, occur in over 80% of uveal melanomas. However, the molecular mechanisms governing this stimulation remain unknown. Using biochemical tools, cellular assays, a small molecule inhibitor, and animal models of uveal melanoma, we tested the hypothesis that the small GTPase, ARF6, is necessary and sufficient for oncogenic GNAQ signaling. Methods: For in vitro experiments, Mel 92.1 and Mel 202 cells were maintained in RPMI 1640 with 10% FBS. Cells were transfected with siRNA against ARF6 or control siRNA using Lipofectamine RNAiMAX (Invitrogen). Transfected cells were then assessed for proliferation using CyQUANT (Invitrogen), anchorage-independent colony growth using CytoSelect 96-Well Cell Transformation Assay (Cell Biolabs), cell invasion using BD BioCoat Tumor Invasion Assay System (BD Bioscience), and ARF6/RhoA/Rac1/PLC levels using pull-down kits (Cells Biolabs, Millipore, Invitrogen). For in vivo experiments, nude mice (Jackson) were anesthetized with ketamine/xylazine. Then, 105 cells transfected with either ARF6 siRNA or control siRNA were injected into the posterior chamber. In a subset of mice, non-transfected cells were injected and the mice were subsequently treated with either 30mg/kg of NAV-2729 or DMSO control. All mice were euthanized after 5 weeks, and eyes were collected, fixed, embedded, sectioned, stained with H&E, and examined histologically for primary tumors by a pathologist who was blinded to the treatment regimen. Results: Compared to controls, uveal melanoma cells with ARF6 knockdown exhibited decreased proliferation, decreased anchorageindependent colony growth, decreased cell invasion, and decreased downstream signaling to PLC-PKC and Rac1/Rho-MAPK pathways (n=3, p<0.01). In vivo, mice with uveal melanoma xenografts using ARF6 knocked down cells exhibited lower tumor incidence and smaller tumor size compared to mice treated with control xenograft cells (n=12, p<0.05). Mice with uveal melanoma xenografts also had decreased tumors when treated with a novel ARF6 inhibitor, NAV2729 (n=9, p<0.05). Conclusions: In this study, we demonstrate that an activated GNAQARF6 complex orchestrates the activity of distinct tumorigenic pathways in uveal melanoma. This suggest that targeting ARF6 may inhibit all of the currently known GNAQ-mediated oncogenic signaling pathways and presents a new strategy for treating uveal melanoma. Arf6 in oncogenic GNAQ signaling. Commercial Relationships: Dallas S. Shi, None; Jae Hyuk Yoo, None; Weiquan Zhu, None; Dean Li, Navigen LLC (C) Support: NH Grants RO1CA163970, RO1NS080893, U54HL112311, RO1HL077671, ROLHL084516, and RO1AR064788 Program Number: 5314 Poster Board Number: A0163 Presentation Time: 8:30 AM–10:15 AM Effects of ranibizumab and amfenac on the functional abilities of uveal melanoma cells Vasco Bravo-Filho1, Patrick T. Logan1, Sultan Aldrees1, Natalia Vila1, Ayman Oweida2, Miguel N. Burnier1. 1Ophthalmology, McGill University, Montreal, QC, Canada; 2McGill University, Montreal, QC, Canada. Purpose: Uveal Melanoma (UM) is the most common primary intraocular tumor in adults and even with recent progress in treating the primary tumor, mortality rate is still high. Also, some tumors are too large at presentation and do not qualify for radiation therapy, which is the standard treatment. Therefore, there is a need for alternative treatment options. Our purpose was to evaluate the effects of ranibizumab in association with amfenac in human uveal melanoma cell lines. Moreover, we tested the ability of these compounds to sensitize uveal melanoma cells to radiation therapy. Methods: Proliferation and migration of the 92.1 uveal melanoma cell line were assessed after pretreatment with ranibizumab (125 microg/ml) or amfenac (150 nM), and the combination of both compounds. In addition, proliferation rates were assessed after treatment with ranibizumab and amfenac and subsequent radiation exposure. After treatment with ranibizumab and amfenac, cells were exposed to various doses of radiation: 0, 4, and 8 Gy. An MTT assay was used to assess proliferation rates 48 hours after radiation exposure, and these values were compared to control cells (without radiation and without treatment, but with radiation). Results: Cells treated with ranibizumab and amfenac had lower proliferation rates compared to controls (P=0.016), and to the group treated only with ranibizumab (P=0.033). Migration was only statistically lower in the group treated with amfenac relative to the control (P=0.014). Treatment with ranibizumab, amfenac, and the combination of both prior to the 8 Gy radiation dose led to a ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology marked reduction in proliferation rates (P=0.009; P=0.01; P=0.034; respectively). There was no statistical difference between the three treatment groups with the 8 Gy dose. Conclusions: The combination of ranibizumab and amfenac decreased the proliferation rate of uveal melanoma cells; however, only amfenac monotherapy significantly decreased cell migration. Other studies as an animal model, would help us to clarify if using this association we could avoid tumor growth. The radiosensitivity of the 92.1 UM cell line was increased by the administration of ranibizumab, amfenac, and combination therapy. Further investigation is warranted to determine if this treatment is a viable pretreatment strategy to render large tumors amenable to radiotherapy. Commercial Relationships: Vasco Bravo-Filho, None; Patrick T. Logan, None; Sultan Aldrees, None; Natalia Vila, None; Ayman Oweida, None; Miguel N. Burnier, None Program Number: 5315 Poster Board Number: A0164 Presentation Time: 8:30 AM–10:15 AM Influence of VEGF isoforms and VEGF inhibition on uveal melanoma cell lines – implications for therapeutic VEGF inhibition Alexa K. Klettner1, Michaela Dithmer1, Anna M. Kirsch1, Lidia Graefenstein1, Sarah E. Coupland2, Johann Roider1. 1Ophthalmology, Univ of Kiel, Univ Medical Center, Kiel, Germany; 2University of Liverpool, Liverpool, United Kingdom. Purpose: The influence of VEGF and its potential inhibitory impact on uveal melanoma (UM) cell growth is controversial. In this study, we investigated the effect of the VEGF isoforms VEGF165 (proangiogenic) and VEGF165b (anti-angiogenic) as well as of VEGFantagonist bevacizumab on UM cells. Methods: Five UM cell lines were used, derived from both primary tumors (92.1, Mel 270) and metastases (OMM2.5, OMM2.3, OMM1). Secretion of VEGF-A was evaluated in ELISA. Influence of 10 ng/ml and 100 ng/ml VEGF165 and VEGF165b, respectively, on UM cells was assessed. Proliferation and toxicity was assessed by WST assay, cell migration using a scratch assay. Cell death was induced by hydrogen peroxide. The expression and phosphorylation of the mitogen activated kinase, Erk1/2, and the expression of the apoptosis-related proteins, Bax and Bcl2, were evaluated in Western blots. Results: All evaluated cell lines secreted VEGF-A. Application of VEGF165 resulted in a significant reduction in cell proliferation in all lines, while VEGF165b only displayed minimal (but significant) reducing effects on the proliferation of OMM1 and OMM2.3. The application of bevacizumab (250mg/ml) had no effect. VEGF165 and VEGF165b were able to accelerate wound healing in OMM1 cells; however, they displayed a slight decelerating effect in OMM2.3 cells, with VEGF165 additionally having some effect on both OMM2.5 and Mel270 cells. Bevacizumab again displayed no effect in either cell line. In Mel270 cells, VEGF165 and VEGF165b reduced the Bax/ Bcl2 ratio. The susceptibility of UM cells to hydrogen peroxide-induced cell death varied. In all cell lines, VEGF165 protected cells from hydrogen peroxide-induced cell death; however bevacizumab was also protective. We found that in Mel 270 cell line, bevacizumab increased the pErk/Erk ratio. Conclusions: The inconsistent effect of VEGF and VEGF inhibition found in the literature can be supported by these results. VEGF was able to protect UM cells from oxidative stress-induced cell death, but, contrary to expectations, proangiogenic VEGF inhibited the proliferation rate of UM cells, while the effect of VEGF and VEGF165b on wound healing was cell line dependent. Bevacizumab, on the other hand, protected UM cells from oxidative stress-induced cell death. These data indicate that a therapy targeting VEGF only in UM is not likely to be beneficial to patients. Commercial Relationships: Alexa K. Klettner, Novartis Pharma (C), Novartis Pharma (R); Michaela Dithmer, None; Anna M. Kirsch, None; Lidia Graefenstein, None; Sarah E. Coupland, None; Johann Roider, None Program Number: 5316 Poster Board Number: A0165 Presentation Time: 8:30 AM–10:15 AM Chrysin reduces cell viability and induces apoptosis in cultured human uveal melanoma cells Codrin E. Iacob. Pathology, The New York Eye &Ear Infirmary of Mount Sinai, New York, NY. Purpose: Chrysin is a natural flavnoid and has been reported to inhibit proliferation and induce apoptosis in various tumor cells. However, the effect of chrysin on uveal melanoma cells has not been reported. The present study investigated the effects of chrysin on the viability of cultured human uveal melanoma cells and compared with normal ocular cells. Methods: Cell viability of two immortal cell lines of uveal melanoma (SP6.5 and M17) treated by chrysin (0, 10, 30 and 100 μM) was studied using the MTT test. Normal uveal melanocytes and retinal pigment epithelial cells (ARPE19) were used as the controls. Cell apoptosis was determined by annexin V-FITC staining. All studies were conducted in triplicate. Results: Chrysin showed a dose-dependent inhibitory effect on the cell viability in two uveal melanoma cell lines treated. Cell viability in cells treated with 30-100 μM chrysin was significantly (P < 0.05) lower than that in cells not treated with chrysin. The ID50 in SP6.5 and M17 cell lines were 28.3 and 35.8 μM, respectively. Annexin V-FITC staining showed that chrysin (30-100 μM) could induce apoptosis of uveal melanoma cells. Chrysin at 10-100 μM did not change the cell viability in normal uveal melanocytes or retinal pigment epithelial cells. Conclusions: Chrysin reduced the cell viability and caused apoptosis of uveal melanoma cells at 30-100 μM, but did not influence the cell viability of normal uveal melanocytes and retinal pigment epithelial cells at identical dosages. These findings suggest that chrysin has a specific cytotoxic effect on uveal melanoma cells. Commercial Relationships: Codrin E. Iacob, None Program Number: 5317 Poster Board Number: A0166 Presentation Time: 8:30 AM–10:15 AM MicroRNA-135b Inhibits Uveal Melanoma Cell Proliferation and Migration Xiaoyan Chen1, Jiao Wang1, Lihua Wang1, Dan-Ning Hu2, Dongsheng Yan1. 1Sch of Ophthal & Optometry, Wenzhou Medical College, Wenzhou, China; 2The New York Eye and Ear Infirmary, Tissue Culture Center, New York Medical College, NY. Purpose: MicroRNAs (miRNAs) can act as either oncogenes or tumor suppressors in tumorigenesis. Evidence indicates that miRNAs play important roles in uveal melanoma cell proliferation and migration. The role of miR-135b in uveal melanoma, however, remains unclear. Here, we investigated the function of miR-135b in uveal melanoma cells. Methods: Realtime RT-PCR was performed to detect the expression of miR-135b in uveal melanoma specimens as well as normal controls. Transfection of miR-135b into uveal melanoma cells was carried out by Lipofectamine RNAiMAX reagent. The proliferation of uveal melanoma cells was measured by MTS assay. Cell cycle was analyzed by flow cytometry. Cell migration was examined by transwell migration assay. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Results: miR-135b was decreased in uveal melanoma specimens as compared with normal controls. Transfection of miR-135b into uveal melanoma cells dramatically inhibited cell proliferation and blocked cell cycle at G1 phase. Furthermore, miR-135b suppressed cell migration. Conclusions: Our results demonstrated that miR-135b may act as a tumor suppressor in uveal melanoma cell proliferation and migration. Commercial Relationships: Xiaoyan Chen, None; Jiao Wang, None; Lihua Wang, None; Dan-Ning Hu, None; Dongsheng Yan, None Support: National Natural Science Foundation of China(81077682 & 81272286) Program Number: 5318 Poster Board Number: A0167 Presentation Time: 8:30 AM–10:15 AM The Importance of Sox-10 Expression in Uveal Melanoma Sarah Alghamdi, Ana Beatriz T. Dias, Mohammed F. Qutub, Julia Caminal, Josep M. Marti, Miguel N. Burnier. Henry C. Witelson Ocular Pathology Lab, McGill University, Montreal, QC, Canada. Purpose: An immunohistochemical panel of S100 protein, Melan A and HMB-45 is commonly used to confirm the diagnosis of malignant melanoma due to the lack of adequate specificity and sensitivity of a single marker. Sox10 is a neural crest transcription factor crucial for specification of Schwann cells and melanocytes. Sox-10 has been shown to be a sensitive marker of cutaneous melanoma, including spindle and desmoplastic subtypes, which are known to be negative for melanocytic markers such as Melan A and HMB-45. This study aimed to evaluate Sox-10 expression in uveal melanoma. Methods: Thirty-nine uveal melanoma cases over a period of 35 years (1980–2014) were retrieved from the Henry C. Witelson Ocular Pathology Laboratory. Formalin-fixed, paraffin-embedded blocks of enucleated eyes with uveal melanoma were cut and stained using an anti-Sox-10 mouse monoclonal antibody. The staining was scored based on the extent of the nuclear expression: diffuse when staining was seen in more than 50% of cells, and focal when it was seen in less than 50% of cells. Results: Of the 39 uveal melanomas studied, 12 were epithelioid (31%), 13 were spindle (33%), and 14 were mixed (36%) subtypes. The mean age of diagnosis was 69 with no gender predilection. Thirty-five showed diffuse nuclear positivity for Sox-10 (90%), two showed focal nuclear staining (5%), while two were negative (5%). Positivity for Sox-10 was also noted in the inner and outer nuclear layers of the retina in 78% of enucleated eyes. Overall, 17% of cases showed Sox-10 nuclear staining in retina pigmented epithelium. The uninvolved ciliary body, choroid, and iris showed focal positivity in 48%, 44%, and 11%, respectively. Five cases expressed Sox-10 in Schwann cells of the scleral nerves (13%). Conclusions: To the best of our knowledge, Sox-10 expression in uveal melanoma has never been investigated. Sox-10 expression was a sensitive, easily recognizable marker for uveal melanoma; however, it was also positive in normal ocular structures. This study suggests that Sox-10 can be incorporated in the panel for diagnosing uveal melanoma tumors. Furthermore, the observation of distinct, diffuse nuclear Sox-10 expression in retinal inner and outer nuclear layers, is a finding that warrants further investigation. Commercial Relationships: Sarah Alghamdi, None; Ana Beatriz T. Dias, None; Mohammed F. Qutub, None; Julia Caminal, None; Josep M. Marti, None; Miguel N. Burnier, None Program Number: 5319 Poster Board Number: A0168 Presentation Time: 8:30 AM–10:15 AM Programmed cell death ligand 1 is highly expressed in uveal melanoma Pablo Zoroquiain1, 2, Dominique F. de Souza1, Joao Mansure1, Mohammed F. Qutub1, Juliana Portela Passos1. 1Pathology, McGill University, Montreal, QC, Canada; 2Pathology, Pontificia Universidad catolica de Chile, Santiago, Chile. Purpose: Programmed cell death-1/ ligand (PD1/PDL1) is a pathway that negatively regulates T-cell activity. A targeted therapy blocking PD1/PDL1 has shown unprecedented tumor response in metastatic cancers. PDL1 expression in tumors has been associated with therapy response; however, this association is controversial due to the lack of a validated commercially available antibody. Despite advances in uveal melanoma (UM) treatment, 40% of patients will develop metastases from which 90% will die. The aim of this study was to analyze PDL1 expression and its prognostic value in UM. Methods: Placenta sections and 92.1 UM cells were used as a positive control to validate a newly PDL1 antibody (E1L3N clone). Negative controls were established by silencing the PDL1 gene with siRNA in UM cell lines. After validation, 40 eyes with UM and clinical information were analyzed. The retinal pigmented epithelium present in each case was used as an internal positive control. Immunohistochemical expression of PDL1 was scored based on extent:(0-3) and intensity (1= less than internal control; 2= higher or equal to internal control). A final score(IRS)was obtained multiplying both values. Low expression was considered an IRS 0-3 and high 4-6. Chi-square, and survival analysis using log-rank test were used to determine statistical significance. Results: Placenta showed staining limited to the trophoblast. UM 92.1 cells showed positive PDL1 staining. After gene silencing, there was a 9-fold decrease in PDL1 mRNA, which corresponded with a significant decrease in IRS (P<0.0001). In UM samples, 4 cases were excluded due to absence of staining in the inner positive controls. PDL1 expression was seen in 88.6% of the cases, with 38.9% of cases showing high expression. Lower PDL1 IRS was observed in epithelioid cells compared to spindle and mixed cell type (P=0.03). No differences were seen in tumor size, vascular loops, lymphocytic infiltration, and metastases. Conclusions: The commercially available anti PDL1 monoclonal antibody (E1L3N) shows high specificity. To the best of our knowledge, this is the first report showing positivity for PDL1 in UM cases. In our series, PDL1 is expressed in most cases. These results support the evaluation of anti-PD/PDL1 therapy in UM. Further studied are needed to determine the importance of this marker for predicting response to treatments targeting this pathway. Commercial Relationships: Pablo Zoroquiain, None; Dominique F. de Souza, None; Joao Mansure, None; Mohammed F. Qutub, None; Juliana Portela Passos, None Program Number: 5320 Poster Board Number: A0169 Presentation Time: 8:30 AM–10:15 AM Deferoxamine inhibits the growth of uveal melanoma cells in vitro Thanos D. Papakostas1, Fotini Nicolaou2, Ahmad Al-Moujahed1, John B. Miller1, Haijiang Lin1, Evangelos S. Gragoudas1, Demetrios Vavvas1. 1Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA; 2Surgery, Massachusetts General Hospital, Boston, MA. Purpose: To evaluate the effect of deferoxamine, an iron chelator used in clinical practice, on the growth of uveal melanoma cell lines. Methods: Two different uveal melanoma cell lines (MEL 270 and MEL285) were treated with various doses of deferoxamine (0.05 - 0.2 mg/ml). Cell growth was assessed by 3-(4,5-dimethylthiazol- ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology 2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins and cell growth transcription factors, was determined by Western blot. Results: Treatment with deferoxamine inhibited cell growth in a dose dependent manner and caused cell cycle arrest in G1 phase after 1 and 2 days of treatment and in S phase after 4 days of treatment. There was a significant increase in the population of apoptotic cells, as determined by flow cytometry. Deferoxamine decreased phosphorylation of ribosomal S6 kinase and decreased expression of cyclin D1 and cyclin dependent kinase 2. Furthermore, it resulted in activation of pro-apoptotic molecule Bcl2 and pro-senescence molecule p21. Conclusions: Deferoxamine inhibits proliferation of uveal melanoma cells in vitro via cell cycle arrest and pro-apoptotic mechanisms. These effects were seen at doses encountered in clinical use of Deferoxamine. In vivo animal studies are needed to further examine its potential for treatment of patients suffering from uveal melanoma. Commercial Relationships: Thanos D. Papakostas, None; Fotini Nicolaou, None; Ahmad Al-Moujahed, None; John B. Miller, None; Haijiang Lin, None; Evangelos S. Gragoudas, QLT (P); Demetrios Vavvas, Genentech (C), Kala Pharmaceuticals (P), Roche (C) Program Number: 5321 Poster Board Number: A0170 Presentation Time: 8:30 AM–10:15 AM The effects of polarized macrophages on melanoma growth characteristics in vitro and in vivo Marta M. Kilian1, Karin U. Loeffler1, Christiane Pfarrer2, Christian Kurts3, Frank G. Holz1, Martina C. Herwig1. 1Ophthalmology, University of Bonn, Bonn, Germany; 2Anatomy, University of Veterinary Medicine Hannover, Hannover, Germany; 3Experimental Immunology, University of Bonn, Bonn, Germany. Purpose: To investigate the influence of different tumor microenvironments on melanoma growth characteristics in vitro and in vivo. The effects of age as well as of M1- and M2- polarized macrophages on tumor growth characteristics shall be verified. Methods: Murine macrophages were polarized in vitro into M1- and M2- macrophages. The result was verified by a multiple cytokine ELISA and immunocytology. The murine melanoma cell line HCmel12 was incubated with the supernatant of M1- or M2polarized macrophages, respectively. In vitro proliferation of M1- and M2- conditioned HCmel12 cells was examined by a BrdU assay. In vivo, 26 CX3CR1+/GFP mice (M1 young n=7, M1 old n=6, M2 young n=7, M2 old n=6; young 8-12 wk, old >10 m) received an intravitreal injection of M1- or M2- conditioned HCmel12 cells. Enucleated eyes were processed for histological (H&E) and immunohistochemical (CD31, collagen 4, laminin, GFP) evaluation of tumor size and different tumor growth characteristics. Results: In vitro polarization of macrophages was effective and was confirmed by a multiple cytokine ELISA and by immunocytology. Intraocular tumor growth was determined in all mice by H&E stains. By immunohistochemistry, M2- conditioned tumors showed a higher mean vascular density (CD31 positive vessels), more frequent collagen 4- positive structures and an increased infiltration by immune cells, particularly lymphocytes and macrophages, in comparison to M1- conditioned intraocular tumors. Tumor size did not differ between M1- and M2- conditioned tumors. Age did not have any impact on tumor size or on other tumor growth characteristics. Conclusions: After in vitro M1- or M2- polarization of macrophages and subsequent M1- or M2- conditioning of HCmel12 melanoma cells, tumor growth characteristics revealed a more aggressive phenotype in M2- conditioned tumors since a high mean vascular density, infiltration of inflammatory cells and extracellular matrix structures are described as poor prognostic factors in uveal melanoma in humans. This animal study indicates that a high mean vascular density and increased collagen 4 expression might be attributed to a M2- characteristic local proangiogenic tumor microenvironment and supports the concept that M2 macrophages have a modulating effect on angiogenesis in melanoma. Commercial Relationships: Marta M. Kilian, None; Karin U. Loeffler, None; Christiane Pfarrer, None; Christian Kurts, None; Frank G. Holz, None; Martina C. Herwig, None Support: BONFOR Research Grant, intramural research fund of the University of Bonn Program Number: 5322 Poster Board Number: A0171 Presentation Time: 8:30 AM–10:15 AM Role of Chemokine Receptor 7 in Uveal and Conjunctival Melanoma Ludwig M. Heindl, Nasrin Refaian, Claus Cursiefen, Simona L. Schlereth. Department of Ophthalmology, University of Cologne, Cologne, Germany. Purpose: Conjunctival (CM) and uveal melanoma (UM) show a different metastatic behavior. While CM spreads mainly via the lymphogen routes to the draining lymph nodes, UM spreads primarily via the hematogen routes into the liver. In this study, we investigated the role of chemokine receptor 7 (CCR7) in relation to the metastatic behavior of UM and CM. Methods: Different human cell lines of uveal (Mel202, Mel270, OM431 and Mel290) and conjunctival melanoma (CRMM1, CRMM2 and CM2005) were investigated for their expression levels of CCR7 by using real time PCR and immunocytochemistry. Results: CM cell lines clearly expressed CCR7 histologically in all cell lines. The highest signal was detectable in CRMM2. UM cell lines expressed CCR7 very weakly. In some UM cell lines, nearly no signal was detectable. Real time PCR showed the highest CCR7 expression in CM2005 (p<0.01 compared to Mel270 and p<0.05 compared to CRMM2). From the tested UM cell lines OM431 showed the highest CCR7 expression level. Conclusions: Metastatic behavior of CM cells to the draining lymph nodes might be influenced by CCR7 expression, whereas the tested UM cells express very little CCR7. Commercial Relationships: Ludwig M. Heindl, None; Nasrin Refaian, None; Claus Cursiefen, None; Simona L. Schlereth, None Support: German Research Foundation (HE 6743/2-1, CU 47/61, CU 47/4-1, Priority Research Project SFB 643: B10), GEROK program by University of Cologne Program Number: 5323 Poster Board Number: A0172 Presentation Time: 8:30 AM–10:15 AM Loss of BAP1 expression is associated with increased angiogenesis in uveal melanoma Gülçin Gezgin1, Inge H. Bronkhorst1, T H. Van Essen1, Sake van Pelt1, Robert M. Verdijk2, Pieter A. van der Velden1, Gregorius P. Luyten1, Thorbald van Hall3, Sjoerd H. van der Burg3, Martine J. Jager1. 1Ophthalmology, Leiden University Medical Center, Leiden, Netherlands; 2Pathology, Erasmus University Medical Center, Rotterdam, Netherlands; 3Clinical Oncology, Leiden University Medical Center, Leiden, Netherlands. Purpose: High risk uveal melanoma (UM) is associated with an inflammatory infiltrate and show an increased amount of vessel growth. UM with monosomy 3 have an increased inflammatory infiltrate in comparison to UM with disomy 3. Although monosomy ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology 3 is associated with loss of BAP1, there are monosomy 3 cases that still have BAP1 expression. We wondered whether loss of BAP1 is associated with higher inflammatory infiltrate and more vessel growth. Therefore, the aim of this study was to analyse the association of BAP1 with inflammatory infiltrate, vessel density and pro-angiogenic factors in uveal melanoma. Methods: Fifty-four eyes with uveal melanoma obtained by enucleation between 2001 and 2007 were included and were analysed for a variety of infiltrating cells, vessel density and pro-angiogenic factors by immunostaining and RNA sequencing with Illumina. These parameters were compared with BAP1 immunohistochemistry staining. Results: Immunohistochemical staining of BAP1 was negative in 30 tumors, whereas 24 tumors stained BAP1 positive. Negative BAP1 staining showed a significant increase of HCA2, HC10, HLA-DR, CD68, CD163, CD4, FOXP3, CD8 and CD3 compared to BAP1 positive tumors (p=0.003, p=0.000, p=0.035, p=0.000, p=0.000, p=0.003, p=0.000, p=0.003, p=0.002). Also, a significantly increased CD34 staining was observed in tumors with negative BAP1 compared to BAP1 positive tumors (p=0.005), indicating that these tumors contain a higher vessel density. The immunostained BAP1 tumors were also compared with gene expression of infiltrating cells. Again, negative BAP1 tumors showed a significant increased gene expression of CD3, CD4, CD8, HLA-A, HLA-B, CD68 and CD163L1 compared to positive BAP1 tumors (p=0.015, p=0.042, p=0.016, p=0.000, p=0.000, p=0.002 and p=0.001). Gene expression of pro-angiogenic factors, such as HIF1A, PECAM1, CDH1, vWF, PDGFRB, MMP14, MMP25 and MMP9 were significantly increased in BAP1 negative tumors compared to BAP1 positive tumors (p=0.000, p=0.004, p=0.000, p=0.013, p=0.004, p=0.002, p=0.024 and p=0.025). However, some other factors, such as VHL and VEGFB, showed the opposite (p=0.003 and p=0.001). Conclusions: Loss of BAP1 expression in UM is not only associated with an increased inflammatory infiltrate, but also with an increase in vessel density and pro-angiogenic factors, suggesting that BAP1 associated leukocyte influx may stimulate angiogenesis. Commercial Relationships: Gülçin Gezgin, None; Inge H. Bronkhorst, None; T H. Van Essen, None; Sake van Pelt, None; Robert M. Verdijk, None; Pieter A. van der Velden, None; Gregorius P. Luyten, None; Thorbald van Hall, None; Sjoerd H. van der Burg, None; Martine J. Jager, None Support: ANVVB, SNOO, Stichting Blinden-Penning, LSBS Program Number: 5324 Poster Board Number: A0173 Presentation Time: 8:30 AM–10:15 AM Effect of Anti-angiogenic Agents Targeting Integrins ανβ3 on human uveal melanoma and vascular endothelia Hua Yang1, Hans E. Grossniklaus1, Wayne Harris2, Qing Zhang1, Edmund K. Waller2, Ravi Chakra3, Zhi-Ren Liu3. 1Ophthalmology, Emory University Eye Center, Atlanta, GA; 2Hematology and Medical Oncology, Emory University, Atlanta, GA; 3Biology, Georgia State University, Atlanta, GA. Purpose: Angiogenesis is a prerequisite for the growth and metastasis of solid tumors. Targeting the proliferation of tumor neovasculature will form an effective anti-cancer therapy. ProAgio is a noval anti-angiogenesis agent that targets Integrins ανβ3 of endothelial cells. In this study, we investigate the effect of the noval anti-angiogenesis agent on human uveal melanoma, mouse melanoma and human/mouse vascular endothelia cells, and screen the sensitivity of tumor cells for the further in vivo experiment. Methods: Primary human uveal melanoma cells Mel290, Mel270, OMM3, 02-1486, mouse melanoma Queens, mouse vascular endothelium sVEC and human vascular endothelium HUVEC were subcultured in 6-well-plates and treated with 5μm ProAgio or equal volume of PBS as control for 24 hours after more than 90% confluence. Harvested cells were stained with Caspase 3, CXCR4 and VEGFR2. The stained cells were acquired by FACScanto and analyzed by FlowJo software. Results: Comparing with the PBS control, ProAgio resulted in the elevated level of caspase 3 in vascular endothelia HUVEC and sVEC, the unchanged level of caspase 3 in human uveal melanoma and mouse melanoma cells. ProAgio decreased the level of CXCR4 in human uveal melanoma OMM3 and mouse vascular endothelium sVEC, also reduced the level of VEGFR2 in human uveal melanoma OMM3 and 02-1486, and vascular endothelia HUVEC, sVEC. Conclusions: ProAgio effectively caused the apoptosis in human and mouse vascular endothelia, not human uveal melanoma in vitro, suppressed the expression of VEGFR2 related with integrins ανβ3 and the migration indicator CXCR4 in vascular endothelia and some types of human uveal melanoma cells. We are able to select human uveal melanoma cells whose expressions of VEGFR2 and CXCR4 can be inhibited by ProAgio for the animal model, in purpose to know the effect of ProAgio on tumor growth and metastasis in vivo. Commercial Relationships: Hua Yang, None; Hans E. Grossniklaus, None; Wayne Harris, None; Qing Zhang, None; Edmund K. Waller, None; Ravi Chakra, None; Zhi-Ren Liu, None Support: NIH Grant R01 CA12644, R01 EY13165 and P30 EY03630, and RPB, Inc. Program Number: 5325 Poster Board Number: A0174 Presentation Time: 8:30 AM–10:15 AM The Effects of Gold Nanoparticles on the Choroidal Melanoma Cells: An In Vitro Study Hamid Ahmadieh1, Somayeh Asadi4, 3, Sahar Balagholi4, 2, Mozhgan Rezaeikanavi4, 1, Mostafa Olfat3, Narges Fazili4, Mehdi Vaez-zadeh3. 1 Ophthalmology, Ophthal Rsch Ctr, Shahid Beheshti Univ Med Sci, Tehran, Iran (the Islamic Republic of); 2Iranian Blood Transfusion Organizatio, Tehran, Iran (the Islamic Republic of); 3K.N.Toosi University of Technology, Tehran, Iran (the Islamic Republic of); 4 Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran (the Islamic Republic of). Purpose: To study the effects of different sizes and concentrations of gold nanoparticles (GNPs) on the cell viability in choroidal melanoma. Methods: GNPs were synthesized following the Fern’s method. Two different diameters of 20 and 40 nm were prepared by making changes in the citrate volume. The uveal melanoma (UM) tissue was obtained after enucleation of an eye. UM cells were cultivated in DMEM with 20% FBS. In the seventh passage, cells were grown on two 24-well plates in which the first five wells were incubated with different concentrations of GNPs and the sixth well saved as control in both plates. The first five wells in the first and second plates had the concentrations of 200, 150, 100, 50 and 25 mg for NPs with the diameter of 20nm and 600, 400, 200, 100 and 50 mg for NPs with the diameter of 40nm, respectively. The MTT assay was done after incubating the cells for 48 hours at 37°C. Results: The larger diameter of GNPs correlated with a higher reduction in cell viability. At a fixed size of GNPs, cell viability decreased with increasing GNPs concentration. The concentrations of 600 and 400 mg for GNPs with the diameter of 40 nm obviously affected the cell viability . For 20 nm diameter, nearly similar behavior for all concentrations of GNPs was observed although the concentration of 200 mg showed a more obvious decrease in the cell viability of the choroidal melanoma cells ( Fig. 1). ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Conclusions: Higher concentrations have more cytotoxic effects on the choroidal melanoma cells in GNPs with a larger diameter compared with those with a smaller diameter. The concentrations lower than 200 mg show better biocompatibility for both sizes of GNPs. Further in vivo cytotoxicity studies are required before high-concentration GNPs can be used for the treatment of choroidal melanoma . Figure 1: Cytotoxicity of gold nanoparticles with different concentrations and sizes following incubation with uveal melanoma cells after 48 hours. Commercial Relationships: Hamid Ahmadieh, None; Somayeh Asadi, None; Sahar Balagholi, None; Mozhgan Rezaeikanavi, None; Mostafa Olfat, None; Narges Fazili, None; Mehdi Vaezzadeh, None Program Number: 5326 Poster Board Number: A0175 Presentation Time: 8:30 AM–10:15 AM Characterizing the role of the p53 effector PERP in cell migration: Insights from uveal melanoma cells Raheela Awais, Lyndsay Davies, Luminita Paraoan. Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom. Purpose: PERP (p53 apoptosis-effector related to PMP-22) is a tetra-span membrane protein specifically expressed during p53mediated apoptosis. PERP also plays a pivotal role in cell-cell adhesion by enabling desmosome function. Previously, we identified PERP as an important molecular determinant of apoptosis in primary uveal melanoma (UM) tumours. We demonstrated that PERP is significantly down-regulated in the highly metastatic (monosomy 3)-type compared with the less aggressive (disomy 3) tumours. We hypothesize that down-regulation of PERP results in enhanced migratory and invasive properties of UM tumour cells due to decreased cell- cell adhesion in the absence of functional PERP. Methods: PERP expression was characterized in UM cell lines OCM1, MEL202, MEL285 and MEL 290 using qPCR and western blotting. The effect of PERP expression on the migration of GFPPERP transfected UM cells was assessed using an in vitro scratch assay and time-lapse microscopy, with non-transfected (NT) and GFP-only transfected cells as controls. The kinetic behaviour of cells was analyzed in real time with the Essen Bioscience Cell Player Migration and Kinetic Cell Invasion assay and data analyzed using IncuCyte software. Experiments were done in triplicates and statistical analysis was performed using the Student’s T-test. Results: Endogenous PERP expression was significantly reduced both at transcriptional and protein level compared to ARPE19 cells. Migration of NT and GFP-only transfected OCM1 cells led to the reduction in the gap created by the scratch. However, GFP-PERP transfected cells migrated significantly less than NT (p=0.005) or GFP-only cells (p=0.02). Kinetic quantification in four UM cell lines also demonstrated a reduction in cell migration capacity following GFP-PERP expression, with highest (71% reduction) in MEL202 and the lowest (13.5% reduction) in MEL285 compared to NT control. Conclusions: The results are consistent with the hypothesis that reduced levels of PERP lead to enhanced migratory and invasive properties of UM tumour cells resulting in metastasis. Further studies will examine whether PERP’s role in cell adhesion in UM is via direct association with the focal adhesion assembly or by indirect regulation of focal adhesion components. Acknowledgements – The work has been supported by The Humane Research Trust, UK. Commercial Relationships: Raheela Awais, None; Lyndsay Davies, None; Luminita Paraoan, None Program Number: 5327 Poster Board Number: A0176 Presentation Time: 8:30 AM–10:15 AM Fine-Needle Aspiration Biopsy of Solitary Amelanotic Choroidal Tumors in 200 Cases Chloe T. Khoo, Wasim A. Samara, Adel A. Set, Arman Mashayekhi, Emil A. Say, Jerry A. Shields, Carol L. Shields. Ocular Oncology Service, Wills Eye Hospital, Philadelphia, PA. Purpose: To analyze outcomes of fine-needle aspiration biopsy (FNAB) of indeterminate solitary amelanotic choroidal tumors. Methods: Retrospective case series of 200 eyes with solitary amelanotic choroidal tumors that underwent FNAB. Results: Of 200 consecutive solitary amelanotic choroidal tumors that underwent FNAB for diagnosis, sufficient sample for a conclusive diagnosis was obtained in 170 (85%). The most frequent diagnoses were choroidal metastasis (81/200, 41%), choroidal melanoma (74/200, 37%), non-specific inflammatory cells (8/200, 4%) and atypical lymphoid cells (3/200, 2%). There were 33 (33/81, 41%) patients with FNAB-proven choroidal metastasis without a known history of primary cancer and further evaluation revealed primary lung carcinoma in 21 (21/33, 64%) and 17 (17/21, 81%) of these patients were smokers. Further, the majority (16/17, 94%) of patients who smoked and did not have prior history of cancer were found to have smoking-related primary cancers (lung = 14/17, 82%; esophagus = 1/17, 6%; kidney = 1/17, 6%) following FNAB. Subgroup analysis showed patients with tumors cytopathologically proven to be choroidal metastasis (versus choroidal melanoma) were more likely to be older (64 vs 59, p = 0.021), have prior history of lung carcinoma (19% vs 0%, p = 0.001), better presenting visual acuity (20/40 vs 20/60, p = 0.024), and have smaller (thickness 4.5 vs 5.4 mm, p = 0.028) tumors. Prior history of breast, kidney, and other systemic cancers, as well as smoking history were not statistically different between both groups. Conclusions: FNAB is a reliable diagnostic modality for indeterminate solitary amelanotic choroidal tumors. Older patients with prior history of lung carcinoma and smaller solitary amelanotic tumors were more likely to have choroidal metastasis rather than melanoma on cytopathology. Commercial Relationships: Chloe T. Khoo, None; Wasim A. Samara, None; Adel A. Set, None; Arman Mashayekhi, None; Emil A. Say, None; Jerry A. Shields, None; Carol L. Shields, None Program Number: 5328 Poster Board Number: A0177 Presentation Time: 8:30 AM–10:15 AM Rates of Sample Acquisition in Uveal Melanoma Fine Needle Aspiration Biopsies Using Transscleral and Transvitreal Techniques Amy C. Schefler1, 2, Maru E. Bretana1, Patricia Chevez-Barrios2, Bin Teh2, Neda Nikpoor3, Daniel Gologorsky3. 1Ophthalmology, Retina Consultants of Houston, Houston, TX; 2Ophthalmology, Houston Methodist Hospital, Houston, TX; 3Ophthalmology, Bascom Palmer Eye Institute, Miami, FL. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Purpose: The purpose of this study was to compare the rate of successful sample acquisition during uveal melanoma fine needle aspiration biopsies using transscleral versus transvitreal techniques. Methods: This was a single-center, retrospective review of patients who chose to undergo fine needle aspiration biopsies for cytopathologic and genomic analyses of uveal melanoma concurrent or before definitive treatment with Iodine-125 plaque or biopsy in 2014. Only patients with 3 months or more of follow-up and adequate data for analysis were included. Clinical variables were reviewed including: patient demographic data (age, sex, right/left eye); clinical tumor data (size of tumor, presence of subretinal fluid, location of tumor); biopsy technique details (transscleral versus transvitreal, number of passes needed, adequacy of sample obtained, GEP class and discriminant value). Results: There were 30 patients who met the inclusion criteria. Ten were females and 20 were males. Seventeen of the eyes in the study were right eyes, and 13 were left eyes. The mean age of the patients was 61 (range, 19-82). Seventeen underwent biopsies with a transvitreal approach and 13 underwent biopsies via a transscleral approach. Of the 30 biopsies, only two patients who underwent transvitreal biopsies had an inadequate sample (93% success rate). Sixteen were Gene Expression Profiling Class 1A; Three patients were Class 1B; and 11 were Class 2. Mean discriminant value was 0.86 for the gene expression profiling sample and all patients except one in the study had a discriminant value > 0.1. The vast majority of the time, only one biopsy pass was needed. No patients developed a retinal detachment and transient vitreous hemorrhage occurred in nearly all patients. There were no cases of extraocular extension. Various techniques were used to ensure biopsy success including adequacy checks with the ophthalmic pathologist present in the operating room for the procedure, use of the 27 g vitreous cutter for lesions < 1.5 mm, direct visualization with a retinal viewing system, and others. Conclusions: Very high success rates can be achieved with both transscleral and transvitreal approaches to fine needle aspiration biopsy for uveal melanoma using the technical approaches described in this study. Commercial Relationships: Amy C. Schefler, None; Maru E. Bretana, None; Patricia Chevez-Barrios, None; Bin Teh, None; Neda Nikpoor, None; Daniel Gologorsky, None Program Number: 5329 Poster Board Number: A0178 Presentation Time: 8:30 AM–10:15 AM Comparison of Gene Expression Profiling and Chromosome 3 Analysis by Fluorescent In Situ Hybridization in Fine Needle Aspiration Biopsy Specimens of Uveal Melanoma Elizabeth Richter, Sujit Itty, Tara A. McCannel. Retina, Jules Stein Eye Institute, Los Angeles, CA. Purpose: To assess the concordance between results of DecisionDxUM uveal melanoma specific Gene Expression Profiling (GEP) (Castle Biosciences, Phoenix, AZ) and Fluorescent in Situ Hybridization (FISH) for chromosome 3 analysis in uveal melanomas undergoing intraoperative fine needle aspiration biopsy (FNAB) for metastatic prognostication during brachytherapy. Methods: Consecutive patients diagnosed with uveal melanoma who underwent intraoperative transscleral or transvitreal fine needle aspiration biopsy with 30-gauge needle prior to placement of iodine-125 radioactive plaque between November 2012 and January 2014, were retrospectively reviewed. Patient demographics and tumor characteristics were obtained. The results of biopsy specimens, analyzed by cytopathology, FISH for monosomy 3 and 6p gain, and for GEP analysis with the DecisionDx-UM assay, were reviewed. Results: A total of 99 intraocular tumors underwent brachytherapy with intraoperative biopsy, including 90 choroidal melanomas, 6 iris melanomas, and 3 uveal metastatic lesions. FISH and GEP results were both available in 44 (44%) patients all with iris or choroidal melanoma. Of these 44, FISH and GEP results were discordant in 7 tumors (15.9%). Six tumors were classified as “Class 1” (four 1A, two 1B), or low risk-for metastasis designation, by GEP but monosomy 3 by FISH; and one tumor was found to be “Class 2” by GEP and disomy 3 by FISH analysis. There was no significant difference with regard to tumor height (p=0.94), patient age (p=0.95), or ciliary body involvement (p=0.97) between discordant and concordant cases. No patients with discordant tumors had confirmed metastatic disease with limited follow up (mean 8.4 months, range 1- 15.5 months). Conclusions: Discordance between GEP and Chromosome 3 status by FISH occurred in our series at a rate of 15.9%, which is similar to previously published reports. No patient or tumor characteristics were identified as risk factors for discordance. The metastatic prognosis of uveal melanoma patients with discordant results is unclear from our study due to limited follow up. Commercial Relationships: Elizabeth Richter, None; Sujit Itty, None; Tara A. McCannel, None Program Number: 5330 Poster Board Number: A0179 Presentation Time: 8:30 AM–10:15 AM Genetic status of chromosome 3 in posterior uveal melanoma in relation to the development of second primary malignancies Mette Bagger2, 1, Karin Wadt2, Morten T. Andersen2, Steffen Heegaard3, 1, Mette K. Andersen2, Jens Kiilgaard1. 1Dept of Ophthalmology, Rigshospitalet/ Glostrup hospital, Copenhagen, Denmark; 2Dept of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark; 3Inst. of Eye Pathology, Copenhagen University, Copenhagen, Denmark. Purpose: To evaluate if the genetic profile of chromosome 3 in posterior uveal melanoma is indicative of the development of second primary malignancies after treatment for posterior uveal melanoma. Methods: Charts from a retrospective consecutive cohort of 153 Patients with posterior uveal melanoma treated from 2009 through 2012 were reviewed. Information on genetic analysis of chromosome 3 and synchronous cancers including histopathological descriptions were collected. Results: A total number of 28 patients (18.3 %) presented at least one second primary malignancy (excluding non-melanoma skin cancers and carcinoma in situ). In 15 patients (9.8 %) a second primary malignancy was diagnosed after treatment of posterior uveal melanoma during a median follow-up time of 3.2 years (range: 0.2-5.7, interquartile range: 2.2-4.3). All secondary cancers were histologically verified. Breast cancer, Prostate cancer, lung cancer and cutaneous melanoma were the most common. Cause of death included metastatic spread of uveal melanoma (30 ptt.), metastatic spread of other primary cancers (7 ptt.) and other causes (7 ptt.). The group of patients who developed a second primary malignancy after treatment demonstrated a significantly (p=0.03) higher frequency of chromosome 3 aberrations compared to patients who did not show any signs of new primary malignant lesions during follow-up (Figure 1). Furthermore, an increased risk of second primary malignancies was observed in patients with gain of chromosome 3 (p=0.0006). Conclusions: Our data demonstrates the importance of surveillance for second primary malignancies in patients treated for posterior uveal melanoma. Furthermore, the determination of chromosome 3 status in posterior uveal melanoma could identify a subgroup of patients with an increased risk of second primary malignancies. However this finding needs to be reproduced in a larger dataset. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology incorporated GEP class and largest basal diameter was associated with strong independent significance of each of the factors. Conclusions: Although the currently available GEP test for prognostic testing of uveal melanoma samples has asserted that recognized clinical prognostic factors for metastasis do not modify the metastatic risk of a patient with a GEP class 2 tumor, our single center study that included a substantial proportion of smaller tumors showed that both GEP class and LBD of the tumor are independent prognostic factors for metastasis and metastatic death in multivariate analysis. Commercial Relationships: Zelia M. Correa, None; James J. Augsburger, None Support: Research to Prevent Blindness Figure 1 Chromosome 3 status of posterior uveal melanoma in patients who developed a second primary malignancy during followup compared to the rest of the cohort (2). Commercial Relationships: Mette Bagger, None; Karin Wadt, None; Morten T. Andersen, None; Steffen Heegaard, None; Mette K. Andersen, None; Jens Kiilgaard, None Support: Grant information: This study was funded by research grants from Fight for Sight Denmark, The Research Fund of Rigshospitalet and The Danish Medical Research Grant/The Højmosegård Grant Program Number: 5331 Poster Board Number: A0180 Presentation Time: 8:30 AM–10:15 AM Independent Prognostic Significance of Gene Expression Profile Class and Largest Basal Diameter of Posterior Uveal Melanomas Zelia M. Correa, James J. Augsburger. Ophthalmology, University of Cincinnati, Cincinnati, OH. Purpose: To determine whether any conventional clinical prognostic factors for metastasis from uveal melanoma retain prognostic significance in multivariate models that includes gene expression profile (GEP) class of the tumor cells. Methods: Prospective IRB-approved clinical study of GEP testing and other conventional prognostic factors for metastasis and metastatic death in 299 patients with primary posterior uveal melanoma evaluated by fine-needle aspiration biopsy (FNAB) of the tumor at the time of or shortly prior to initial treatment with GEP testing of some of the aspirated tumor cells. Univariate prognostic significance of all evaluated potential prognostic variables (patient age, largest basal diameter of tumor, thickness of tumor, intraocular location of tumor, cytopathological class of tumor cells, and GEP class) was performed by comparison of Kaplan-Meier event rate curves and univariate Cox proportional hazards modeling. Multivariate prognostic significance of combinations of significant prognostic factors identified by univariate analysis was performed using step-up and step-down Cox proportional hazards modeling. Results: GEP class of the tumor cells was the strongest prognostic factor for metastatic death in this series. However, largest basal diameter of the tumor, thickness of the tumor and intraocular tumor location also proved to be significant individual prognostic factors in this study. On multivariate analysis, a two-term model that Program Number: 5332 Poster Board Number: A0181 Presentation Time: 8:30 AM–10:15 AM Uveal melanoma: clinical features and intraocular radiation treatment response based on the gene expression profiling assay Euiyong Kweon, Namchun Cho, Min Ahn, Dongwook Lee, Incheon You, Youra Kim, Woojin Kim. Chonbuk National University Medical School, Jeonju, Korea (the Republic of). Purpose: To characterize the clinical features and intraocular radiation treatment response of class 1 and class 2 uveal melanomas based on gene expression profiling assay. Methods: The charts of one hundred thirty patients who diagnosed uveal melanoma by tumor biopsy from a single institution were reviewed. The medical records of the ninety patients who underwent intraocular radiation treatment were analyzed for clinical characteristics and radiation response based upon molecular pattern of uveal melanomas. Results: Fifty-eight patients (64.4%) had class 1 tumors and thirtytwo patients (35.6%) had class 2 tumors. The mean age of patients at the time of diagnosis was significantly greater in patients with class 2 tumors (68.8±15.3 years) as compared to patients with class 1 tumors (60.1±15.2 years) (P=0.047). There was no difference in fundus findings and fluorescein angiographic features (presence of orange pigment, drusen, and subretinal fluid) (P=0.197, 0.285, 0.334) and statistically borderline difference in the rate of enucleation between 2 classes during follow-up periods (P=0.052). Mean pretreatment ultrasound thickness was significantly greater for class 2 patients (6.61±3.0 mm) as compared to class 1 patients (4.80±2.4 mm) (P=0.003). Moreover, there was significant difference between class 1 and class 2 tumors based on the extent of change in ultrasound thickness at approximately 6, 12 months after intraocular radiation treatment (P=0.018). Conclusions: Age and ultrasound tumor thickness were pretreatment findings to predict molecular pattern, but there was a large variability in most clinical parameters (gender, tumor location, chorioretinal findings, metastasis and adjunctive treatments) in both classes in determination of molecular characteristics of the uveal melanomas. This study demonstrated that class 2 uveal melanomas are generally greater in tumor thickness at diagnosis and had significantly greater decrease in thickness after intraocular radiation treatment than class 1 uveal melanomas. Commercial Relationships: Euiyong Kweon, None; Namchun Cho, None; Min Ahn, None; Dongwook Lee, None; Incheon You, None; Youra Kim, None; Woojin Kim, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 5333 Poster Board Number: A0182 Presentation Time: 8:30 AM–10:15 AM Validation of the Liverpool Uveal Melanoma Prognosticator Online for predicting survival of patients with choroidal melanoma Sarah W. DeParis, Bertil E. Damato. Ophthalmology, University of California San Francisco, San Francisco, CA. Purpose: Almost 50% of patients with choroidal melanoma develop metastatic disease despite successful eradication of the primary tumor. Accurate prognostication for patients with choroidal melanoma enhances care planning and improves patient quality of life, even when the prognosis is found to be poor. However, univariate prognostication based on anatomic, histologic or genetic predictors is not accurate enough to be relevant to individual patients. Further, the accuracy of various prognostic models is diminished by missing data. The Liverpool Uveal Melanoma Prognosticator Online (LUMPO) performs multivariate analysis of genetic, histologic and anatomical data, taking the patient’s age and sex into account and compensating for any missing data, providing prognostications that are accurate enough to be relevant to individual patients. This tool has been validated in the United Kingdom, but it remains to be seen whether it is generalizable to other patient populations. In this study, we seek to validate LUMPO in a group of patients who received care for choroidal melanoma at the University of California, San Francisco. Methods: A retrospective chart review was performed for all patients who were treated for choroidal melanoma at the University of California, San Francisco between 2002 and 2007. Tumor characteristics were recorded including largest basal tumor diameter and thickness, extraocular extension, and ciliary body involvement. In patients who underwent enucleation, histopathological data was gathered, including the presence of epithelioid cells, vascular loops, and mitotic count. Survival data nonspecific to cause of death were gathered from the UCSF Cancer Center tumor database. The data were analyzed using the method previously outlined by Eleuteri et al. Results: A total of 409 patients were compared to the established database of patients in the United Kingdom. Patient characteristics including age at diagnosis, race, gender, and laterality were found to be similar across both patient groups. Time from diagnosis to death was as well similar between the two groups. LUMPO accurately predicted survival in our cohort of patients, even when information was missing including histopathological and genetic data. Conclusions: These results confirm that LUMPO is a valuable method of prognostication for choroidal melanoma and is generalizable to patients in the United States. Commercial Relationships: Sarah W. DeParis, None; Bertil E. Damato, None Program Number: 5334 Poster Board Number: A0183 Presentation Time: 8:30 AM–10:15 AM Sirtuin 2 expression in uveal melanoma correlates with metastasis in an animal model Juliana Portela Passos, Ana Beatriz T. Dias, Pablo Zoroquiain, Christina Mastromonaco, Sarah Alghamdi. Henry C. Witelson Ocular Pathology Laboratory, McGill University, Montreal, ON, Canada. Purpose: Sirtuins (Sirt) are a family of seven enzymes that are involved in the cell cycle. Previous studies have shown that Sirt2 acts both as a tumor suppressor and as an oncogene. In uveal melanoma, we have shown that Sirt2 is preferentially expressed in malignant, but not in normal, uveal melanocytes. The purpose of this study was to analyze Sirt2 expression in primary tumors and metastases from an animal model of uveal melanoma. Methods: The rabbit model of uveal melanoma with human uveal melanoma cells injected into the suprachoroidal space used in this study has been previously described. Seventeen formalin-fixed, paraffin-embedded uveal melanoma eyes from the rabbits were immunostained for Sirt2 and were analyzed. In addition, three lung metastases were all stained and scored. Immunostaining was scored based on the intensity and distribution of the immunoreaction. Intensity was classified as 0, 1, 2, or 3, which corresponds to negative, weak, moderate, or strong staining, respectively. Extent was classified as 0 for negative, 1 for less than 50% positive cells, 2 for cells with positivity between 50% to 80%, or 3 for cells with >80% positivity. A total immunoreactive score (IRS) was then generated by multiplying intensity by extent. Results: Sirt2 was expressed in 14 of 17 primary uveal melanoma tumors analyzed with an average IRS of 1.71. Three of these animals also had metastases, which had a mean IRS of 2.33. The difference in Sirt2 staining was not statistically different between the primary tumors and metastases (P>0.05). We also compared the IRS in primary tumors from rabbits that did not have metastases (n = 14; IRS = 1.93) to those animals that had metastases (n = 3; IRS = 0.67; P = 0.19). Conclusions: Primary tumors that generated metastases had a higher expression of Sirt2 than tumors that did not metastasize. Moreover, metastastic uveal melanoma showed higher expression of Sirt2 than primary tumors. These results strongly suggest that Sirt2 acts as an oncogene in this uveal melanoma animal model. Although our data did not reach statistical significance, this may be a consequence of the limited number of metastases investigated. Future studies are warranted to confirm these findings in human ocular tumors and metastases. Commercial Relationships: Juliana Portela Passos, None; Ana Beatriz T. Dias, None; Pablo Zoroquiain, None; Christina Mastromonaco, None; Sarah Alghamdi, None Program Number: 5335 Poster Board Number: A0184 Presentation Time: 8:30 AM–10:15 AM Analysis of DNA hydroxymethylation in uveal melanoma Cindy Weidmann1, 2, Christine Yao1, 2, Jade Pomerleau1, 2, Solange Landreville1, 2. 1Ophthalmology, Université Laval, Quebec City, QC, Canada; 2Axe médecine régénératrice, Centre de recherche du CHU de Québec, Quebec City, QC, Canada. Purpose: Epigenetic regulation of cancer involves posttranslational modifications of histones and DNA methylation as a means of controlling gene expression without modifying the DNA sequence. These dynamic mechanisms allow tumor cells to adapt faster to their changing microenvironment. The enzymatic conversion of cytosine (C) to 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5hmC) silences the methylation and reactivates gene transcription. Hypoxia plays a role in the etiology of uveal melanoma (UM) according to increased expression of HIF1A by tissue microarray immunostaining. Our general hypothesis is that hypoxia modulates DNA methylation in UM. The objectives of our study are to compare the overall level of hydroxymethylation between choroidal melanocytes and UM cells, as well as to evaluate the influence of hypoxia on this epigenetic mark. Methods: Skin (N=1) and eye (N=6) melanoma tissue sections were used for immunofluorescence analyses using 5-mC and 5-hmC antibodies to study the overall levels of methylation and hydroxymethylation in situ. Melanocytes from the choroid (N=3) and UM cells (N=6) were then exposed to oxygen levels of 21% and 1%, and DNA was isolated to perform immunofluorescence analyses and slot blot using the same antibodies. Levels of 5-mC and 5-hmC were ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology quantified by measuring the densitometric mean (normalization with DAPI or an anti-ssDNA antibody; Mann-Whitney test). Results: In situ, there was no staining for 5-hmC in UM tumors while neural cells of the retina were positive. The 5-mC staining was fainter or absent in tumor cells compared to retina cells. These results were similar to the positive control (skin). In vitro, both methylation and hydroxymethylation levels were decreased in UM cells compared to normal melanocytes (P < 0.0001). Hypoxic conditions also impaired the overall levels of 5-mC and 5-hmC in all cell types (P < 0.0005 and P < 0.05, respectively). Conclusions: Overall levels of methylation and hydroxymethylation were reduced in the UM genome, and hypoxia accentuated the loss of these epigenetic marks. Unlike genetic mutations, alterations of DNA methylation are reversible, and represent potential therapeutic targets for restoring the epigenetic balance in tumor cells. Commercial Relationships: Cindy Weidmann, None; Christine Yao, None; Jade Pomerleau, None; Solange Landreville, None Support: Doctoral Research Training Award from Université Laval, Fondation du CHU de Québec, Fondation de l’Université Laval, Fondation des maladies de l’oeil, Fondation “Ophtalmologie, recherche et développement”, Canada Foundation for Innovation, Vision Health Research Network Uveal Melanoma Quebec Database and Eye Tissues Bank (FRQS), ThéCell Network (FRQS) Program Number: 5336 Poster Board Number: A0185 Presentation Time: 8:30 AM–10:15 AM The effects of acetylsalicylic acid as an anti-tumor agent in a metastatic ocular melanoma cell line Dominique Fausto de Souza, Sultan Aldrees, Mohammed F. Qutub, Sarah Alghamdi, Ana Beatriz T. Dias, Miguel N. Burnier. The Henry C. Witelson Ocular Pathology Laboratory, McGill University, Montreal, QC, Canada. Purpose: Acetylsalicylic acid (ASA) was shown to inhibit proliferation, angiogenesis and induce apoptosis in colorectal, ovarian and endometrial cancer cells. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression, via both COX-2 dependent and independent mechanisms. Currently, ASA is being evaluated in clinical trials as an adjuvant therapy to treat multiple cancer types. The goal of the current study is to evaluate the effects of ASA on metastatic uveal melanoma (UM) cells. Methods: OMM1.5, a well characterized UM cell line derived from a metastatic liver nodule, was used in the current study. OMM1.5 cells were treated with 5 mM ASA for 48 hours and compared to their untreated counterparts for their ability to secrete a panel of 10 proangiogenic cytokines using a sandwich ELISA-based array. Additionally, the functional characteristics of treated and untreated cells were compared using proliferation, invasion, and migration assays. Results: Treatment with ASA caused a significant decrease in angiogenin and PIGF secretion, and an increase in HB-EGF secretion (P<0.05). No significant change was seen for the other seven proangiogenic cytokines, which included ANG-2, EGF, bFGF, HGF, Leptin, PDGF-BB, and VEGF. Moreover, treatment with ASA significantly inhibited the proliferation and invasion capabilities of OMM1.5 cells (P<0.05 for both). Conclusions: These results suggest that ASA may be effective as an adjuvant therapy to reduce the proliferation and invasion of metastatic UM. Future studies are needed to determine the mechanisms underlying how ASA simultaneously increase and decreases some proangiogenic cytokines in metastatic UM cell lines. Commercial Relationships: Dominique Fausto de Souza, None; Sultan Aldrees, None; Mohammed F. Qutub, None; Sarah Alghamdi, None; Ana Beatriz T. Dias, None; Miguel N. Burnier, None Program Number: 5337 Poster Board Number: A0186 Presentation Time: 8:30 AM–10:15 AM Bromodomain (BRD) inhibition as a novel strategy to inhibit the Microphthalmia-associated transcription factor (MITF) axis in uveal melanoma (UM) Vassiliki Poulaki1, 2, Nicholas Mitsiades3, Warren Fiskus3. 1 Ophthalmology, Veterans Affairs Boston Healthcare System, Jamaica Plain/Boston, MA; 2School of Medicine, Boston University, Boston, MA; 3Medicine, Baylor College of Medicine, Houston, TX. Purpose: UM is universally lethal when metastatic, creating an unmet need for novel, effective, targeted systemic therapies. MITF is a critical oncogenic transcription factor in UM, yet no targeted therapies are available to inhibit it. BRDs are epigenetic readers that recognize acetylated lysine on chromatin proteins and regulate gene transcription. Lysine 27-specific acetylation of Histone 3 (H3K27ac) is associated with enhancer sites on chromatin and active transcription. Methods: We performed Chromatin ImmunoprecipitationSequencing (ChIP-Seq) analysis for BRD4, H3K27ac, MITF and RNA Polymerase II (RNAP2) in UM cells. We also examined the transcriptomic and proteomic effects of BRD4 siRNA and of the BRD inhibitor JQ1 alone and in combination with the PKC inhibitors bisindolylmaleimide I (BIM) or AEB071 against a panel of five UM cell lines in vitro and in a UM xenograft model. Results: Integrated ChIP-Seq analysis demonstrated strong genome-wide co-localization of BRD4, H3K27ac, MITF and RNAP2, suggesting that they cooperate to drive MITF-dependent transcription. Treatment of UM cells with JQ1 abolished the recruitment of BRD4 to the c-Myc and MITF genes, and suppressed the expression of c-Myc and MITF mRNAs and proteins, as well as of MITF target genes, in multiple UM cell lines. These effects were also encountered upon treatment with BRD4 siRNA. Global gene expression analysis revealed that JQ1 induces a transcriptional signature that is associated with decreased MITF activity, decreased metastatic potential and improved patient survival when applied to publicly available UM patient datasets. JQ1 increased expression of p21, p27, BCL2L11 and cleaved PARP proteins in UM cells. These effects were enhanced by the combination of JQ1 with BIM or AEB071, which led to synergistic loss of cell viability (by isobologram analysis; CI < 1.0). JQ1 significantly inhibited the growth of UM xenografts in SCID/Beige mice (p < 0.01). Conclusions: BRD4 is a central regulator of MITF-driven oncogenic signaling in UM. BRD inhibition silences c-myc and MITF expression and function, inhibits proliferation and induces apoptosis, holding promise for the treatment of UM, either as monotherapy or in combination with PKC inhibitors. JQ1 significantly inhibited the growth of UM 92.1 xenografts in SCID/Beige mice, as evidenced by bioluminescence (A) and tumor size (B and C). Commercial Relationships: Vassiliki Poulaki, None; Nicholas Mitsiades, None; Warren Fiskus, None Support: Conquer Cancer Foundation of the American Society of Clinical Oncology (ASCO) Career Development Award ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 5338 Poster Board Number: A0187 Presentation Time: 8:30 AM–10:15 AM Modulation of MMP-2 and MMP-9 as Potential Adjuvant Therapy to Limit the Growth of Metastatic Uveal Melanoma Nabil Saleh1, Anderson H. Webb1, Bradley T. Gao1, Ryan P. Lee1, Justin B. Lendermon1, Matthew W. Wilson1, Vanessa M. Morales1, 2 1 . Ophthalmology, University of Tennessee Health Science Center, Memphis, TN; 2Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Purpose: Our study focused on the progression and invasion of uveal melanoma (UM), the most common intraocular malignancy in adults. Degradation of the extracellular matrix is a critical step during tumor progression as it correlates with the ability of the microenvironment to restrict tumor growth. Matrix metalloproteinases (MMPs) are a family of zinc ion-dependent endopeptidases, which digest different substrates present in the tissue. Regulation of MMPs during the interplay of the tumor and the surrounding microenvironment is still unclear. Methods: We cultured three lines of UM cells (Mel 270, OMM1, and 92.1). Each cell lines were exposed to the common phorbol esters Phorbol 12-myristate 13-acetate (PMA) and 12-O-Tetradecanoylphorbol-13-Acetate (TPA) at varying concentrations, and to pharmacological inhibitors of MMP-2 and MMP-9 (ARP100 and AG-L-66085, Santa Cruz Biotechnology, Dallas, TX). Genomic studies and protein analyses were conducted to determine the expression of MMP-2 and MMP-9. To study effect of drug on tumor (spheroid) size, Nano3D BioPrinting assays (Nano3D Biosciences, Inc, Houston, TX) were performed. Results: Our results show MMP-2 and MMP-9 mRNA expression in UM cell lines. The expression levels are higher in the metastatic UM cell line OMM1. Gene expression was reduced upon use of inhibitors, even when phorbol esters were used. Tumor shrinkage was observed when MMP-9 inhibitors were used in the metastatic UM cell line as measured by the Nano3D BioPrinting assay. Conclusions: In this study, we investigated the changes in uveal melanoma progression in vitro after pharmacological inhibition of MMP-2 and MMP-9. Both inhibitors reduced expression of the expected targeted genes, however only the MMP-9 slowed the reduction in spheroid size. Our studies revealed pharmacological inhibition of MMP-9 reduced the size of the tumor spheroids of metastatic UM cell line in vitro. Commercial Relationships: Nabil Saleh, None; Anderson H. Webb, None; Bradley T. Gao, None; Ryan P. Lee, None; Justin B. Lendermon, None; Matthew W. Wilson, None; Vanessa M. Morales, None Program Number: 5339 Poster Board Number: A0188 Presentation Time: 8:30 AM–10:15 AM Protein Candidate Biomarkers of Uveal Melanoma Metastasis John W. Crabb1, 2, Bo Hu2, Jack S. Crabb1, Arun D. Singh1. 1Cole Eye Institute, Cleveland Clinic, Cleveland, OH; 2Lerner Research Institute, Cleveland Clinic, Cleveland, OH. Purpose: Proteomic analysis of metastasized and non-metastasized uveal melanoma primary tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis. Methods: Fifteen uveal melanoma specimens collected from enucleated eyes at the Cole Eye Institute, Cleveland Clinic were subjected to global quantitative proteomic analysis using LC MS/ MS iTRAQ technology. Bruch’s membrane choroid complex protein extracts from nine normal human postmortem eyes were pooled and utilized as a reference control specimen. Tumor and control protein was extracted in SDS and digested with trypsin. Peptides were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/SwissProtein database with the false discovery rate £ 1%. iTRAQ tags were quantified by the weighted average method (Matrix Science, 2.4.1). Differentially expressed proteins exhibited group differences with p £ 0.05 (t-test) in a sample set composed of five metastasized and five non-metastasized ocular tumors. Logistic regression modeling and principal component analysis was used to test the quantitative capability of differentially expressed proteins to classify the metastatic status of five independent ocular tumor specimens Results: Over 1700 proteins were quantified with two or more peptides from 15 uveal melanoma primary tumors. Thirty-six differentially expressed proteins were identified from quantitative comparison of proteins in 5 metastatic and 5 non-metastatic ocular tumors. Among differentially expressed proteins, the levels of collagen alpha-3 (VI) and heat shock protein beta-1 correctly classified metastasis in five independent tumor samples. The metastatic status of one independent tumor specimen was also classified correctly by 13 other differentially expressed proteins and by principal component analysis. Conclusions: While additional uveal melanoma specimens must be studied to confirm the identification of differentially expressed proteins and to establish their discriminatory capabilities between metastatic and non-metastatic tumors, the current results suggest collagen alpha-3 (VI) and heat shock protein beta-1 as candidate biomarkers of metastasis. Commercial Relationships: John W. Crabb, None; Bo Hu, None; Jack S. Crabb, None; Arun D. Singh, None Support: Supported in part by NIH grants EY021840, EY022134, Research to Prevent Blindness (RPB) Unrestricted Grant to the Cole Eye Institute, RPB Senior Investigator Award, and the Cleveland Clinic. Program Number: 5340 Poster Board Number: A0189 Presentation Time: 8:30 AM–10:15 AM Insights into Genetic Alterations of Liver Metastases from Uveal Melanoma Conni Hanke1, Andrew Dodson2, Sarah Lake1, Helen Kalirai1, Sarah E. Coupland1. 1Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom; 2 Institute of Cancer Research, London, United Kingdom. Purpose: The liver is the main organ affected by metastatic uveal melanoma (MUM), but current treatments of metastases rarely prolong life and most patients die within a year of diagnosis. In this study, the genomic aberrations present in MUMs were identified and compared with those found in matched primary UMs (PUMs) to shed light into the molecular characteristics of these tumors. Methods: DNA was extracted from 13 liver MUMs and six matched PUMs. The Affymetrix SNP Array 6.0 platform was used to detect copy number variations (CNVs), and data analysis was performed using the Partek Genomic Suite™ software. During analysis the samples were divided into different groups: (i) MUMs only, (ii) PUMs only, and (iii) matched PUM/MUM pairs. Results: Across the whole genome a broad spectrum of CNVs, mainly amplifications (87%), was observed, which confirmed the main chromosomal changes, i.e. deletions on chromosome (chr.) 3 and amplifications on chr. 8q. In total > 17000 genes with CNVs were detected for MUMs and > 20000 for PUMs. A group comparison of the MUMs with the PUMs found that the similarities between these groups varied, e.g., 94% of the amplifications on chr. 8q, but only 34% of the amplifications on chr. 9 were shared. Within the MUMs the most common CNVs were gene amplifications on chr. 8q, 20, 17, and 19 and gene deletions on chr. 3. Amplifications of the genes ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology GNAQ and GNA11 (commonly mutated in PUM), were observed in 100% of PUMs but only 58% of MUMs; the BAP1 gene was deleted in 42% of MUMs and 40% of PUMs. CNVs of the SF3B1 and EIF1AX genes were not identified in MUMs, but the former was amplified in 60% and the latter deleted in 20% of PUMs. Other interesting genes like PTP4A3 or WISP1 were amplified in 100% of both, MUMs and PUMs. For the PUM/MUM pairs between > 4500 and > 20000 genes with CNVs were identified, and between 30-55% of commonly altered genes were found. However, the comparison also revealed that the more similar the CNVs on chr. 3 and chr. 8q in these pairs, the fewer CNVs were present on other chromosomes. Conclusions: The genomic characterization of MUMs revealed a broad spectrum of CNVs with a high prevalence of amplifications on chr. 8q. Our data underline the importance of chr. 8q in UM metastatic disease. By validating the biological importance of specific gene amplifications in MUMs, novel therapeutic targets are likely to be identified. Commercial Relationships: Conni Hanke, None; Andrew Dodson, None; Sarah Lake, None; Helen Kalirai, None; Sarah E. Coupland, None Support: Liverpool RLBUHT Charitable Funds to cover salary and bench fees Program Number: 5341 Poster Board Number: A0190 Presentation Time: 8:30 AM–10:15 AM MIRNA PROFILING IN VITREOUS HUMOR, VITREAL EXOSOMES AND SERUM FROM UVEAL MELANOMA PATIENTS: PATHOLOGICAL AND DIAGNOSTIC IMPLICATIONS Michele Reibaldi, Teresio Avitabile, Andrea Russo, Antonio Longo, Mario D. Toro, Caterina Gagliano, marco ragusa, Santo Stella, Vincenza Bonfiglio, Cesare Mariotti. Catania University, Catania, Italy. Purpose: Uveal melanoma (UM) is the second most common form of melanoma, as it represents approximately 5–6 % of all melanoma diagnoses. Actual therapeutic impact on patients survival is debatable at best, given that up to 50% of patients succumb to their disease. Although several methods are available, accurate diagnosis is not always easily feasible due to potential accidents (e.g., intraocular hemorrhage). Accordingly, there is a great need for improved, minimally invasive diagnostic methods for UM. Based on the assumption that the profile of circulating miRNAs is often altered in human cancers, we sought to verify whether UM patients show different serum or vitreous humor (VH) miRNAs profiles respect to healthy controls. Methods: By using TaqMan Low Density Arrays, we analysed 754 miRNAs from VH, vitreal exosomes, and serum of 6 UM patients and 6 healthy donors. Results: Our data demonstrate that VH miRNAs profile from UM patients is unique and only partially overlaps with that from serum of the same patients.Moreover, 90% of miRNAs are shared between VH and vitreal exosomes. Interestingly, also the alterations of miRNAs expression profiles in VH and vitreal exosomes of UM patients overlap in a statistically significant manner. This could suggest that miRNAs profile alterations in VH result from the dysregulation of the exosomal molecular cargo. We reported 32 miRNAs differentially expressed in UM patients in at least two different types of samples analyzed. Comparable modifications were detected in an independent cohort of twelve ocular melanoma patients. Most alterations were common to VH and vitreal exosomes. Interestingly, miR-146a was upregulated in the serum of UM patients. Upregulation of miR-21 and miR-146a was also detected in formalin-fixed and paraffinembedded UM, suggesting that VH and serum alterations in UM patients could be the consequence of molecular mutations arising in tumoral cells. Computational functional analysis on miR-146a (the only miRNA dysregulated according to all biological matrices) showed an overrepresentation of biological functions related to cancer and immunity. Conclusions: Our findings suggest the possibility to screen the blood of UM patients to identify diagnostic miRNAs released by the affected eye: based on this, miR-146a could be considered a potential non invasive blood marker of UM. Commercial Relationships: Michele Reibaldi, None; Teresio Avitabile, None; Andrea Russo, None; Antonio Longo, None; Mario D. Toro, None; Caterina Gagliano, None; marco ragusa, None; Santo Stella, None; Vincenza Bonfiglio, None; Cesare Mariotti, None Program Number: 5342 Poster Board Number: A0191 Presentation Time: 8:30 AM–10:15 AM Ultrasonography versus Transillumination to Localize Choroidal Melanomas in Preparation for Proton Beam Irradiation Jonathan Lu1, 2, Ellen Redenbo2, Susanna S. Park2, 1. 1UC Davis School of Medicine, Sacramento, CA; 2Department of Ophthalmology & Vision Science, University of California Davis Eye Center, Sacramento, CA. Purpose: Proton beam therapy is a highly effective, globe saving local treatment for ocular melanoma, but efficacy of treatment is dependent on proper localization and placement of tantalum rings in preparation for irradiation. The ring placement currently relies on tumor localization intraoperatively using transillumination of the globe. The goal of this study was to evaluate the relative accuracy of this localization method compared to ultrasonography, and to identify tumor characteristics that would affect the relative accuracy of these two methods. Methods: This study is a retrospective chart review of all eyes with ocular melanoma from 2006 to 2014 who had tantalum rings placed in preparation for proton beam therapy where the placement of the rings were localized using both transillumination and ultrasonography. All tantalum rings were placed under general anesthesia by the same surgeon. Each eye had four rings placed at the edges of the tumor. Transillumination through the globe was performed intraoperatively to determine the distance between the rings to tumor edge. The distance between the ring and tumor edge was reconfirmed by ultrasonography postoperatively among the identified patients. Results: Among 53 patients with ocular melanoma treated with proton beam irradiation, we identified 18 patients (18 eyes) who had tumor localization performed using both ultrasonography and transillumination. The mean age was 63 years (range 22 to 89 years) and 44% were female. Among 71 rings placed around 18 tumors, 30 rings (42%) had tumor to ring distance of 1mm or greater difference between the two methods (range 0 to 4 mm difference, mean 1.1 mm + 0.3 mm). The tumor feature associated with a greater difference in tumor localization between the two imaging techniques was increasing tumor height (p value = 0.04). Tumor pigmentation (p value = 0.77) and tumor location (p value = 0.16) did not significantly affect the difference in tumor localization using the two imaging methods in this study. Conclusions: Tumor localization with tantalum rings in preparation for proton irradiation for treatment of choroidal melanoma may have some limitation in accuracy when using transillumination method alone. Postoperative ultrasonography may be a useful supplemental method of reconfirming the accuracy of tumor localization especially in tumors of increasing thickness. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Commercial Relationships: Jonathan Lu, None; Ellen Redenbo, None; Susanna S. Park, None Support: UC Davis Medical Student Research Fellowship Program Number: 5343 Poster Board Number: A0192 Presentation Time: 8:30 AM–10:15 AM Irradiation enhances chromosomal aberrations in uveal melanoma Mehmet Dogrusoz1, Wilma G. M. Kroes2, Sjoerd V. Duinen3, Carien L. Creutzberg4, Mieke Versluis1, Jaco C. Bleeker1, Marina Marinkovic1, Gregorius P. Luyten1, Martine J. Jager1. 1Department of Ophthalmology, Leiden University Medical Center, Leiden, Netherlands; 2Department of Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands; 3Department of Pathology, Leiden University Medical Center, Leiden, Netherlands; 4Department of Clinical Oncology, Leiden University Medical Center, Leiden, Netherlands. Purpose: We wondered whether uveal melanoma (UM) would show different chromosomal aberrations after previous irradiation. Chromosome analysis of UM was performed by karyotyping and fluorescence in situ hybridization (FISH). We determined the spectrum of chromosomal aberrations in a retrospective cohort of enucleated UM, some of which had previously undergone brachytherapy or proton beam irradiation. Furthermore, we analyzed which tumor features determined success or failure of karyotyping and FISH. Methods: Material from 327 UM-containing enucleated eyes was submitted for karyotyping, while FISH analysis for chromosome 3 was performed in 240 samples. 36 UM had previously undergone irradiation. The outcome of karyotyping was studied, and the success rate of both techniques was evaluated and compared to clinicopathologic characteristics of the tumor and to prior irradiation treatment. Results: Karyotypes showed that aberrations occur in all chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16 and Y being altered in at least 15% of the tumors. Aberrations were more common in previously irradiated tumors, compared to primarily enucleated cases, and reached significance for chromosomes 5 and 13 (p=0.004 and p=0.04, respectively, Table 1). Karyotyping was successful in 79% of primarily enucleated tumors, but only in 25% of previously irradiated tumors (p<0.001). In primarily enucleated cases, both a large tumor prominence (p=0.004) and a high mitotic count (p=0.007) were associated with successful karyotyping. In previously irradiated tumors, this was only true for a high mitotic count (p=0.03). FISH analysis for monosomy 3 failed significantly more often in irradiated tumors as well (p=0.04), and was more often successful when primarily enucleated tumors contained epithelioid cells (p=0.002); in previously irradiated cases, FISH analysis was more often successful in tumors with a large diameter (p=0.015) and prominence (p=0.001), and a high mitotic count (p=0.019). Conclusions: Karyotyping demonstrated that all chromosomes in UM can be aberrant, with an increased frequency after irradiation. Prior irradiation and histopathologic features characteristic of low tumor aggressiveness were associated with both unsuccessful karyotyping and FISH. The frequent lack of success in irradiated tumors emphasizes the shortcomings of these techniques and shows the relevance of taking biopsies for chromosomal testing prior to irradiation treatment. Commercial Relationships: Mehmet Dogrusoz, None; Wilma G. M. Kroes, None; Sjoerd V. Duinen, None; Carien L. Creutzberg, None; Mieke Versluis, None; Jaco C. Bleeker, None; Marina Marinkovic, None; Gregorius P. Luyten, None; Martine J. Jager, None Program Number: 5344 Poster Board Number: A0193 Presentation Time: 8:30 AM–10:15 AM Diffusion-Weighted Magnetic Resonance Imaging And Ultrasound Evaluation Of Choroidal Melanomas After Proton Beam Therapy Livio Giulio Marco Franco1, Teresio Avitabile1, Michele Reibaldi1, Antonio Longo1, Vincenza Bonfiglio1, Seby Flavio Gulisano1, Pietro Valerio Foti2, marco ragusa3, Rosario Caltabiano4, Andrea Russo1. 1Institute of Ophthalmology, University of Catania, Catania, Italy; 2Department of Radiology, University of Catania, Catania, Italy; 3Department Gian Filippo Ingrassia, Unità di BioMedicina Molecolare Genomica e dei Sistemi Complessi, Genetica, Biologia Computazionale, University of Catania, Catania, Italy; 4Department Gian Filippo Ingrassia, Unità di Anatomia Patologica, University of Catania, Catania, Italy. Purpose: To compare the ultrasound and magnetic resonance imaging parameters of ocular melanoma and to assess their variation after proton-beam therapy. Methods: Fifteen choroidal melanoma patients treated with protonbeam therapy were enrolled in the study. All patients underwent ophthalmologic evaluations, ultrasound, conventional magnetic resonance (MR) imaging and diffusion-weighted MR imaging before the start of therapy and 3 and 6 months after therapy. Basal diameters, thickness, internal reflectivity, tumor volumes and apparent diffusion coefficient (ADC) values of ocular melanomas were measured at each examination. Correlations between internal reflectivity and ADC were investigated. Results: No significant changes were seen in tumor diameters and tumor height as assessed by B-scan and A-scan respectively. Significant increase in mean tumor internal reflectivity was detected at 6 months (baseline: 35 % ± 11; 6 months: 48 % ± 8, Tukey-Kramer p=0.005). By MRI, compared to baseline (mean 547 ± 262 mm3), a ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology significant reduction in volume was seen at 6 months (Tukey-Kramer p= 0.045) (mean volume 339 ± 170 mm3, mean reduction 38%). A significant increase in ADC (baseline 1002 ± 109 mm2/sec) was detected both at 3 and 6 months after proton-therapy (respectively 1454 ± 90 and 1833±261, mm2/sec, both p<0.001). Conclusions: By MRI, in particular by ADC assessment, it is possible to detect early variations in melanoma treated by proton beam therapy. This examination could be used together with ultrasound in the follow-up of this treatment. Commercial Relationships: Livio Giulio Marco Franco, None; Teresio Avitabile, None; Michele Reibaldi, None; Antonio Longo, None; Vincenza Bonfiglio, None; Seby Flavio Gulisano, None; Pietro Valerio Foti, None; marco ragusa, None; Rosario Caltabiano, None; Andrea Russo, None Program Number: 5345 Poster Board Number: A0194 Presentation Time: 8:30 AM–10:15 AM Diffuse Intraocular Proliferation of uveal melanoma following radiotherapy Amir Mohsenin1, Vivek A. Rudrapatna2, George J. Harocopos3, Sander R. Dubovy1, J William Harbour1. 1Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, FL; 2Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY; 3Department of Ophthalmology, Washington University School of Medicine, St. Louis, MO. Purpose: To describe the presentation and management of diffuse intraocular proliferation of uveal melanoma following radiotherapy Methods: A retrospective chart review was conducted identifying patients with uveal melanoma treated with radiotherapy who later developed vitreous and retinal invasion by melanoma cells. Results: 5 patients were identified. All patients had a collarbutton tumor configuration. 1 patient had a biopsy performed at time of radiotherapy. 1 patient was managed with vitrectomy alone. 2 patients that were initially treated with vitrectomy and intravitreal methotrexate required enucleation. Enucleated specimens demonstrated retinal and vitreous invasion by melanoma cells. Conclusions: Intraocular proliferation of uveal melanoma is a rare and challenging sequela after radiotherapy that may warrant aggressive intervention. Commercial Relationships: Amir Mohsenin, None; Vivek A. Rudrapatna, None; George J. Harocopos, None; Sander R. Dubovy, None; J William Harbour, None Support: NIH Center Core Grant P30EY014801, Research to Prevent Blindness Unrestricted Grant Program Number: 5346 Poster Board Number: A0195 Presentation Time: 8:30 AM–10:15 AM Uveal Melanoma Regression After Brachytherapy: Relationship with Monosomy Chromosome 3 Hassan A. Aziz1, Sachin Salvi2, Nakul Singh3, Suhail Dar3, brandy hayden1, Arun D. Singh1. 1Cole Eye Institute, Miami, FL; 2Royal Hallamshire Hospital, Sheffield, United Kingdom; 3Case Western Reserve University School of Medicine, Cleveland, OH. Purpose: To explore relationship between regression rate of ciliary/ choroidal melanoma after plaque radiotherapy and percentage composition with chromosome 3 monosomy tumor cells. Methods: One hundred fifty consecutive patients underwent fine needle aspiration biopsy at the time of plaque radiation therapy to sample tumor cells for prognostication. Fluorescence in situ hybridization (FISH) was performed to evaluate the percentage of tumor cells with chromosome 3 monosomy (200 cell count). Regression rate was calculated as percent change in tumor height (measured by ultrasonography). Relationship between regression rate and tumor location, initial tumor height, and percentage chromosome 3 monosomy was assessed by univariate linear regression at months 3, 6, and 12 following plaque radiation therapy (R version 3.1.0). Results: Of the 150 patients, 75 patients were excluded because treatment (enucleation and resection, 48) or location of the tumor (irido ciliary, 16 ) or lack of cytological confirmation (11) of the diagnosis. At time of diagnosis the mean tumor height was was 5.15 mm (1.90-13.00mm). Percent chromosome 3 monosomy ranged from 0-20% (35) to 81-100 (10%). The percentage of tumor height at months 3, 6, and 12 did not statistically correlate with tumor location (ciliray or choroidal), initial tumor height, and percentage chromosome 3 monosomy (Figure 1). Conclusions: Regression rate in height of uveal melanoma following brachytherapy does not correlate with the percentage composition of tumor cells with chromosome 3 monosomy. Commercial Relationships: Hassan A. Aziz, None; Sachin Salvi, None; Nakul Singh, None; Suhail Dar, None; brandy hayden, None; Arun D. Singh, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 5347 Poster Board Number: A0196 Presentation Time: 8:30 AM–10:15 AM Sorafenib in metastatic uveal melanoma : A multicentric prospective phase II study Frederic Mouriaux1, Vincent Servois2, Sophie Piperno-Neumann2, Thierry Lesimple3, Antoine Thyss4, Thomas Jouary5, Eve-Marie Neidhart-Berard6, Nicolas Penel7, Corinne Delcambre8, Françoise Joly8. 1Ophthalmology, CHU, Rennes, France; 2Institut Curie, Paris, France; 3Centre Eugène Marquis, Rennes, France; 4Centre Antoine Lacassagne, Nice, France; 5Dermatology, CHU, Bordeaux, France; 6 Centre Antoine Berard, Lyon, France; 7Centre Oscar Lambret, Lille, France; 8Centre François Baclesse, Caen, France. Purpose: Uveal melanoma is the most common primary ocular malignancy in adults, accounting for 70 % of all eye malignancy. Despite adequate local treatment, about 30-50 % of patients develop metastasis and the survival rate is 50% after 10 years and the median survival time is 5-7 months after development of metastasis. There is no consensual and effective treatment in metastatic uveal melanoma. Preclinical data suggest that Sorafenib, a multikinase inhibitor of cell proliferation and angiogenesis may be a potential treatment of metastatic uveal melanoma. The primary objective of this study was the non-progression rate at 6 months with 800 mg/day of Sorafenib in patient with metastatic uveal melanoma. Methods: Multicenter, non-comparative, non-randomized, phase II study was conducted. The primary objective was to determine the non-progression rate at 6 months for patients receiving Sorafenib at dose of 400 mg twice per day orally according the RECIST criteria. Quality of Life and adverse events were evaluated. Secondary efficacy endpoints included progression-free survival, overall survival, quality of life and adverse events Results: 32 patients were enrolled in this study and 29 patients were evaluable. Non-progression at 6 months was observed in 11 patients (38%). Non-progression at 12 months was observed in 4 patients (13%). The overall survival was 10 months. Fatigue was a frequent adverse event and limited the quality of life. Conclusions: Sorafenib at dose of 400 mg twice per day orally showed non-progression rate at 6 months in some patients with metastatic uveal melanoma Commercial Relationships: Frederic Mouriaux, None; Vincent Servois, None; Sophie Piperno-Neumann, None; Thierry Lesimple, None; Antoine Thyss, None; Thomas Jouary, None; Eve-Marie Neidhart-Berard, None; Nicolas Penel, None; Corinne Delcambre, None; Françoise Joly, None Clinical Trial: N° EudraCT : 2010-022527-29 Program Number: 5348 Poster Board Number: A0197 Presentation Time: 8:30 AM–10:15 AM Unsuspected and misdiagnosed ciliary body and choroidal melanoma after enucleation and evisceration: review of cases in the Ottawa-Gatineau region from 1996-2012 Solin Saleh1, 2, Seymour Brownstein1, 2, André Jastrzebski1, 2, David Jordan1, Steven Gilberg1, Brian Leonard1, Bernard Hurley1. 1 Ophthalmology, University of Ottawa, Ottawa, ON, Canada; 2 Pathology, University of Ottawa, Ottawa, ON, Canada. Purpose: There is little in the recent literature on the topic of clinically unsuspected and misdiagnosed ciliary body and choroidal melanomas with a calculation of the frequency of these events for a set geographical area. We present 2 cases unexpectedly diagnosed with a melanoma of the ciliary body and choroid after enucleation and evisceration, respectively, and 2 cases of clinically misdiagnosed ciliary body and choroidal melanoma. As the only ophthalmic pathology laboratory in our region serving a population of 1,236,300, we determined the rate of these outcomes over a period of 16 years. Methods: We carried out a retrospective single centred, population based case review in the Ottawa-Gatineau region. We reviewed 3,320 total cases, of which 98 were ciliary body and choroidal melanomas. Four cases were included in the study group. We calculated the frequency of clinically unsuspected melanoma of the ciliary body and choroid in enucleated and eviscerated eyes and of clinically misdiagnosed melanoma of the ciliary body and choroid in enucleated eyes in our region. We also determined the rate of uveal melanoma undergoing enucleation in our region by calculating the annual population and the mean number of cases within this area over a 16-year-span. Results: Out of 98 diagnoses of ciliary body and choroidal melanoma, we identified 2 cases where the diagnosis was clinically unsuspected. We also identified 2 cases clinically misdiagnosed as a ciliary body or choroidal melanoma, with one of these being a nonneoplastic lesion. In our region, 2.0% of ciliary body and choroidal melanomas were clinically unsuspected and 2.0% of clinically diagnosed ciliary body and choroidal melanomas were misdiagnosed. We also calculated the rate of uveal melanoma undergoing enucleation in our region of 5.4 cases/1,000,000 population/year. Conclusions: We present the first and only single centred, population based data on the rates of unsuspected and misdiagnosed ciliary body and choroidal melanoma in the current era of modern diagnostic imaging. Unsuspected melanoma of the ciliary body or choroid is a rare occurrence, with a rate significantly lower than previously published. The rate of clinically misdiagnosed ciliary body or choroidal melanoma at our centre is within the range of recent reports of this encounter. Commercial Relationships: Solin Saleh, None; Seymour Brownstein, None; André Jastrzebski, None; David Jordan, None; Steven Gilberg, None; Brian Leonard, None; Bernard Hurley, None Program Number: 5349 Poster Board Number: A0198 Presentation Time: 8:30 AM–10:15 AM First description of a melanoma-associated paraneoplastic syndrome with retinopathy and cochleopathy Armin Mohi1, Mahdy Ranjbar1, Salvatore Grisanti1, Henning Frenzel2, Martin Rudolf1. 1Department of Ophthalmology, University of Luebeck, Luebeck, Germany; 2Department of ENT, University of Luebeck, Luebeck, Germany. Purpose: Melanoma-associated retinopathy (MAR) is a known paraneoplastic syndrome in some patients with cutaneous malignant melanoma. It is characterized by the presence of serum auto-antibodies (IgG) against retinal proteins, affecting retinal photoreceptor function and leading to retinal damage. While a reaction of melanoma-associated auto-antibodies against various neuronal proteins is already known, an association with symptoms of other sensory organs has not been published yet. In this case-report we are able to demonstrate, that a symptomatic cochleopathy due to paraneoplastic serum auto-antibodies can be present in patients with cutaneous malignant melanoma and MAR. Methods: A 60 year old women with cutaneous malignant melanoma and visual affection was presented in our hospital for further diagnostics and treatment. She reported flickering photopsias and night-blindness. We were able to detect a reduction of visual acuity, a distinct loss of the visual field and reduced b-waves in the ERG. MAR-auto-antibodies were detected in an independent referencecentre. An advanced staging examination revealed a metastatic recurrence of the melanoma. Noteworthy was an early development of a tinnitus. We initiated an immune-suppressive therapy with prednisolone, performed weekly clinical examinations and took blood samples in a 14 days interval. The blood samples were used ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology for indirect-immune-fluorescence on macaque retina and cochlea sections, to detect auto-antibodies. Results: We were able to observe a quick improvement of BCVA and field of view after one month of prednisolone treatment, while the b-waves in the ERG were still reduced. The tinnitus remained unchanged. A cerebral MRI showed no affection of the auditory pathway as an explanation for the tinnitus. The indirect-immunefluorescence showed a specific pattern, staining the cell-bodies of photoreceptors and hair-cells. Under therapy this pattern showed a reduction of intensity. Controls with immune sera of healthy individuals showed no staining at all. Conclusions: To our knowledge, we demonstrated for the first time the presence of specific auto-antibodies against hair cells of the cochlea which was associated with MAR. Together with the tinnitus it is the first description of symptomatic melanoma-associated cochleopathy (MAC). Commercial Relationships: Armin Mohi, None; Mahdy Ranjbar, None; Salvatore Grisanti, None; Henning Frenzel, None; Martin Rudolf, None 527 New insights in myopia Thursday, May 07, 2015 12:00 PM–1:45 PM 2C/3C Mile High Blrm Paper Session Program #/Board # Range: 5841–5847 Organizing Section: Anatomy and Pathology/Oncology Contributing Section(s): Biochemistry/Molecular Biology Program Number: 5841 Presentation Time: 12:00 PM–12:15 PM Rates of change of ocular properties during emmetropization in the developing chick eye vary linearly with retinal blur Melanie C. Campbell1, 2, Zheng Shao1, 2. 1Physics & Astronomy, University of Waterloo, Waterloo, ON, Canada; 2School of Optometry and Vision Science, University of Waterloo, Waterloo, ON, Canada. Purpose: Emmetropization is an active process involving retinal feedback. We examine changes in ocular properties during emmetropization in the normal chick eye to determine which changes are proportional to the retinal blur due to defocus. Methods: Mean ocular refraction (spherical equivalent, MOR), dioptric length (Kʹ), eye power, pupil radius and cornea and lens powers were calculated from literature values of ocular components (including our results). They and their rates of change were then fitted versus MOR, rate of change in optical axial length (OAL-cornea to retina) and calculated retinal blur due to defocus. The relationships of corneal radius, corneal and lens powers to other parameters and to angular (EB) and linear retinal blur (LRB) were examined. Results: Because of its exponential decrease with age during emmetropization, the rate of change of MOR (D/day) varies linearly with MOR. However, it also varies approximately linearly with retinal blur (both EB and LRB; p<0.001) before emmetropization is complete (up to day 30). During emmetropization, the rate of increase of OAL decreases, as a linear function of decreasing retinal blur (EB p=0.004 and LRB p<0.02). This relationship breaks down around the time that emmetropization is complete (~day 30). Similarly, during emmetropization, the rate of increase in corneal radius varies linearly with retinal blur (EB p=0.002 and LRB p<0.01). The rate of change in lens power is almost proportional to the rate of change of retinal blur (p<0.0001) before the completion of emmetropization. As expected from the above results, the rate of change of corneal radius (p<0.0001) and lens power (p<0.0001) are proportional to the rate of change of OAL during emmetropization. Conclusions: In chick, emmetropization occurs with a rate of ocular elongation proportional to the size of the hyperopic defocus blur on the retina until about day 30. When retinal blur approaches cone spacing, it no longer decreases, MOR is stable and many relationships above no longer apply during slower eye growth to day 75. Thus emmetropization appears to be driven by the angular defocus blur directly causing proportional changes in OAL, corneal radius and lens power until blur reduces sufficiently and emmetropization is complete. Many of these results are consistent with findings during emmetropization in children but here we show a direct link to retinal blur. Commercial Relationships: Melanie C. Campbell, None; Zheng Shao, None Support: NSERC Canada 35321 Program Number: 5842 Presentation Time: 12:15 PM–12:30 PM The Effect of Discontinuous Wear of 2-zone Concentric Bifocal Spectacle Lenses on Refractive Error Development & Eye Growth in Young Chicks Nevin W. El-Nimri, Christine F. Wildsoet. Vision Science, UC Berkeley, Berkeley, CA. Purpose: To characterize in young chicks the myopia control effects of two-zone, distance or near center concentric multifocal lens designs worn on an alternate day schedule interleaved with standard single vision corrective lenses. Methods: Monocular -10 Diopter (D) single vision (SV) lenses were worn by ten-day-old chicks for 4 days to induce myopia, after which the SV lenses were replaced by one of 2 multifocal lens designs (-10 D center/ -5 D periphery, MFDC) or -5 D center/ -10 D periphery (MFDP)) every other day for a total of 6 days. A control group wore -10 D SV lenses every day. A minimum of 4 birds was included in each treatment group. Refractive error and axial ocular dimensions were monitored every three days with retinoscopy and high frequency A-scan ultrasonography respectively. Results: The MFDC lens had greater myopia control effects than MFNC lenses although both groups recorded less myopia than the SV group and intergroup differences in on-axial dimensions were consistent with refractive errors. Mean baseline interocular optical axial length differences (± SD) were 0.29 ± 0.14 mm (SV), 0.26 ± 0.14 mm (MFNC), and 0.32 ± 0.17 mm (MFDC). After 6 days of lens wear, equivalent values were 0.46 ± 0.27 mm (SV), 0.39 ± 0.19 mm (MFNC), and 0.28 ± 0.17 mm (MFDC). Mean interocular refractive error differences (± SD) on day 6 were -12.18 D ± 1.77 (SV), -11.01 ± 1.14 D (MFNC), and -7.60 ± 0.97 D (MFDC) compared to baseline values of -12.32 ± 0.95 D (SV), -12.17 ± 1.56 D (MFNC), and -12.48 ± 0.41 D (MFDC). Conclusions: The results demonstrate that discontinuous wear of multifocal lenses, e.g., every other day, interleaved with standard single vision correction of refractive errors, may be sufficient to control the progression of myopia. Commercial Relationships: Nevin W. El-Nimri, None; Christine F. Wildsoet, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Program Number: 5843 Presentation Time: 12:30 PM–12:45 PM Melanopsin knock-out mice have abnormal refractive development and increased susceptibility to form-deprivation myopia Ranjay Chakraborty1, 3, Duk C. Lee1, 3, Erica G. Landis1, 3, Michael A. Bergen1, 3, Han na Park1, Curran Sidhu1, Samer Hattar5, P M. Iuvone1, 2 , Richard A. Stone4, Machelle T. Pardue3, 1. 1Ophthalmology, Emory University School of Medicine, Atlanta, GA; 2Pharmacology, Emory University, Atlanta, GA; 3Rehab R&D Center of Excellence, Atlanta VA Medical Center, Decatur, GA; 4Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, PA; 5Departments of Biology and Neuroscience, Johns Hopkins University, Baltimore, MD. Purpose: Melanopsin, a photopigment in intrinsically photosensitive retinal ganglion cells (ipRGCs), is involved in regulating visual signaling, retinal dopaminergic cell activity, and circadian activity. Here, we examined the contribution of melanopsin to normal refractive development and to visual form-deprivation (FD) in a mutant mouse lacking melanopsin (Opn4-/-). Methods: Refractive development of Opn4-/- and age-matched wildtype (WT) mice, both on mixed C57BL/6 and 129S1/Sv background, were measured every 2 weeks from 4 to 16 weeks of age. Weekly measurements were performed on a separate cohort of mice that underwent monocular FD in the right eye from 4 weeks of age using head-mounted diffuser goggles. Refraction, corneal curvature, and ocular biometry were obtained using photorefraction, keratometry and 1310 nm spectral-domain optical coherence tomography. Results: Under normal visual conditions, Opn4-/- mice (n=10) were significantly more myopic than WT mice (n=10) at early ages (mean refractive error at 4 weeks, Opn4-/-: -3.56 ± 0.35 D; WT: +0.94 ± 0.35 D, p<0.05), and they became relatively more hyperopic than the WT mice by the end of 16 weeks (Opn4-/-: +6.53 ± 0.25 D, WT: +4.52 ± 0.64 D, p<0.05). The axial length of Opn4-/- mice (mean at 12 weeks, 3.29 ± 0.01 mm) was significantly shorter (p<0.001) than that of WT animals (3.35 ± 0.01 mm). After 3 weeks of FD, goggled Opn4-/- mice (n=6) showed a significant myopic shift (difference between the right and left eyes) of -4.08 ± 0.89 D compared to non-goggled controls (n=6, + 0.43 ± 0.52 D, p<0.05). No significant refractive shift was observed in WT mice goggled for 3 weeks (-1.65 ± 0.65 D). Also, Opn4-/- mice showed a significant increase (p<0.05) in axial length with FD at 3 weeks (difference of right and left eyes) of 0.03 ± 0.01 mm in comparison with WT animals (-0.01 ± 0.004 mm). There were no significant differences in corneal curvatures of Opn4-/- and WT mice under either normal or FD conditions. Conclusions: Our findings suggest that melanopsin signaling pathways contribute to normal refractive development, and the development of FD myopia in mice. Future studies are needed to determine other mechanisms underlying abnormal ocular development in Opn4-/- mice, and the extent to which these findings reflect an interaction of refractive development and circadian biology. Commercial Relationships: Ranjay Chakraborty, None; Duk C. Lee, None; Erica G. Landis, None; Michael A. Bergen, None; Han na Park, None; Curran Sidhu, None; Samer Hattar, None; P M. Iuvone, None; Richard A. Stone, None; Machelle T. Pardue, None Support: NEI Grant EY022342, NEI Grant EY004864, NEI Grant EY016435, NIH Core grant P30EY006360, Research to Prevent Blindness Grant and Mackall Foundation Trust Program Number: 5844 Presentation Time: 12:45 PM–1:00 PM Expression of soluble Bmp antagonists from the zebrafish RPE leads to eye enlargement and myopia Ross F. Collery, Brian A. Link. Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI. Purpose: Bmp signaling is involved in early eye development, but negatively regulates growth in the adult eye. bugeye zebrafish lacking the large endocytic receptor Lrp2, which is expressed in the RPE and binds Bmp4, show grossly enlarged eyes and high myopia. Here we investigate the effects of expressing antagonists to Bmp (Nog3, Grem2) as well as a soluble domain of Lrp2 found to bind Bmp4, and analyze the effects on eye size and refractive state. Methods: An RPE-specific promoter was used with the bipartite Gal4/UAS system to express Nog3, Grem2, and the first LDLA1 domain of Lrp2. Zebrafish expressing transgenes and control siblings were imaged using a Bioptigen SD-OCT system at 2 months of age, measuring eye axial length, retinal radius and lens diameters. Using these metrics, eye sizes were normalized and degrees of refractive errors were calculated. Results: Zebrafish expressing Nog3 from the RPE showed enlarged eyes and increased myopia compared to their control siblings. lrp2-/zebrafish show enlarged and myopic eyes relative to wild-types, and this phenotype is exacerbated with RPE-driven Nog3 expression. Similarly, expression of Grem2 from the RPE also increased eye size and degree of myopia, but only in lrp2-/- zebrafish. Finally we demonstrated that Bmp4 binds in vivo to the LDLA1 extracellular domain of Lrp2, and found that expression of soluble Lrp2 (LDLA1)eGFP from the RPE also increased eye size and degree of myopia. Conclusions: Bmp antagonists exert their effect by binding extracellular ligands and prevent them from activating their cognate receptors, thus modulating signaling. We propose that overexpression of Bmp antagonists in the RPE leads to a large, myopic eye phenotype in part by inhibiting Bmp signaling. In ongoing experiments, we will investigate whether Lrp2 is endogenously cleaved to generate a soluble LDLA1-containing ectodomain, and examine whether such putative processing alters its effects on Bmp signaling during emmetropization and with myopia. Commercial Relationships: Ross F. Collery, None; Brian A. Link, None Support: NIH/NEI RO1EY016060; P30EY001931 Program Number: 5845 Presentation Time: 1:00 PM–1:15 PM Increased retinaldehyde dehydrogenase activity during recovery from induced myopia Angelica Harper1, Gennadiy P. Moiseyev2, Jody A. Summers1. 1Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK. Purpose: Previous studies have identified significant increases in retinaldehyde dehydrogenase 2 (RALDH2) steady state mRNA in the chick choroid during recovery from myopic defocus. Thus, RALDH2 may be responsible for the increase in choroidal all-transretinoic acid (atRA) synthesis observed during recovery from myopic defocus. The present study examines RALDH activity and RALDH2 protein expression in control and recovering chick ocular tissues to further elucidate the link between RALDH2, atRA, and visually guided ocular growth. Methods: Myopia was induced in chicks by form deprivation for 10 days, followed by up to 15 days of recovery. Retina/RPE, choroid, and/or sclera were isolated from control (not treated) and recovering (treated) eyes at various points during recovery. RALDH catalytic ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. RALDH2 protein expression in control and recovering homogenates was compared by western blotting. Results: RALDH activity was increased in recovering choroids as compared to controls by 66.5% (p > 0.01, paired t-test) at day 3 of recovery and by 49.7% (p > 0.05, paired t-test) at day 7 (n = 6). When RALDH activity in control and day 4 recovering retina/RPE, choroid, and sclera was compared, RALDH activity could only be detected in the choroid (n = 9). Western blot analyses indicated that RALDH2 protein expression was highest in the choroid and minimal in retina/ RPE and sclera. Choroidal RALDH2 expression was 281.1% greater (p > 0.05, paired t-test) in recovering eyes as compared with controls (n = 3). Conclusions: These results demonstrate that the choroid is the major ocular tissue exhibiting RALDH activity, which is responsible for increased RALDH activity during recovery. These increases correspond to increases in choroidal RALDH2 protein expression, suggesting that increased RALDH activity in the recovering choroid is due to increased RALDH2 protein expression. These results suggest that RALDH2 may be useful as a therapeutic target for the treatment of myopia. Commercial Relationships: Angelica Harper, None; Gennadiy P. Moiseyev, None; Jody A. Summers, None Support: NEI Grant R01 EY09391 Program Number: 5846 Presentation Time: 1:15 PM–1:30 PM RNA sequencing of sclera from form-deprived guinea pigs identifies multiple signalling pathways underlying myopia Nethrajeith Srinivasalu1, 2, Sally A. McFadden2, Callan Medcalf2, Gayle Philip3, Michael Zhang1, Paul N. Baird1. 1Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital., Melbourne, VIC, Australia; 2School of Psychology, University of Newcastle., Callaghan, NSW, Australia; 3Victorian Life Sciences Computation Initiative, Melbourne, VIC, Australia. Purpose: Remodelling of the posterior sclera accompanies myopia, and in young guinea pig eyes, early changes occur in the peri-papillary zone (PPZ) around the optic nerve. We examined the differential expression of genes in the PPZ sclera to identify significant pathways associated with myopia. Methods: One eye of guinea pigs (n=3) was form-deprived for 2 weeks (day 6-20) using a translucent diffuser to induce myopia. The other eye was untreated and served as a matched control. At the end of 2 weeks, refractive error was measured in cyclopleged eyes using a Nidek auto-refractor. Posterior scleral punches (4 mm) were collected from the PPZ from myopic and control eyes. Total RNA was extracted using an RNeasy® Fibrous tissue mini kit. RNA was sequenced by a commercial provider using an Illumina HiSeq 2000 platform. RNA-seq data were analysed using three different statistical programs (CuffDiff, edgeR and Voom) and the most differentially expressed genes (p value < 10-3) were used to identify significant pathways. Results: Form-deprivation induced relative myopia in all animals. The mean difference between myopic and control eyes was -3.76±0.6 D at the end of the treatment period. The number of genes differentially expressed between myopic and control sclera were 26,088, 14,301, and 14,298 using CuffDiff, edgeR and Voom analysis tools, respectively. Differences in gene expression (p<10-3) were found for 850 (CuffDiff), 794 (edgeR) and 270 (Voom) genes using the different analysis programs. Pathway analysis conducted on significant genes identified more than 50 different pathways, of which four have previously been reported as associated with myopia. Conclusions: Number of differentially expressed genes were identified in the posterior myopic sclera compared to non-myopic eyes, which varied between analysis packages. Several signalling pathways were consistently identified across the different analysis programs with some of these having previously been identified as associated with myopia in humans and animal models. These findings provide insights into potential pathways involved in active scleral remodelling. Further targeted investigation of these genes may provide potential avenues for development of treatment therapies. Commercial Relationships: Nethrajeith Srinivasalu, None; Sally A. McFadden, None; Callan Medcalf, None; Gayle Philip, None; Michael Zhang, None; Paul N. Baird, None Support: NHMRC Senior Research Fellowship 1028444, Coal Port Authority and Hunter Medical Research Institute, Melbourne International Fee Remission Scholarship Program Number: 5847 Presentation Time: 1:30 PM–1:45 PM Eye Elongation and Retinal Degeneration in the IRBP Knockout Mouse Natecia Williams, Shannon Getz, Curran Sidhu, Jeffrey H. Boatright, Machelle T. Pardue, P M. Iuvone, J M. Nickerson. Ophthalmology, Emory University, Atlanta, GA. Purpose: Interphotoreceptor retinoid-binding protein (IRBP) is abundant in the subretinal space and binds retinoids and lipophilic molecules. Despite initial expression at embryonic day 12 (E12), the role of IRBP in eye development is unknown. Congenic IRBPdeficient mice (KO) show eye elongation starting at postnatal day 8 (P8), abnormal pruning indicated by reduced TUNEL-positive cell death in the inner nuclear layer by P12, increased TUNEL-positive cell death of photoreceptors at P23-P27, and profound myopia by P30 followed by slow retinal degeneration (RD) (Wisard et al. IOVS. 2011; 52:5804-11). Based on experimental myopia (Stone et al. PNAS 1989; 86:704-6), we propose KO mice will show a decrease in dopamine (DA) and the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) and number of retinal dopaminergic cells (DA-cells) coinciding with abnormal eye elongation, abnormal pruning of inner retinal cells, and RD. Methods: Using immunohistochemistry for tyrosine hydroxylase (TH) on retinal flat mounts, we counted the DA-cells in retinas of C57BL/6J (WT) and KO mice at P12-P30. HPLC was used to analyze the amounts of DA and DOPAC at P12-P30. t-tests for agematched mice determined P-values. Results: Although no difference was observed at P12, DOPAC levels in KO retinas increased by 64% at P23 (N=9 WT, 8 KO, P < 0.001) and 73% by P30 compared to WT mice (N=9 WT, 7 KO, P < 0.01). Similarly, KO retinal DA levels increased by 25% at P23 (N=9 WT, 8 KO, P < 0.01) and 21% at P30 compared to WT mice (N=9 WT, 7 KO, P < 0.05). The number of retinal TH+ cells was 28% greater in KO retinas compared to WT at P30 (N=4 WT, 4 KO, P<0.05). Conclusions: Despite development of myopia, KO mice have increased levels of DA and DOPAC contrary to the expectation that high levels of DA should slow eye growth. The data would suggest that myopia observed in the KO mice is atypical to refractive myopia, independent of dopaminergic activity and visual input. The excess of TH+ cells in the KO retina may result from insufficient pruning of retinal DA-cells in the development of the KO inner retina. In addition, this high dopaminergic activity seems to be characteristic of KO retinas after P23, the age in which we begin to see a rise in cell death in the KO retina, which may suggest a role for DA or DA-cells in this RD. Commercial Relationships: Natecia Williams, None; Shannon Getz, None; Curran Sidhu, None; Jeffrey H. Boatright, None; ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Anatomy and Pathology/Oncology Machelle T. Pardue, None; P M. Iuvone, None; J M. Nickerson, None Support: NIH Grant R01EY021592, NIH Grant R01EY016470, NIH Grant T32EY007092, NIH Grant P30EY006360, NIH Grant R01EY004864 ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].