Primary Uveal Melanoma Research Articles

Serum biomarkers of metastatic disease: current practice and future perspectives

AbstractPurpose Molecular genetic testing of primary uveal melanoma (UM) specimens is being integrated into clinical practice for prognostication purposes. However, the current screening protocol for metastatic disease (MD) it is not able to detect micrometastases. There is a clear need for easily assessable biomarkers that would allow earlier detection of MD and aid monitoring of systemic adjuvant therapy. As UM disseminates haematogeneously, it is reasonable to search for blood‐borne biomarkers as indicators of MD.Methods Review of the literature to provide an overview of blood biomarkers studies in UM. Outline of the workflow, which could lead to new biomarker discovery using a discovery proteomics approach.Results Conventional biomarkers described in other cancer types have been investigated in blood samples from UM patients. These markers have failed to provide reproducible results and therefore their usefulness in the clinical senario has been questioned. The enumeration of circulating tumour cells shows good potential, but results are influenced by the technique used for detection. Proteomic technology has been applied to UM tumour tissue, secretome and cell lines, mainly using 2D gels. Quantitative mass spectrometry‐based discovery proteomics comparing high risk tumours versus low risk ones, followed by system biology analysis to select a panel of meaningful candidates among the differential expressed proteins, bears the potential to be a more accuare and effective approach.Conclusion None of the blood biomarkers tested in UM patients have demonstrated the necessary sensitivity and specificity to guide clinical decisions. A rational development of clinically relevant blood biomarkers can be achieved applying proteomic technologies.

Genetics of Primary Intraocular Tumors

Primary intraocular neoplasms are tumors that originate within the eye. The most common malignant primary intraocular tumor in adults is uveal melanoma and the second is primary intraocular lymphoma or vitreoretinal (intraocular) lymphoma. The most common malignant intraocular tumor in children is retinoblastoma. Genetics plays a vital role in the diagnosis and detection of ocular tumors. In uveal melanoma, monosomy 3 is the most common genetic alteration and somatic mutations of BAP1, a tumor suppressor gene, have been reported in nearly 50% of primary uveal melanomas. The retinoblastoma gene RB1 is the prototype tumor suppressor gene—mutations in RB1 alleles lead to inactivated RB protein and the development of retinoblastoma. Immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangement is observed in B-cell or T-cell primary vitreoretinal lymphoma, respectively. Other factors related to the genetics of these three common malignancies in the eye are discussed and reviewed.

Differential Expression of Fourteen Proteins between Uveal Melanoma from Patients Who Subsequently Developed Distant Metastases versus Those Who Did Not

To compare the proteomic profiles of two categories of primary uveal melanoma tissue samples; those from patients who have subsequently developed metastatic disease and those who have not. Two-dimensional difference gel electrophoresis (2D DIGE) was performed on 25 uveal melanoma tissue specimens (minimum follow-up of 7 years) comparing nine uveal melanoma tumors from patients who developed metastatic disease and 16 from those who did not. Most of the tumors which metastasized also exhibited chromosome 3 monosomy. Selected differentially expressed proteins were further followed up by immunohistochemistry and functional validation in vitro using siRNA. Proteomic analysis revealed 14 statistically significant differentially expressed proteins, with nine showing increased expression (PDIA3, VIM/HEXA, SELENBP1, ENO1, CAPZA1, ERP29, TPI1, PARK7, and FABP3) and five showing decreased expression (EIF2S, PSMA3, RPSA, TUBB, and TUBA1B) in uveal melanomas that subsequently metastasized compared with those that did not. Immunohistochemical analysis was performed for six of the differentially expressed proteins and gave similar results to the 2D DIGE study for two of these proteins, fatty acid-binding protein, heart-type (FABP3) and triosephosphate isomerase (TPI1). siRNA knockdown in the 92.1 uveal melanoma cell line confirmed a functional role for FABP3 and TPI1 in invasion in vitro. Proteomic analysis identified proteins differentially expressed in uveal melanoma that will subsequently metastasize, some of which appear to have a functional role in invasion. These results may contribute to better predictive tests (along with genetic analysis) and to the identification of new therapeutic targets.

778 Development and Pharmacological Assessment of New Models of Orthotopic Primary Human Uveal Melanoma and Retinoblastoma Xenografts

778 Development and Pharmacological Assessment of New Models of Orthotopic Primary Human Uveal Melanoma and Retinoblastoma Xenografts

Adjuvant sunitinib in high-risk patients with uveal melanoma: A pilot study.

8560 Background: Uveal melanoma is the most common primary intraocular cancer in adults. Despite the successful treatments for primary tumors, up to 50% of the patients later die of distant metastases. Currently no effective adjuvant treatment is available for these patients. Our group previously reported that sunitinib stabilized systemic metastases in 65% of uveal melanoma patients who failed prior treatments. In this pilot study, we tested sunitinib in an adjuvant setting in high-risk patients with primary uveal melanoma. Methods: Patients with estimated metastatic death rate ≥ 50% based on the following criteria were eligible: (1) monosomy 3 and 8q amplification; (2) large tumor (≥ 15 mm in diameter and ≥7 mm in thickness); (3) monosomy 3 and other risk factors (large tumor, epithelioid dominant cell type, local recurrence, or extra-scleral extension). Sunitinib was given at 25 mg PO daily for at least 6 months. Primary endpoint was disease-free survival (DFS) and overall survival (OS), estimated with Kaplan-Meier analysis (SPSS 17.0). Secondary endpoint was safety. Results: A total of 23 Caucasian patients (median, 54 years; range: 25 – 77) were enrolled. All patients received sunitinib for at least 6 months (range: 6 – 12). Eighteen patients had confirmed monosomy 3, from whom 13 (72%) also showed 8q amplification. The median follow-up was 24 months (range 12 – 54 months). Only one patient died of unknown cause (OS: 30.4 months). A total of seven patients (30%) developed systemic metastases, all of which were liver metastases. The DFS and OS rates at 2 years were 70% (95% CI: 47 – 86%) and 100%, respectively. The OS rate at 2 years was better than historical control with monosomy 3 patients (51%, 95% CI: 31 – 71%) (Invest Ophthalmol Vis Sci 2003; 44:1008). In those patients who relapsed, the median time to progression after finishing sunitinib was 2 months (range: 0 – 8). The most common adverse events were: diarrhea (47%), fatigue (39%), dermatitis (30%), stomatitis (13%), leucopenia (8%), and nausea (4%). Most were grade 1 or 2 (88%) and only one was considered grade 4 (diarrhea). Conclusions: In high-risk uveal melanoma patients with estimated metastatic death rate of ≥ 50%, adjuvant sunitinib showed promising results and deserves further investigation.

Modeling Human Choroidal Melanoma Xenograft Growth in Immunocompromised Rodents to Assess Treatment Efficacy

To evaluate potential treatments of primary uveal melanoma in rodent xenograft models, it is necessary to track individual tumor growth during treatment. Previously, high-frequency ultrasound (HF-US) was used to measure tumor volume in nude rats for up to 2 weeks. This study tests the hypothesis that HF-US can be used to repeatedly measure tumor volume for at least a month in both nude rat and severe combined immunodeficiency (SCID) mouse xenograft models of human uveal melanoma, with the goal of modeling tumor growth to evaluate treatment efficacy. C918 human uveal melanoma spheroids were implanted in the choroids of six nude rats and six severe combined immunodeficiency mice. OCM-1 human uveal melanoma spheroids were implanted in six nude rats. Every 4-7 days thereafter for up to 5 weeks, HF-US images of the tumor-bearing eye were captured every 100 or 250 μm. Tumor areas were measured on each image and integrated to calculate volume. Tumor growth was modeled using a logistic curve, and parameters characterizing growth, including the time to reach a target volume (t(T)), were evaluated as potential measures of treatment efficacy. Tumor volume could be measured for up to 5 weeks in all models, and the logistic curve described the growth well. The parameter t(T) was shown to be a suitable endpoint to evaluate treatments. HF-US is a practical method to track uveal melanoma growth in the same nude rat or SCID mouse for up to a month. Such growth data can be used to evaluate treatments in these xenograft models.

Higher Percentage of FISH-Determined Monosomy 3 and 8q Amplification in Uveal Melanoma Cells relate to Poor Patient Prognosis

To investigate the relation between patient survival and incrementally increasing percentages of fluorescence in situ hybridization-determined complete loss of chromosome 3 (monosomy 3) and gain of chromosome 8q in primary uveal melanoma cells. Clinicopathological factors were related to disease-free survival. Fluorescence in situ hybridization was performed using probes on chromosomes 1, 3, 6, and 8. The percentages of UM cells with monosomy 3 or chromosome 8q gain were classified in groups with incrementally increasing percentages and related to disease-free survival. Correlations between clinical factors and cytogenetic aberrations were also analyzed. Two-hundred twenty choroidal and ciliary body melanomas were analyzed. The following proved to be significant predictors of survival in univariate analysis: older patient age (P = 0.003); large tumor diameter (P < 0.001); mixed cell type (P = 0.001); presence of closed microvascular loops (P < 0.001); loss of chromosome 1p (P = 0.006); monosomy 3 (P < 0.001); gain of 6p (P < 0.001); and gain of chromosome 8q (P < 0.001). Multivariate Cox analysis displayed monosomy 3 (Hazard ratio [HR] 2.83, P = 0.002) and gain of chromosome 8q (HR 3.13, P = 0.002) as the most important independent prognostic factors of poor survival, followed by older patient age (HR 1.02, P = 0.017). Increasing percentages of monosomy 3 and gain of chromosome 8q in tumor cells showed a correlation with worse prognosis (Log-rank test 49.9 and 40.4, both P < 0.001) and increased number of additional copies of 8q correlated with shorter disease-free interval (Log-rank test 45.7, P < 0.001). A high percentage monosomy 3 and chromosome 8q gain in primary UM cells showed a strong relation with poor disease-free survival compared with low percentage aberrations.

Collaborative Ocular Oncology Group Report Number 1: Prospective Validation of a Multi-Gene Prognostic Assay in Uveal Melanoma

This study evaluates the prognostic performance of a 15 gene expression profiling (GEP) assay that assigns primary posterior uveal melanomas to prognostic subgroups: class 1 (low metastatic risk) and class 2 (high metastatic risk). Prospective, multicenter study. A total of 459 patients with posterior uveal melanoma were enrolled from 12 independent centers. Tumors were classified by GEP as class 1 or class 2. The first 260 samples were also analyzed for chromosome 3 status using a single nucleotide polymorphism assay. Net reclassification improvement analysis was performed to compare the prognostic accuracy of GEP with the 7th edition clinical Tumor-Node-Metastasis (TNM) classification and chromosome 3 status. Patients were managed for their primary tumor and monitored for metastasis. The GEP assay successfully classified 446 of 459 cases (97.2%). The GEP was class 1 in 276 cases (61.9%) and class 2 in 170 cases (38.1%). Median follow-up was 17.4 months (mean, 18.0 months). Metastasis was detected in 3 class 1 cases (1.1%) and 44 class 2 cases (25.9%) (log-rank test, P<10(-14)). Although there was an association between GEP class 2 and monosomy 3 (Fisher exact test, P<0.0001), 54 of 260 tumors (20.8%) were discordant for GEP and chromosome 3 status, among which GEP demonstrated superior prognostic accuracy (log-rank test, P = 0.0001). By using multivariate Cox modeling, GEP class had a stronger independent association with metastasis than any other prognostic factor (P<0.0001). Chromosome 3 status did not contribute additional prognostic information that was independent of GEP (P = 0.2). At 3 years follow-up, the net reclassification improvement of GEP over TNM classification was 0.43 (P = 0.001) and 0.38 (P = 0.004) over chromosome 3 status. The GEP assay had a high technical success rate and was the most accurate prognostic marker among all of the factors analyzed. The GEP provided a highly significant improvement in prognostic accuracy over clinical TNM classification and chromosome 3 status. Chromosome 3 status did not provide prognostic information that was independent of GEP.

Abstract 1585: Primary and metastatic uveal melanoma cell lines express MelanA that can be used as a target antigen for transfection of mature dendritic cells

Abstract The most common intra-ocular malignancy is uveal melanoma (UM). Metastatic disease occurs in approximately 50% of UM patients and presents most frequently in the liver. Novel therapies that prevent the development of metastases are needed and immunotherapy is a potential option. Because of its origin in the immune-privileged eye, UM may be particularly responsive to T cell-based immunotherapy. MelanA is frequently over-expressed by human cutaneous melanomas. MelanA expression has also been detected in UM primary and metastatic tumor-tissue sections and could serve as a target antigen for immunotherapy of UM. Here we report that primary and metastatic UM cell lines express MelanA and that this protein can be used as a target antigen for transfection of mature dendritic cells (mDC). MelanA expression was confirmed in primary and metastatic UM cell lines using intracellular flow cytometry and quantitative PCR (qPCR). In addition, sequencing of UM-derived MelanA demonstrated that MelanA in primary and metastatic UM cells has an identical sequence as compared to the published MelanA consensus sequence. As an initial step towards activating UM-specific T cells by UM-derived mRNA transfected DC we are generating MelanA mRNA transfected mDC. To facilitate both MHC class I and class II presentation of MelanA epitopes after mRNA electroporation into mDC, we included the endosomal targeting sequence DCLamp linked to the MelanA protein. Monocytes were isolated from leukapheresis products of healthy donors by density centrifugation and plastic adherence. Monocytes were differentiated to monocyte-derived DC by culturing with IL-4 and GM-CSF for 6 days to obtain immature DC (iDC). iDCs were matured with a cytokine mixture containing IL-4, GM-CSF, IL-1α, IL-6, TNFα, and PGE2 for 48 hours. The resulting mDC expressed cell surface maturation markers CD25, CD83, and CD86 as confirmed by flow cytometry. mDC were transfected with either MelanA-mRNA of MelanA-DCLamp-mRNA, using an established electroporation protocol. To assess transfection efficiency and monitor MelanA expression in transfected mDC, MelanA-mRNA and MelanA-DCLamp-mRNA expression was demonstrated by qPCR and MelanA-protein expression was shown by intracellular flow cytometry at different time points post-transfection using variable mRNA concentrations. The detected amount of MelanA-mRNA and protein was dependent on the amount of transfected mRNA and presented transiently. Subsequently, total RNA was isolated from MelanA+ primary and metastatic UM cell lines, then reversely transcribed into cDNA, amplified by PCR and in vitro transcribed to mRNA. As confirmed by qPCR, MelanA-mRNA was present in the pool of UM-derived mRNA and can be used for transfection of mDC. Collectively, these data demonstrate that mDC can be transfected with MelanA-mRNA and suggest MelanA could serve as a target for immunotherapy of UM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1585. doi:1538-7445.AM2012-1585

Metastatic Uveal Melanoma

Uveal melanoma is the most common primary intraocular cancer in adults. Nearly half of primary uveal melanoma tumors metastasize, but there are currently no effective therapies for metastatic uveal melanoma. The recent discovery of mutations that underlie uveal melanoma metastasis, growth, and survival provide a key to the molecular understanding of this disease. Much work is now underway to leverage this knowledge to develop effective therapies. This review summarizes recently discovered molecular features of uveal melanoma and therapies being explored to capitalize on this knowledge.

Notch Signaling Promotes Growth and Invasion in Uveal Melanoma

To determine whether uveal melanoma, the most common primary intraocular malignancy in adults, requires Notch activity for growth and metastasis. Expression of Notch pathway members was characterized in primary tumor samples and in cell lines, along with the effects of Notch inhibition or activation on tumor growth and invasion. Notch receptors, ligands, and targets were expressed in all five cell lines examined and in 30 primary uveal melanoma samples. Interestingly, the three lines with high levels of baseline pathway activity (OCM1, OCM3, and OCM8) had their growth reduced by pharmacologic Notch blockade using the γ-secretase inhibitor (GSI) MRK003. In contrast, two uveal melanoma lines (Mel285 and Mel290) with very low expression of Notch targets were insensitive to the GSI. Constitutively active forms of Notch1 and Notch2 promoted growth of uveal melanoma cultures and were able to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited anchorage-independent clonogenic growth and cell invasion and reduced phosphorylation levels of STAT3 and extracellular signal-regulated kinase (Erk)1/2. Suppression of canonical Notch activity using short hairpin RNA targeting Notch2 or CBF1 was also able to reduce tumor growth and invasion. Finally, intraocular xenograft growth was significantly decreased by GSI treatment. Our findings suggest that Notch plays an important role in inducing proliferation and invasion in uveal melanoma and that inhibiting this pathway may be effective in preventing tumor growth and metastasis.

BAP1tism of a Tumor Suppressor

Driving cancer cells into a more differentiated state is a rational therapeutic approach. Primary uveal melanoma cells with a propensity to metastasize have less-differentiated features than their less aggressive counterparts. Treatment of uveal melanoma cells with histone deacetylase inhibitors induces a more differentiated phenotype with resultant lower growth capacity.

Mda-9/Syntenin Is Expressed in Uveal Melanoma and Correlates with Metastatic Progression

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound–healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

Uveal Melanoma—Clinical and Pathological Update

Although ocular treatments of the primary uveal melanoma are effective to eradicate the primary tumor locally, its impact on survival remains unclear. The reason we believe local treatment has limited or no impact on survival is because, despite the increase in diagnostic accuracy over the past several decades and development of globe-sparing treatments, the mortality rates of patients with uveal melanomas of any given size have not changed appreciably during this period. Development of metastases is a complex multi-step process that occurs almost exclusively via a hematogenous route. Due to the complexity of this process it is difficult to isolate individual events in patients that are important in the overall progression of the disease from primary tumor to metastasis. Because most uveal melanomas are currently managed by eye-preserving methods that do not yield a pathologic specimen, a number of investigators are currently performing biopsy (usually fine needle aspiration biopsy) of these tumors prior to, or at the time of, ocular treatment to obtain representative tumor specimens for cytopathologic, cytogenetic, and/or molecular biologic analysis. Many new discoveries about the cytogenetic and molecular biologic abnormalities in uveal melanoma cells, and the ways these features influence development and prognosis of metastatic tumors have been published. Hopefully these findings will help us understand the process and improve patient survival since the impact of surveillance testing for metastasis and systemic therapy on the survival of patients with metastatic uveal melanoma is still uncertain.

Expression of cancer-testis antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1) in primary human uveal and conjunctival melanoma

AimMetastatic disease in ocular melanoma remains untreatable, is associated with late detection and is resistant to conventional systemic therapies. Many tumours including cutaneous melanoma express specific cancer-testis (CT) antigens and...

Carcinoembryonic Antigen Cell Adhesion Molecule-1 (CEACAM1) in Posterior Uveal Melanoma: Correlation with Clinical and Histological Survival Markers

Carcinoembryonic antigen cell adhesion molecule (CEACAM)-1 is a multi-functional protein, with strong predictive value for poor prognosis when found in primary cutaneous melanoma lesions. In this study, the expression of CEACAM1 in uveal melanoma was correlated with clinicopathologic parameters. CEACAM1 expression was immunohistochemically evaluated in 79 primary uveal melanomas and 21 liver metastases of patients who were treated at the Hadassah-Hebrew University Medical Center between the years 1986 and 2006. The findings were correlated with location, cell type, extracellular matrix patterns, tumor size, and metastatic disease. CEACAM1 was expressed in 45% of the primary tumors compared with 81% of the metastases (Fisher's exact test, P = 0.003). There was no significant association between CEACAM1 and location of the primary tumors. Histologically, CEACAM1 was associated with epithelioid-type tumors (69.6%), but not with spindle-type tumors (25.0%) (Cramer's V = 0.354; P = 0.019). Also it was significantly associated with network extracellular matrix pattern (73.3%), but not with silent pattern (11.8%) (Cramer's V = 0.510; P = 0.004). CEACAM1-positive tumors were not statistically different in size from CEACAM1-negative tumors. The higher frequency of CEACAM1 in patients who ultimately developed metastases (58.8% vs. 41.7%) was not statistically significant (likelihood ratio χ(2) = 2.069; P = 0.1503). This report describes CEACAM1 expression in uveal melanoma. Correlation with poor prognostic factors such as epithelioid cell type and networks of extracellular matrix pattern was found, but definitive prognostic conclusions still cannot be deduced. Additional validation studies on the use of CEACAM1 expression as a prognostic marker are warranted.

Prognostic associations of insulin-like growth factor-1 receptor in primary uveal melanoma

Objective To study the association between immunoreactivity for insulin-like growth factor-1 receptor (IGF-1R) in primary ciliary body and choroidal melanoma and metastatic death in a consecutive, population-based data set. Design Retrospective, consecutive, population-based cohort study. Participants A total of 167 patients with choroidal and ciliary body melanoma, enucleated from 1972 to 1981, with long-term survival data. Methods Specimens were immunostained by using the avidin-biotinylated peroxidase complex method and polyclonal antibodies to IGF-1R. The percentage of tumour area that was immunopositive was recorded. Survival was assessed by Cox multivariate regression analysis. Results The tumour area could be reliably measured from 129 (78%) of the 167 choroidal or ciliary body melanomas. More heavy pigmentation ( p = 0.001), larger number of macrophages ( p = 0.003) and higher microvascular density ( p = 0.060) were associated with a higher percentage of tumour area that was immunopositive for IGF-1R, the reverse being true of extrascleral extension ( p = 0.049). No significant association was observed with ciliary body extension, largest basal tumour diameter, cell type, mean diameter of the 10 largest nucleoli, and presence of extravascular matrix loops and networks ( p = 0.61-0.96). The percentage of tumour area that was immunopositive for IGF-1R was not associated with survival. Conclusions In our data set, immunoreactivity for IGF-IR did not independently predict metastasis from primary uveal melanoma. Partial loss of antigenicity can not be ruled out as a confounding factor because no frozen sections were available. Results of previous studies have likewise been variable, suggesting that immunohistochemical determination of IGF-1R from formalin-fixed, paraffin-embedded specimens is not practical as a routine test.

Abstract B217: Antitumor activity of the new Bcl-2/Bcl-xl inhibitor S44563 in primary human uveal melanoma xenografts.

Abstract Background: Uveal melanoma (UM) is the most common primary malignancy of the eye in adults. Although over 95% of patients have disease limited to the eye at diagnosis, 50% will develop liver metastases and ultimately die of their disease. Because of the limited efficacy of current treatments, new therapeutic strategies need therefore to be developed. Reduced capacity for apoptosis induction is one potential obstacle to therapy but, consequently, regulators of apoptosis may constitute an attractive approach to anticancer therapeutics, such as the Bcl-2 family proteins. Human uveal melanomas are characterized by a high amount of Bcl-2 expression, ranging between 50% and 100% of studied cases. Such an observation has been confirmed in our panel of 16 primary human uveal melanoma xenografts obtained from patient's tumors (Némati et al, CCR 2010). We have therefore investigated the efficacy of the new Bcl-2/Bcl-XL inhibitor S44563 on 4 primary human UM xenografts. Materials and methods: Four well characterized models of primary human UM, obtained from patients after enucleation (MP41 and MP77) or liver metastasis surgery (MM26 and MM66) (Némati et al, CCR 2010), were used for the in vivo experiments. S44563 (50 or 100 mg/kg/day, days 1–5/8–12/22–26/29–33) was administered IP alone or combined with fotemustine, either concomitant (15 or 30 mg/kg days 1 and 22), or after chemotherapy (100 mg/kg) at days 43–47/50–54/64–68/71–75. Tumor Growth Inhibition (TGI) was calculated to measure the efficiency of various tested compounds. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were determined by immunohistochemistry (IHC). Results: The expression of Bcl-2, defined by IHC, was highly positive for the 4 models used (75% to 100% of tumor cells). S44563 administered alone at 50 and 100 mg/kg induced a moderate TGI of about 50% in 1 model (MP41) among all without dose effect. When S44563 was combined to fotemustine, we observed a synergistic activity in 2 models (MP77 and MM66) among the 4 tested, without impact on the proportion of complete remission. Finally, when S44563 was concomitantly and/or administered after fotemustine, we found a delay of tumor growth in 2 among the 3 tested xenografts (MP77, and MM26). IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expressions were not modified after S44563 administration. Conclusion: We have shown that S44563, despite a relative low efficacy when administered alone, increased the efficacy of chemotherapy in concomitant combination or after fotemustine. Such preliminary results therefore underline the therapeutic potential of this new Bcl-2/Bcl-xl inhibitor in human uveal melanoma. Némati et al. Clin Cancer Res 2010;16:2352–2362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B217.

Abstract A20: Development and pharmacological assessment of new models of orthotopic primary human uveal melanoma and retinoblastoma xenografts.

Abstract Background: The treatment of both uveal melanoma and retinoblastoma could require enucleation in unfavorable prognostic disease. In order to allow pharmacological assessment of innovative intraocular treatments in more relevant and predictive models, we have developed preclinical orthotopic xenografts of both primary human uveal melanoma (UM) and retinoblastoma (Rb) into immunodeficient mice. Materials and methods: Orthotopic models of human UM and Rb have been developed from two panels of subcutaneous xenografts previously established and characterized in the laboratory (Némati et al 2010; Aerts et al 2010), i.e. 6 UM models (MP34, MP41, MP42, MP55, MP65, and MM26) and 3 Rb models (RB102, RB111, and RB200). Mice bearing xenografts were sacrificed and tumors were dissected to obtain a suspension of fresh tumor cells at a concentration of 8000 cells/l in DMEM serum-free medium. Under intraperitoneal anesthesia, 2 l of cell suspension was injected into the subretinal space of the right eye for 3 groups of mice using a 32G needle via a Hamilton syringe. Each group was constituted by 3 to 6 SCID mice. After sub-retinal injection, ophthalmic examination of the mice was performed weekly. When tumor cells invaded vitreal cavity and anterior chamber, the mice were sacrificed for ophthalmological pathological analyzes. Finally, using the RB200 model, we have then evaluated the efficacy of 2 μl intraocular administration of bevacizumab (25 mg/kg/week for 4 weeks), melphalan (500 μg/kg/week for 4 weeks), and carboplatin (100 μg/kg/week for 4 weeks). Results: The 6 UM cells developed in all injected eyes, 6 to 10 weeks after orthotopic transplantation. Pathological analyzes confirmed UM diagnosis and conservation of histological sub-type, i.e. epithelioid and/or spindle cell, than in corresponding patient's tumors and subcutaneous xenografts. Liver tissues examination is on-going. Rb tumor cells developed in all injected eyes 4 to 6 weeks after orthotopic transplantation for RB102 and RB200. In contrast, no tumor growth was observed in injected eyes of the RB111 model. Pathological examination of the injected eyes confirmed the presence of a massive infiltration of the retina, vitreous and anterior chamber by retinoblastoma cells. Finally, we have observed a high efficacy of intraocular administration of carboplatin (17% of intraocular tumors), but not bevacizumab (100%) nor melphalan (67%), in the RB200 model than in the control group. Conclusions: We have developed relevant and available preclinical models of human UM and Rb that allow pharmacological assessment of intraocular standard therapies. We will now evaluate innovative therapeutic approaches in such a tumor situation. Némati et al. Clin Cancer Res 2010;16:2352–2362. Aerts et al. Photodiagnosis Photodyn Ther 2010;7:275–283. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A20.

Abstract B16: Genomic, genetic, and gene expression profile characterization of a panel of primary human uveal melanoma xenografts.

Abstract Background: Uveal melanoma (UM), which is the most common primary malignancy of the eye in adults, is marked by the occurrence of non resectable liver metastases in about 50% of patients, without any curative treatment. New therapeutic strategies are therefore warranted, and require to be evaluated in relevant and predictive preclinical models in this rare tumor. Primary human cancer xenografts, directly obtained from patient's tumors, constitute such models available for pharmacological assessments. We have developed a panel of 16 UM xenografts obtained from intraocular or metastatic tumor lesions and performed a first comparative characterization with their corresponding patient's tumors (Némati et al, CCR 2010). In this study, we have completed this characterization by the determination of their genomic, genetic, and gene expression profiles. Materials and methods: Sixteen UM xenografts at very early (P1), early (P4), and late (P9) in vivo passages and their corresponding patient's tumors have been included in the study. After extraction, available DNA/RNA tumor samples were hybridized and scanned at the Institut Curie microarray core facility. Affymetrix GeneChip® Genome-Wide Human SNP6.0 arrays and protocols were used for DNAs, and Affymetrix GeneChip® Human Exon 1.0 ST arrays and protocols for RNA. Gene mutations of GNAQ, GNA11, and BRAF were detected on tumors, metastasis, xenografts, and derived cell lines by PCR, de novo sequencing using forward and reverse primers (BigDye v1.1, Life Technologies) and capillary electrophoresis on GNAS and GNA15 were screened on tumors and xenografts when mutation of GNAQ, and GNA11 were not seen. Results: Detection of copy number alterations revealed chromosomal aberrations of high risk data sets, including loss of chromosome 1p (46%), gain of 1q (56%), loss of 3 (73%), gain of 6p (37%), loss of 6q (75%), gain of 8q (80%), and multiple genomic alterations in 31/42 samples (73%). Comparison of patient's tumors and corresponding xenografts showed high similarities, with a total correlation score of 0.89. Among the 22517 studied genes, about 3% were differentially expressed between patient's tumors and their corresponding xenografts, in which 2 thirds due to the loss of the human stroma cells during the in vivo transplantation process. The remaining third is due to genes up-expressed in xenografts and related to mitosis, DNA repair, and kinase activity. No differentially expressed genes were found between xenografts at different passages. Finally, with total concordance between patient's tumors and xenografts, GNAQ, GNA11, and BRAF genes were found to be mutated in 31%, 62%, and 0%, respectively. Conclusion: Genomic, genetic, and gene expression profile characterization of our primary human UM xenografts showed a high conservation of their molecular features during their in vivo transplantation process and in vivo maintain into mice. We therefore consider that they constitute relevant preclinical tools for pharmacological experiments. Reference: 1. Némati et al. Clin Cancer Res 2010;16:2352–2362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B16.