Faculty Bibliography

BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16

Background: Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. Here we use gene expression profiling of metastatic melanoma samples to search for a molecular basis for this observation. Methods: We analyzed gene expression profiles of 44 metastatic melanoma specimens collected from 38 patients who were followed clinically for a median of 20 months after surgery. RNA was processed using standard methods and hybridized to Affymetrix Human Genome HU133 Plus 2.0 arrays. Data were analyzed using GeneSpring and PathwayAssist software, normalizing using a PLIER algorithm and utilizing ANOVA statistical tests. Results: Unsupervised clustering yielded 4 distinct groups of subjects, 2 of which had large differences in overall survival. We then used supervised clustering to compare patients whose post-surgery survival was less then 1.5 years (11 subjects, 9.5 month median survival) to those who survived greater than 1.5 years (23 subjects, 26 month median survival, 17 alive at last follow-up). Only genes with changes in expression of > 2 with a p value of < 0.05 were accepted, resulting in a group of approximately 200 genes. Genes associated with immune response (TNFa, IRF1, Granzyme B, CD8a, CXCL11, IL27AR, GBP1, CXCL10, CCBP2, GBP2, CCR9 and IFIT4) or with cell proliferation (FGFR1, MET, MDM2, CDC25A, RFC2, SOS1, HOXA3, MCM4, MCM7, ORC5L, KIF23 and VAV3) were highly represented. Prolonged survival was associated with elevated expression of immune response genes and decreased expression of genes associated with cell proliferation. Conclusions: Gene expression profiling of metastatic melanoma samples identified a set of genes associated with patient survival, and suggests that immune surveillance is a mechanism for prolonged survival in these patients

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need for novel approaches to identify new effective treatments. In this study, we screened 2,000 compounds from the Spectrum Library at a concentration of 1 micromol/L using two chemoresistant melanoma cell lines (M-14 and SK-Mel-19) and a spontaneously immortalized, nontumorigenic melanocyte cell line (melan-a). We identified 10 compounds that inhibited the growth of the melanoma cells yet were largely nontoxic to melanocytes. Strikingly, 4 of the 10 compounds (mebendazole, albendazole, fenbendazole, and oxybendazole) are benzimidazoles, a class of structurally related, tubulin-disrupting drugs. Mebendazole was prioritized to further characterize its mechanism of melanoma growth inhibition based on its favorable pharmacokinetic profile. Our data reveal that mebendazole inhibits melanoma growth with an average IC(50) of 0.32 micromol/L and preferentially induces apoptosis in melanoma cells compared with melanocytes. The intrinsic apoptotic response is mediated through phosphorylation of Bcl-2, which occurs rapidly after treatment with mebendazole in melanoma cells but not in melanocytes. Phosphorylation of Bcl-2 in melanoma cells prevents its interaction with proapoptotic Bax, thereby promoting apoptosis. We further show that mebendazole-resistant melanocytes can be sensitized through reduction of Bcl-2 protein levels, showing the essential role of Bcl-2 in the cellular response to mebendazole-mediated tubulin disruption. Our results suggest that this screening approach is useful for identifying agents that show promise in the treatment of even chemoresistant melanoma and identifies mebendazole as a potent, melanoma-specific cytotoxic agent

PURPOSE: Extracellular matrix remodeling during tumor growth plays an important role in angiogenesis. Our preclinical data suggest that a newly identified cryptic epitope (HU177) within collagen type IV regulates endothelial and melanoma cell adhesion in vitro and angiogenesis in vivo. In this study, we investigated the clinical relevance of HUI77 shedding in melanoma patient sera. EXPERIMENTAL DESIGN: Serum samples from 291 melanoma patients prospectively enrolled at the New York University Medical Center and 106 control subjects were analyzed for HU177 epitope concentration by a newly developed sandwich ELISA assay. HU177 serum levels were then correlated with clinical and pathologic parameters. RESULTS: Mean HU177 epitope concentration was 5.8 ng/mL (range, 0-139.8 ng/mL). A significant correlation was observed between HU177 concentration and nodular melanoma histologic subtype [nodular, 10.3 +/- 1.6 ng/mL (mean +/- SE); superficial spreading melanoma, 4.5 +/- 1.1 ng/mL; all others, 6.1 +/- 2.1 ng/mL; P = 0.01 by ANOVA test]. Increased HU177 shedding also correlated with tumor thickness (< or =1.00 mm, 3.8 +/- 1.1 ng/mL; 1.01-3.99 mm, 8.7 +/- 1.3 ng/mL; > or =4.00 mm, 10.3 +/- 2.4 ng/mL; P = 0.003 by ANOVA). After multivariate analysis controlling for thickness, the correlation between higher HU177 concentration and nodular subtype remained significant (P = 0.03). The mean HU177 epitope concentration in control subjects was 2.4 ng/mL. CONCLUSIONS: We report that primary melanoma can induce detectable changes in systemic levels of cryptic epitope shedding. Our data also support that nodular melanoma might be biologically distinct compared with superficial spreading type melanoma. As targeted interventions against cryptic collagen epitopes are currently undergoing phase I clinical trial testing, these findings indicate that patients with nodular melanoma may be more susceptible to such targeted therapies

BACKGROUND: Different Insulin-like Growth Factor Binding Proteins (IGFBPs) have been investigated as potential biomarkers in several types of tumors. In this study, we examined both IGFBP-3 and -4 levels in tissues and sera of melanoma patients representing different stages of melanoma progression. METHODS: The study cohort consisted of 132 melanoma patients (primary, n = 72; metastatic, n = 60; 64 Male, 68 Female; Median Age = 56) prospectively enrolled in the New York University School of Medicine Interdisciplinary Melanoma Cooperative Group (NYU IMCG) between August 2002 and December 2006. We assessed tumor-expression and circulating sera levels of IGFBP-3 and -4 using immunohistochemistry and ELISA assays. Correlations with clinicopathologic parameters were examined using Wilcoxon rank-sum tests and Spearman-rank correlation coefficients. RESULTS: Median IGFBP-4 tumor expression was significantly greater in primary versus metastatic patients (70% versus 10%, p = 0.01) A trend for greater median IGFBP-3 sera concentration was observed in metastatic versus primary patients (4.9 microg/ml vs. 3.4 microg/ml, respectively, p = 0.09). However, sera levels fell within a normal range for IGFBP-3. Neither IGFBP-3 nor -4 correlated with survival in this subset of patients. CONCLUSION: Decreased IGFBP-4 tumor expression might be a step in the progression from primary to metastatic melanoma. Our data lend support to a recently-described novel tumor suppressor role of secreting IGFBPs in melanoma. However, data do not support the clinical utility of measuring levels of IGFBP-3 and -4 in sera of melanoma patients

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients

Cancer/testis (CT) antigens are potential targets for cancer immunotherapy, with NY-ESO-1 being among the most immunogenic. In several clinical trials in malignant melanoma (MM) patients, NY-ESO-1 protein/peptides showed clear evidence of inducing specific immunity. However, little is known about NY-ESO-1 expression in primary and metastatic MM and its relationship to disease progression. We analyzed NY-ESO-1 expression immunohistochemically in a series of primary and metastatic MMs and its relation to prognostic parameters and survival. We studied 61 primary and 63 metastatic MM specimens (from 61 and 56 patients, respectively). The prevalence of NY-ESO-1 expression was significantly higher in metastatic versus primary tumors [18/56 (32%) versus 8/61 (13%), P = 0.015]. There was a significant association between initial stage at presentation and NY-ESO-1 expression [stage I (3.45%), stage II (9.52%) and stage III (45.45%), P = 0.0014]. Primary MMs expressing NY-ESO-1 were significantly thicker than NY-ESO-1 negative cases (median thickness 4.7 mm versus 1.53 mm respectively, P = 0.03). No significant difference was seen in overall survival. In conclusion, NY-ESO-1 is more frequently expressed in metastatic than in primary MM and its expression is associated with thicker primary lesions and a higher frequency of metastatic disease, indicative of a worse prognosis. Our study suggests that patients with metastatic MM who express NY-ESO-1 may benefit from NY-ESO-1-based immunotherapy

ABSTRACT: BACKGROUND: Treatment options for patients with recurrent superficial bladder cancer are limited, necessitating aggressive exploration of new treatment strategies that effectively prevent recurrence and progression to invasive disease. We assessed the effects of belinostat (previously PXD101), a novel histone deacetylase inhibitor, on a panel of human bladder cancer cell lines representing superficial and invasive disease, and on a transgenic mouse model of superficial bladder cancer. METHODS: Growth inhibition and cell cycle distribution effect of belinostat on 5637, T24, J82, and RT4 urothelial lines were assessed. Ha-ras transgenic mice with established superficial bladder cancer were randomized to receive either belinostat or vehicle alone, and assessed for bladder weight, hematuria, gene expression profiling, and immunohistochemistry (IHC). RESULTS: Belinostat had a significant linear dose-dependent growth inhibition on all cell lines (IC50 range of 1.0-10.0 micromolar). The 5637 cell line, which was derived from a superficial papillary tumor, was the most sensitive to treatment. Belinostat (100 mg/kg, intraperitoneal, 5 days each week for 3 weeks) treated mice had less bladder weight (p<0.05), and no hematuria compared with 6/10 control mice that developed at least one episode. IHC of bladder tumors showed less cell proliferation and a higher expression of p21WAF1 in the belinostat-treated mice. Gene expression profile analysis revealed 56 genes significantly different in the treated group; these included the upregulation of p21WAF1, induction of core histone deacetylase (HDAC), and cell communication genes. CONCLUSION: Our data demonstrate that belinostat inhibits bladder cancer and supports the clinical evaluation of belinostat for the treatment of patients with superficial bladder cancer

BACKGROUND: In patients with T1b-T3b cutaneous melanoma the utility of radiologic imaging at the time of diagnosis is unclear. Whether initial imaging led to a change in stage or treatment plan was investigated. METHODS: The melanoma database was searched for patients with T1b-T3b primary lesions, clinically N0, and asymptomatic for metastatic disease. Radiologic studies conducted before wide local excision +/- sentinel lymph node biopsy as well as all further imaging and investigations were analyzed. Outcome measures included upstaging, change in initial surgical management, true-positive, false-positive, true-negative, and false-negative rates of each imaging modality. RESULTS: In all, 344 preoperative imaging studies (chest x-ray [CXR], computed tomography [CT], positron emission tomography [PET]/CT) were performed on 158 patients, resulting in 49 findings suspicious for metastatic melanoma and 134 findings suggestive of nonmelanoma pathology. Only 1 of 344 (0.3%) studies, a PET/CT, correlated with confirmed metastatic melanoma. The false-positive rates were CXR 5 of 7 (71.4%), chest CT 21 of 24 (87.5%), abdomen/pelvis CT 10 of 11 (90.9%), head CT 2 of 2 (100.0%), PET/CT 3 of 5 (60.0%). No patient was upstaged or had a change in initial surgical management based on preoperative imaging. The cost of all initial imaging and imaging to follow-up abnormal findings was estimated as $555,308 for the 158 patients studied. CONCLUSIONS: Imaging at the time of initial diagnosis of T1b-T3b, clinically N0, M0 melanoma was of low yield with a high false-positive rate, and did not lead to upstaging or change in initial surgical management. These findings suggest that imaging of asymptomatic patients at the time of diagnosis may not be warranted

BACKGROUND: Overexpression of Neutral Endopeptidase (NEP) has been reported in metastatic carcinomas, implicating NEP in tumor progression and suggesting a role for NEP inhibitors in its treatment. We investigated the role of NEP expression in the clinical progression of cutaneous melanoma. METHODS: We screened 7 melanoma cell lines for NEP protein expression. NEP-specific siRNA was transfected into the lines to examine the role of gene transcription in NEP expression. Immunohistochemistry was done for 93 specimens and correlated with clinicopathologic parameters. Thirty-seven metastatic melanoma specimens were examined for NEP transcript expression using Affymetrix GeneChips. In a subset of 25 specimens for which both transcript and protein expression was available, expression ratios were used to identify genes that co-express with NEP in GeneChip analysis. RESULTS: NEP was overexpressed in 4/7 human melanoma cell lines, and siRNA knock-down of NEP transcripts led to downregulation of its protein expression. NEP protein overexpression was significantly more common in metastatic versus primary tumors (P = 0.002). Twelve of 37 (32%) metastatic tumors had increased NEP transcript expression, and an association was observed between NEP transcript upregulation and protein overexpression (P < 0.0001). Thirty-eight genes were found to significantly co-express with NEP (p < 0.005). Thirty-three genes positively correlated with NEP, including genes involved in the MAP kinase pathway, antigen processing and presentation, apoptosis, and WNT signaling pathway, and 5 genes negatively correlated with NEP, including genes of focal adhesion and the notch signaling pathways. CONCLUSION: NEP overexpression, which seems to be largely driven by increased transcription, is rare in primary melanoma and occurs late in melanoma progression. Functional studies are needed to better understand the mechanisms of NEP regulation in melanoma