Faculty Bibliography

Background: Genetic risk factors of human cancer emerge as promising markers of clinical outcome. The recent melanoma genome-wide scans (GWAS) have identified loci associated with the disease risk, nevi or UV/pigmentation, but the prognostic potential of these variants is yet to be determined. In this study, we performed the first-to-date systematic evaluation of the association between established melanoma risk loci and melanoma progression. Methods: 891 melanoma patients prospectively accrued and followed up at NYU Medical Center were studied. We examined the association of 108 melanoma susceptibility single nucleotide polymorphisms (SNPs), selected or imputed from recent GWASs on melanoma, nevi or pigmentation, with recurrence-free survival (RFS) and overall survival (OS). The genotyping was performed using Sequenom I-plex. Cox PH model was used to test the association between each SNP and RFS and OS adjusted by age at diagnosis, gender, tumor stage and histological subtype. ROC curves were used to measure predictive utility of SNPs in predicting 3-year recurrence. Results: The strong association was observed for rs7538876 (RCC2) with RFS (HR=2.445, 95% CI 1.57 - 3.8, p=0.0006) and rs9960018 (DLGAP1) with both RFS and OS (HR=4.7, 95% CI=2.11-10.43, p=0.0061, HR=1.55, 95% CI=1.11-2.17, p=0.0094, respectively) using adjusted multivariate analysis. In addition, we identified the classifier with rs7538876 and rs9960018, stage and histological type at primary tumor diagnosis, achieving a higher area under the ROC curve (AUC=84%) compared to the baseline (AUC=78%) in predicting 3-year recurrence. Univariate survival analyses have identified associations of several SNPs with ulceration and/or tumor thickness. Conclusions: Our data revealed an association between specific melanoma susceptibility variants and worse clinicopathological variables at the time of diagnosis as well as worse disease outcome. The strength of associations observed for rs7538876 and rs9960018 suggest biological implication of!

Background: Cell surface glycans, oligosaccharides present on proteins and lipids, play important roles in a range of biological processes. Our group recently reported on the role of aberrant glycosylation in promoting melanoma cell invasion and immunosuppression (Cancer Cell, 2011). In this study, we further exploit the ability of glycomic profiling of melanoma, using lectin microarrays, to identify differences in melanoma-associated glycosylation for new melanoma biomarkers and/or targets of treatment. Methods: We utilized a ratiometric dual colored 90-lectin microarray to examine differences in glycosylation between 13 primary and 14 metastatic melanoma cell lines, 5 human melanoma tissues and 4 human epidermal melanocytes (HEM). One HEM was used as a biological reference for hybridization. A subset of melanoma cell lines (n=4) was used for comparison with the glycomic profiles of the NCI-60 lines using a mixed reference. Hierarchical clustering of lectins with significant differences in binding to the sample sets (t-tests, p<0.05) was used to visualize and infer differences in glycosylation patterns. Results: A shift in the glycome related to reduction in expression of high mannose and core fucosylated complex N-glycans was seen in melanoma cell lines and tissues compared to HEMs. Glycomic profiles of primary and metastatic melanoma were similar. Interestingly, enrichment of high mannose and potentially a-2,3 sialic acid was seen in BRAF mutant (n=19) compared to BRAF wild type (n=8) cell lines. Our tested melanoma cell lines showed similar glycosylation patterns compared to melanoma lines of the NCI-60 panel, which all show higher expression of high mannose and complex N-glycans compared to other cancers. Comparison of melanoma cell lines to tissues shows similar glycomic profiles, suggesting that glycosylation of melanoma cells is not significantly altered (or blunted) due to culture. Conclusions: Our data suggest that alterations in cell surface glycosylation may be an early event in the!

Background: Increased tumor infiltrating lymphocytes (TILs) in primary (P) and locoregional melanoma tissue correlate with improved clinical outcome. Our recent data have suggested that matrix metalloproteinase 23 (MMP 23) expression (exp) in P result in lower prevalence of TILs and correlate with poor clinical outcome. On this basis, we examined P and metastatic (M) melanoma tissues to assess for concordance between the presence of TILs, MMP 23 protein levels and clinical response(resp) to anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) therapy (tx). Methods: 21 melanoma patients (pts) with M specimens were analyzed. 17 matched P specimens were also evaluated. Immunohistochemical (IHC) staining for TILs of the pre-anti-CTLA4 specimens were conducted and confirmed by 2 pathologists. IHC TILs were graded- 2+: >10% TILs present in multiple foci in both peri- and through the tumor; 1+: 1-10% TILs present in one or more foci in the tumor and predominantly peri-tumor; 0: no TILs were present or if the lymphocytes did not infiltrate the tumor. TILs in P and M were analyzed for concordance and potential for predictability of resp to anti-CTLA4 tx. Staining to identify lymphocyte subtypes and MMP 23 exp in M is being completed. Results: 20 pts received anti-CTLA4 tx. M analysis- 6 pts with 0 TILs in M (5 no response [NR], 1 partial response[PR]); 8 pts with 1-2+ TILs in M (1 complete response [CR], 5 PR, 2 progressive disease [PD]); 6 pts with 2+ TILs in M ( 3 CR, 2 PR, 1PD). 1 pt with 2+ TILs in M resected, no tx and 4 years disease free. TILs present in 13 P, absent in 4 P and not evaluable in 4 pts with unknown P melanoma. MMP 23 protein scores in P (range 2-4) correlated with melanoma recurrence. MMP 23 exp in M will be reported. Conclusions: TILs in P do not appear to correlate with TILs in M or predict for resp to anti-CTLA4 tx. TILs in M may be an indicator of responsiveness to anti-CTLA4 tx. Identification of the type of M TIL subsets may further refine tx recommendations

Background: We hypothesize that BRAF mutations result in microRNA (miRNA) alterations which contribute to orchestrating the mutant BRAF's oncogenic effects in melanoma. Our study is the first to examine the association between the BRAF mutation status in primary melanomas and the expression of miRNAs that target known tumor suppressors. Methods: 84 prospectively accrued melanoma patients at New York University Langone Medical Center were studied. DNA and total RNA were extracted from consecutive sections of formalin-fixed paraffin-embedded primary tissues. BRAF mutation status was determined by DNA sequencing. RNA was hybridized to miRCURY miRNA microarrays containing 1314 probes. Normalized miRNA data were analyzed using the t-test (p<0.05) to identify differentially expressed miRNAs between BRAFmut vs. BRAFwt cases. Those with an average fold change (FC) > 2 were selected for predicted (TargetScan, PicTar) and validated (miRWalk) gene target analysis, and overlapping genes targeted by 2 miRNAs were analyzed using pathway-mapping algorithms (KEGG, BioCarta, PANTHER). Results: 48 (57%) primaries were BRAFwt and 36 (43%) were BRAFmut (26 V600E, 4 V600K, 1 V600R, 1 V600D, 4 other). 30 miRNAs met the criteria for statistically significant differential expression and FC thresholding: let-7i, miR-23c, -26a/b, -27b, -34a, -98, -126*, -141, -148a, -181b, -195, -199a-3p, -199a/b-5p, -200a/b/c, -203, -205, -455-3p, -491-3p, -606, -641, -646, -1297, -4301; miRPlus-C1070, -C1110, -G1246-3p (average FC: 2.3-3.5, all increased in BRAFmut vs. BRAFwt). Predicted and validated target gene analysis revealed 317 genes, of which 110 (35%) were convergent targets of 2 miRNAs. Pathway analyses of the predicted, validated, and convergent target genes pointed to the potential impact of BRAFmut-associated miRNAs on known tumor suppressors FAS, PTEN, and TNF and the p53 pathway. Conclusions: Differentially expressed miRNAs in BRAFmut vs. BRAFwt primaries target genes with known roles in melanoma biology and/or treatmen!

Chromatin dynamics exert a critical function in a number of cancers, and only recently has the role of histone variants in cancer initiation and/or progression begun to be unraveled.Wepreviously probed the H2A variant profile in malignant melanoma (MM) and revealed that macroH2A levels are significantly decreased during melanoma progression, exerting a tumor-suppressive function mediated by direct transcriptional regulation of CDK8. Here, we demonstrate that global protein levels of another variant, H2A.Z, follow an opposite pattern compared to macroH2A - H2A.Z levels increase as cells become increasingly malignant. We differentiate the two isoforms of H2A.Z, namely H2A.Z.1 and H2A.Z.2 (encoded by H2AFZ and H2AFV, respectively), and we show that expression of both is higher in metastatic melanoma specimens from patients as compared to benign nevi. In addition, higher H2A.Z.1 and 2 levels significantly correlate with shorter time to recurrence and lower overall survival in patients followed up for 3 years after excision of the metastatic lesion. Our combined FISH (Fluorescent In Situ Hybridization) and CGH (Comparative Genomic Hybridization) analyses implicate gene amplification as a likely mechanism underlining H2AFZ and H2AFV overexpression. Moreover, loss of function studies revealed that H2A.Z.2-depleted cells were profoundly delayed in the progression through the cell cycle, in particular during DNA replication. Gene expression profiling showed that many cell cycle-regulating genes were significantly down-regulated upon H2A.Z.2 depletion. Collectively, our data strongly suggest that H2A.Z.2 drives MM progression through the regulation of cell-cycle-regulating genes and we anticipate that our studies will provide imperative knowledge for rationally guided epigenetic therapies as well as for the identification of novel diagnostic and prognostic markers for MM

ABSTRACT: Angiogenesis is essential for promoting growth and metastasis of solid tumors by ensuring blood supply to the tumor mass. Targeting angiogenesis is therefore an attractive approach to therapeutic intervention of cancer. Tumor angiogenesis is a process that is controlled by a complex network of molecular components including sensors, signaling transducers, and effectors, leading to cellular responses under hypoxic conditions. Positioned at the center of this network are the hypoxia-inducible factors (HIFs). HIF-1 is a major transcription factor that consists of two subunits, HIF-1alpha and HIF-1beta. It mediates transcription of a spectrum of gene targets whose products are essential for mounting hypoxic responses. HIF-1alpha protein level is very low in the normoxic condition but is rapidly elevated under hypoxia. This dramatic change in the cellular HIF-1alpha level is primarily regulated through the proteosome-mediated degradation process. In the past few years, scientific progress has clearly demonstrated that HIF-1alpha phosphorylation is mediated by several families of protein kinases including GSK3beta and ERKs both of which play crucial roles in the regulation of HIF-1alpha stability. Recent research progress has identified that Polo-like kinase 3 (Plk3) phosphorylates HIF-1alpha at two previously unidentified serine residues and that the Plk3-mediated phosphorylation of these residues results in destabilization of HIF-1alpha. Plk3 has also recently been found to phosphorylate and stabilize PTEN phosphatase, a known regulator of HIF-1alpha and tumor angiogenesis. Given the success of targeting protein kinases and tumor angiogenesis in anti-cancer therapies, Plk3 could be a potential molecular target for the development of novel and effective therapeutic agents for cancer treatment.

Melanoma incidence and associated mortality continue to increase worldwide. The lack of treatments with durable responses for stage IV melanoma may be due, at least in part, to an incomplete understanding of the molecular mechanisms that regulate tumor initiation and/or progression to metastasis. Recent evidence supports miRNA dysregulation in melanoma impacting several well-known pathways such as the PI3K/AKT or RAS/MAPK pathways, but also underexplored cellular processes like protein glycosylation and immune modulation. There is also increasing evidence that miRNA can improve patient prognostic classification over the classical staging system and provide new therapeutic opportunities. The integration of this recently acquired knowledge with known molecular alterations in protein coding genes characteristic of these tumors (i.e., BRAF and NRAS mutations, CDKN2A inactivation) is critical for a complete understanding of melanoma pathogenesis. Here, we compile the evidence of the functional roles of miRNAs in melanomagenesis and progression, and of their clinical utility as biomarkers, prognostic tools and potential therapeutic targets. Characterization of miRNA alterations in melanoma may provide new angles for therapeutic intervention, help to decipher mechanisms of drug resistance, and improve patient classification for disease surveillance and clinical benefit.

ABSTRACT: BACKGROUND: Identification of melanoma patients at high risk for recurrence and monitoring for recurrence are critical for informed management decisions. We hypothesized that serum microRNAs (miRNAs) could provide prognostic information at the time of diagnosis unaccounted for by the current staging system and could be useful in detecting recurrence after resection. METHODS: We screened 355 miRNAs in sera from 80 melanoma patients at primary diagnosis (discovery cohort) using a unique quantitative reverse transcription-PCR (qRT-PCR) panel. Cox proportional hazard models and Kaplan-Meier recurrence-free survival (RFS) curves were used to identify a miRNA signature with prognostic potential adjusting for stage. We then tested the miRNA signature in an independent cohort of 50 primary melanoma patients (validation cohort). Logistic regression analysis was performed to determine if the miRNA signature can determine risk of recurrence in both cohorts. Selected miRNAs were measured longitudinally in subsets of patients pre-/post-operatively and pre-/post-recurrence. RESULTS: A signature of 5 miRNAs successfully classified melanoma patients into high and low recurrence risk groups with significant separation of RFS in both discovery and validation cohorts (p = 0.0036, p = 0.0093, respectively). Significant separation of RFS was maintained when a logistic model containing the same signature set was used to predict recurrence risk in both discovery and validation cohorts (p < 0.0001, p = 0.033, respectively). Longitudinal expression of 4 miRNAs in a subset of patients was dynamic, suggesting miRNAs can be associated with tumor burden. CONCLUSION: Our data demonstrate that serum miRNAs can improve accuracy in identifying primary melanoma patients with high recurrence risk and in monitoring melanoma tumor burden over time.

The sentinel lymph node is the initial site of metastasis. Downregulation of antitumor immunity has a role in nodal progression. Our objective was to investigate the relationship between immune modulation and sentinel lymph node positivity, correlating it with outcome in melanoma patients. Lymph node/primary tissues from melanoma patients prospectively accrued and followed at New York University Medical Center were evaluated for the presence of regulatory T cells (Foxp3(+)) and dendritic cells (conventional: CD11c(+), mature: CD86(+)) using immunohistochemistry. Primary melanoma immune cell profiles from sentinel lymph node-positive/-negative patients were compared. Logistic regression models inclusive of standard-of-care/immunological primary tumor characteristics were constructed to predict the risk of sentinel lymph node positivity. Immunological responses in the positive sentinel lymph node were also compared with those in the negative non-sentinel node from the same nodal basin and matched negative sentinel lymph node. Decreased immune response was defined as increased regulatory T cells or decreased dendritic cells. Associations between the expression of these immune modulators, clinicopathological variables, and clinical outcome were evaluated using univariate/multivariate analyses. Primary tumor conventional dendritic cells and regression were protective against sentinel lymph node metastasis (odds ratio=0.714, 0.067; P=0.0099, 0.0816, respectively). Antitumor immunity was downregulated in the positive sentinel lymph node with an increase in regulatory T cells compared with the negative non-sentinel node from the same nodal basin (P=0.0005) and matched negative sentinel lymph node (P=0.0002). The positive sentinel lymph node also had decreased numbers of conventional dendritic cells compared with the negative sentinel lymph node (P<0.0001). Adding sentinel lymph node regulatory T cell expression improved the discriminative power of a recurrence risk assessment model using clinical stage. Primary tumor regression was associated with prolonged disease-free (P=0.025) and melanoma-specific (P=0.014) survival. Our results support an assessment of local immune profiles in both the primary tumor and sentinel lymph node to help guide therapeutic decisions.