Faculty Bibliography

T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a unique self-antigen system comprising seven human melanoma gp100(209-217)-specific TCRs spanning physiological affinities (1-100 muM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 microM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.

The incidence of melanoma is increasing faster than that of any other cancer, and predicted to double every 10-20 years. Surgery can be curative in Stage I, II, or III disease, but 75% of patients with deep primary lesions develop extensive recurrence or distant metastases and have dismal prognosis. In fact, there is no curative treatment for stage IV melanoma. Although novel targeted therapies, such as BRAF inhibitors and anti-CTLA4 antibodies are showing promising results in melanoma clinical trials, resistance to these agents and patient relapse rapidly ensue. Therapeutic resistance has been commonly attributed to functional redundancy between intimately hardwired cellular pathways responsible for tumor cell maintenance and survival. In order to avoid redundancy, we propose to directly inhibit the transcription of multiple genes required for the establishment or maintenance of tumors. First, we analyzed the expression of Bromodomain (BrD)-containing proteins, a family of epigenetic readers that bind acetylated lysine. BrDs are present in histone acetyl transferases (i.e. CBP/p300, PCAF, GCN5) and transcriptional regulators (i.e. BET family members: BRD2, 3, and 4). mRNA expression arrays showed several BrD-containing genes as upregulated in primary and metastatic melanoma cell lines compared to normal melanocytes. Analysis of available expression profiles also revealed higher levels of BRD2 and BRD4 in melanoma tissues relative to nevi or normal skin (P<0.001). Furthermore, immunohistochemistry staining of a melanoma tissue microarray confirmed overexpression of BRD4 protein in primary (P<0.001) and metastatic tumors (P<0.001) compared to nevi. BRD4 knockdown using siRNA or shRNA suppressed the proliferation and colony formation capacity of several metastatic melanoma cell lines. Moreover, melanoma cells stably infected with shBRD4 carrying lentivirus displayed reduced tumor growth in vivo compared with their counterpart infected with a non-silencing control. To identify cellular pathways mo!

the molecular characterization of melanoma has ex- panded to include studies of microRNA (miRNA) ex- pression. As miR-16 has been utilized as a normalizer in serum-based miRNA studies in several cancers, we evaluated miR-16 expression as a potential reference for normalization of serum miRNA expression in melanoma patients. Methods: 143 primary cutaneous melanoma patients who presented to New York Uni- versity (NYU) Langone Medical Center for surgical resection of AJCC stage I-III disease were studied. In addition, sera samples from 60 control subjects were utilized including 22 healthy volunteers, 13 rheuma- toid arthritis patients, 20 non-melanoma cancer pa- tients (10 renal cell carcinoma and 10 bladder cancer), and 5 Atypical Mole Syndrome patients. The Kruskal- Wallis test (k = 6) or Wilcoxon test (k = 2) with Bon- ferroni correction was used for analyses of miR-16 expression in melanoma patients compared to various control groups, using raw Ct values directly. The Kruskal-Wallis test was used to compare miR-16 ex- pression across stages of melanoma. The equivalence test for independent samples was used to test the equivalence of miR-16 expression among different groups. Results: No significant differential expression of miR-16 was observed between melanoma patients and healthy volunteers (Wilcoxon test, p = 0.37). How- ever, miR-16 did show a significant difference in ex- pression as it related to stage of melanoma (p = 0.015). Additionally, the equivalence test was unable to con- firm equivalent expression of miR-16 in any melanoma versus control group pair. Conclusion: Our data in- dicate that miR-16 cannot be used as a universal normalizer in sera studies of melanoma patients

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!