Faculty Bibliography

T-cells have evolved the unique ability to discriminate "self" from "non-self" with high sensitivity and selectivity. However, tissue-specific autoimmunity, tolerance or eradication of cancer does not fit into the self/non-self paradigm because the T-cell responses in these situations are most often directed to non-mutated self-proteins. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a novel self-antigen system comprised of seven human melanoma gp100209-217-specific TCRs spanning physiological affinities (1 to 100 muM). We found that in vitro and in vivo T cell responses are determined by TCR affinity. Strikingly, we found that T cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 muM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Our results suggest a relatively low affinity threshold is necessary for the immune system to avoid selfdamage given the close relationship between antitumor activity and autoimmunity. This, in turn, indicates that treatment strategies focusing on TCRs in the intermediate affinity range (KD ~10 muM) or targeting or targeting shared tumor antigens would dampen the potential for autoimmunity during adoptive T cell therapy for the treatment of cancer

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor-treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma. Cancer Res; 73(20); 6264-76. (c)2013 AACR.

Objectives: Age is an understudied factor when considering treatment options for melanoma. Here, we examine the impact of age on primary melanoma treatment in a prospective cohort of patients. Methods: We used logistic regression models to examine the associations between age and initial treatment, using recurrence and melanoma-specific survival as endpoints. Results: 444 primary melanoma patients were categorized into three groups by age at diagnosis: 19-45 years (24.3%), 46-70 (50.2%), and 71-95 (25.5%). In multivariate models, older patients experienced a higher risk of recurrence (hazard ratio 3.34, 95% confidence interval, CI, 1.53-7.25; p < 0.01). No significant differences were observed in positive biopsy margin rates or extent of surgical margins across age groups. Patients in the middle age group were more likely to receive adjuvant therapy than those in the older group (odds ratio 2.78, 95% CI 1.19-6.45; p = 0.02) and showed a trend to longer disease-free survival when receiving adjuvant therapy (p = 0.09). Conclusion: Our data support age as an independent negative prognostic factor in melanoma. Our data suggest that age does not affect primary surgical treatment but may affect decisions of whether or not patients receive postoperative treatment(s). Further work is needed to better understand the biological variables affecting treatment decisions and efficacy in older patients.

Background: Manipulation of key epigenetic regulators in melanoma proliferation is emerging as a new therapeutic strategy. Bromodomain-containing proteins such as the extraterminal domain (BET) family are components of transcription factor complexes and determinants of epigenetic memory. We investigated the expression of BRD4, a BET family member in melanoma cell lines and tissues, and the effects of its inhibition with the small molecule compounds MS436 and MS417 in in vitro and in vivo models of melanoma. Methods: BRD2 and BRD4 expression were analyzed by immunohistochemistry. We tested the effects of pharmacological or RNAi-mediated inhibition of BRD4 in melanoma cells using crystal violet-based assays for proliferation/colony formation and flow-cytometry for cell cycle analysis. The molecular effects of BRD4 suppression were examined using RNA sequencing, Real-Time quantitative PCR and western blots for p27, p21, MYC, ERK1 and SKP2. In the in vivo xenograft experiments NOD/SCID/IL2R-/-mice were injected with melanoma cells and treated with MS417. Statistical significance was determined by unpaired t-test (GraphPad). Results: BRD4 was found significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi (p<0.001). Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. Rapidly after BET displacement, key cell cycle genes (SKP2, ERK1 and c-MYC) were downregulated concomitantly with the accumulation of CDK inhibitors (p21, p27), followed by melanoma cell cycle arrest. BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status. Conclusions: Our results demonstrate for the first time a role for BRD4 in melanoma maintenance and support the role of BET proteins as novel targets in melanoma. Further investigation in the clinical setting is warranted

Background: Although patient age at diagnosis is not currently included in guidelines for treatment of primary melanoma, several lines of evidence suggest that patient age is an important, yet understudied, factor when considering treatment options. Here, we attempt to address the limited knowledge of the impact of age on primary melanoma treatment. Methods: In a prospectively enrolled and followed-up cohort of melanoma patients at NYU, we used logistic regression models to evaluate the association between patient age at diagnosis, tumor baseline characteristics, including BRAF and NRAS mutation status, and likelihood of receiving and responding to adjuvant therapy. We examined adjuvant therapy effectiveness using recurrence and melanoma-specific survival as endpoints. Results: 444 primary melanoma patients were included in the study (median follow-up: 6.3 years; age range: 19-95 years). Age was categorized into three groups spanning the range of age at presentation: younger (19-45 years; 24%), middle (46-70 years; 50%), and older (71-95 years; 26%). Older patients were significantly more likely to have advanced stage, nodular subtype (P < 0.01, both variables), and BRAF wildtype tumors (P = 0.04). Controlling for these factors as well as gender, older patients experienced a higher risk of recurrence (HR older vs. younger 3.34, 95% CI 1.53-7.25; P < 0.01). Of the 128/444 (29%) patients who were eligible for adjuvant treatment (clinical stage IIB), only 67/128 (52%) received treatment. Using a propensity score that accounts for stage at presentation, patients in the middle age group were more likely to receive adjuvant therapy than those in the older group (OR 2.61, 95% CI 1.12-6.08; P = 0.03). In addition, a trend suggesting benefit from adjuvant therapy (defined as longer melanoma-specific survival) was observed only in the middle age group (P = 0.07). Conclusions: Our data suggest that older melanoma patients, despite having a significantly worse prognosis, are less likely to receive and bene!

Background: Patients with metastatic melanoma are eligible for BRAF inhibitor therapy if the BRAF V600E mutation can be identified in their tumor specimen. Patients lacking an available specimen for genotyping are unable to receive inhibitor therapy. We developed two mutation-specific genotyping platforms and tested their ability to detect BRAF and NRAS mutations in archived plasma and tumor samples to determine the potential utility of blood-based tumor genotyping in melanoma. Methods: We analyzed a group of 96 patients with stage III or IV melanoma, prospectively enrolled and followed in the NYU Melanoma Biorepository program. Each patient had a plasma sample and one or more tumor samples available for analysis. We used a combination of allele-specific PCR (Taqman) and SNaPshot assays to identify BRAF V600 and NRAS Q61 mutations in the tumor and plasma samples. Results: Among the 96 patients, 51 had stage III disease at the time of analysis; 45 had stage IV disease. Seventy-two patients had 2 or more tumor samples available for analysis, for a total of 204 tumors analyzed. In total, 52/96 (54%) patients had one or more BRAF or NRAS mutant tumors, including one patient with separate BRAF and NRAS mutant tumors (BRAF, n=35 (36%); NRAS, n=18 (19%)). We successfully amplified plasma DNA from 39/52 (75%) patients with tumor-associated mutations. Among those patients with amplifiable plasma DNA we detected mutations in 7 (18%) patients including 3 BRAF V600E, one V600K, 2 NRAS Q61K and one Q61L. Plasma-based mutations matched tumor-associated mutations in all 7 patients. All 7 patients had active disease at the time of blood draw. There were 32 patients with tumor-associated mutations in which a mutation could not be detected in the plasma. Only 15 of those 32 (47%) had active disease at the time of blood draw. There were no mutations detected in the plasma of the 44 patients whose tumors lacked BRAF or NRAS mutations. Conclusions: These data suggest that plasma-based detection of BRAF and NRAS mut!

The prognostic value of mitotic rate in melanoma is increasingly recognized, particularly in thin melanoma in which the presence or absence of a single mitosis/mm can change staging from T1a to T1b. Still, accurate mitotic rate calculation (mitoses/mm) on hematoxylin and eosin (H&E)-stained sections can be challenging. Antimonoclonal mitotic protein-2 (MPM-2) and antiphosphohistone-H3 (PHH3) are 2 antibodies reported to be more mitosis-specific than other markers of proliferation such as Ki-67. We used light microscopy and computer-assisted image analysis software to quantify MPM-2 and PHH3 staining in melanoma. We then compared mitotic rates by each method with conventional H&E-based mitotic rate for correlation with clinical outcomes. Our study included primary tissues from 190 nonconsecutive cutaneous melanoma patients who were prospectively enrolled at New York University Langone Medical Center with information on age, gender, and primary tumor characteristics. The mitotic rate was quantified manually by light microscopy of corresponding H&E-stained, MPM-2-stained, and PHH3-stained sections. Computer-assisted image analysis was then used to quantify immunolabeled mitoses on the previously examined PHH3 and MPM-2 slides. We then analyzed the association between mitotic rate and both progression-free and melanoma-specific survival. Univariate analysis of PHH3 found significant correlation between increased PHH3 mitotic rate and decreased progression-free survival (P=0.04). Computer-assisted image analysis enhanced the correlation of PHH3 mitotic rate with progression-free survival (P=0.02). Regardless of the detection method, neither MPM-2 nor PHH3 offered significant advantage over conventional H&E determination of mitotic rate.