Faculty Bibliography

BACKGROUND: Metastatic melanoma of unknown primary (MUP) is uncommon, biologically ill defined, and clinically understudied. MUP outcomes are seldom reported in clinical trials. In this study, we analyze responses of MUP patients treated with systemic therapy in an attempt to inform treatment guidelines for this unique population. METHODS: New York University (NYU)'s prospective melanoma database was searched for MUP patients treated with systemic therapy. PubMed and Google Scholar were searched for MUP patients treated with immunotherapy or targeted therapy reported in the literature, and their response and survival data were compared to the MUP patient data from NYU. Both groups' response data were compared to those reported for melanoma of known primary (MKP). RESULTS: The MUP patients treated at NYU had better outcomes on immunotherapy but worse on targeted therapy than the MUP patients in the literature. The NYU MUP patients and those in the literature had worse outcomes than the majority-MKP populations in 10 clinical trial reports. CONCLUSIONS: Our study suggests that MUP patients might have poorer outcomes on systemic therapy as compared to MKP patients. Our cohort was small and limited data were available, highlighting the need for increased reporting of MUP outcomes and multi-institutional efforts to understand the mechanism behind the observed differences.

Tumor infiltrating lymphocytes (TILs) in primary melanomas are thought to represent the host anti-tumor immune response, but controversy exists over whether TILs offer independent prognostication of survival. We studied a cohort of 1241 primary melanoma patients to assess the association of absent, non-brisk, and brisk TIL grade with survival outcomes. We tested whether quantitative TIL counts using immunohistochemical lymphocyte markers CD3, CD45, and FOXP3 add prognostic value to TIL grading compared to histology alone in 15% of the cohort. To assess for inter-group immunologic heterogeneity among TIL grades, we investigated differential expression of 594 immunoregulatory genes in 67 primary melanomas. On histologic evaluation of 1241 primary melanomas, TILs were graded as absent (n=388, 31%), non-brisk (n=330, 27%), and brisk (n=523, 42%). Patients with brisk TILs had improved recurrence-free survival (RFS) (P=.025) and overall survival (OS) (P=.006) compared to patients with non-brisk and absent TILs, for which there were no differences in RFS (P=.40) or OS (P=.41). TIL quantitation by immunohistochemistry did not improve prognostication compared to TIL grading on hematoxylin and eosin stained sections. Melanomas with non-brisk and absent TILs share similar immunoregulatory gene expression profiles. In contrast, melanomas with brisk TILs demonstrate upregulation of T-cell activation pathways and inhibition of upstream immune checkpoint regulators. The presence of TILs in primary melanomas represents a heterogeneous group and caution in prognostic interpretation is warranted. Melanomas with brisk TILs are defined by an immunostimulatory gene expression profile and improved prognosis compared to melanomas with non-brisk or absent TILs.

BACKGROUND: Age has been reported as an independent prognostic factor for melanoma-specific survival (MSS). We tested the hypothesis that age impacts the host anti-tumor immune response, accounting for age-specific survival outcomes in three unique melanoma patient cohorts. METHODS: We queried the U.S. population-based Surveillance, Epidemiology, and End Results Program (SEER), the prospective tertiary care hospital-based Interdisciplinary Melanoma Cooperative Group (IMCG) biorepository, and the Cancer Genome Atlas (TCGA) biospecimen database to test the association of patient age at time of melanoma diagnosis with clinicopathologic features and survival outcomes. Age groups were defined as 65 (older). Each age group in the IMCG and TCGA cohorts was stratified by tumor infiltrating lymphocyte (TIL) measurements and tested for association with MSS. Differential expression of 594 immunoregulatory genes was assessed in a subset of primary melanomas in the IMCG and TCGA cohorts using an integrative pathway analysis. RESULTS: We analyzed 304, 476 (SEER), 1241 (IMCG), and 292 (TCGA) patients. Increasing age at melanoma diagnosis in both the SEER and IMCG cohorts demonstrated a positive correlation with tumor thickness, ulceration, stage, and mortality, however age in the TCGA cohort did not correlate with mortality. Older age was associated with shorter MSS in all three cohorts. When the young age group in both the IMCG and TCGA cohorts was stratified by TIL status, there were no differences in MSS. However, older IMCG patients with brisk TILs and intermediate aged TCGA patients with high lymphocyte scores (3-6) had improved MSS. Gene expression analysis revealed top pathways (T cell trafficking, communication, and differentiation) and top upstream regulators (CD3, CD28, IFNG, and STAT3) that significantly changed with age in 84 IMCG and 43 TCGA primary melanomas. CONCLUSIONS: Older age at time of melanoma diagnosis is associated with shorter MSS, however age's association with clinicopathologic features is dependent upon specific characteristics of the study population. TIL as a read-out of the host immune response may have greater prognostic impact in patients older than age 45. Recognition of age-related factors negatively impacting host immune responses may provide new insights into therapeutic strategies for the elderly.

BACKGROUND: Although 20% to 30% of melanomas are histopathologically 'nevus associated,' the majority of melanomas arise de novo, ie, in clinically normal skin with no associated nevus. We examined whether these forms of melanoma differed in their associations with clinical and histopathologic features and patient survival. METHODS: We analyzed two prospective cohorts from our institution with protocol-driven follow-up information (NYU1, n = 1024; NYU2, n = 1125). We used univariate and multivariable analyses to examine associations between de novo vs nevus-associated melanoma classification and age, anatomic site, tumor thickness, tumor ulceration, mitotic index, histological subtype, clinical stage, and survival. We tested the associations identified in NYU1 using NYU2 as a replication cohort. All tests of statistical significance were two-sided. RESULTS: In NYU1, de novo melanomas were associated with tumor thickness greater than 1.0 mm (odds ratio [OR] = 1.96, 95% confidence interval [CI] = 1.43 to 2.70, P < .001), ulceration (OR = 1.65, 95% CI = 1.10 to 2.54, P = .02), nodular subtype (OR = 3.26, 95% CI = 1.70 to 7.11, P = .001), greater than stage I (OR = 2.35, 95% CI = 1.65 to 3.40, P < .001), older age (OR = 1.64, 95% CI = 1.18 to 2.30, P = .004), and shorter overall survival (HR = 1.63, 95% CI = 1.22 to 2.18, P < .001). In NYU2, de novo melanoma was again statistically significantly associated with thickness greater than 1.0 mm (OR = 2.24, 95% CI = 1.72 to 2.93, P < .001), ulceration (OR = 2.88, 95% CI = 1.95 to 4.37, P < .001), nodular subtype (OR = 2.41, 95% CI = 1.75 to 3.37, P < .001), greater than stage I (OR = 2.42, 95% CI = 1.80 to 3.29, P < .001), older age (OR = 1.68, 95% CI = 1.31 to 2.17, P < .001), and shorter overall survival (HR = 2.52, 95% CI = 1.78 to 3.56, P < .001). In multivariable analysis, de novo classification was an independent, poor prognostic indicator in NYU2 (HR = 1.70, 95% CI = 1.19 to 2.44, P = .004). Male patients had a statistically significantly worse survival than female patients if their melanoma was de novo (NYU1, P < .001; NYU2, P < .001); unexpectedly, there was no sex difference in survival among patients with nevus-associated tumors. CONCLUSIONS: These data suggest that de novo melanomas are more aggressive than nevus-associated melanomas. This classification scheme may also provide a useful framework for investigations into sex differences in melanoma outcomes.

While the role of genetic risk factors in the etiology of uveal melanoma (UM) has been strongly suggested, the genetic susceptibility to UM is currently vastly unexplored. Due to shared epidemiological risk factors between cutaneous melanoma (CM) and UM, in this study we have selected 28 SNPs identified as risk variants in previous genome-wide association studies on CM or CM-related host phenotypes (such as pigmentation and eye color) and tested them for association with UM risk. By logistic regression analysis of 272 UM cases and 1782 controls using an additive model, we identified five variants significantly associated with UM risk, all passing adjustment for multiple testing. The three most significantly associated variants rs12913832 (OR = 0.529, 95% CI 0.415-0.673; p = 8.47E-08), rs1129038 (OR = 0.533, 95% CI 0.419-0.678; p = 1.19E-07) and rs916977 (OR = 0.465, 95% CI 0.339-0.637; p = 3.04E-07) are correlated (r(2) > 0.5) and map at 15q12 in the region of HERC2/OCA2, which determines eye-color in the human population. Our data provides first evidence that the genetic factors associated with pigmentation traits are risk loci of UM susceptibility.

CD8+ T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.

PURPOSE: The identification of personalized germline markers with biological relevance for the prediction of cutaneous melanoma (CM) prognosis is highly demanded but to date it has been largely unsuccessful. As melanoma progression is controlled by host immunity, here we present a novel approach interrogating immunoregulatory pathways using the genome-wide maps of expression quantitative trait loci (eQTL) to reveal biologically relevant germline variants modulating CM outcomes. EXPERIMENTAL DESIGN: Using whole genome eQTL data from a healthy population, we identified 385 variants -significantly impacting the expression of 268 immune-relevant genes. The 40 most significant eQTLs were tested in a prospective cohort of 1,221 CM patients for their association with overall (OS) and recurrence-free survival using Cox regression models. RESULTS: We identified highly significant associations with better melanoma OS for rs6673928, impacting IL19 expression (HR 0.56, 95% CI 0.41-0.77; P= 0.0002) and rs6695772, controlling the expression of BATF3 (HR 1.64, 95% CI 1.19-2.24; P= 0.0019). Both associations map in the previously suspected melanoma prognostic locus at 1q32. Furthermore, we show that their combined effect on melanoma OS is substantially enhanced reaching the level of clinical applicability (HR 1.92, 95% CI 1.43-2.60; P= 2.38e-5). CONCLUSIONS: Our unique approach of interrogating lymphocyte-specific eQTLs reveals novel and biologically relevant immunomodulatory eQTL predictors of CM prognosis that are independent of current histopathological markers. The significantly enhanced combined effect of identified eQTLs suggests the personalized utilization of both SNPs in a clinical setting, strongly indicating the promise of the proposed design for the discovery of prognostic or risk germline markers in other cancers.

Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases. Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra-terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma. Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF-mutant melanoma. We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF-mutant melanoma cell lines. Further, to investigate the molecular mechanisms underlying the effects of combined treatment, we conducted antibody arrays of in vitro drug-treated cell lines and RNA sequencing of drug-treated xenograft tumors. The combination of JQ1 and Vemurafenib acted synergistically in BRAF-mutant cell lines, resulting in marked apoptosis in vitro, with upregulation of proapoptotic proteins. In vivo, combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone. RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination, with several anti-apoptotic genes significantly down-regulated. Collectively, our data provide a rationale for combined BET and BRAF inhibition as a novel strategy for the treatment of melanoma.

Endothelin-1 (ET-1) is a vasoactive peptide that also plays a role in the tanning response of the skin. Animal and cell culture studies have also implicated ET-1 in melanoma progression, but no association studies have been performed to link ET-1 expression and melanoma in humans. Here, we present the first in-vivo study of ET-1 expression in pigmented lesions in humans: an ET-1 immunohistochemical screen of melanocytic nevi, melanoma in situ lesions, invasive melanomas, metastatic melanomas, and blue nevi was performed. Twenty-six percent of melanocytic nevi and 44% of melanoma in situ lesions demonstrate ET-1 expression in the perilesional microenvironment, whereas expression in nevus or melanoma cells was rare to absent. In striking contrast, 100% of moderately to highly pigmented invasive melanomas contained numerous ET-1-positive cells in the tumor microenvironment, with 79% containing ET-1-positive melanoma cells, confirmed by co-staining with melanoma tumor marker HMB45. Hypopigmented invasive melanomas had reduced ET-1 expression, suggesting a correlation between ET-1 expression and pigmented melanomas. ET-1-positive perilesional cells were CD68-positive, indicating macrophage origin. Sixty-two percent of highly pigmented metastatic melanomas demonstrated ET-1 expression in melanoma cells, in contrast to 28.2% of hypopigmented specimens. Eighty-nine percent of benign nevi, known as blue nevi, which have a dermal localization, were associated with numerous ET-1-positive macrophages in the perilesional microenvironment, but no ET-1 expression was detected in the melanocytes. We conclude that ET-1 expression in the microenvironment increases with advancing stages of melanocyte transformation, implicating a critical role for ET-1 in melanoma progression, and the importance of the tumor microenvironment in the melanoma phenotype.