Faculty Bibliography

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.

We examined the microRNA signature that distinguishes the most common melanoma histological subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM). We also investigated the mechanisms underlying the differential expression of histology-specific microRNAs. MicroRNA array performed on a training cohort of 82 primary melanoma tumors (26 SSM, 56 NM), and nine congenital nevi (CN) revealed 134 microRNAs differentially expressed between SSM and NM (P<0.05). Out of 134 microRNAs, 126 remained significant after controlling for thickness and 31 were expressed at a lower level in SSM compared with both NM and CN. For seven microRNAs (let-7g, miR-15a, miR-16, miR-138, miR-181a, miR-191, and miR-933), the downregulation was associated with selective genomic loss in SSM cell lines and primary tumors, but not in NM cell lines and primary tumors. The lower expression level of six out of seven microRNAs in SSM compared with NM was confirmed by real-time PCR on a subset of cases in the training cohort and validated in an independent cohort of 97 melanoma cases (38 SSM, 59 NM). Our data support a molecular classification in which SSM and NM are two molecularly distinct phenotypes. Therapeutic strategies that take into account subtype-specific alterations might improve the outcome of melanoma patients.

Melanoma brain metastasis that develops as the isolated first visceral site challenges the current paradigm of tumor progression in which brain metastasis is regarded as the final stage. Here we test the hypothesis that melanoma patients who develop brain metastasis as the isolated first visceral site have distinct clinicopathological features at the time of primary melanoma diagnosis. Cutaneous melanoma patients enrolled in 2 prospectively collected databases were studied (Cohort 1: 1972-1982, Cohort 2: 2002-2009). Patients who developed brain metastasis as isolated first visceral site were compared with (1) all other patients, (2) patients who developed visceral metastasis: extracranial only or extracranial and brain, and (3) patients who progressed to other isolated visceral sites first. Two hundred seven of 2280 (9.1%) patients developed brain metastasis (median follow-up, 5.2 y). Seventy-four of 207 (35.7%) brain metastasis patients progressed to brain metastasis as the isolated first visceral site. These patients presented with primaries that were thinner and had no mitosis compared with all other visceral metastasis patients (Fisher's combined P = .02, .05, respectively), and there was a significant difference in American Joint Committee on Cancer stage distribution at initial melanoma diagnosis (combined P = .02). Post-visceral metastasis survival, however, was shorter in patients with brain metastasis as isolated first visceral site than in patients with visceral metastasis: extracranial and brain (combined P = .03). Brain metastasis as isolated first visceral site is a distinct clinicopathological entity. Studies are needed to better understand the biological factors driving this phenotype at the time of primary melanoma diagnosis and to determine its clinical implications.

Purpose In this Phase 1, multicenter, open-label study, intetumumab (CNTO 95), a fully human anti-alphav integrin monoclonal antibody was evaluated for safety, pharmacokinetics, and pharmacodynamic activity in patients with melanoma or angiosarcoma. Patients and methods Patients with histologically-confirmed inoperable melanoma or angiosarcoma refractory to standard treatment were allocated to treatment with 10 mg/kg or 20 mg/kg intetumumab, administered once every 3 weeks for up to four cycles unless unacceptable toxicity or disease progression occurred. Extended dosing was available for patients who responded with stable disease or better. Results Eight patients received 10 mg/kg and 11 received 20 mg/kg intetumumab. Baseline patient characteristics were comparable between treatment groups; 18 patients had metastatic malignant melanoma and one had angiosarcoma. No dose-limiting toxicities were observed. Headache was the most common adverse event across both dose groups. Vomiting, nausea and chills were more common, and uveitic reactions lasted longer, in patients treated with 20 mg/kg compared with 10 mg/kg intetumumab. No patient developed antibodies to intetumumab. Intetumumab drug exposure as assessed by area under the curve and maximum serum concentration appeared to increase approximately dose-proportionally from 10 to 20 mg/kg, while volume of distribution remained constant for both doses. Stable disease was observed in two patients with metastatic malignant melanoma (one in each dose group) for at least 6 weeks. Conclusions In patients with metastatic malignant melanoma and angiosarcoma in this study, intetumumab demonstrated manageable toxicity, was well tolerated, and presented approximately dose-proportional pharmacokinetics for the 10 mg/kg and 20 mg/kg doses

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAF(V600E) as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAF(V600E) mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAF(V600E) allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAF(V600E)-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAF(V600E)and BRAF(wild-type) cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAF(V600E) mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAF(V600E) mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful