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.

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.

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.

Melanoma is currently the fifth and sixth most common solid malignancy diagnosed in men and women, respectively. Although accounting for only 4% of cases of all cutaneous malignancies, melanoma accounts for more than 75% of all deaths from skin cancer. This article discusses epidemiology and risk factors, proper biopsy technique, advanced histologic evaluation of biopsy material, assessment of tumor thickness and staging, preoperative metastatic evaluation, excision margin, treatment of regional lymph nodes, treatment of recurrence, and some special clinical situations.

BACKGROUND: Accelerated whole-breast radiotherapy (RT) with tumor bed boost in the treatment of early invasive breast cancer has demonstrated equivalent local control and cosmesis when compared with standard RT. Its efficacy in the treatment of ductal carcinoma in situ (DCIS) remains unknown. METHODS AND MATERIALS: Patients treated for DCIS with lumpectomy and negative margins were eligible for 2 consecutive hypofractionated whole-breast RT clinical trials. The first trial (New York University [NYU] 01-51) prescribed to the whole breast 42 Gy (2.8 Gy in 15 fractions) and the second trial (NYU 05-181) 40.5 Gy (2.7 Gy in 15 fractions) with an additional daily boost of 0.5 Gy to the surgical cavity. RESULTS: Between 2002 and 2009, 145 DCIS patients accrued, 59 to the first protocol and 86 to the second trial. Median age was 56 years and 65% were postmenopausal at the time of treatment. Based on optimal sparing of normal tissue, 79% of the patients were planned and treated prone and 21% supine. At 5 years' median follow-up (60 months; range 2.6-105.5 months), 6 patients (4.1%) experienced an ipsilateral breast recurrence in all cases of DCIS histology. In 3/6 patients, recurrence occurred at the original site of DCIS and in the remaining 3 cases outside the original tumor bed. New contralateral breast cancers arose in 3 cases (1 DCIS and 2 invasive carcinomas). Cosmetic self-assessment at least 2 years after treatment is available in 125 patients: 91% reported good-to-excellent and 9% reported fair-to-poor outcomes. CONCLUSIONS: With a median follow-up of 5 years, the ipsilateral local recurrence rate is 4.1%, comparable to that reported from the NSABP (National Surgical Adjuvant Breast and Bowel Project) trials that employed 50 Gy in 25 fractions of radiotherapy for DCIS. There were no invasive recurrences. These results provide preliminary evidence that accelerated hypofractionated external beam radiotherapy is a viable option for DCIS.

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!