Faculty Bibliography

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

Background: The 'gold standard' for the diagnosis of melanocytic lesions is dermatopathology. Although most of the diagnostic criteria are clearly defined, the interpretation of histopathology slides may be subject to interobserver variability. Objectives: The aim of this study was to determine the variability among dermatopathologists in the interpretation of clinically difficult melanocytic lesions. Methods: This study used the database of MelaFind(R), a computer-vision system for the diagnosis of melanoma. All lesions were surgically removed and sent for independent evaluation by four dermatopathologists. Agreement was calculated using kappa statistics. Results: A total of 1,249 pigmented melanocytic lesions were included. There was a substantial agreement among expert dermatopathologists: two-category kappa was 0.80 (melanoma vs. non-melanoma) and three-category kappa was 0.62 (malignant vs. borderline vs. benign melanocytic lesions). The agreement was significantly greater for patients >/=40 years (three-category kappa = 0.67) than for younger patients (kappa = 0.49). In addition, the agreement was significantly lower for patients with atypical mole syndrome (AMS) (kappa = 0.31) than for patients without AMS (kappa = 0.76). Limitations: The data were limited by the inclusion/exclusion criteria of the MelaFind(R) study. This might represent a selection bias. The agreement was evaluated using kappa statistics. This is a standard method for evaluating agreement among pathologists, but might be considered controversial by some statisticians. Conclusions: Expert dermatopathologists have a high level of agreement when diagnosing clinically difficult melanocytic lesions. However, even among expert dermatopathologists, the current 'gold standard' is not perfect. Our results indicate that lesions from younger patients and patients with AMS may be more problematic for the dermatopathologists, suggesting that improved diagnostic criteria are needed for such patients.

BACKGROUND: Melanoma patients who develop brain metastases (B-Met) have limited survival and are excluded from most clinical trials. In the current study, the authors attempted to identify primary tumor characteristics and clinical features predictive of B-Met development and post-B-Met survival. METHODS: A prospectively accrued cohort of 900 melanoma patients was studied to identify clinicopathologic features of primary melanoma (eg, thickness, ulceration, mitotic index, and lymphovascular invasion) that are predictive of B-Met development and survival after a diagnosis of B-Met. Associations between clinical variables present at the time of B-Met diagnosis (eg, extracranial metastases, B-Met location, and the presence of neurological symptoms) and post-B-Met survival were also assessed. Univariate associations were analyzed using Kaplan-Meier survival analysis, and the effect of independent predictors was assessed using multivariate Cox proportional hazards regression analysis. RESULTS: Of the 900 melanoma patients studied, 89 (10%) developed B-Met. Ulceration and site of the primary tumor on the head and neck were found to be independent predictors of B-Met development on multivariate analysis (P = .001 and P = .003, respectively). Clinical variables found to be predictive of post-B-Met survival on multivariate analysis included the presence of neurological symptoms (P = .008) and extracranial metastases (P = .04). Ulceration was the only primary tumor characteristic that remained a significant predictor of post-B-Met survival on multivariate analysis (P = .04). CONCLUSIONS: Primary tumor ulceration was found to be the strongest predictor of B-Met development and remained an independent predictor of decreased post-B-Met survival in a multivariate analysis inclusive of primary tumor characteristics and clinical variables. The results of the current study suggest that patients with ulcerated primary tumors should be prospectively studied to determine whether heightened surveillance for B-Met can improve clinical outcome. Cancer 2011. (c) 2010 American Cancer Society

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman's rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM

Background Early detection and treatment of melanoma is important for optimal clinical outcome, leading to biopsy of pigmented lesions deemed suspicious for the disease. The vast majority of such lesions are benign. Thus, a more objective and accurate means for detection of melanoma is needed to identify lesions for excision. Objectives To provide proof-of-principle that epidermal genetic information retrieval (EGIR; DermTech International, La Jolla, CA, U.S.A.), a method that noninvasively samples cells from stratum corneum by means of adhesive tape stripping, can be used to discern melanomas from naevi. Methods Skin overlying pigmented lesions clinically suspicious for melanoma was harvested using EGIR. RNA isolated from the tapes was amplified and gene expression profiled. All lesions were removed for histopathological evaluation. Results Supervised analysis of the microarray data identified 312 genes differentially expressed between melanomas, naevi and normal skin specimens (P < 0.001, false discovery rate q < 0.05). Surprisingly, many of these genes are known to have a role in melanocyte development and physiology, melanoma, cancer, and cell growth control. Subsequent class prediction modelling of a training dataset, consisting of 37 melanomas and 37 naevi, discovered a 17-gene classifier that discriminates these skin lesions. Upon testing with an independent dataset, this classifier discerned in situ and invasive melanomas from naevi with 100% sensitivity and 88% specificity, with an area under the curve for the receiver operating characteristic of 0.955. Conclusions These results demonstrate that EGIR-harvested specimens can be used to detect melanoma accurately by means of a 17-gene genomic biomarker

Objective: Previous melanoma studies evaluating prognostic factors of survival at recurrence have focused on primary tumor characteristics and clinical variables at first recurrence. We examined the prognostic relevance of recurrent tumor proliferation. Methods: 114 melanoma patients with available recurrent tissues who were prospectively enrolled at New York University Medical Center were studied. Standard of care prognostic variables (e.g. stage at initial diagnosis and lactate dehydrogenase level) and recurrent tissue expression of proliferative marker Ki-67 were evaluated for their association with overall survival. Results: High Ki-67 expression was observed in 57 (50%) of the 114 recurrent melanomas. On univariate analysis, the median overall survival of patients whose recurrent tumors overexpressed Ki-67 was significantly shorter than that of patients whose recurrent tumors had low Ki-67 expression (3.6 vs. 9.5 years, p = 0.03). On multivariate analysis, a high proliferative index of the recurrent melanoma remained an independent predictor of worse overall survival, controlling for stage at initial diagnosis, disease-free survival, and stage at first recurrence [HR = 2.09 (95% CI 1.24-3.54), p = 0.006]. Conclusions: Our results demonstrate the prognostic relevance of tumor proliferation in recurrent melanoma patients. Data also support restratification of risk assessment upon recurrence that considers tumor biology in addition to clinical variables evaluated as part of the standard of care

BACKGROUND: Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study. In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed. METHODOLOGY/PRINCIPAL FINDINGS: Thirty-six patients treatment-naive advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously. Tumor BRAF(V600E) mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR). Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples. The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation. One patient had a RECIST partial response (PR) lasting 175 days. Three patients experienced stable disease (SD) with a mean duration of 37 weeks. Routine BRAF(V600E) sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors. No correlation was seen between BRAF(V600E) mutational status and clinical activity. No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples. CONCLUSIONS/SIGNIFICANCE: Sorafenib monotherapy has limited activity in advanced melanoma patients. BRAF(V600E) mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib. TRIAL REGISTRATION: Clinical Trials.gov NCT00119249

Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm