Title: Epigenetic Dysregulation in Cutaneous Malignant Melanoma
Description: BACKGROUND: Evidence is rapidly accumulating that epigenetic mechanisms, including DNA methylation and demethylation, histone modifications, and non-coding RNAs, may play a central role in the pathobiology of melanoma. Previous studies have demonstrated the utility of the novel epigenetic mark, 5-hydroxymethylcytosine, to distinguish between benign cutaneous nevi and primary cutaneous melanoma. Further applications of this epigenetic biomarker to enhance diagnostic accuracy and prognostic precision in the evaluation of a unique set of diagnostically-challenging primary cutaneous melanomas and sentinel lymph node biopsies will be explored. In addition, clinically-applicable next-generation sequencing platforms enable us to identify novel gene mutations present in patient melanoma samples. Accordingly, the prevalence and nature of mutations in genes encoding epigenetic regulators (as well as non-epigenetic regulators) in patient melanoma samples will also be explored in this study.

METHODS: Fifty-two histologic sections of primary cutaneous melanoma (NP=52) were obtained from the pathology archives of two academic institutions. These cases were intentionally selected based on the presence of pseudomaturation (nMPM=24) or associated pre-existing nevus (nMPEN=28). Immunohistochemistry for 5-hydroxymethylcytosine was performed on all primary melanoma cases (NP=52). In addition, a collection of twenty eight histologic sections of sentinel lymph node biopsies (NS=28) containing either metastatic melanoma (nMM=18) or nodal nevus (nNN=10) was also retrieved from the pathology archive of one academic institution. Dual-labeling direct immunofluorescence and immunohistochemistry for MART-1/5-hydroxymethylcytosine was performed on all sentinel lymph node biopsy cases. Finally, targeted next generation sequencing was performed on thirty-eight patient melanoma specimens (NM=38) to detect exonic mutations in 275 cancer genes, 41 of which encode known epigenetic regulators.

RESULTS: Collectively, regions containing pseudomaturing cells within primary melanomas with pseudomaturation demonstrated intermediate immunopositivity for 5-hydroxymethylcytosine, in stark contrast to the overlying melanoma, which showed complete, diffuse loss. The staining intensity in pseudomaturing regions was quantifiably distinct and intermediate to that of pre-existing nevi (strong, homogeneous positivity) and bona fide melanoma (complete, diffuse loss), providing further support to the hypothesis that pseudomaturing melanoma cells may reflect a more indolent subpopulation. 5-hydroxymethylcytosine immunoreactivity was strongly retained in 10 of 10 (100%) cases of nodal nevus but ‘lost’ in 18 of 18 (100%) of cases of metastatic melanoma, thus representing a useful adjunctive strategy to definitively diagnose histologically subtle micrometastases. Targeted next generation sequencing demonstrated that 20.2% of all somatic mutations (107 of 530) affected an epigenetic regulator, with 35 of 38 samples (92.1%) harboring at least one mutation in an epigenetic gene. Genes with the highest percentage of UVB-signature mutations encoded epigenetic regulators. In addition, MECOM, a novel, central epigenetic regulator, as well as TET2/IDH1, critical enzymatic and metabolic regulators of DNA 5-hydroxymethylation, were found to be more frequently mutated than previously described.

DISCUSSION/CONCLUSION: The present study provides direct genomic evidence that epigenetic regulators may be involved in the pathobiology of melanoma and that novel, personalized therapeutic targets may be revealed with next generation sequencing. In addition, our immunohistochemical investigations demonstrate that the epigenetic biomarker, 5-hydroxymethylcytosine, can enhance diagnostic and microstaging accuracy in the histopathologic evaluation of pseudomaturing primary cutaneous melanoma and refine prognostic evaluations by enabling the distinction of metastatic melanoma from its diagnostic mimic, nodal nevus, in sentinel lymph node biopsies.