Sentinel lymph node biopsies are conducted to stage patients with newly-diagnosed melanomas that have histopathologic attributes conferring defined levels of metastatic potential.,Because benign nevic cells may also form ‘deposits’ in lymph nodes (nodal nevus), the pathologic evaluation for metastatic melanoma within sentinel lymph nodes can be challenging. 28 sentinel lymph node biopsy cases containing either metastatic melanoma (N=18) or nodal nevi (N=10) were retrieved from the archives of the Brigham and Women’s Hospital Department of Pathology (2011-2014).,In addition, two sentinel lymph node cases that were favored to represent metastatic disease but whose histopathologic features were viewed as equivocal, with melanoma favored, were also included.,Dual-labeling for the melanocyte lineage marker, MART-1, and the epigenetic marker 5-hydroxymethylcytosine, a functionally-significant indicator that has been shown to distinguish benign nevi from melanoma, was performed on all cases using immunohistochemistry and/or direct immunofluorescence.,All (18 of 18) metastatic melanoma cases showed complete loss of 5-hydroxymethylcytosine nuclear staining in MART-1-positive cells and all (10 of 10) nodal nevus cases demonstrated 5-hydroxymethylcytosine nuclear staining in MART-1 positive cells.,In addition, 5-hydroxymethylcytosine staining confirmed the favored diagnoses of metastatic melanoma in the two ‘equivocal’ cases.,Thus, 5-hydroxymethylcytosine may be a useful adjunctive marker to distinguish between benign nodal nevi and metastatic melanoma during the evaluation of sentinel lymph node biopsies for metastatic melanoma.
Metastatic melanoma is a devastating disease with limited therapeutic options.,MicroRNAs (miRNAs) are small non coding RNA molecules with important roles in post-transcriptional gene expression regulation, whose aberrant expression has been implicated in cancer.,We show that the expression of miRNAs from a large cluster on human chromosome 14q32 is significantly down-regulated in melanoma cell lines, benign nevi and melanoma samples relative to normal melanocytes.,This miRNA cluster resides within a parentally imprinted chromosomal region known to be important in development and differentiation.,In some melanoma cell lines, a chromosomal deletion or loss-of-heterozygosity was observed in the cis-acting regulatory region of this cluster.,In several cell lines we were able to re-express two maternally-induced genes and several miRNAs from the cluster with a combination of de-methylating agents and histone de-acetylase inhibitors, suggesting that epigenetic modifications take part in their silencing.,Stable over-expression of mir-376a and mir-376c, two miRNAs from this cluster that could be re-expressed following epigenetic manipulation, led to modest growth retardation and to a significant decrease in migration in-vitro.,Bioinformatic analysis predicted that both miRNAs could potentially target the 3'UTR of IGF1R.,Indeed, stable expression of mir-376a and mir-376c in melanoma cells led to a decrease in IGF1R mRNA and protein, and a luciferase reporter assay indicated that the 3'UTR of IGF1R is a target of both mir-376a and mir-376c.,Our work is the first to show that the large miRNA cluster on chromosome 14q32 is silenced in melanoma.,Our results suggest that down-regulation of mir-376a and mir-376c may contribute to IGF1R over-expression and to aberrant negative regulation of this signaling pathway in melanoma, thus promoting tumorigenesis and metastasis.