Downregulation of Cyclin Kinase Inhibitors p16 INK4a and p27 in Conjunctival Melanomas

Purpose: Loss of cyclin-dependent kinase inhibitors p16INK4a, p27, and p21 has been linked to melanoma progression. We evaluated their expression in conjunctival melanocytic proliferations and explored correlations among genomic, RNA, and protein levels of p16INK4a. Methods: Expression of p16INK4a, p27, and p21 was analyzed by immunohistochemistry in 51 conjunctival nevi, 38 conjunctival melanocytic intraepithelial neoplasia (C-MIN), and 49 conjunctival melanomas (CJMs). Whole-genome/exome sequencing and RNA sequencing were available for 14 and 11 CJMs, respectively. Three CJM cell lines were treated with a DNA methyltransferase inhibitor. Results: p16INK4a was expressed in 92% of nevi, 23.7% of C-MIN, and 42.3% of CJMs; p27 in 98%, 13.5%, and 26.5%, respectively; and p21 in 33.3%, 2.8%, and 28.6%, respectively. Downregulation of p16INK4A and p27 in CJMs versus nevi was significant. No CDKN2A mutations were identified. One CJM showed homozygous CDKN2A loss and two had heterozygous loss, all lacking p16INK4a expression. Among 11 cases with normal ploidy, p16 mRNA was detected in eight (72.3%), but protein in only four (36.4%), suggesting post-transcriptional and post-translational regulation. Treatment with 5-aza-2'-deoxycytidine upregulated p16INK4a and reduced proliferation in two CJM cell lines. Conclusions: p16INK4a and p27 are significantly downregulated in CJM and may help in the histopathological differential diagnosis between melanoma and conjunctival nevus. p16INK4a loss involves genomic, epigenetic, and post-translational mechanisms. Restoring p16 function may reduce CJM aggressiveness.