Hypermethylation-induced inactivation of IRF6 and RARβ genes as a potential prognostic biomarker in vulvar squamous cell carcinoma

Objectives: Vulvar squamous cell carcinoma (VSCC) represents 5% of gynecological malignancies. About 80% of VSCCs arises from inflammatory diseases, such as lichen sclerosus (LS). The molecular alterations involved in onset/progression of LS-associated VSCC are unknown. Interferon regulatory factor 6 (IRF6) and retinoic acid receptor β (RARβ) tumor-suppressor genes have been found to be downregulated by promoter methylation in different carcinomas.1,2 In this study, we aimed to evaluate the involvement of the IRF6 and RARβ promoter methylation in the onset of VSCC from LS. Methods: IRF6 and RARβ mRNA expression and promoter methylation profiles were investigated by quantitative PCR (qPCR) and sequencing of PCR-amplified bisulfite-treated DNA, respectively, in VSCC (n=20) and the adjacent LS (n=20), vulvar intraepithelial neoplasia (VIN; n=5, only for IRF6), cancer-free LS (cfLS, n=20) and normal skin (NS, n=20). IRF6 and RARβ pathway-related genes p63 and c-Jun mRNAs were also investigated by qPCR.3,4 Results: IRF6 expression decreased 2.2-, 2.9-, 4.5- and 6.6-fold from cfLS, VIN, LS to VC, respectively, whereas p63 was overexpressed in all specimens compared to NS (p<0.05). IRF6 promoter was hypermethylated in 9/20 (45%) LS, 1/5 (20%) VIN, 16/20 (80%) VSCC, 2/20 (10%) cfLS, and 0 NS.3 Unlike IRF6, RARB was significantly down-expressed, 4.8-fold, only in VC (p<0.01, versus NS). Consistently, c-jun expression was 2.6-fold up-expressed in VC (p<0.01, versus NV). RARβ gene promoter was hypermethylated in 18/20 (90%) VSCC, 11/20 (55%) cfLS, 10/20 (50%) LS and 5/20 (25%) NS. Interestingly, 2/18 (11.1%) VSCC from females with tumor recurrences showed full methylation of RARβ gene promoter.4 Conclusions: IRF6 and RARB gene expressions are hampered by promoter hypermethylation in vulvar diseases and vulvar cancer. While IRF6 promoter hypermethylation occurs in a stepwise manner from vulvar LS to cancer, RARB promoter hypermethylation was found to be mainly associated to vulvar cancer. IRF6 and RARB dysregulations may play a role in LS development and progression, respectively. Therefore, IRF6 and RARβ promoter hypermethylations may be biomarkers of cancer-risk in LS patients, and prognostic biomarkers of cancer progression in patients affected by LS-associated-VSCC.3,4 References: 1.Ivanova T et al. BMC Cancer.2:4,2002. 2.Botti E et al. PNAS.108:13710-15,2011. 3.Rotondo JC et al. JAMA Dermatol.152:928-33,2016. 4.Rotondo JC et al. JAMA Dermatol.154:1-5,2018.