Association of retinoic acid receptor ß gene with onset and progression of lichen sclerosus-associated vulvar squamous cell carcinoma

IMPORTANCE Molecular alterations in lichen sclerosus-associated vulvar squamous cell carcinoma (LS-VSCC) are largely unknown. OBJECTIVE To determine whether the retinoic acid receptor ß (RARß) tumor-suppressor gene is involved in the onset and/or progression of LS-VSCC. DESIGN, SETTING, AND PARTICIPANTS The case-control study, conducted at University- Hospital of Ferrara, Italy, included 20 LS-VSCC (mean [SD] age, 75 [3] years) and 20 cancer-associated vulvar LS (caVLS; mean [SD] age, 62 [11] years) formalin-fixed embedded tissue specimens, 20 cancer-free vulvar LS (cfVLS), and 20 normal skin fresh specimens from diagnostic biopsies and women surgically treated for nonmalignant skin lesions, respectively. RARß gene expression and promoter methylation were investigated in LS-VSCC and caVLS adjacent to VSCC specimens, and in cfVLS and normal skin specimens, as controls, by RT-Q real-time polymerase chain reaction (PCR) analysis, and sequencing of PCR-amplified bisulfite-treated DNA. C-Jun expression, an RARß pathway-related gene, was also investigated. MAIN OUTCOMES AND MEASURES RARß expression, correlation with its promoter methylation and c-Jun expression, and association with onset or progression of LS-VSCC. RESULTS In LS-VSCC, RARß messenger RNA was 3.4-, 3.6-, and 4.8-fold lower than in caVLS (P = .001), cfVLS (P = .005), and normal skin (P < .001), respectively. The RARß mRNA levels were similar in caVLS, cfVLS, and normal skin. The RARß promoter was hypermethylated in 18 (90%) of 20 LS-VSCC, 11 (55%) of 20 cfVLS, 10 (50%) of 20 caVLS, and 5 (25%) of 20 in the normal skin group. The degree of methylation of RARß promoter was higher in LS-VSCC, ranging from 5 to 9 (full promoter methylation) CpGs methylated, than in caVLS (P = .02), cfVLS (P = .03), or normal skin (P < .001), which was up to 5 CpGs methylated. Importantly, 0 of 8 LS-VSCC with 5 to 6 CpGs methylated and 5 (63%) of 8 LS-VSCC with 7 to 8 CpGs methylated were from patients with lymph node metastasis at diagnosis, respectively, whereas there were 2 of 2 (100%) LS-VSCC samples with 9 CpG methylated from patients with lymph node metastasis at diagnosis and subsequent recurrence. In LS-VSCC c-Jun mRNA was 4.3-, 1.4-, and 2.6-fold higher than in caVLS (P < .001), cfVLS (P = .001), and normal skin (P < .001), respectively. The expression of c-Jun was similar in caVLS, cfVLS, and normal skin. CONCLUSIONS AND RELEVANCE Hypermethylation-induced RARß down-expression was associated with LS-VSCC and correlates with the upregulation of c-Jun. The degree of methylation of RARß promoter increased with the malignancy of LS-VSCC. Therefore, RARß gene dysregulation may play a role in progression of LS-VSCC, and RARß promoter methylation status may be used as a prognostic marker in clinical treatment of patients with LS-VSCC.