John Charles Rotondo1, Alessandro Borghi2, Rita Selvatici3, Eros Magri4, Enzo Bianchini4, Elena Montinari4, Monica Corazza2, Annarosa Virgili2, Mauro Tognon1, Fernanda Martini1. 1. Section of Pathology, Oncology, and Experimental Biology, Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara, Ferrara, Italy. 2. Section of Dermatology and Infectious Diseases, Department of Medical Sciences, University of Ferrara, Ferrara, Italy. 3. Department of Medical Sciences, University of Ferrara, Ferrara, Italy. 4. Section of Anatomic Pathology, Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara and S. Anna University Hospital, Ferrara, Italy.
Abstract
IMPORTANCE: The molecular mechanism leading to the development of vulvar squamous cell carcinoma (VSCC) from vulvar lichen sclerosus (VLS) is unknown. OBJECTIVE: To assess the possible involvement of the IRF6 tumor-suppressor gene in the development of VSCC from VLS. DESIGN: In laboratories at the University of Ferrara, Ferrara, Italy, IRF6 gene expression and promoter methylation were investigated in paraffin-embedded VSCC and adjacent vulvar intraepithelial neoplasia (VIN) and VLS specimens, in cancer-free VLS (cfVLS) specimens, and in healthy skin specimens by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis and by sequencing of PCR-amplified bisulfite-treated DNA. Methylation-induced dysregulation was assessed by expression of p63, the IRF6-transactivator gene. MAIN OUTCOMES AND MEASURES: IRF6 expression, correlation with promoter methylation and p63 expression, and association with development of VSCC from VLS. RESULTS: Specimens from 60 participating women (1 specimen from each) were analyzed for the study (mean [SD] age, 66.3 [12.1] years): 20 paraffin-embedded specimens of VSCC (patient age, 75.3 [8.3] years) with adjacent VLS lesions, in 5 of which VIN preneoplastic tissue was also present (patient age, 64.3 [15.3] years); 20 cfVLS specimens (patient age, 62.1 [11.4] years) obtained from diagnostic biopsies; and 20 normal skin specimens from noncancer surgical patients (patient age, 61.4 [9.1] years). IRF6 messenger RNA was found to be reduced 4.5-, 2.9-, 6.6-, and 2.2-fold in VLS, VIN, VSCC, and cfVLS specimens, respectively, compared with controls, although p63 was still expressed in all specimens. IRF6 promoter was hypermethylated in 9 (45%) of 20 VLS specimens, 1 (20%) of 5 VIN specimens, 16 (80%) of 20 VSCC specimens, 2 (10%) of 20 cfVLS specimens, and 0 normal skin specimens. CONCLUSIONS AND RELEVANCE: IRF6 dysregulation may be involved in the development of VSCC from VLS. Methylation of the IRF6 promoter may be a marker of cancer risk in patients with VLS.
IMPORTANCE: The molecular mechanism leading to the development of vulvar squamous cell carcinoma (VSCC) from vulvar lichen sclerosus (VLS) is unknown. OBJECTIVE: To assess the possible involvement of the IRF6tumor-suppressor gene in the development of VSCC from VLS. DESIGN: In laboratories at the University of Ferrara, Ferrara, Italy, IRF6 gene expression and promoter methylation were investigated in paraffin-embedded VSCC and adjacent vulvar intraepithelial neoplasia (VIN) and VLS specimens, in cancer-free VLS (cfVLS) specimens, and in healthy skin specimens by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis and by sequencing of PCR-amplified bisulfite-treated DNA. Methylation-induced dysregulation was assessed by expression of p63, the IRF6-transactivator gene. MAIN OUTCOMES AND MEASURES: IRF6 expression, correlation with promoter methylation and p63 expression, and association with development of VSCC from VLS. RESULTS: Specimens from 60 participating women (1 specimen from each) were analyzed for the study (mean [SD] age, 66.3 [12.1] years): 20 paraffin-embedded specimens of VSCC (patient age, 75.3 [8.3] years) with adjacent VLS lesions, in 5 of which VIN preneoplastic tissue was also present (patient age, 64.3 [15.3] years); 20 cfVLS specimens (patient age, 62.1 [11.4] years) obtained from diagnostic biopsies; and 20 normal skin specimens from noncancer surgical patients (patient age, 61.4 [9.1] years). IRF6 messenger RNA was found to be reduced 4.5-, 2.9-, 6.6-, and 2.2-fold in VLS, VIN, VSCC, and cfVLS specimens, respectively, compared with controls, although p63 was still expressed in all specimens. IRF6 promoter was hypermethylated in 9 (45%) of 20 VLS specimens, 1 (20%) of 5 VIN specimens, 16 (80%) of 20 VSCC specimens, 2 (10%) of 20 cfVLS specimens, and 0 normal skin specimens. CONCLUSIONS AND RELEVANCE: IRF6 dysregulation may be involved in the development of VSCC from VLS. Methylation of the IRF6 promoter may be a marker of cancer risk in patients with VLS.
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