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Brief Report |

Hypermethylation-Induced Inactivation of the IRF6 Gene as a Possible Early Event in Progression of Vulvar Squamous Cell Carcinoma Associated With Lichen Sclerosus

John Charles Rotondo, PhD1; Alessandro Borghi, MD2; Rita Selvatici, PhD3; Eros Magri, DBS4; Enzo Bianchini, MD4; Elena Montinari, MD4; Monica Corazza, MD2; Annarosa Virgili, MD2; Mauro Tognon, PhD1; Fernanda Martini, PhD1
[+] Author Affiliations
1Section of Pathology, Oncology, and Experimental Biology, Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara, Ferrara, Italy
2Section of Dermatology and Infectious Diseases, Department of Medical Sciences, University of Ferrara, Ferrara, Italy
3Department of Medical Sciences, University of Ferrara, Ferrara, Italy
4Section of Anatomic Pathology, Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara and S. Anna University Hospital, Ferrara, Italy
JAMA Dermatol. 2016;152(8):928-933. doi:10.1001/jamadermatol.2016.1336.
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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.

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Figure 1.
IRF6 Expression Measured by Reverse Transcriptase–Quantitative Polymerase Chain Reaction

Relative expression was calculated with the ΔΔCt method. Data are expressed as the ratio of the number of copies of the target gene relative to GAPDH. The results are expressed as mean (SEM) relative fold change (2-ΔΔCt) over the value of normal skin specimens. cfVLS indicates cancer-free vulvar lichen sclerosus; VIN, vulvar intraepithelial neoplasia; VLS, vulvar lichen sclerosus; VSCC, vulvar squamous cell carcinoma.

aP < .005 for comparison of VLS vs cfVLS.

bP < .005 for comparisons of VSCC vs VIN and VSCC vs cfVLS.

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Figure 2.
IRF6 Promoter Methylation Analysis

A, Bisulfite–polymerase chain reaction (PCR) sequencing of the IRF6 promoter region in representative specimens of normal skin, cancer-free vulvar lichen sclerosus (cfVLS), vulvar lichen sclerosus (VLS), vulvar intraepithelial neoplasia (VIN), and vulvar squamous cell carcinoma (VSCC). On the left, the filled-in and clear circles represent methylated and unmethylated CpG islands, respectively. The CpG islands within the IRF6 locus are numbered across the top of the grid. Each row represents 1 PCR product. On the right is 1 representative sequence showing 4 CpGs (11-14) within the IRF6 PCR product. B, Frequencies of IRF6 hypermethylation in normal skin, cfVLS, VLS, VIN, and VSCC samples. Only clones showing 50% or more methylated CpGs were considered hypermethylated in this analysis.

aFor comparisons of VLS vs normal skin (P = .002) and VLS vs cfVLS (P = .03).

bFor comparisons of VSCC vs normal skin (P < .001), VSCC vs cfVLS (P < .001), VSCC vs VLS (P = .049), and VSCC vs VIN (P = .02).

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Figure 3.
Comparison of Altered IRF6 and p63 Expression Measured by Reverse Transcriptase–Quantitative Polymerase Chain Reaction

Relative expression was calculated by use of the ΔΔCt method. Data are expressed as the ratio of copies of target gene relative to GAPDH. The results are expressed as mean (SEM) relative fold change (2-ΔΔCt) over the value of normal skin specimens.

aP < .001 for comparison of VLS vs cfVLS.

bP < .001 for comparison of VIN vs cfVLS.

cP = .15 for comparison of VIN vs VLS.

dP < .005 for comparisons of VSCC vs VIN and VSCC vs cfVLS.

eP < .001 for comparisons of VSCC vs VIN and VSCC vs VLS.

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