Association of Retinoic Acid Receptor β Gene With Onset and Progression of Lichen Sclerosus–Associated Vulvar Squamous Cell Carcinoma

Abstract

IMPORTANCE Molecular alterations in lichen sclerosus-associated vulvar squamous cell carcinoma (LS-VSCC) are largely unknown.OBJECTIVE To determine whether the retinoic acid receptor β (RARβ) tumor-suppressor gene is involved in the onset and/or progression of LS-VSCC. DESIGN, SETTING, AND PARTICIPANTSThe 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. RARβ 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 RARβ pathway-related gene, was also investigated. MAIN OUTCOMES AND MEASURESRARβ expression, correlation with its promoter methylation and c-Jun expression, and association with onset or progression of LS-VSCC. RESULTSIn LS-VSCC, RARβ 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 RARβ mRNA levels were similar in caVLS, cfVLS, and normal skin. The RARβ 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 RARβ 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 RARβ down-expression was associated with LS-VSCC and correlates with the upregulation of c-Jun. The degree of methylation of RARβ promoter increased with the malignancy of LS-VSCC. Therefore, RARβ gene dysregulation may play a role in progression of LS-VSCC, and RARβ promoter methylation status may be used as a prognostic marker in clinical treatment of patients with LS-VSCC.

Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients

“…Specific primers sets used in real‐time PCRs were used to analyze the expression of 84 genes involved in different pathways, such as osteogenic differentiation, cartilage condensation, ossification, bone metabolism, bone mineralization, binding to Ca 2+ and homeostasis, extracellular matrix (ECM) protease inhibitors, adhesion molecules, cell‐to‐cell adhesion, adhesion molecules of the ECM, and growth factors. For data analysis, the fold change (FC) of each gene expression was calculated by using the 2 −ΔΔCt method, whereas housekeeping genes, used as controls, were used to normalize results and Log 2 FC; <1 or >1 was considered significant …”

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women

“…The PCR conditions were: 10 min of denaturation at 95 • C, 40 cycles of 1 min at 95 • C, 1 min at 65 • C, and 2 min at 72 • C, final extension for 5 min at 72 • C. PCR products were directly sequenced using the automated ABI Prism 3730 × l-Genetic Analyzer (Applied Biosystems, Monza, Italy) (Rotondo et al, 2017). SERPINA1 sequences showing more than or equal to 5 out of 8 methylated CpGs (≥50%) were considered hypermethylated (Rotondo et al, 2018).…”

Role of human papillomavirus infection in the etiology of vulvar cancer in Italian women

“…vulvar high-grade squamous intraepithelial lesion (VHSIL) [9,10]. HPV-independent VSCC occurs mainly in older women [1,11] and arises from a chronic inflammatory dermatosis, referred to as lichen sclerosus [12,13], that can evolve into differentiated vulvar intraepithelial neoplasia (VIN), a precursor lesion of VSCC [14].…”