BACKGROUND: Vulva squamous cell carcinoma (VSCC) develops through two separate molecular pathways-one involving high-risk human papilloma virus infection (HPV-associated), and the other without HPV infection (HPV-independent) often involving TP53 mutation. HPV-associated VSCC generally has a better progression-free survival than HPV-independent VSCC. The aim of this study was to determine TP53 mutation status using immunohistochemistry, compare different methods of HPV detection and correlate both with survival in a retrospective cohort of 123 patients with VSCC.
METHODS: Immunohistochemistry for p53, Ki67 and p16INK4A (a surrogate marker for HPV infection) was performed on formalin-fixed paraffin-embedded tissues from a cohort of surgically treated VSCC patients to identify molecular subtypes of VSCC. Presence of HPV infection was detected by HPV DNA PCR and HPV mRNA in situ hybridization (ISH). The Pearson chi-square test and multivariable Cox regression model were used to investigate the association of different parameters with progression-free survival and disease-specific survival (DSS), and Kaplan-Meier curves were used to show the association of different parameters with survival.
RESULTS: The results of p53 and p16INK4A immunohistochemistry confirmed three VSCC subtypes associated with different prognosis. The TP53 mutation status was identified as an independent prognostic factor of worse progression-free survival (p = 0.024) after adjustment for FIGO stage. p16INK4A immunohistochemistry, mRNA ISH, and DNA PCR had excellent concordance in terms of HPV detection. According to the multivariable Cox regression model, the presence of hrHPV mRNA correlated significantly with increased progression-free survival (p = 0.040) and DSS (p = 0.045), after adjustment for other confounders.
CONCLUSIONS: p53 and p16INK4A immunohistochemistry stratify VSCC cohort into three subtypes with TP53mutated patients having the worst prognosis. The detection of hrHPV mRNA by ISH was an independent predictor of increased survival. Thus, the combined detection of p53 and HPV mRNA might improve risk stratification in VSCC.

Vulvar squamous cell carcinoma arises from precursor lesions: vulvar intraepithelial neoplasias (VIN). Most of them are caused by the human papillomavirus (HPV), and the precursor lesion for this group is VIN usual type/high grade squamous intraepithelial lesion (uVIN/HSIL) of variable clinical presentation and having a light invasive potential. Some VIN are HPV-independent and arise in older women against the background of chronic dermatoses, mostly lichen sclerosus. Their histological diagnosis is more subtle. They have a higher invasive potential. A third precursor, leading to well-differentiated, or even verrucous, carcinomas, is still ill-defined to this day. We detail these lesions\' clinics, histology, and biomarkers (immunohistochemical and molecular).

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing.