Cancer risk assessment in vulvar intra-epithelial neoplasia

High-grade vulvar intraepithelial neoplasia (VIN) is the precursor of vulvar cancer and is divided into human papillomavirus (HPV)-associated high-grade squamous intraepithelial lesion (HSIL) and HPV-independent VIN. HPV-independent VIN is often referred to as differentiated VIN (dVIN) and arises independent from HPV infection, mostly on a background of lichen sclerosus. High-grade VIN is a heterogeneous disease with a varying risk of progression to cancer, shows frequent recurrences upon treatment, and is accompanied by decreased quality of life. To date, there are no prognostic tests stratifying patients into low or high vulvar cancer risk. Instead, all patients are treated similarly, with overtreatment as a result, leading to morbidity. Hence, this thesis aimed to investigate the potential of objective biomarkers for cancer risk stratification of VIN patients.
Most of the data in this thesis is based on a longitudinal, population-based, historical cohort including 894 patients, which was created with a PALGA search on high-grade VIN diagnosed between 1991 and 2011. These patients did not have previous or concurrent vulvar cancer. In this cohort series, the 10-year vulvar cancer risk was 10% for HSIL and 50% for HPV-independent VIN.
After retrieving 84% of the tissue blocks of the historical cohort (751/894), the cohort was categorized by histopathological reassessment, integrating results of immunohistochemistry of p16INK4a, p53, and Ki-67, and HPV DNA testing. Reassessment showed that 83% of the HPV-independent VIN was incorrectly classified, as immunohistochemical markers had not been widely performed during the study period. This showed the importance of standard immunohistochemical stains p16 and p53 in making an initial diagnosis of high-grade VIN. In this revised series we also showed that cancer risk within the HPV-independent group strongly depended on p53 status, with 10-year cancer risks of 8%, 67% and 28% in patients with HSIL, p53 mutant HPV-independent VIN, and p53 wild-type HPV-independent VIN, respectively. As a result, we proposed a three-tiered classification of high-grade VIN , analogous to the vulvar carcinomas.
We further tested 12 methylation markers (ASCL1/CADM1/ FAM19A4/GHSR/LHX8/MAL/miR124-2/PHACTR3/PRDM14/SST/ZIC1/ZNF582) on a pilot series of 192 vulvar lesions, showing that methylation increased with lesion severity, both in HPV-associated and HPV-independent lesions. The methylation markers were subsequently validated on the revised historical cohort including 113 controls, maintaining the promising results of the pilot series. A three-gene marker panel (ZNF582/SST/miR124-2) was determined, which showed a detection of 50% (1/2) of VSCC, 87% (34/39) of dVIN, 72% (392/541) of vHSIL, 36% (26/74) of vLSIL, 21% (6/28) of non-dysplastic and 5% (6/113) of controls as methylation positive. Finally, we demonstrated the prognostic value of methylation in vulvar HSIL, as methylation-positive HSIL harboured a 4.9 times higher vulvar cancer risk after five years compared to methylation-negative HSIL. In HPV-independent VIN, p53 status was the sole prognostic risk factor (HR 7.67) for progression to cancer.
The prognostic value of methylation and p53 immunohistochemistry will be further validated in a prospective series that we have set up at the beginning of this PhD trajectory. This prospective series is called the VENUS (Vulvar intraEpithelial Neoplasia in sitU Study), which is a national, multicenter, prospective study, including patients with HSIL and HPV-independent VIN.
To conclude, thesis underscores the importance of biomarker-based testing of high-grade VIN, which is of utmost importance for effective risk stratification of HSIL and HPV-independent VIN. The ultimate goal is more patient-individualized therapy aiming for decreased morbidity and increased quality of life of patients with VIN.