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 (LS). 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, there is an urgent clinical need for objective biomarkers for cancer risk stratification of high-grade VIN. In this thesis (Chapter 2), the incidence of high-grade VIN was calculated from a longitudinal, population-based, historical cohort series including 1,148 patients with an original diagnosis of high-grade VIN between 1991 and 2011. Vulvar cancer risk and associated risk factors were studied in the patients with high-grade VIN without previous or concurrent vulvar cancer (n=894). During the study period, the 10-year vulvar cancer risk was 10% for HSIL and 50% for HPV-independent VIN. In a systematic literature search on HPV-independent VIN (Chapter 3), the high cancer risk of this patient group was confirmed, and it was also shown that the time to progression to cancer was short. In Chapter 4, twelve candidate DNA methylation markers (ASCL1/CADM1/ FAM19A4/GHSR/LHX8/MAL/miR124-2/PHACTR3/PRDM14/SST/ZIC1/ZNF582) were tested for high-grade VIN and vulvar cancer detection with qMSP in a cross-sectional series, including 192 vulvar samples: 58 vulvar cancers, 30 VIN adjacent to vulvar cancer, 41 VIN without associated vulvar cancer (37 HSIL and 4 HPV-independent VIN) and 63 healthy vulvar tissues. Methylation markers showed significantly higher methylation levels with increasing severity of disease, both in HPV-associated and HPV-independent lesions. In Chapter 6, 751 of the 894 original high-grade VIN without associated vulvar cancer from the historical cohort (Chapter 2), were retrieved and categorized by histopathological reassessment, integrating results of immunohistochemistry of p16INK4a, p53, and Ki-67, and HPV DNA testing. Integrated analyses resulted in 88% HPV-associated lesions, 11% HPV-independent lesions, and 1% inconclusive lesions. Immunohistochemical markers and HPV genotyping were useful for a correct diagnosis, but also showed pitfalls one needs to be aware of. In the revised groups, the 10-year cancer risks were 8%, 67% and 28% in patients with HSIL, p53 mutant HPV-independent VIN, and p53 wild-type HPV-independent VIN, respectively. Moreover, an exploratory study on 12 patients with multifocal vulvar HSIL (Chapter 5) demonstrated that biomarker results (methylation markers, HPV genotype, and immunohistochemical markers) varied between patients, but were comparable within most patients. The 12 DNA methylation markers were validated in a cross-sectional analysis on the 751 vulvar tissue samples, together with 113 healthy vulvar controls (Chapter 7). SST was the best-performing individual marker, detecting only 2% of controls and 80% of high-grade VIN, including 95% of HPV-independent VIN. Selection of a marker panel, including ZNF582, SST and miR124-2, resulted in a comparably high accuracy. In Chapter 8, it was shown that HSIL with a positive methylation status harboured a 4.87 times higher vulvar cancer risk after five years compared to HSIL with a negative methylation status. In HPV-independent VIN, p53 status was the sole prognostic risk factor (HR 7.67) for progression to cancer. In a series of 150 vulvar lesions (Chapter 9), validating the performance of immunohistochemical markers CK17 and SOX2, significantly more CK17- and SOX2-positive cases were observed in HPV-independent VIN compared to non-dysplastic cases. Highest diagnostic accuracy (89%) for HPVi VIN was obtained when combining p53 and CK17 IHC markers.