Nikki B Thuijs

High‐grade vulvar intraepithelial neoplasia: comprehensive characterization and long‐term vulvar carcinoma risk

AimsAdequate diagnosis of human papillomavirus (HPV)‐associated high‐grade squamous intraepithelial lesion (HSIL) and HPV‐independent vulvar intraepithelial neoplasia (VIN) is essential but can be challenging. We comprehensively characterized a large population‐based series of vulvar lesions, originally reported as high‐grade VIN, and assessed the cancer risk.Methods and resultsBaseline high‐grade VIN of 751 patients were categorized by histopathological reassessment, integrating the results of immunohistochemistry (p16INK4a, p53, Ki‐67) and HPV DNA testing. Integrated analyses resulted in 88.4% HPV‐associated lesions (77.0% HSIL, 10.9% low‐grade SIL [LSIL], and 0.4% vulvar squamous cell carcinoma [VSCC]), 10.9% HPV‐independent lesions (6.1% HPV‐independent VIN, 4.7% nondysplastic lesions, and 0.1% VSCC) and 1.1% inconclusive lesions. HSIL demonstrated p16INK4a block‐positivity in 99.0%, increased Ki‐67 in ≥2/3rd of the epithelium in 93.6%, and HPV positivity in 99.6%. In HSIL, a p53 wildtype mid‐epithelial staining pattern was common (51.6%) while this was not observed in HPV‐independent lesions. HPV‐independent VIN harboured mutant p53 patterns in 65.2% and showed a wide morphological spectrum, ranging from differentiated to nondifferentiated (‘HPV‐associated‐like’, in 41.3%). Kaplan–Meier analyses showed a 10‐year cancer risk of 8.0% in HPV‐associated HSIL, 67.4% in HPV‐independent VIN/p53mutant, and 27.8% in HPV‐independent VIN/p53wildtype. Strikingly, the 10‐year cancer risk was 73.3% in HPV‐independent VIN with nondifferentiated (‘HPV‐associated‐like’) morphology.ConclusionImmunohistochemistry by p16INK4a and p53 is highly recommended for optimal categorization into HPV‐associated and HPV‐independent VIN, which is of utmost importance given the different cancer risk. The high cancer risk of HPV‐independent VIN underscores the need for surgical treatment and close follow‐up, especially in case of a p53 mutant pattern and/or nondifferentiated morphology.

Clinical validation of methylation biomarkers for optimal detection of high‐grade vulvar intraepithelial neoplasia

Abstract The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)‐associated and HPV‐independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high‐grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high‐grade VIN, were reassessed and categorized into HPV‐associated or HPV‐independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation‐specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high‐grade VIN was determined by logistic regression analysis. SST was the best‐performing individual marker (AUC 0.90), detecting 80% of high‐grade VIN cases, with excellent detection of HPV‐independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST . Selection of a marker panel, including ZNF582 , SST and miR124‐2 , resulted in a comparably high accuracy for detection of high‐grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high‐grade VIN. SST , as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high‐grade VIN in need of treatment, particularly HPV‐independent VIN, from low‐grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.

Vulvar intraepithelial neoplasia: Incidence and long‐term risk of vulvar squamous cell carcinoma

AbstractThe risk of vulvar squamous cell carcinoma (VSCC) in patients with high‐grade vulvar intraepithelial neoplasia (VIN) is considered lower in high‐grade squamous intraepithelial lesion (HSIL) compared to differentiated VIN (dVIN), but studies are limited. Our study investigated both the incidence of high‐grade VIN and the cumulative incidence of VSCC in patients with HSIL and dVIN separately. A database of women diagnosed with high‐grade VIN between 1991 and 2011 was constructed with data from the Dutch Pathology Registry (PALGA). The European standardized incidence rate (ESR) and VSCC risk were calculated, stratified for HSIL and dVIN. The effects of type of VIN (HSIL vs dVIN), age and lichen sclerosis (LS) were estimated by Cox regression. In total, 1148 patients were diagnosed with high‐grade VIN between 1991 and 2011. Between 1991‐1995 and 2006‐2011, the ESR of HSIL increased from 2.39 (per 100 000 woman‐years) to 3.26 and the ESR of dVIN increased from 0.02 to 0.08. The 10‐year cumulative VSCC risk was 10.3%; 9.7% for HSIL and 50.0% for dVIN (log rank P < .001). Type of VIN, age and presence of LS were independent risk factors for progression to VSCC, with hazard ratios of 3.0 (95% confidence interval [CI] 1.3‐7.1), 2.3 (95% CI 1.5‐3.4) and 3.1 (95% CI 1.8‐5.3), respectively. The incidence of high‐grade VIN is rising. Because of the high cancer risk in patients with dVIN, better identification and timely recognition are urgently needed.

DNA methylation and copy number alterations in the progression of HPV‐associated high‐grade vulvar intraepithelial lesion

AbstractHuman papillomavirus (HPV)‐associated high‐grade vulvar intraepithelial lesion (HSIL) is a precursor of vulvar squamous cell carcinoma (VSCC). Because of the 8% cancer risk, many vulvar HSIL patients undergo aggressive and mutilating treatments. Characterizing HSIL by their progression risk can help individualize treatment strategies. Accordingly, copy number alterations (CNAs) and DNA methylation have been identified as biomarkers for cancer risk stratification of HSIL. Here, we assessed their potential correlation, and relation to HPV16 (sub)lineages and progression to vulvar cancer. Eighty‐two vulvar formalin‐fixed paraffin‐embedded (FFPE) samples, including controls, HSIL, HSIL adjacent to VSCC and VSCC, with previously determined DNA methylation profiles, were analysed for CNAs using mFAST‐SeqS. Genome‐wide z‐scores were calculated to determine overall aneuploidy (aneuploidy scores), and compared to the methylation levels and status of marker panel ZNF582/SST/miR124‐2. For 52 HPV16‐positive cases, HPV (sub)lineages were determined by Sanger sequencing. HPV16 lineage A was predominant (86.4%), followed equally by lineages B, C, and D. Frequent chromosomal alterations included chr1pq, chr3q, chr9q gains, and chr2q, chr4q losses. Median aneuploidy scores increased across disease categories, from 0 in controls, to 3 in HSIL, 16 in HSIL adjacent to VSCC and 29 in VSCC. A positive relationship between aneuploidy scores and DNA methylation levels was found (ρ = 0.61, Spearman's rank correlation test). Aneuploidy scores were significantly higher in methylation‐positive samples (p < .001). In conclusion, we showed that DNA methylation and CNAs both rise with increasing severity of disease, indicating their prognostic value for cancer risk stratification of HSIL, while no relation to HPV16 (sub)lineages was found.