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

## TL;DR

This study shows that DNA methylation and copy number changes increase with the severity of HPV-related vulvar lesions, suggesting they could help predict cancer risk in patients.

## Contribution

The study demonstrates a novel positive correlation between DNA methylation and copy number alterations in HSIL progression, independent of HPV16 lineages.

## Key findings

- DNA methylation and copy number alterations increase with disease severity from HSIL to vulvar cancer.
- Aneuploidy scores and methylation levels are significantly higher in methylation-positive samples.
- No association was found between HPV16 (sub)lineages and progression to vulvar cancer.

## Abstract

Human 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.

What's new?

Patients with human papillomavirus (HPV)‐associated high‐grade vulvar intraepithelial lesion (HSIL) often undergo damaging treatments to prevent progression to cancer. Molecular characterization of HSIL to assess progression risk could reduce the need for aggressive interventions. Here, cancer risk stratification of HSIL was investigated by copy number alterations (CNAs) and DNA methylation analysis in vulvar specimens. Analyses reveal a positive relationship between CNAs and DNA methylation, wherein both increase with progression to vulvar cancer. HPV16 lineages, which influence viral oncogenicity, had no relationship with progression to cancer. The novel insights underscore the prognostic potential of CNAs and DNA methylation for cancer risk stratification in HSIL patients.