Molecular heterogeneity in human papillomavirus-dependent and -independent vulvar carcinogenesis

Abstract

Vulvar squamous cell carcinoma (VSCC) and precancerous vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)-dependent and -independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinicopathological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV-status and CNA by means of whole-genome next-generation shallow-sequencing were assessed in VSCC and VIN. The latter included VIN of women with associated VSCC (VIN^VSCC ) and women who did not develop VSCC during follow-up (VIN^noVSCC ). HPV-testing resulted in 41 HPV-positive (16 VIN^VSCC , 14 VIN^noVSCC , and 11 VSCC) and 24 HPV-negative (11 VIN^VSCC and 13 VSCC) lesions. HPV-positive and -negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV-negative lesions. HPV-negative VIN^VSCC had less CNA than HPV-negative VSCC (P = .009), but shared chromosome 8 alterations. HPV-positive VIN^noVSCC had less CNA than VIN^VSCC (P = .022). Interestingly, 1pq gain was detected in 81% of HPV-positive VIN^VSCC and only in 21% of VIN^noVSCC (P = .001). In conclusion, HPV-dependent and -independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Cancer risk in VIN seems to be reflected by the extent of CNA, in particular chromosome 1 gain in HPV-positive cases.

Keywords: copy number alterations; human papillomavirus; progression risk; vulvar intraepithelial neoplasia; vulvar squamous cell carcinoma.

Figure 1

Copy number profiles and frequency plots of copy number alterations in vulvar squamous cell carcinomas (VSCC) and vulvar intraepithelial neoplasias (VIN). Representative copy number profiles are shown for (A) HPV‐negative VSCC, (B) HPV‐positive VSCC, (C) HPV‐negative VIN^VSCC, (D) HPV‐positive VIN^VSCC, and (E) HPV‐positive VIN ^{no VSCC}. The arrow in (C) indicates 11q13/cyclin D1 amplification in this sample. Frequency plots are shown for (F) HPV‐negative VSCC, (G) HPV‐positive VSCC, (H) HPV‐negative VIN^VSCC, (I) HPV‐positive VIN^VSCC, and (J) HPV‐positive VIN ^{no VSCC}. Gains are indicated in red (upper panel) and losses are indicated in blue (lower panel). Figures were generated using QDNAseq and CGHcall (see methods)

Figure 2

Clustering of HPV‐positive vulvar intraepithelial neoplasias (VIN). A, Weighted Clustering of Called Array CGH data (WECCA) output including a dendrogram, showing gains (green), and losses (red). Alternating chromosomes are indicated on the left. The panel below indicates the corresponding sample types. Three main clusters can be discerned. B, Importance score plot comparing vulvar intraepithelial neoplasia (VIN) lesions in the 3 clusters. For each chromosomal region the maximum pair‐wise symmetrized Kullback‐Leibler divergence was determined, revealing gain of chromosome 1pq as the most striking difference of the VIN lesions in the 3 clusters

Figure 3

Schematic representation of HPV‐induced and HPV‐independent routes of vulvar carcinogenesis and the most frequently affected chromosomal regions. The involvement of specific copy number aberrations in the progression of vulvar intraepithelial neoplasia (VIN) towards vulvar squamous cell carcinoma (VSCC) is indicated for HPV‐negative (HPV^NEG; upper panel) and HPV‐positive (HPV^POS; lower panel) lesions. Chr., chromosome; dVIN, differentiated VIN; uVIN, usual VIN