Subtypes of HPV-Positive Head and Neck Cancers Are Associated with HPV Characteristics, Copy Number Alterations, PIK3CA Mutation, and Pathway Signatures

Abstract

Structured Abstract Purpose There is substantial heterogeneity within human papillomavirus (HPV) associated head and neck cancer (HNC) tumors that predispose them to different outcomes, however the molecular heterogeneity in this subgroup is poorly characterized due to various historical reasons. Experimental Design We performed unsupervised gene expression clustering on deeply-annotated (transcriptome and genome) HPV(+) HNC samples from two cohorts (84 total primary tumors), including 18 HPV(−) HNC samples, to discover subtypes and characterize the differences between subgroups in terms of their HPV characteristics, pathway activity, whole-genome somatic copy number alterations and mutation frequencies. Results We identified two distinct HPV(+) subtypes (namely HPV-KRT and HPV-IMU). HPV-KRT is characterized by elevated expression of genes in keratinocyte differentiation and oxidation-reduction process, whereas HPV-IMU has strong immune response and mesenchymal differentiation. The differences in expression are likely connected to the differences in HPV characteristics and genomic changes. HPV-KRT has more genic viral integration, lower E2/E4/E5 expression levels and higher ratio of spliced to full length HPV oncogene E6 than HPV-IMU; the subgroups also show differences in copy number alterations and mutations, in particular the loss of chr16q in HPV-IMU and gain of chr3q and PIK3CA mutation in HPV-KRT. Conclusion Our characterization of two subtypes of HPV(+) HNC tumors provides valuable molecular level information that point to two main carcinogenic paths. Together, these results shed light on stratifications of the HPV(+) HNCs and will help to guide personalized care for HPV(+) HNC patients.

Integrated Analysis of Whole-Genome ChIP-Seq and RNA-Seq Data of Primary Head and Neck Tumor Samples Associates HPV Integration Sites with Open Chromatin Marks

“…Consistent with our findings, recent publications have established two subtypes of HPV+ HNSCC from gene expression profiles (29, 30), called HPV-KRT and HPV-IMU in (30) or mesenchymal and classical in (29). The HPV-KRT was associated with keratinocyte differentiation and episomal HPV, while HPV-IMU was associated with strong immune response, mesenchymal differentiation and HPV viral integration within the DNA (30). Moreover, that study also found that the episomal HPV infection correlated with the HPV-positive mesenchymal subtype from (29).…”

“…In our cohort, HPV integration was detected only in one tumor specimen of ten in Cluster 1, whereas samples within the Cluster 2 were enriched for positive HPV integration (13 out of 29, Fisher Exact Test p-value of 0.064). Gene set enrichment analysis showed that genes with tumor-specific H3K27ac-associated enrichment were significantly associated with HPV-KRT (30) (Table S11) and with the classical subtypes (29) (Fig. S15A–D), confirming the relationship of H3K27ac enrichment to HPV integrated HNSCC subtypes.…”

“…If this hypothesis is true, the human-viral fusion sites, that we called integration sites in this study, could instead represent chimeric human-viral episomes described in other studies (32, 47). The fact that the episomal and integrated HPV can co-exist in the same tissue or even the same cell (30, 32, 47, 48), and both can be transcribed, adds another twist to the complex picture of HPV infection in human cancers. Southern blots or FISH-based technologies followed by high-resolution microscopy might help to resolve this scientific dispute (32, 49), and should be evaluated in conjunction with H3K27ac binding sites in future studies.…”

“…Although the role of HPV-integration is controversial, recent studies have demonstrated that HPV+ HNSCC samples with episomal and integrated HPV have unique signatures of gene expression, and can be separated based on them (22, 30, 32, 47–49). Clustering of gene expression data described two HPV+ HNSCC subtypes: mesenchymal and classical (29).…”

“…The R/Bioconductor package GeneOverlap was used to associate the disease-specific gene set for H3K27ac (29, 30). A one-sided Wilcoxon gene set test was further applied to assess the enrichment of the disease-specific H3K27ac gene set with the continuous weights of the gene classifier for HPV+ HNSCC subtypes from (29).…”