Human Papillomavirus Integration Strictly Correlates with Global Genome Instability in Head and Neck Cancer

Abstract

Human papillomavirus (HPV)-positive head and neck cancers, predominantly oropharyngeal squamous cell carcinoma (OPSCC), exhibit epidemiologic, clinical, and molecular characteristics distinct from those OPSCCs lacking HPV. We applied a combination of whole-genome sequencing and optical genome mapping to interrogate the genome structure of HPV-positive OPSCCs. We found that the virus had integrated in the host genome in two thirds of the tumors examined but resided solely extrachromosomally in the other third. Integration of the virus occurred at essentially random sites within the genome. Focal amplification of the virus and the genomic sequences surrounding it often occurred subsequent to integration, with the number of tandem repeats in the chromosome accounting for the increased copy number of the genome sequences flanking the site of integration. In all cases, viral integration correlated with pervasive genome-wide somatic alterations at sites distinct from that of viral integration and comprised multiple insertions, deletions, translocations, inversions, and point mutations. Few or no somatic mutations were present in tumors with only episomal HPV. Our data could be interpreted by positing that episomal HPV is captured in the host genome following an episode of global genome instability during tumor development. Viral integration correlated with higher grade tumors, which may be explained by the associated extensive mutation of the genome and suggests that HPV integration status may inform prognosis.

Implications: Our results indicate that HPV integration in head and neck cancer correlates with extensive pangenomic structural variation, which may have prognostic implications.

Figure 1.

Copy number and polymorphisms of viral genomes in OPSCCs. Shown underneath a map of the HPV16 genome are copy number values as a function of genome position of virus from eleven of the twelve OPSCCs examined in this study. Copy number was determined from the total WGS read counts at each position of the virus, normalized to the average read count over unique human genome sequences in the same sample. Positions of single nucleotide polymorphisms relative to reference HPV16 (NC_001526.4) in each virus are designated, color coded to indicate the nucleotide substitution (T, red; C, blue; A, green; G, orange). Tumors with integrated virus are shown in teal and those with only extrachromosomal virus are shown in violet.

Figure 2.

Genome structure surrounding of the sites of viral integration. Shown are diagrams of the regions surrounding the sites of viral integration in eight OPSCCs. The upper four contained only a single integration site while the lower four contained two separate sites. Virus is shown as a bar or dot in yellow and regions of the genome that become duplicated following integration are shown in color. The upper (or leftward for 3922T) portion of each diagram indicates the location of integration, with nearby genes shown above, while the lower (or rightward) portion represents the local structure following integration and focal amplification. Gray segments indicate regions unmappable by OGM. Diagrams are not to scale.

Figure 3.

Global genome structural variation accompanies viral integration. Circos plot diagrams of somatic SVs in all the OPSCC genomes, relative to the patients’ normal genome, showing translocations and inversions in the center, copy number on the inner ring and insertions (green), deletions (orange) and duplications (light blue) on the third most outer ring. Chromosomes are ordered sequentially in the outer ring on which are indicated cytologic banding patterns and the centromere (red bar).

Figure 4.

Significantly mutated genes in integrated versus episomal tumors. Frequently mutated genes in OPSCCs with integrated HPV (left) and only episomal HPV (right) are shown on the heat map for each of the tumor samples. The mutation classes are indicated by color. Total number and type of exonic mutations in each sample are shown in the graph above the heat map. Mutation percentage of each gene in the cohort is shown immediately to the right of the heat map. Graph on the far right shows mutation percentage of the gene in COSMIC (upper aerodigestive tract, head and neck, squamous cell carcinoma).