Human papilloma virus integration sites and genomic signatures in head and neck squamous cell carcinoma

Abstract

A prevalence of around 26% of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has been previously reported. HPV induced oncogenesis mainly involving E6 and E7 viral oncoproteins. In some cases, HPV viral DNA has been detected to integrate with the host genome and possibly contributes to carcinogenesis by affecting the gene expression. We retrospectively assessed HPV integration sites and signatures in 80 HPV positive patients with HNSCC, by using a double capture-HPV method followed by next-generation Sequencing. We detected HPV16 in 90% of the analyzed cohort and confirmed five previously described mechanistic signatures of HPV integration [episomal (EPI), integrated in a truncated form revealing two HPV-chromosomal junctions colinear (2J-COL) or nonlinear (2J-NL), multiple hybrid junctions clustering in a single chromosomal region (MJ-CL) or scattered over different chromosomal regions (MJ-SC) of the human genome]. Our results suggested that HPV remained episomal in 38.8% of the cases or was integrated/mixed in the remaining 61.2% of patients with HNSCC. We showed a lack of association of HPV genomic signatures to tumour and patient characteristics, as well as patient survival. Similar to other HPV associated cancers, low HPV copy number was associated with worse prognosis. We identified 267 HPV-human junctions scattered on most chromosomes. Remarkably, we observed four recurrent integration regions: PDL1/PDL2/PLGRKT (8.2%), MYC/PVT1 (6.1%), MACROD2 (4.1%) and KLF5/KLF12 regions (4.1%). We detected the overexpression of PDL1 and MYC upon integration by gene expression analysis. In conclusion, we identified recurrent targeting of several cancer genes such as PDL1 and MYC upon HPV integration, suggesting a role of altered gene expression by HPV integration during HNSCC carcinogenesis.

Keywords: MYC; PDL1; HPV copy number; HPV integration; carcinogenesis; head and neck squamous cell carcinoma.

Fig. 1

Distribution of HPV genomic signatures in 80 patients with HPV positive head and neck squamous cell carcinoma. 2J‐COL: two hybrid colinear junctions; 2J‐NL: two hybrid nonlinear junctions; 2J‐UN: two hybrid junctions with a lost junction; EPI: episomal; MJ‐CL: multiple hybrid junctions clustered in one locus; MJ‐SC: multiple hybrid junctions scattered at distinct loci.

Fig. 2

Chromosomal distribution of the 267 HPV‐human chromosome junction sequences found in 49 patients with HPV positive head and neck carcinoma. The orange dots represents 4 to 8 different breakpoints in the region, the light green dots 2 or 3 breakpoints, the dark green dots depict unique breakpoint in the region. The dark red star represents the 27 breakpoints in PDL1 region, the light red star the 20 breakpoints in MYC region, the orange star represents the 5 breakpoints in MACROD2 region and the light green star the 3 breakpoints in KLF5 region.

Fig. 3

HPV integration breakpoints in the chromosomal region 9p24.1 in four patients with head and neck carcinoma. We represented PDL1 7 exons, the 3’ UTR and the 5’UTR with the starting codon (ATG) and the stop codon (TAA). Each color corresponds to a patient: R295, R299, R613 and R650. Each Arrow is a breakpoint position. 9ptel indicates the telomere and Cen the centromere. The numbers indicate the genomic positions.

Fig. 4

mRNA expression level of PDL1, PDL2 and PLGRKT (A) and MYC and PVT1 (B) and in 44 patients with HPV positive head and neck squamous cell carcinoma. Each dot represents a patient. The horizontal bars display the median for each data set. (A) Patients R295, R299 and R650 have integration in the 9p24.1 chromosomal region. (B) Patients R654 and R661 have integration in the 8q24.21 chromosomal region.