Abstract PO-026: Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer
Authors: Sasa Noah , Eguchi Hirotaka , Tanaka Hidenori , Fukusumi Takahito , Suzuki Motoyuki , Takenaka Yukinori , Okada Yukinori , Inohara Hidenori 
Published: September 2023

Abstract
Human papillomavirus (HPV) integration has been considered as one of the driver factors for cancer progression, but its pattern and role in carcinogenesis is still unclear. Whole genome sequencing data of 14 Japanese HPV16+ OPSCC and 13 HPV+ OPSCC from Pan-Cancer Analysis of Whole Genomes were used to identify integrations and somatic mutations. The phylogenetic analysis of HPV16 revealed that in tumors of 14 Japanese subjects, the A4 variant was the most common (8/14, 57.1%), followed by the A5 variant (2/14, 14.3%), which is considered unique to HPV16 in East and Southeast Asian, and the A2/A3/D2/D3 variants (1/14, 7.1%), showing a significant difference from the distribution of the HPV16 sublineages in Europe and the United States, where the A1 variant accounts for half. A total of 250 integration breakpoints (BPs) in 20 of the 27 tumors (74.1%) were detected. We estimated the cancer cell fraction of integration BPs and found that HPV integration also shows intra-tumor heterogeneity, indicating that nearly half of the integration events occurred after carcinogenesis. Clonal BPs were more likely to occur in the E1 (P = 0.036), confirming that the disruption of the E1 gene may result in abnormal negative regulation of the E6/E7 oncogenes and promote OPSCC carcinogenesis. Subclonal BPs were less likely to occur in the E6 (P = 0.045), suggesting that the E6 may also play an important role in the process of cancer evolution after carcinogenesis. Four states of the HPV genome were identified: (1) episomal-only (7/27, 25.9%), (2) integrated-only (4/27, 14.8%), (3) clonally-mixed (episomal + clonally integrated) (11/27, 40.7%), and (4) subclonally-mixed (episomal + subclonally integrated) (5/27, 18.5%). Since nearly half of these cancers have developed with just episomal copies of HPV, integration itself is not essential for carcinogenesis and can occur during and after carcinogenesis. Interestingly, the E6/E7 were conserved in all four cancers with integrated-only HPV, while the E2 and E1 were disrupted or deleted. This confirms that the constitutive expression of E6/E7 is essential for the carcinogenesis of HPV+ OPSCC. These results suggest that some integrations may be drivers of carcinogenesis with disruption of E1/E2, as in these four cancers, and others may arise randomly due to genomic instability, as in the cancers with the E6/E7 deleted integrants. The number of BPs of structural variations (SVs) occurred in the overlapping 100 kb regions flanking integration BPs (ITG regions) was positively correlated with the number of integration BPs (Spearman’s rank correlation ρ = 0.78; Permutation test P = 9.9 × 10−5). The genomic instability that causes clonal integrations during carcinogenesis is considered to cause clonal SVs, mainly large duplications, and subclonal integrations and SVs such as large deletions. Clonal SVs, mainly large duplications, were also observed in the regions where only subclonal integrations occurred, suggesting that large duplications occur before integrations.
Citation Format: Noah Sasa, Hirotaka Eguchi, Hidenori Tanaka, Takahito Fukusumi, Motoyuki Suzuki, Yukinori Takenaka, Yukinori Okada, Hidenori Inohara. Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-026.