Intratumoral heterogeneity and clonal evolution induced by HPV integration 1

49 The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive 50 cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read 51 sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed 52 form of structural variation, “heterocateny,” characterized by diverse, interrelated, and repetitive 53 patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints 54 shared across structural variants facilitated stepwise reconstruction of their evolution from a 55 common molecular ancestor. This analysis revealed that virus and virus-host concatemers are 56 unstable and, upon insertion into and excision from chromosomes, facilitate capture, 57 amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of 58 heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings 59 indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of 60 an oncogenic DNA virus, thereby extending known consequences of HPV integration to include 61 promotion of intratumoral heterogeneity and clonal evolution.