Abstract Cancer-associated papillomavirus (HPV) infection often leads to genomic rearrangements such as the integration of viral DNA into the human genome or formation of extrachromosomal DNA (ecDNA) circles with human-HPV DNA fusions, copy number alterations, translocations, inversions, or all at once. Such structures play a key role in cancer formation and progression by altering the expression of human and viral genes. They can also potentially increase genomic instability promoting new rearrangements, but little is known about how these rearrangements evolve during cancer. Unfortunately, short-read sequencing and standard assembly methods often fail to reconstruct HPV-induced rearrangements because of their repetitive nature. Long-read technology can potentially help to overcome this limitation.Here, we used Nanopore long-read whole genome sequencing to explore HPV structures in head and neck cancer cell line UPCI-SCC-090, known for its complex HPV rearrangements. We tested several protocols and methods on this cell line to estimate their impact on rearrangement detection. To study the dynamics of HPV rearrangements, we compared data from UPCI-SCC-090 to another cell line isolated later from the same patient (UPCI-SCC-152).As a result, we discovered a core set of UPCI-SCC-090 rearrangements involving chr 2,3,6 and 9, which remain robust across different tested protocols and methods, and these structures were consistent with previous studies. At the same time, we identified new minor rearrangements in UPCI-SCC-090 indicating heterogeneity within this cell line. The comparison with the UPCI-SCC-152 cell line demonstrated that most rearrangements were kept, but the overall profile of rearrangements changed. For example, UPCI-SCC-152 missed all HPV-induced alterations on chr 6.We concluded that the long-read approach failed to reconstruct all rearrangements completely but allowed us to study them in more detail on a single-molecule level and detect heterogeneity of HPV integrations within and between cell lines. We demonstrated that HPV-induced rearrangements formed a stable core that could persist for a long time with a minor group of variable rearrangements. Citation Format: Vera Mukhina, Alexa Anderson, Daria A. Gaykalova. HPV-related genome rearrangements in head and neck cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 315.