Abstract 2937: Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology
Authors: Parida Preetiparna , Mukherjee Nivedita , Singh Agastya , Singh Ashima , Sharan Krishna , Rao Mahadev , Lewis Shirley , Radhakrishnan Sabarinathan , Damerla Rama Rao 
Published: March 2024

Introduction: Integration of human papillomavirus (HPV) DNA into the human genome is considered as a key event in cervical carcinogenesis. Different methods have been employed in exploring HPV integration sites, such as APOT, DIPS-PCR, and NGS. However, these methods have their drawbacks. APOT heavily relies on the quality of the sample due to its dependence on RNA, while DIPS-PCR mostly identifies known sites and has limited capability in uncovering new integration sites. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Also, it requires large amounts of sequencing data, and thus is not applicable in clinical usage, which requires fast and accurate results. Therefore, the development of a new method for HPV integration detection with high accuracy and prompt reporting capacity is crucial for future clinical application.

Methods: We established a novel library preparation strategy for the detection of HPV integration in the human genome using Oxford Nanopore sequencing. Using this novel technology, Nanopore libraries were prepared from HPV positive cell lines HeLa and SiHa followed by sequencing on the Oxford Nanopore. We also determined HPV integration sites in five patients with cervical cancer patients with HPV positive tumors using Ilumina WGS. HPV integrations and breakpoints in human genome from both Nanopore and Ilumina WGS were extracted using Minimap and ViFi tool respectively. All HPV integration sites from cell lines and patient samples were validated using Sanger sequencing.

Results: The novel library preparation approach for detection of HPV integrations with Nanopore sequencing technology were sensitive and efficient in detection of commonly reported HPV breakpoints in HPV positive cell lines HeLa and SiHa. Additionally, we applied this method to clinical samples from five patients subjected to Whole Genome Sequencing, consistently yielding concordant results.

Conclusion: Our Nanopore based assay for detecting HPV integrations without the need for WGS demonstrated high efficacy. These data highlight the sensitivity and efficiency of this methodology in detecting HPV integrations events, emphasizing its potential as a reliable diagnostic tool in understanding pathophysiology of HPV-related malignancies and its use as prognostic markers in clinical settings.

Citation Format: Preetiparna Parida, Nivedita Mukherjee, Agastya Singh, Ashima Singh, Krishna Sharan, Mahadev Rao, Shirley Lewis, Sabarinathan Radhakrishnan, Rama Rao Damerla. Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2937.