82. Development and validation of a liquid biopsy next generation sequencing assay for HPV detection cancer patients

ABSTRACT

Introduction Human papillomavirus (HPV) is a common sexually transmitted infection with high-risk genotypes integrating into the host genome, leading to oncogenesis. HPV infections account for over 90% of anal and cervical cancers, making detection and monitoring of HPV status crucial. Liquid biopsy is a minimally invasive procedure that detects circulating tumor HPV-DNA (cfHPV-DNA) in blood plasma, serving as a biomarker for early detection, prognosis, and disease monitoring. Our aim was to develop and validate a liquid biopsy Next Generation Sequencing (NGS) assay for HPV detection, copy number analysis and integration sites identification in cancer patients. Methods We developed a molecular barcoded hybridization capture-based NGS panel covering 193 HPV types (including all 13 high-risk types), using Twist Biosciences Alpha Chemistry. Libraries were prepared from cell line controls and patient samples per manufacturer’s instructions. Amplified libraries were hybridized to the HPV panel and sequenced on the Illumina platform. A custom bioinformatics pipeline was developed to analyze the sequencing data, reporting HPV type, copy number and genome integration sites. The pipeline includes demultiplexing reads for alignment, error correction using Unique Molecular Identifiers (UMI) to collapse reads by family sizes, coverage assessment and copy number determination by HPV type followed by integration site detection through chromosomal junctions. Analytical validation was performed to establish minimum input, limit of detection (LOD), precision, specificity and accuracy of the assay. Results The assay demonstrated 100% specificity, as no HPV types were detected in healthy donor samples. The limit of detection (LOD) was established at 15 copies using serially diluted HPV16 positive cell line SiHa. The platform's accuracy was validated with 20 patients previously tested through ddPCR with known HPV counts, showing a 95% sensitivity and strong linear correlation (R2 > 0.9) between the two methodologies. Integration site detection into chromosome 13 of the human genome (chr3:74087560-HPV16:3131 and chr13:73788868-HPV16:3387) in the SiHa cell line, was confirmed. Conclusion The HPV assay is effective for detecting HPV type, copy number, and integration sites in plasma samples for high-risk types commonly found in anal and cervical cancers