人乳头瘤病毒 (HPV) 感染是导致口咽鳞状细胞癌 (OPSCC) 的一个日益重要的原因。病毒基因组整合到宿主基因组中被认为会影响致癌作用，然而，与 OPSCC 患者预后的相关性仍不清楚。目前的整合检测技术及其不适合福尔马林固定石蜡包埋 (FFPE) 组织阻碍了 HPV 整合的研究。本研究旨在开发和验证一种基于靶向邻近连接的新型测序方法（靶向基因座扩增/捕获 [TLA/TLC]），用于细胞系和 FFPE OPSCC 中的 HPV 整合检测。为了鉴定 HPV 整合，将 TLA/TLC 应用于 7 个细胞系和 27 个 FFPE OPSCC。在预处理步骤之后，对细胞系进行基于聚合酶链式反应 (PCR) 的 HPV 富集，并在双端测序之前对 FFPE 组织进行基于捕获的 HPV 富集。 TLA 能够对目标周围数百 kb 的区域进行测序，检测准确的 HPV 整合位点、结构变异和染色体重排。根据检测整合乳头瘤病毒序列的PCR数据和文献，在所有细胞系中，鉴定出一个或多个整合位点。 TLC 在 15/27 FFPE OPSCC 中检测到整合的 HPV，并确定了简单和复杂的整合模式。一般来说，TLA/TLC 确认了 PCR 数据并检测到了其他整合位点。总之，TLA/TLC 能够可靠、稳健地检测细胞系和 FFPE OPSCC 中的 HPV 整合，从而能够对 HPV 整合的临床相关性进行大规模、基于人群的研究。此外，这种方法对于临床诊断中 HPV 相关肿瘤的克隆性评估可能很有价值。© 2024 作者。 《医学病毒学杂志》由 Wiley periodicals LLC 出版。

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.