Abstract：
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.
摘要：
人乳头瘤病毒（HPV）感染是口咽鳞状细胞癌（OPSCC）的增加原因。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患者预后的相关性尚不清楚。目前的整合检测技术及其对福尔马林固定石蜡包埋(FFPE)组织的不适用性阻碍了对HPV整合的研究。本研究旨在开发和验证一种新的基于靶向邻近连接的测序方法（靶向基因座扩增/捕获[TLA/TLC]），用于细胞系和FFPEOPSCC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FFPEOPSCC。按照预处理步骤，在配对末端测序之前,对细胞系进行基于聚合酶链反应(PCR)的HPV富集,并对FFPE组织进行基于捕获的HPV富集.TLA能够在目标周围进行数百kb的测序，检测确切的HPV整合位点，结构变体，和染色体重排.在所有细胞系中，确定了一个或多个集成站点，根据PCR数据和文献检测整合的乳头状瘤病毒序列。TLC在15/27FFPEOPSCC中检测到整合的HPV，并鉴定了简单和复杂的整合模式。总的来说,TLA/TLC确认PCR数据并检测到额外的整合位点。总之，TLA/TLC可靠且稳健地检测细胞系和FFPEOPSCC中的HPV整合，启用大型，关于HPV整合的临床相关性的基于人群的研究。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有价值.
