Xu Bo, Chotewutmontri Sasithorn, Wolf Stephan, Klos Ursula, Schmitz Martina, Dürst Matthias, Schwarz Elisabeth
Research Program Infection and Cancer, DKFZ, Heidelberg, Germany.
PLoS One. 2013 Jun 18;8(6):e66693. doi: 10.1371/journal.pone.0066693. Print 2013.
Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.
宫颈癌由高危型人乳头瘤病毒（HPV）引起，在全球超过一半的病例中是由HPV16导致的。病毒DNA整合到宿主基因组是宫颈癌发生过程中常见的突变。由于整合发生在不同的基因组位置，所以在每个病例中都会产生独特的病毒-细胞DNA连接。这种独特性极大地增加了精确鉴定HPV整合位点的难度。我们在此报告一种用于确定HPV16 DNA整合位点序列的新型多重策略的开发。它包括DNA片段化和接头标记、HPV16早期区域的PCR富集、Illumina下一代测序、数据处理以及通过接头PCR验证候选整合位点。该策略应用于51个宫颈癌样本（47个原发性肿瘤和4个细胞系）。总共鉴定出75个HPV16整合位点（3'端连接）并分配到各个样本。通过将DNA连接与病毒癌基因融合转录本的存在情况进行比较，44个肿瘤可分为四组：有一个转录活跃的HPV16整合的肿瘤（n = 12）、有转录和沉默DNA连接的肿瘤（n = 8）、携带游离型HPV16 DNA的肿瘤（n = 10）以及有一到六个DNA连接但无融合转录本的肿瘤（n = 14）。整合的HPV16 DNA的3'断点在主要剪接受体上游的早期区域片段内显示出具有统计学意义（p<0.05）的优先分布，这突出了病毒癌基因表达失调在致癌过程中的重要性。一半的已定位HPV16整合位点靶向细胞基因，表明HPV整合对宿主基因有直接影响（插入诱变）。总之，用于确定HPV16整合位点的多重策略非常有效。它将为全面绘制HPV整合位点以及在癌症患者治疗后将HPV整合位点用作个体化生物标志物以早期诊断残留和复发性疾病开辟新途径。
