Luft F, Klaes R, Nees M, Dürst M, Heilmann V, Melsheimer P, von Knebel Doeberitz M
Division of Molecular Diagnostics and Therapy, Department of Surgery, University of Heidelberg, Germany.
Int J Cancer. 2001 Apr 1;92(1):9-17.
Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrated into the host cell genome in HPV-related cancers cells. This suggests that malignant conversion of HPV-infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise sequence information on viral-cellular junctions were obtained only for few cell lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation-mediated PCR assay for the detection of integrated papillomavirus sequences (DIPS-PCR). DIPS-PCR was initially used to amplify genomic viral-cellular junctions from HPV-associated cervical cancer cell lines (C4-I, C4-II, SW756, and HeLa) and HPV-immortalized keratinocyte lines (HPKIA, HPKII). In addition to junctions already reported in public data bases, various new fusion fragments were identified. Subsequently, 22 different viral-cellular junctions were amplified from 17 cervical carcinomas and 1 vulval intraepithelial neoplasia (VIN III). Sequence analysis of each junction revealed that the viral E1 open reading frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Chromosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n), suggesting random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS-PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS-PCR might allow efficient therapy control and prediction of relapse in patients with HPV-associated anogenital cancers.
人乳头瘤病毒（HPV）基因组通常以游离分子形式存在于HPV相关的癌前病变中，而在HPV相关的癌细胞中它们常整合到宿主细胞基因组中。这表明HPV感染上皮细胞的恶性转化与细胞和病毒序列的重组有关。由于技术限制，仅在少数细胞系和原发性病变中获得了病毒 - 细胞连接处的精确序列信息。为了促进基因组HPV整合的分子分析，我们建立了一种连接介导的PCR检测方法来检测整合的乳头瘤病毒序列（DIPS-PCR）。DIPS-PCR最初用于从HPV相关的宫颈癌细胞系（C4-I、C4-II、SW756和HeLa）和HPV永生化角质形成细胞系（HPKIA、HPKII）中扩增基因组病毒 - 细胞连接处。除了公共数据库中已报道的连接处外，还鉴定出了各种新的融合片段。随后，从17例宫颈癌和1例外阴上皮内瘤变（VIN III）中扩增出22种不同的病毒 - 细胞连接处。对每个连接处的序列分析表明，在22例中的20例（91%）中病毒E1开放阅读框（ORF）与细胞序列融合。染色体整合位点定位于染色体1（2条）、2（3条）、7（2条）、8（3条）、10（1条）、14（5条）、16（1条）、17（2条）和线粒体DNA（1条），提示染色体整合位点随机分布。通过DIPS-PCR获得的精确序列信息进一步用于监测4例宫颈癌、1例复发性癌前病变和1例淋巴结转移的单克隆起源。因此，DIPS-PCR可能有助于HPV相关肛门生殖器癌患者的有效治疗控制和复发预测。
