Development of a high-resolution mapping platform for HPV DNA integration in premalignant lesions

摘要 绝大多数人宫颈癌是由人乳头瘤病毒（HPV）引起的。这些 病毒还与一部分其他类型的癌症（头颈部、肛门、阴道、外阴）有关。 子宫颈癌是全世界妇女死亡的第二大癌症原因。超过40种不同类型 的HPV感染生殖道，近一半的人口至少感染过一次HPV。 然而，绝大多数HPV感染者感染后不会因抗病毒而发生侵袭性肿瘤。 免疫反应。宫颈癌是通过一系列的宫颈上皮内瘤变（CIN）发展而来的 步骤，CIN1，CIN2和CIN3，但只有少数妇女，即使CIN3进展到侵入性 癌HPV DNA基因组作为一个环状的染色体外附加体复制， 每一个被感染的细胞都有数千个拷贝然而，在大多数浸润性癌中，HPV DNA整合到 由于异常的宿主细胞DNA修复机制而导致的人类基因组DNA的损伤。这导致病毒致癌基因 （特别是E6和E7）与宿主细胞及其后代永久结合。通常 病毒DNA通常仅作为病毒基因组的一部分整合到人类致癌基因中。整合的病毒 DNA改变癌基因的表达，导致该细胞的克隆扩增。宫颈疾病传统上 通过巴氏涂片进行筛查和监测，但HPV检测被证明具有上级特异性， 敏感性，并取代巴氏涂片作为主要工具。然而，目前的HPV临床检测 通常只检测最常见的HPV类型，并且通常只搜索病毒的一个亚组分。 基因组我们建议在这里开发一种检测大量不同HPV类型的方法 即使只有一小部分病毒基因组存在，也能成功。该检测试剂盒将使用 通过DNA探针组对整个HPV进化枝（目前143种）进行杂交捕获，以富集 组织样本中的HPV DNA，然后进行深入的下一代DNA测序。它还将使用一种独特的 我们建立的宫颈CIN 1 - 3、肿瘤和对照样本的生物储存库具有高度多样性， 我们服务的布朗克斯病人群体。在使用15种HPV类型探针组的初步研究中，我们的方法 在26例CIN 1 - 3病变和肿瘤中检测了8种不同的HPV类型，并检测了整合的HPV DNA 在24例CIN 2/3和肿瘤中有22例。我们的单一检测将同时1）产生明确的HPV类型 特异性，因为获得了广泛的病毒序列，2）检测常见和罕见的HPV类型，3） 发现HPV，即使只有部分病毒基因组存在，4）确定是否整合HPV DNA， 存在，和5）辨别整合的HPV DNA是否接近人类致癌基因。我们进一步建议发展一个 荧光显微镜方法（Junc-FISH）检测临床中患者特异性整合的HPV 样品我们的工作将提供上级HPV检测方法， 更广泛的HPV类型特异性和敏感性，也产生了关于HPV DNA的疾病相关见解 一体化

Abstract The vast majority of human cervical cancers are caused by human papillomaviruses (HPVs). These viruses are also implicated in a fraction of other types of cancer (head & neck, anus, vagina, vulva). Cervical cancer is the second leading cancer cause of death of women worldwide. Over 40 different types of HPV infect the genital tract, and nearly half of the human population is infected by an HPV at least once. However, the vast majority of HPV-infected people infected do not develop invasive tumors due to antiviral immune responses. Cervical carcinomas develop through a series of cervical intraepithelial neoplasia (CIN) steps, CIN1, CIN2, and CIN3, but only a minority of women even with CIN3 progress to invasive carcinomas. The HPV DNA genome replicates as a circular, extra-chromosomal episome with up to many thousands of copies per infected cell. However, in most invasive carcinomas, HPV DNA is integrated into human genomic DNA due to aberrant host cell DNA repair mechanisms. This results in the viral oncogenes (notably E6 and E7) becoming permanently associated with the host cell and its descendents. Usually, the viral DNA is integrated into a human oncogene, often as only a fraction of the viral genome. Integrated viral DNA alters oncogene expression resulting in clonal expansion of that cell. Cervical disease has traditionally been screened and monitored by Pap smears, but HPV testing is proving to have superior specificity and sensitivity, and is supplanting Pap smears as the primary tool. However, current HPV clinical testing generally detects only the most common HPV types, and often searches for only a subfraction of the viral genome. We propose here to develop a method for detection of a massive set of different HPV types that will succeed even when only a fraction of the viral genome is present. The assay will use hybridization capture by a DNA probe set for the entire a clade of HPVs (currently 143 types) to enrich for HPV DNA in tissue samples, followed by deep, next generation DNA sequencing. It will also use a unique biorepository of cervical CIN1-3, tumor and control samples that we established featuring the highly diverse Bronx patient population that we serve. In preliminary studies using a 15 HPV type probe set, our method detected 8 different HPV types in a set of 26 CIN1-3 lesions and tumors, and detected integrated HPV DNA in 22 of 24 CIN2/3’s and tumors. Our single assay will simultaneously 1) yield unambiguous HPV type specificity because of the extensive viral sequences obtained, 2) detect common and rare HPV types, 3) find HPV even when only part of the viral genome is present, 4) determine if integrated HPV DNA is present, and 5) discern if integrated HPV DNA is near a human oncogene. We further propose to develop a fluorescence microscopy approach (Junc-FISH) to detect patient-specific integrated HPV in clinical samples. Our proposed work should provide superior HPV detection methods with higher and much broader HPV type specificity and sensitivity that also yields disease-relevant insight about HPV DNA integration.