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

摘要 绝大多数人类宫颈癌是由人类乳头瘤病毒(HPV)引起的。这些 病毒还与一小部分其他类型的癌症(头颈部、肛门、阴道、外阴)有关。 宫颈癌是全球女性死亡的第二大癌症原因。超过40种不同的类型 人类乳头状瘤病毒感染生殖道，近一半的人口至少感染过一次。 然而，绝大多数感染HPV的人由于服用了抗病毒药物而不会发展为侵袭性肿瘤 免疫反应。宫颈癌发生于一系列宫颈上皮内瘤变(CIN) STEPS、CIN1、CIN2和CIN3，但即使是CIN3，也只有一小部分女性进展为侵袭性 癌症。HPV DNA基因组复制为一个环状的、染色体外的Episome，有多达许多 每个被感染的细胞有数千个拷贝。然而，在大多数浸润性癌中，HPV DNA整合到 人类基因组DNA由于宿主细胞DNA修复机制的异常。这导致了病毒致癌基因。 (特别是E6和E7)变得与宿主细胞及其后代永久相关。通常， 病毒DNA整合到人类致癌基因中，通常只是病毒基因组的一小部分。集成病毒 DNA改变癌基因的表达，导致该细胞的克隆性扩张。传统上，宫颈疾病 已经通过巴氏涂片进行筛查和监测，但HPV检测被证明具有更好的特异性和 敏感度，并正在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小部分 基因组。我们建议开发一种方法来检测大量不同类型的HPV 即使只有一小部分病毒基因组存在，这也会成功。化验结果将使用 DNA探针捕获整个HPV分支(目前为143种)的杂交捕获以进行浓缩 组织样本中的HPV DNA，随后进行深层次的下一代DNA测序。它还将使用唯一的 我们建立的宫颈CIN1-3、肿瘤和对照样本的生物库具有高度多样性 我们所服务的布朗克斯患者群体。在使用15型HPV型探针组的初步研究中，我们的方法 在26个CIN1-3病变和肿瘤中检测到8种不同的HPV类型，并检测到整合的HPV DNA 22/24例CIN2/3‘S和肿瘤。我们的单一检测将同时产生明确的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) 即使只存在部分病毒基因组也能找到HPV，4)确定整合的HPV DNA是否 目前，以及5)识别整合的HPV DNA是否接近人类致癌基因。我们进一步建议制定一项 荧光显微镜技术(JUNC-FISH)检测临床患者特异性整合型HPV 样本。我们提出的工作将为HPV检测方法提供更高和更多的 更广泛的HPV类型特异性和敏感度，还可提供对HPV DNA的疾病相关洞察 整合。

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.