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

开发用于癌前病变中 HPV DNA 整合的高分辨率绘图平台

• 批准号: 10018826
• 负责人: JACK R. LENZ
• 金额: $ 21.79万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10018826
• 关键词: Advisory Committees; African American; Age; Anatomy; Antiviral Agents; Anus; Archives; Bacterial Artificial Chromosomes; Benchmarking; Biological Assay; Biological Markers; Cancer Etiology; Carcinoma; Carcinoma in Situ; Cause of Death; Cells; Cervical; Cervical Cancer Screening; Cervical Intraepithelial Neoplasia; Cervix Uteri; Cervix carcinoma; Clinical; Clonal Expansion; Color; Custom; DNA; DNA Integration; DNA Probes; DNA Repair; DNA sequencing; Detection; Development; Disease; Episome; Epithelial; Epithelium; Etiology; Event; FDA approved; Fluorescence Microscopy; Fluorescent in Situ Hybridization; Genetic Markers; Genome; Genomic DNA; Genomic Instability; Goals; HPV-High Risk; Head Cancer; Head and neck structure; Hepatitis B Virus; Hepatitis C virus; High-Throughput DNA Sequencing; High-Throughput Nucleotide Sequencing; Human; Human Genome; Human Herpesvirus 4; Human Herpesvirus 8; Human Papillomavirus; Human T-lymphotropic virus 1; Human papilloma virus infection; Hybrids; Image; Immune response; In Situ; Infection; Lesion; Malignant neoplasm of anus; Malignant neoplasm of cervix uteri; Maps; Methodology; Methods; Minority; Monitor; Neck Cancer; Nucleotides; Oligonucleotides; Oncogenes; Oncogenic Viruses; Pap smear; Patients; Play; Population; Preventive service; Process; Resolution; Rhadinovirus; Risk; Role; Sampling; Sensitivity and Specificity; Series; Side; Specificity; Technology; Testing; Tissue Banks; Tissue Sample; United States; Vagina; Viral; Viral Genome; Viral Oncogene; Virus; Virus Integration; Vulva; Woman; Work; analysis pipeline; base; biobank; biomarker identification; cancer type; carcinogenesis; clinical practice; design; high risk; human DNA; immune clearance; insight; neoplastic cell; next generation; patient population; premalignant; reproductive tract; research clinical testing; screening; screening guidelines; tool; tumor; tumor progression; viral DNA

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.

抽象的 绝大多数人类宫颈癌是由人乳头瘤病毒（HPV）引起的。这些 病毒还与一小部分其他类型的癌症（头颈癌、肛门癌、阴道癌、外阴癌）有关。 宫颈癌是全世界女性死亡的第二大癌症原因。超过 40 种不同类型 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 感染者由于抗病毒药物的作用，不会发展为侵袭性肿瘤。 免疫反应。宫颈癌是通过一系列宫颈上皮内瘤变（CIN）发展而来的 步骤，CIN1、CIN2 和 CIN3，但即使患有 CIN3，也只有少数女性进展为侵入性 癌。 HPV DNA 基因组以环状、染色体外附加体的形式复制，其中包含多达许多 每个受感染的细胞有数千个拷贝。然而，在大多数浸润性癌中，HPV DNA 被整合到 由于宿主细胞 DNA 修复机制异常而导致的人类基因组 DNA。这导致病毒癌基因 （特别是 E6 和 E7）与宿主细胞及其后代永久相关。通常， 病毒 DNA 被整合到人类癌基因中，通常只是病毒基因组的一部分。综合病毒式 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 通过子宫颈抹片检查进行筛查和监测，但 HPV 检测被证明具有卓越的特异性和 敏感性，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 通常仅检测最常见的 HPV 类型，并且通常仅搜索病毒的一小部分 基因组。我们在此建议开发一种方法来检测大量不同的 HPV 类型 即使只存在病毒基因组的一小部分，这种方法也会成功。该测定将使用 通过 DNA 探针组对整个 HPV 进化枝（目前有 143 种类型）进行杂交捕获，以富集 组织样本中的 HPV DNA，然后进行下一代深度 DNA 测序。它还将使用独特的 我们建立的宫颈 CIN1-3、肿瘤和对照样本的生物样本库具有高度多样化的特点 我们服务的布朗克斯患者群体。在使用 15 HPV 型探针组的初步研究中，我们的方法 在一组 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 在 24 个 CIN2/3 和肿​​瘤中的 22 个中。我们的单一检测将同时 1) 产生明确的 HPV 类型 由于获得了广泛的病毒序列，因此具有特异性，2) 检测常见和罕见的 HPV 类型，3) 即使仅存在部分病毒基因组，也能找到 HPV，4) 确定整合的 HPV DNA 是否存在 5) 辨别整合的 HPV DNA 是否靠近人类致癌基因。我们进一步建议开发一个 荧光显微镜方法 (Junc-FISH) 在临床中检测患者特异性整合 HPV 样品。我们提出的工作应该提供更好的 HPV 检测方法，具有更高和更多的性能。 更广泛的 HPV 类型特异性和敏感性，还可获得有关 HPV DNA 的疾病相关见解 一体化。