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

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Molecular & Cellular Analysis Technologies
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Abstract The vast majority of human cervical cancers are caused by human papillomaviruses (HPVs). Theseviruses 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 typesof 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 antiviralimmune 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 invasivecarcinomas. The HPV DNA genome replicates as a circular, extra-chromosomal episome with up to manythousands of copies per infected cell. However, in most invasive carcinomas, HPV DNA is integrated intohuman 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, theviral DNA is integrated into a human oncogene, often as only a fraction of the viral genome. Integrated viralDNA alters oncogene expression resulting in clonal expansion of that cell. Cervical disease has traditionallybeen screened and monitored by Pap smears, but HPV testing is proving to have superior specificity andsensitivity, and is supplanting Pap smears as the primary tool. However, current HPV clinical testinggenerally detects only the most common HPV types, and often searches for only a subfraction of the viralgenome. We propose here to develop a method for detection of a massive set of different HPV typesthat will succeed even when only a fraction of the viral genome is present. The assay will usehybridization capture by a DNA probe set for the entire a clade of HPVs (currently 143 types) to enrich forHPV DNA in tissue samples, followed by deep, next generation DNA sequencing. It will also use a uniquebiorepository of cervical CIN1-3, tumor and control samples that we established featuring the highly diverseBronx patient population that we serve. In preliminary studies using a 15 HPV type probe set, our methoddetected 8 different HPV types in a set of 26 CIN1-3 lesions and tumors, and detected integrated HPV DNAin 22 of 24 CIN2/3?s and tumors. Our single assay will simultaneously 1) yield unambiguous HPV typespecificity 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 ispresent, and 5) discern if integrated HPV DNA is near a human oncogene. We further propose to develop afluorescence microscopy approach (Junc-FISH) to detect patient-specific integrated HPV in clinicalsamples. Our proposed work should provide superior HPV detection methods with higher and muchbroader HPV type specificity and sensitivity that also yields disease-relevant insight about HPV DNAintegration.