Using Next-Generation Sequencing Technology to Clarify If there Is HPV-Negative Cervical Adenocarcinoma and Its Mechanism of Carcinogenesis

Cervical cancer is the third in incidence and the fourth in mortality cancer of women worldwide. There is strong epidemiological and molecular biological evidence indicating that human papillomavirus (HPV) plays a central role in the etiology of cervical cancer. Over 90% of cervical cancers and their precursor lesions are attributable to HPV infection, and using multiple polymerase chain reactions (PCRs) targeting different genes of the virus, assuring adequacy of specimens, and optimizing experimental procedure can obtain nearly 100% HPV-positive results. The detection of HPV DNA tends to be lower in adenocarcinoma than in squamous cell carcinoma. In the recently held 13th IGCS Biennial Meeting, a Japanese group as well as a European study detected 0% HPV in minimal deviation adenocarcinoma of cervix, in the former and 0% HPV detected in clear cell or serous carcinoma of the latter. The next-generation DNA sequencing (NGS) technologies have enabled the sequencing of the entire human genome in a few weeks.Whole genome sequencing determines the complete DNA sequence of an organism's genome at a single time. In addition, high-throughput sequencing technologies generate millions of short reads from a library of nucleotide sequences. It is feasible to catalogue all classes of somatically acquired mutation in a cancer, including base substitutions, insertions and deletions, copy number changes and genomic rearrangements. From optimization of our procedures the sensitivity of HPV detection and cross validation with type-specific PCRs and direct sequencing in invasive cervical cancer can be improved from 96.6% up to 99.3%. In our data bank, there are 10 cases with HPV-negative cervical adenocarcinoma by multiple methods. In the first year study, histology slides review will be performed independently by the two gynecological pathologists (S.H. and C.R.L.).We will check with the so-called CUT PCR first, if the sample remains HPV-negative then will further submit it to whole genome resequencing.We intend to resequence the whole genome of those designated HPV-negative cervical adenocarcinomas using 30X coverage. After subtracting the human sequences, the remaining sequences can be aligned to all the HPV sequences published in the public domains. If the answer is “HPV sequences are present”, then what genotype and cause of false negative of the conventional approaches can be determined. In contrast, if there are not any HPV sequences annotated, then the coverage will be doubled to 60X coverage to confirm the absence of HPV in the cervical carcinoma tested. This work will take 1.5 years.When there are no HPV sequences of any know type been identified, these samples will then be compared with HPV-positive adenocarcinoma to elucidate the molecular mechanism using exome, SNP, RNAseq to investigate HPV-independent carcinogenesis of uterine cervix in the 2nd half of the second and the third year. This project will provide definite conclusion for the presence of HPV-negative cervical adenocarcinoma and also shed light on the mechanism of HPV-independent carcinogenesis of cervical adenocarcinoma if such tumors exist. It will significantly impact the future role Taiwanese status on HPV research in the world.

Project ID:PC10007-1133
External Project ID:NSC100-2314-B182-017