Prognostic significance of HPV physical status and integration sites in cervical cancer.

Human papilloma virus (HPV) infection is the major cause of cervical cancer and integration of HPV DNA into the host cell genome is believed to be essential for malignant transformation. MiRNAs are a class of 19-24 nt non-coding RNAs that regulate gene expression primarily through post transcriptional repression or m-RNA degradation in a sequence specific manner. The aim of this study was to determine the frequency of HPV16 and 18 integrated and episomal forms and to evaluate its prognostic significance in invasive cervical carcinoma cases and to detect by in-silico approach MiRNAs near HPV integration sites (within <3Mb). HR-HPV 16 and 18 typing was performed by Nested Multiplex PCR (NMPCR) and HPV 16 and 18 physical status (integrated and episomal forms) was determined by Amplification of Papillomavirus Oncogene Transcripts (APOT) assay. Nested PCR products of the APOT assay were resolved on a 2% agarose gel and the PCR products of interest were excised and sequenced. In silico analysis was done to identify the Fragile sites and MiRNAs' near integration sites of the HPV. Episomal forms were more common with the HPV16 type and integrated forms with the HPV18 type (p= 0.011). Patients with tumors having the episomal forms had a better disease free survival than those with integrated forms of HPV16 type, but this did not reach statistical significance. We detected 53 miRNAs near integration sites, of which 39 have been reported to be associated with cancers. The incidence of miRNAs near HPV integration sites was 78.3%, being more common with HPV16. This is the first study from India to provide the physical status of HPV16 and HPV 18 in cervical cancers, to assess their prognostic importance and to identify FRA and MiRNAs' near HPV integration sites.