Identification and Characterization of Human Papillomavirus Causing Cervical Cancer in Pakistani Women

Abstract

Cervical cancer is the most prevalent gynecological malignancy in low to middle income countries. In Pakistan, cervical cancer ranks as the 3rd leading cause of death in females. The important reason for this higher mortality rate in country is due to lack of valuable screening programs. Cancer of the cervix is a preventable disease. In Pakistan, HPV screening is relatively neglected and information on HPV prevalence and its molecular epidemiology is limited among Pakistani women. The purpose of this study was to determine the socio-demographic factors influencing cervical cancer screening and to assess the role of screening should be test for early detection of cervical carcinoma in the Pakistani female. Beside this, molecular characterization, genotyping and detection of human papillomavirus from formalin fixed paraffin embed cervical biopsies obtained from patients with cervical cancer to determine most prevalent HPV type in Pakistan. Furthermore, it demonstrates additional information regarding role of HPV infection in early events of epithelial wound healing an in vitro model system. Between, January 2013 to December 2014 cervical smears were taken from married females with gynecological problems visiting Pakistan Institute of Medical Sciences. Pap smears were also obtained from Armed Forces Institute of Pathology, Rawalpindi. In the present study, a total of 118 cervical smears were screened using Pap smear test. All patients were also subjected to visual inspection with acetic acid (VIA). The mean age of the female patients was 35.78 ± 9.8 years. There were 42(35.5%) normal Pap smear, 33(27.9%) inflammatory, 11(9.3%) LSIL,5(4.2%)HSIL and 18(15.2%) abnormal Pap smear. Acetic acid (VIA) was positive in 27/118 (22.8%) patients. The present study also determined and characterized HPV infections and to evaluated the HPV DNA in subjects with abnormal cervical cytology. A total of 105 histopathological confirmed formalin fixed paraffin embedded (FFPE) biopsies were obtained from cervical cancer patients and were screened for HPV DNA by nested PCR using primers MY09/MY11 and GP5+/GP6+. The HPV positive samples were also screened for high risk HPV genotypes by HPV genotyping PCR and further confirmed and characterized by nucleotide sequencing. HPV DNA was iv detected in 90 (96.8 %) FFPE biopsies. Out of positive HPV, 81 (90%) and 1 (1.2%) were infected with HPV16 and HPV18 respectively, while 8 (8.8 %) remained untypeable on genotyping HPV PCR. DNA sequencing revealed HPV16 (n= 41) was the most frequent type followed by HPV35, 18, 56, 59, 67 and 73 respectively. To the best of our knowledge, molecular epidemiology of HPV 35, 56, 59, 67 and 73 were first time reported in Pakistan. This study provides important information on the HPV prevalence and its high-risk type distribution causing cervical cancer in Pakistan. It contributes information related to molecular epidemiology of HPV types in the country that will be fundamental for local decision-makers to consider cervical cancer screening programs and useful globally for the understanding of HPV variant distribution around the world Another important part of present study was establishment of Normal Immortalized Human Keratinocytes (NIKS) and NIKS HPV 16 cell lines to know early events of HPV infection and wound healing process and to determine role of epidermal growth factor (EGF). As EGF is an important proliferative agent and is necessary in tissue culture experiments to stimulate the proliferation of NIKS cells. HPV 16 viral copy number increased when using 500 ng/ml, hence high EGF increase in viral copy number but do not have impact on morphology of keratinocytes and proliferation of cells. Expression levels of E6 indicate >2-3 fold increase at day 5 in the 100ng/ml and 500ng/ml samples respectively. The findings of this thesis have great implementation and provide an over view on prevalence, molecular epidemiology and in vitro model studies. Therefore, it is need of time to monitor virus for early detection and better characterization which will not only help the physicians, surgeons, oncologist for rapid diagnosis but also help researchers for investigating early events in viral infection and wound healing process.