Molecular Genotyping and Immunophenotyping of High Risk Human Papilloma Virus Types among Yemeni Women Diagnosed with Cervical Cancer

Abstract

This is a retrospective analytical study was conducted in Yemen during the period from January 2012 to January 2015. The study aimed to determine the most frequent HPV genotypes among Yemeni women diagnosed with cervical cancer. One hundred and fifty Formalin-fixed paraffin-embedded (FFPE) tissue blocks of patients previously diagnosed with cervical cancer and fifty FFPE tissue blocks of patients with non neoplastic lesions who had undergone hysterectomy for any causes other than cervical cancer as (control group) were included in this study and investigated for the presence of HR-HPV infection by immunohistochemistry techniques and molecularbiology technique using PCR. All samples and data were obtained from the archive of national cancer centers, different hospitals and private histopathology laboratories in Sana'a, Aden and Hadhramout during the period from 2009 to 2013. In these three governorates exist the only three national cancer centers in Yemen and all cancer patients coming from different governorates of Yemen are referred to these centers for diagnosis and treatment. The age of patients ranged from 21 to 75 years with the mean ages of 46.73 years, most of patients were in the age group 41-50 representing 88/200 (44%), squamous cell carcinoma (SCC) was the most common among cases representing 80/150 (53.3%) and Most of them 35/80 (43.7%) were found in the age group 41-50. Three sections from each sample were prepared, one stained with haematoxyline and eosin (H&E) to confirm the histopathological diagnosis, the second section was immunostained using avidin biotin technique to detect the HPV by the use of monoclonal mouse anti-human papillomavirus clone (K1H8) from Dako company which is used to demonstrate HPV type 6, 11, 16, 18, 31, 33, 42, 51, 52, 56 and 58 and the third section was prepared for PCR in which the paraffin removed, then the DNA was extracted from each sample according to the steps described in DNA extraction kit purchased from Sacace biotechnologies-Casera –Italy. The obtained DNA was amplified by PCR according to HPV High Risk Typing Kit from Sacace biotechnologies. Type specific primers (primer for HR-HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66) were used. The PCR products were visualized in 3% Agarose gel with 0.5 µg/ml Ethidium bromide staining. The study found that immunohistochemical detection of HPV gave positive results in 52% (78/150) of cases and 12% (6/50) of controls while by PCR HPV types were detected in 114/150 (76%) of cases and 8/50 (16%) of controls, so the prevalence of HR-HPV was114/150 (76%) among cases and 8/50 (16%) among control. Among the positive patients, 89/122 (73%) had single type infections, and 33/122 (27%) was multiple HPV types involving mainly HPV 16, HPV 18 and HPV 31. Consequently, the risk associated with HPV infection was found to be statistically significant. Ten HR-HPV types were detected in this study, the most common genotypes among cases were HPV 16: 72/150 (48%), (51/72 as single infection, 21/72 as multiple infections), most common associated with SCC in women less than 50 years old, the second most common was HPV 18: 31/150 (20.7%), (16/31 as single infection and 15/31 as multiple infection) and always in association with adenocarcinoma, followed by HPV 31: 9/150 (6%) (that was mainly detected as co-infection with other types), HPV 45: 9/150 (6%), HPV 58: 6/150 (4%), HPV 33: 5/150 (3.3%), HPV 35: 5/150 (3.3%), HPV 39: 5/150 (3.3%), HPV 59: 4/150 (2.7%) and HPV52: 1/150 (0.7%). Among the control group HPV 16: 2/50 (4%), HPV 31, 33, 35, 18, 45, and 58 were seen, 1/50 (2%) each. In this study there was no association between types of cervical cancer and types of HR-HPV with the regions of Yemen. Also there was no statistically significant association between type of cervical cancer, regions and age groups. The study concluded that the prevalence of HR-HPV infection is high among Yemeni women, with 76% prevalence among cases. So, HR-HPV proved to be the most important factor that leads to cervical cancer. HPV 16 had the highest prevalence and is commonly associated with SCC in women less than 50 years old, followed by HPV 18 that was mainly associated with adenocarcinoma. HPV 16, 18, 45, 31 and 58 were the most common five HPV types among Yemeni patients. Multiple infections with more than one type of HPV were found in about 27% of all positive cases involving mainly HPV16, 18 and 31. Immunohistochemistry using clone K1H8 (anti-HPV) is considerably lower-priced, and has reasonable specificity to be applied in Yemen for screening for HR-HPV, but the PCR is more sensitive than immunohistochemistry in the detection of HPV.