Oncogenic Human Papillomavirus involvement in epithelial ovarian carcinoma among women in Morocco

Epithelial Ovarian cancer (EOC) although rare is the most lethal gynecological cancer in women worldwide. Despite its high prevalence few studies have been performed to evaluate the prevalence and determinants of HPV infection worldwide. The aim of the present study was to investigate the presence of HPV-DNA in Moroccan patients with EOC using PCR among women in Casablanca, and to examine the prevalence of some HPV genotypes in Moroccan population. We performed a study of HPV detection on Fresh biopsies of 70 epithelial ovarian cancer patients. PCR was realized using the MY09/11 and GP5+/6+ primers. Genotyping of HPV was performed by PCR typespecific for HPV 6, 11, 16, 18, 31, and 33.Data was statistically analyzed using SPSS software. Hence, the mean age was 48.9 years (range,21-76 years). Serous adeno carcinoma (75.71%) and stage III of the disease represent the majority of cases. eight patients were HPV positive (11.42%).Results of HPV genotyping revealed predominance of two genotypes: HPV 16 (87.5%) and HPV 31(12.5).No co-infection identified. Approximately 75% of positive cases had a serous cystadeno carcinoma and more than 62,5% had FIGO advanced stage (III or IV).Our study showed that high-risk HPV infection could play a major role among patients with EOC in Morocco.


Background:
EOC although rare is the most lethal gynecological cancer in women worldwide and represent the fifth most common malignancy, leading to 22.400 newly diagnosed cancer cases and over 14,300 deaths every year in the US Good management of EOC depends on an early detection of the disease; unfortunately, the late and poor prognosis is a result of (a) the nature of this malignancy which is insidious asymptomatic in its early onset, (b) deficiency of effective methods of detection at an early stage of the disease, and (c) resistance of chemotherapy [1].
In Morocco, ovarian cancer is the 5th female cancer and accounts for 4, 9 % of cases recorded during the period 2008-2012, in terms of incidence this malignancy represent brute incidence of 5.6 per 100,000 women, standardized incidence on the Moroccan population of 5.4 per 100,000 women and a standardized incidence on the world population of 6.2 per 100,000 women [2] with more ©Biomedical Informatics (2019) than 70% of cases discovered in advanced stages of the disease (III or IV). More than half of the cases occur between 45 and 64 years with serous adeno carcinoma as the predominant histological type [3]. Efforts for early detection of epithelial ovarian tumors are ineffective, because of the ambiguity of both the origin and the pathogenesis of this malignancy.
Infectious agents, mainly viruses, are among the few known causes of cancer and contribute to a variety of malignancies. Human papilloma virus (HPV), Hepatitis B and C virus, and Epstein bar virus, are implicated in the cervical cancer, the liver cancer and Burkitt's lymphoma respectively as found by Moss et Blaser [4]. The Link between HPV and the genesis of cervical cancer has been indisputably proven and identified [5], whereas, its involvement in ovarian cancer still controversial despite the anatomical proximity of the two organs. Some studies provide evidence of the involvement of the viral agents in the carcinogenesis [6, 7] and others deny it [8,9]. Thus, the role of HPV in ovarian cancer tumorigenesis remained a big challenge. Because the ovarian cancer etiology is multi-factorial and HPV infection is related to sexual behavior, a more precise assay should take the covariates such as age, ethnicity, and lifestyle into consideration. The aim of the present study was to determine viral etiology of EOC especially HPV, thus we investigate the presence of HPV DNA in Moroccan patients with EOC using the highly sensitive technique of polymerase chain reaction (PCR) among women in Casablanca area, Morocco and we examine the prevalence of some HPV genotypes in Moroccan population.

Methodology:
The study involved a series of 70 fresh biopsies from patients aging from 21 to 76 years, with histo pathologically confirmed EOC obtained after surgical intervention in the department of gynecology and obstetrics "A", Ibn Rochd University Hospital, Casablanca, Morocco. Informed consent was obtained from all participants and the study protocol was approved by the local ethics committee of Faculty of Medicine and Pharmacy of Casablanca Morocco.  DNA Extraction was performed using the phenol/chloroform method routinely used in the LVMQB/ETB. Briefly, small sections of 5µm obtained from frozen tissue samples using a scalpel, were placed in 1.5 mL sterile Eppendorf tube. Depending on the size of the biopsy, 250 to 500µL of lysis buffer (10mM Tris-HCl pH 7.5, 10mM EDTA, 10%SDS) containing 200µg/ml of proteinase K were added and digested for 3 h at 55°C. Purification was performed by phenol/chloroform/iso amyl alcohol (25:24:1). Then the DNA was precipitated using 7.4 M ammonium acetate and absolute ethanol. The DNA pellet was washed in 70% cold ethanol and dried at 37°C for 15 min before being re-suspended in 30 to 50µL of ultrapure water and stored at-20°C until PCR amplification.
Before the PCR amplification of the viral DNA, the extract was dosed in Nano drop 8000 Spectrophotometer (Nano drop Technologies, Wilmington, DE, USA).To evaluate the efficiency of the extraction, integrity of specimen and absence of PCR inhibitors, all extracted DNA were subject to an amplification of β-globin reference gene using the primers pair PCO4/GH20 as described previously [12]. All amplification was carried out with 100 ng/µl of DNA in Perkin Elmer 2400 GeneAmp PCR thermal Cycler (Scientific Support, Inc, Hayward, CA). DNA from the SiHa cell line was used as positive PCR control and ultrapure water as negative control.
Typing was carried out type-specific PCR for HPV 6, 11, 16, 18, 31 and 33 as described previously [13]. Statistical analysis for obtained data was performed using SPSS software. P-Values are calculated using tree-ways ANNOVA. The significance level was considered when p < 0.05.  Between 70 EOC samples analyzed, HPV-DNA was detected in 11.42% (8/70) of cases. Only two high-risk genotypes were identified: HPV 16 was the most prevalent with 87.5% (7/8) followed by HPV 31 with 12.5% (1/8). None of the patients had more than one type of HPV ( Table 2). The distribution of positive cases according to the age, histological type and FIGO stage is reported in Figure 2. Moreover, these results shows that more than 80% of HPV positive patients were 45 years and older exclusively for HPV type 16. 75% of positive cases had a serous cyst adenocarcinoma and more than 62.5% of positive cases had FIGO advanced stage (III or IV) of the disease. The only patient with HPV Type 31 had mucinous cyst adeno carcinoma. There was no statistical association between clinical features and HPV infection ©Biomedical Informatics (2019) 58 (p>0.05).Whereas, statistical analysis showed that there is correlation between age and clinical stage (p < 0.05) (Figure 3). Endo-metrium and fallopian tubes are a continuation of anatomical endo-cervical glands, and the infection can spread this way or (iv) the sperm may be responsible for this transition by absorbing HPV DNA and transmit these nuclear entities to cells of the reproductive system or may be carriers of the virus during passage of the endocervical canal and thus reach the ovarian cortex after ovulation [26]. In sum, although the relationship between HPV and malignant tumors of the upper genital tract remains controversial, the involvement of viruses in carcinogenesis even in the most unsuspected cancers is becoming increasingly evident.

Conclusion:
The present study showed for the first time, the presence of highrisk HPV in Moroccan patients with an epithelial ovarian carcinoma, suggesting that this virus may play an important role in ©Biomedical Informatics (2019) 59 ovarian carcinogenesis considered as a tumorigenic virus. These studies will be interesting to develop advanced tools for early diagnosis and a better prognosis of cancer. In view of the findings, it may be interesting to examine the possible link HPV-EOC in the large case-control studies among Moroccan patients to better elucidate this association.