Frequency of Human Papilloma Virus (HPV) subtypes 31,33,35,39 and 45 among Yemeni women with cervical cancer

Background Identification of different HPV subtypes in unidentified communities provides sufficient information for screening and monitoring potential impact of a vaccination program. Therefore, the aim of this study was to screen for the presence of HPVs subtypes 31,33,35,39 and 45 among Yemeni women with Cervical Cancer. Methodology A total of 200 (150 malignant and 50 benign) tissue samples were obtained from Yemeni women with cervical cancer, were investigated for the presence of HPV subtypes 31,33,35,39 and 45 by Polymerase Chain Reaction (PCR). Results Of the 150 cervical cancer tissue specimens, HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were identified in 10/150 (6.7 %), 6/150 (4 %), 6/150 (4 %), 5/150 (3.3 %) and 10/150 (6.7 %), respectively. The frequency of these HPV subtypes among Yemeni women with cervical cancer was 24 %. Conclusion: HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were prevalent among Yemeni women with cervical cancer.


Introduction
Cervical cancer is the third most common cancer in women Worldwide [1]. Oncogenic human papillomavirus (HPV) is the most important risk factor associated with cervical cancer [2]. HPV have been divided into high-and low-risk on the basis of their oncogenic potential. High risk HPV is considered to be the leading etiological cause for cervical cancer [3]. Cervical cancer is one of the most common cancers among women worldwide [4]. Cervical cancer is a very slow progressive disease which needs prolonged period to progress from normal to pre-cancer and to the potentially fatal invasive cancer. There is a gap of 10-20 years between precancer and cancer which offers an opportunity to screen, detect and treat pre-cancer and avoid its progression to cancer. Immunocompromised women, however, progress more frequently and more quickly to pre-cancer and cancer [5,6].
Infection with high-risk human papillomavirus (HR-HPV) is established as the main cause of cervical carcinoma [7]. HPV has been involved in 99.7 % of cervical squamous cell cancer cases worldwide [8]. Human Papilloma Virus (HPV) is small DNA virus commonly infecting mucosa and cutaneous keratinocytes. Up to now, at least 200 HPV genotypes are known. HPV have been divided into high-and low-risk on the basis of their oncogenic potential. High risk HPV is considered to be the leading etiological cause for cervical cancer [3]. Low-risk HPV types include types 6, 11, 42, 43, and 44. High-risk HPV types include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 [9].
The L1 and L2 proteins form icosahedral capsids for progeny virion generation. Based on sequence variation of the L1, L2, HPV variants differ in biological and chemical properties, as well as geographical distribution [10][11][12]. Intratypic sequence variation has also been found in the E2, E4, E5, E6, and E7 genes of HPV-16 [10]. The E6 and E7genes encode viral oncoproteins that target Rb and p53, respectively. During the viral life cycle, these proteins facilitate stable maintenance of episomes and stimulate differentiated cells to reenter the S phase [12]. The oncogenicity of specific HPV variants appears to vary geographically ethnically [10,11]. In 2012, the International Agency for Research on Cancer concluded that there was consistent and sufficient epidemiological, experimental and mechanistic evidence of carcinogenicity to humans for HPV31, HPV33, HPV35, HPV39, HPV45, for cervical cancer. Consequently, these types were regarded as 1A carcinogens. They all belong to the family of the α-Papillomaviridae, in specific to the species, α7 (HPV39, HPV45) and α9 (HPV31, HPV33, HPV35) [13].
However, there are very limited studies about HPVs and their related cancers in the majority of developing countries such as the greater Middle East which including Yemen and its neighboring countries, such as Saudi Arabia, Qatar, United Arab Emirates [14]. However, most of the studies in this context have focused on HPV subtypes 16 and 18 ignoring other high risk or low risk HPV subtypes. Therefore, the aim of this study was to find out the prevalence of HPV 31, HPV 33, HPV35, HPV 39 and HPV45 among Yemeni women with cervical cancer.

Materials and methods
In this study, 200 formalin fixed paraffin wax processed tissue samples of cervical lesions were obtained from formerly operated patients from National Cancer Centers and different histopathology laboratories in Sana'a, Hadhramout and Aden, Yemen. All specimens and data were obtained from the archive of National Cancer Centers, and different hospitals and different private histopathology laboratories in the three governorates mention above during the period from 2008 to 2012 (full coverage). Cancer specimens included: Squamous Cell Carcinoma (SCC), Adenocarcinoma (Ad), Adenosquamo-Carcinoma (AdSC).

DNA extraction
DNA was extracted from paraffin-embedded samples, by immersing tissue section in xylene to dissolve the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases were digested by proteinase K. Buffer containing denaturing agent (sodium dodecyl sulfate (SDS)), was added to facilitate digestion [15]. Nucleic acids were purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation. Following phenol extractions, RNase A was added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A were used to remove any remaining enzyme. Sodium acetate and isopropanol were added to precipitate DNA, and high speed centrifugation was used to pellet the DNA and facilitate isopropanol removal. Washing with 70 % ethanol was performed to remove excess salts, followed by centrifugation to re-pellet the DNA [16,17]. DNA is re-suspended in distilled water, quantified and stored at −20°C Purified DNA was subsequently used in downstream applications of PCR.

DNA quantification
To evaluate the DNA quantification after DNA extraction, we had analyzed DNA measurement using a Nano-Drop spectrophotometer.
Samples and controls were amplified using Gene Amp PCR system 9700. The PCR programis described in Table 1.

Gel-electrophoresis
The PCR products were visualized in 2 % Agarose gel with 0.5 μg/ml Ethidium bromide. Ten micro liters of 100 bp DNA ladder and PCR product was loaded on the gel. Gel electrophoresis was performed at 120 V and 36 mA for 60 min. Pictures were taken by Gel documentation system (Gel mega, digital camera and software in a computer).

Data analysis
Data management was done by using the Statistical Package for Social Sciences (SPSS version 16). SPSS was used for analysis and to perform Fisher exacttest for statistical significance (P value <0.05 was considered significant). The 95 % confidence level and confidence intervals were used.

Ethical consideration
Before the study conducted the proposal of the study were ethically approved by ethical committee of the Sudan University of Science and Technology. Then the inform consent were agreed by the general managers of all national cancer centers, hospitals and private histopathology laboratories in which the study were performed.

Discussion
The mortality of cervical cancers is high in Yemen and most cases are diagnosed late stage of the disease. In fact, there is a complete paucity of data in this context from Yemen; due to the absence of sufficient national registries. Although, the prevalence of HPV is unknown in Yemen, but data from the neighboring countries showed increased burden. In present study, the relationship between cervical cancer and the presence of the high risk HPV (HR-HPV) subtypes HPV31, HPV33, HPV35, HPV39, and HPV45 genome was tested. Data on HPV type spreading is essential to assess the possible impact of present and future HPV preventive strategies. The prevalence and distribution of HPV genotypes differs significantly globally, and these variances might be related to the complex geographical and biological interaction between different HPV types and host immunogenetic factors.
However, there is a lack of literature regarding the burden of HPV in Yemen. To the best of our knowledge there was only two study investigated the prevalence of HPV in cervical cancer. The first one examined the presence of HPV16 and HPV18 in 84 cervical cancer tissue samples that were previously categorized as harboring HPV by immunohistochemistry using K1H8 (anti-HPV). In the use of PCR testing for HPV subtypes 16 [19]. Another study from Yemen found that the prevalence of HPV subtypes 52,56,58,59, and 66, among cases was 0.6, 0, 4, 3.3 and 0 % respectively [20].
Again the presence of the high risk HPV (HR-HPV) subtypes HPV31, HPV33, HPV35, HPV39, and HPV45 was previously reported in a number of studies. Prospective follow-up studies have proposed that persistent HR-HPV infections play a crucial role in the progression of CIN lesions and in the development of cervical cancer. The mean clearance time for the distinct HR-HPV type was 16.5 months. HPV16 and HPV31 were the most persistent infections (clearance times = 18.1 and 16.2 months, respectively), whereas HPV39 infections cleared quickly. The mean copies per cell in HPV18/45, HPV31, HPV33/52/58, and HPV39 infections were higher in persisting HPV infections than in HPV infections that cleared, but the difference was not significant [21]. In a study from Kuwait, investigated the distribution of HPV in 298 women with abnormal cervical cytology, found that the prevalence of HPV33 was 9.9 % [22], which was very high compared to our findings. Although we found that the most prevalent HPV subtypes 9% 14