Prevalence of human papillomavirus genotypes in cervical cancer in Maiduguri, Nigeria

Introduction Cervical cancer is the commonest gynaecological malignancy and the second most common cancer among women worldwide. Several epidemiological, clinical and molecular studies have strongly implicated oncogenic high-risk human papillomavirus infection in the aetiopathogenesis of cervical cancer. The objectives of this study were to determine the cervical HPV prevalence and genotype distribution in cervical cancer in Maiduguri, Nigeria. Methods This was a descriptive and retrospective study. Sixty-three archived paraffin-embedded tissue blocks with confirmed diagnoses of cervical cancer during the study period (2013-2015) were retrieved and examined. The procedure included deparaffinization of tissue samples, DNA extraction, PCR, gel electrophoresis, and HPV genotyping by reverse hybridization line probe assay. Results Sixty-three cervical cancer cases were subjected to genomic DNA extraction and HPV-DNA detection by PCR. Fifty-eight samples showed PCR positivity while 5 samples were PCR negative. HPV-specific DNA was detected in 44 of the 58 PCR-positive samples and thus the prevalence was 69.8%. Ten different high-risk HPV genotypes were detected. Both single and multiple high-risk HPV infections were observed. The most prevalent type of the human papillomavirus detected was HPV16. Conclusion HPV-DNA was prevalent in majority of the examined cervical cancer tissues and that HPV16, HPV18, HPV45, HPV51 and HPV52 were the predominant HPVs detected in both single and multiple HPV infections. The results of this study and further studies will provide more detailed information about HPV and may contribute significantly to the prevention of cervical cancer through primary high-risk HPV testing and HPV vaccination against the oncogenic viruses.


Introduction
Cervical cancer is a malignant neoplasm arising from the uterine cervix-the part of the uterus connecting the body of the uterus to the vagina [1]. The burden of cervical cancer is quite high in the developing countries and constitutes a major health problem [2]. This is a source of great concern considering the fact that cervical cancer is preventable and curable at low cost with currently available methods [2]. Worldwide, it is second only to breast cancer in incidence and is the third leading cause of cancer mortality among the female population [3]. It accounts for about 8% of both total cancer cases and total cancer deaths in the world [4]. In 2012, it was estimated that there were 528,000 cases of cervical cancer, and 266,000 deaths [4]. An estimated 80% of cases of cervical cancer occur in the developing countries, and remains the second most common cancer among women in those countries [2]. In Africa, the sub-Sahara is the region with the highest incidence of cervical cancer in the world with concomitant high mortality affecting women at their prime age [2].
In Nigeria, cervical cancer is currently the second most common cancer in women after breast cancer with about 14,089 new cases diagnosed annually and 8240 deaths [5]. Most studies from different parts of Nigeria have also indicated that cervical cancer is the commonest gynaecological cancer. In Maiduguri, Northeastern Nigeria, it was the commonest gynaecological malignancy representing 70.5% of the total gynaecological cancers [6]. Molecular, clinical and epidemiological studies have shown that human papillomavirus infection plays a necessary role in the aetiopathogenesis of cervical carcinoma [3]. More than 90% of cervical carcinoma contains DNA sequences of specific HPV types [3].
A study carried out on more than 1000 invasive cervical cancer specimens at 32 hospitals in 22 different countries revealed that HPV DNA was detected in 93% of the tumours [7]. HPV-16 was present in 50%, HPV-18 in 14%, HPV-45 in 8% and HPV-31 in 5% [7]. In another study conducted to investigate regional variations in the contribution made by different HPV types to invasive cervical carcinoma, a total of 10,058 cases from 85 studies using PCR indicated that the most common HPV types in almost all the regions, in order of decreasing prevalence were: HPV- 16, 18, 45, 31, 33, 58, 52, 35, 59, 56, 51, 68, 39, 82, 73, 66, and 70 [8]. The index study utilized the PCR to detect the presence of HPV-DNA in cervical cancer tissue biopsies from the University of Maiduguri Teaching Hospital.
Furthermore, genotyping of the HPV-positive samples was also carried out using reverse hybridization line probe assay.

Methods
The present study was conducted at the University of Maiduguri Teaching Hospital (UMTH) which is the apex referral centre for persons or specimens in the northeastern region of Nigeria. UMTH is located within the city of Maiduguri. This was a retrospective study of 63 cases of confirmed cervical cancer diagnosed in the Department of Histopathology of UMTH between January 2013 and December 2015.
The histopathology request cards and slides were retrieved and reviewed. All the archival samples of formalin-fixed, paraffinembedded tissues of confirmed cervical cancer within the study period were selected and subjected to the laboratory procedures. The study protocol was approved by the Research and Ethics Committee of the Hospital.
DNA extraction: DNA was extracted from 8-10 micron sections of FFPE tissues using QIAamp ® DNA FFPE Tissue kit from Qiagen (Hamburg, Germany) according to the manufacturer's instruction. The protocol was slightly modified to allow overnight digestion with Proteinase K. The tissue blocks were sectioned and processed under strict conditions to avoid potential contamination. For each block, a separate and sterile microtome blade was used after carefully and thoroughly cleaned with xylene and 70% alcohol after each cut.
DNA was quantified using NanoDrop2000C spectrophotometer (Thermos Scientific, USA). Concentration was determined based on absorbance at 260nm. Purity was estimated as ratio of absorbance at 260nm to absorbance at 280nm (A260:A280). of extracted DNA as template DNA was added. Amplification was carried out using Mastercycler Nexus (Eppendorf, Germany) using the following thermocycler conditions: initial denaturation at 94°C for 3min, followed by 35 cycles of denaturation at 94°C for 30secs, annealing at 56°C for 45sec and extension at 68°C for 45sec followed by final extension at 68°C for 5min.
Gel electrophoresis: to confirm amplification of the 122-bp β-globin, agarose gel electrophoresis was performed on 2% agarose in TAE buffer. Electrophoresis was carried out at 90V for 30min and viewed under UV trans-illuminator. A 100-kb size ladder (Promega) was used as the standard size DNA marker, and staining was done with Ethidium Bromide.
Detection of HPV using SPF10 primers: the β-globin-positive DNA samples were subjected to PCR to detect the HPV-DNA. The of extracted DNA as template DNA was added. Amplification was carried out using the following thermocycler conditions: initial denaturation at 94°C for 3min, followed by 40cycles of denaturation at 94°C for 1min, annealing at 52°C for 1min and extension at 68°C for 1min followed by final extension at 68°C for 5min.  (1.0%) as shown in Table 1 As a single HPV infection, HPV16 and HPV18 were identified. They accounted for 14 (31.8%) and 3 (6.8%) of the total cases respectively.
In this study, ten different high-risk HPV types (HPVs- 16,18,31,35,45,51,52,58,59 and 73) were identified in cervical cancer cases with positive HPV-DNA status. Along with these high-risk HPVs, co-infection with low-risk HPV11 was observed in six of the analysed samples. In addition, both single and multiple HPV infections were observed.
Overall, the five most prevalent high-risk HPV genotypes in this study in decreasing order of frequency were HPV16, HPV18, HPV45, HPV52 and HPV51. This finding is a global phenomenon most especially for the first three HPVs as reported in several studies across the world [10,12,16,20,[33][34][35]. HPV16 and HPV18 were the most common HPVs detected in this study and accounted for 39.6% and 19.8% respectively and 59.4% combined. These two high-risk HPVs are the most frequently studied and consequently implicated in the causation of cervical cancer worldwide [10,34,[36][37][38][39]. They are the most prevalent and most potent carcinogenic viruses [10,40]. Their probability of disease progression and persistence is significantly higher than other HPVs [10]. Together, HPV16 and HPV18 account for 60-80% of the HPVs implicated in cervical cancer in the sub-Saharan Africa and most other countries in the world [10,12,34]. However, few studies have reported that HPV16 was not the most common HPV detected in cervical cancer due to geographical variation [25,41]. A study by Zohoncon et al. [41] in Parakou, Benin Republic reported no HPV16 in cervical cancer and that HPV39 was the most prevalent genotype in their study.
Although most of the studies from Africa have observed multiple HPV infections, the finding in this study was as expected. In our environment, there is high rate of polygamous marriages; a man can marry up to four wives. In addition, divorcees, widows and widowers also remarry. In all these circumstances, the tendency of transmitting multiple HPV types is quite high. In other African countries, HIV infection, invasiveness of cervical lesions and geographical variation were suggested as factors responsible for multiple HPV infections [20,25,46]. The prevalence of multiple HPV infections has been attributed to multiple sexual partners, immunosuppression, smoking and pre-existing HPV [25,41,43,[46][47][48]. The significance of multiple HPV infection in cervical intraepithelial lesions and cervical cancer has conflicting reports [49]. However, most studies have identified that multiple HPV types exhibit synergism and additivity and were associated with persistent infection, higher risk of disease progression, disease severity and poor survival [17,43,47,48].
Conversely, Salazar et al. [42] and Wang et al. [50] have shown no significant difference between single and multiple HPV infection, but a reduced rate of high-grade cervical lesions. Intergenotypic competition or effective immune response has been suggested as a possible mechanism for a reduced rate of high-grade cervical lesions in multiple HPV infections [42].
The recently introduced nonavalent HPV vaccine was granted market authorization in the USA and Europe in the mid-2015 [51]. It is considered as a safe and effective vaccine and will further reduce the incidence of HPV infection and HPV-related cancers. It can also protect unvaccinated individuals through herd immunity [51,52].
Studies have shown the remarkable benefits of effective screening, early diagnosis and curative therapy of cervical cancer [53]. Such remarkable gains are credited to the effectiveness of the Pap test in detecting cervical precursor lesions, some of which would have progressed to cancer if not treated [53]. Despite its relatively high specificity and wide application in screening of cervical cancer, Pap smear is not very sensitive [53]. A study was conducted by Manga et al. [54] on comparative analysis of cervical HPV DNA testing and cytological Pap smear findings among women in tertiary health centre in northern Nigeria. It was observed that cytology had relatively high specificity but low sensitivity in detecting HPV infection, and hence suggested the introduction of HPV DNA testing to improve efficiency and maximize the sensitivity of cytology-based cervical cancer screening for women above 30 years. In another study carried out by Kolawole et al. [53] it was shown that high-risk HPV was present in 100% of cases of Pap smear samples with abnormal cytology, and noted that the use of HPV DNA technique would be an effective and rapid means of detecting HPV in cervical cytology specimens.
Wright et al. [54] reported that primary high-risk HPV screening in women older than 25 years is significantly more sensitive for detection of CIN III than either cytology or hybrid strategy, although this increase is associated with more colposcopies. However, a negative result of primary high-risk HPV screening offers better reassurance of low cancer risk compared to cytology-only screening conducted at the same interval [54].

Conclusion
This study has shown that HPV-DNA was prevalent in majority of the examined cervical cancer tissues and that HPV16, HPV18, HPV45, HPV51 and HPV52 were the predominant HPVs detected in both single and multiple HPV infections. The results of this study and further studies will provide more detailed information about HPV and may contribute significantly to the prevention of cervical cancer through the currently available HPV vaccines and HPV DNA-based screening tests against the oncogenic viruses.

What is known about this topic
• Strong association between cervical cancer and high-risk HPVs; • High-risk HPV types 16 and 18 are the most prevalent oncogenic viruses worldwide; • Sensitive HPV-based tests and effective HPV vaccines have been developed.

What this study adds
• Being the first of its kind in Maiduguri and Northeast Nigeria, the findings of this study will serve as baseline data; • The observed high prevalence of HPV infection in cervical cancer along with the predominance of HPV16 and HPV18 is consistent with several studies worldwide; • The high proportion of multiple HPV infections observed in this study could be explained by the high rate of polygamous marriage in our environment.