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Prevalence of human papillomavirus type 16 in Sudanese women diagnosed with cervical carcinoma


1 Department of Zoology, University of Khartoum, Khartoum, Sudan
2 Department of Public Health, Communicable Diseases Control Programs, Ministry of Public Health, Doha, Qatar
3 Department of Virology, Central Laboratory, Ministry of Higher Education and Scientific Research, Khartoum, Sudan

Correspondence Address:
Elmoubasher Abu Baker Abd Farag,
Department of Public Health, Communicable Diseases Control Programs, Ministry of Public Health, P. O. Box: 42, Doha
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_656_18

 > Abstract 


Objectives: Squamous cell carcinoma (SCC) of the cervix is one of the leading causes of death in developing countries. Infection with high-risk human papillomavirus (HR-HPV) is the major risk factor to develop malignant lesions HR types (HPV16 and HPV18) account for about 70% of all invasive cervical cancers worldwide. It is estimated that 833 Sudanese women are diagnosed with cervical cancer and 534 die from the disease every year. The present study aimed to detect HPV 16, and determine the association of HPV16 with age and various grades of cervical carcinoma in patients with clinically confirmed cervical SCC.
Materials and Methods: A total of 158 formalin fixed paraffin embedded tissues blocks from Sudanese women diagnosed as cervical cancer and benign were collected between 2012 and 2016 at Omdurman Maternity Hospital and National Laboratories, Khartoum, Sudan. HPV DNA detection was done using HPV 16 specific primers in real-time polymerase chain reaction.
Results: The frequency of HPV 16 was identified among 10.34% (n = 6) and 6% (n= 6) women with abnormal cytology and normal cytology, respectively. Based on age, high prevalence rate of HPV 16 was observed among age group 61–70 in women with malignant cases. The degree of differentiation, an important classification in SCC cases revealed that 5% = 3) cases had moderately differentiated SCC and two of them were keratinized SCC. In addition, 3.4% (n = 2) SCC cases were keratinized and well differentiated.
Conclusion: Overall, the prevalence of HPV types 16 was higher but had no significant association with cervical SCC in Sudanese women.

Keywords: Cervical cancer, human papillomavirus type 16, real time-polymerase chain reaction, squamous cell carcinoma, Sudan



How to cite this URL:
Elhasan LM, Bansal D, Osman OF, Enan K, Farag EA. Prevalence of human papillomavirus type 16 in Sudanese women diagnosed with cervical carcinoma. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 21]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269748




 > Introduction Top


Cervical cancer, a second most prevalent cancer of women, is a major public health problem in African and Asian countries with annual incidence of >550,000 new cases and approximately 270,000 deaths worldwide.[1],[2],[3] It is well established that human papillomavirus (HPV), a common sexually transmitted virus, is one of the main causative agent for cervical cancer.[4] In addition, high-risk (HR) HPV genotypes have been reported as major risk factor of cervical cancer and its precursors.[4] It is the most common cancer among women in Asia and Africa.[3] In developed world, cervical cancer frequency and mortality rate is 9.0% and 3.2%, respectively, which increased two to three fold (17.8% and 9.8%, respectively) in developing countries.[3] Significantly lower incidence and mortality rates in the developed world might be due to successful implementation of cervical cancer screening programs include cytological (Pap smear) and HPV DNA testing.[5] However, the current screening methods have limitations particularly difficult to detect early morphological neoplastic changes as a result reduces its sensitivity. In developing countries, where majority of new cases occur, survival rates are extensively lower as a result of diagnosis at advanced stages.[1],[6] Since up to 90% cases are curable with early detection, therefore, it is important to diagnose cervical cancer at an early stage.[7],[8]

Cervical cancer is the second most diagnosed cancer and leading cause of death among women in Sudan, with more than two-thirds of all women with invasive cervical cancer being diagnosed at an advanced stage.[9] The reasons for this may be attributed the lack of organized screening program, but other factors potentially associated with advanced stages of cervical cancer at diagnosis are remain unknown.[10] It was shown that most cervical cancers in Sudan are squamous cell carcinoma (SCC) (90.9%), followed by adenocarcinoma (4.8%) and other epithelial tumors (4.3%). Of the SCC, 98.8% were invasive and 1.2% intraepithelial (cervical intraepithelial neoplasia).[11]

Identification of HPV types in communities provides baseline information, which is essential component in both the development and evaluation, as well as in the effective implementation and monitoring of HPV vaccination programs. Therefore, the present study was aimed to screen the presence of HPV16 in formalin-fixed paraffin embedded (FFPE) tissues from SCC cases in Sudanese women, evaluate association of HPV16 with cervical SCC and investigate the association of HPV16 with various degree of differentiation of SCC.


 > Materials and Methods Top


Study area, design and sample collection

This is a case control study with a total of 158 FFPE cervical SCC or benign blocks obtained from patients diagnosed at the department of histopathology, Omduran maternity hospital and National Laboratories, Khartoum, Sudan between years 2012 and 2016. This study was conducted according to the principles expressed in the Declaration of Helsinki. This study was reviewed and approved by Research Committee of department of zoology, faculty of science, university of Khartoum, Khartoum, Sudan.

Subjects were enrolled irrespective of race, religion, socioeconomic status, and geographic distribution. Clinical and sociodemographic data such as: Age, clinical information, tumor differentiation, and diagnosis were obtained from the patient's records.

Histopathological analysis

FFPE specimens were sectioned using microtome (Microtome Leica, LECA RM2125RT) and examined under the light microscope for hematoxylin and eosin staining by experienced histopathologist. The morphological alterations (keratinized and nonkeratinized) of SCC were recorded using the recommended classification and assessed histologically for the degree of differentiation. To prevent potential cross contamination of tissue during sample sectioning the microtome blade cleaned with HCL.

DNA extraction and genotyping of human papillomavirus 16

For all the samples included in this study, DNA was extracted from each sample (40 μm sections) using guanidine chloride method after samples deparaffinization as previously described[12] and stored at −20°C till further use. To determine the presence of HPV type 16 primers specific for the E6 gene (forward 5'-ATTAGTGAGTATAGACATTA-3' and reverse 5'-GGCTTTTGACAGTTAATACA-3') was used as previously described.[13] Real time-polymerase chain reaction (PCR) assay was performed in T Professional thermo cycler (Biometra Anlytica Jena Co., German) using the following protocol: 10 μl of SYBR green mix (Real MOD Green GH qpcr iNtRON Biotechnology Co., Korea) along with 1 μL of (5 pmol/μL) each of forward and reverse primers, 2 μL extracted DNA and the mixture was made up to 20 μL volume with nuclease free water. The PCR amplification was initiated at 94°C for 20 s and followed by 35 amplification cycles (denaturation at 94°C for 20 s, annealing at 52°C for 1 min and extension at 62°C for 30 s).

All the samples were analyzed in duplicates on PCR tubes. A positive control (a samples which was positive by Fluorescence Diagnostic (Fluorescence Probing) kits, San sure Biotech Co, China) and a negative control (nuclease free water) were included in each amplification reaction. HPV 16 positive samples were detected by analyzing the amplicon dissociation curve and samples showing a melting temperature (Tm) at 52°C was considered as positive.

Statistical analysis

Statistical analysis was performed using IBM SPSS Version 16.0. Chicago, SPSS Inc. The P values, confidence interval (CI) and odd ratios (OR) were calculated for cases and control to determine the prevalence of HPV16. A P< 0.05 (OR = 95% CI: 0.948–1.139) was considered statistically significant.


 > Results Top


Demographic profile and clinical characteristics of the study population

This study includes 58 cases of cervix tissues and 100 control tissues from Sudan. The age distribution among cases and controls was relatively similar and age range was 29–93 years with a mean age of 54.7 (±12.5) years. The studied population had different clinical symptoms such as postmenopausal bleeding, supra pubic pain, vaginal bleeding, fungating cervical mass, burning micturition, lower abdominal pain, offensive watery vaginal discharge, and Bleeding on Touch [Table 1].
Table 1: Demographic, clinical profile and prevalence of human papillomavirus 16 among different age groups of cases and controls Sudanese women

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Human papillomavirus type 16 prevalence in squamous cell carcinoma cases and controls

The overall HPV 16 prevalence in cervical cancer and noncancerous samples were 10.3% (n = 6/58) and 6% (n = 6/100), respectively. Furthermore, HPV 16 prevalence was found to be highest (20%, n = 2) in age group >70 years among women diagnosed with cervical carcinoma, however, among women with normal cytology HPV 16 prevalence was observed higher in the 61–70 age group (15%, n = 3). HPV 16 prevalence appeared to increase slightly with age in SCC cases, while no such trend was seen in control cases [Table 1].

The relation between presence of human papillomavirus 16 and degree of cell differentiation in cases

The degree of differentiation, an important classification in SCC cases revealed that 5% (n = 3) cases had moderately differentiated SCC and two of them were keratinized SCC [Figure 1]. In addition, 3.4% (n = 2) SCC cases were keratinized and well differentiated [Table 2].
Figure 1: Representative pictures of Hematoxylin & Eosin (H&E) in squamous cell carcinoma (SCC), (a) non-keratinizing SCC (H&E) and (b) keratinizing well-differentiated SCC (H&E)

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Table 2: The relation between presence of human papillomavirus 16 and degree of cell differentiation (keratinized and nonkeratinized) of squamous cell carcinoma cases

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Furthermore, Chi-square test was used to analyze the association between HPV16 infection and cervical SCC, however no significant association was found in HPV16 infection versus cervical carcinoma.


 > Discussion Top


HPV is a sexually transmitted infection and its consequence cervical cancer is the most common cancer among women globally. Cervical cancer is well known for its high mortality and morbidity worldwide, as well as, its link to HR-HPV etiology was well established.[14]

In the present study, 10.3% and 6.0% samples were found to be positive for HPV16 in women with abnormal (SCC cases) and normal (benign or control cases) cytology, respectively. Although significant regional variability in the prevalence of HPV seen even in regions of close proximity and common ancestry, which may be due to differences in sexual and sociocultural norms.[15] The prevalence of HPV varied from 1.4% to 41.9%,[16] however the relatively low prevalence of HPV 16 infection found in our study compared to previous reports from Sudan,[17] Africa[18] and globally.[11],[19],[20],[21],[22] The reasons for these variations might be due to quality of the specimens tested and the sensitivity of the HPV detection assay used in this study.

HPV type distribution in Sudan appears to differ from that in other countries and not associated with cancer. The current study detected HPV 16 in 10.3% of SCC cases which is lower than previously reported globally.[23],[24] In addition, HPV16 incidence was not significantly associated with clinical stage and degree of differentiation in the present study; hence it cannot be a determinant of the tumor behavior in cervical carcinoma in Sudan.

In regard to the relationship between age and HPV infection, high frequency of HPV16 infections were identified in the age group with >70 years, which is in contrast with previous reports from Sudan, where prevalence was found to be highest in age group 31–45 years.[17] However, the flat distribution of HPV was reported in EMENA.[25],[26],[27],[28],[29] Among the previous studies, HPV 16 and 18 prevalence has been reported higher in women aged ≥45 compared to women with age group ≤44, the reason of lower prevalence may be due to the conservative nature of the Sudanese society.[11]

The integration of the E6 gene of the HPV 16 has cohesive within the host DNA. It is well accepted that DNA from FFPE tissue lacks large fragments of sufficient integrity, which allow for a robust PCR amplification, demonstrated that DNA isolated from FFPE tissue itself exhibits an inhibitory effect on PCR leading to an instable amplification. The small DNA fragments from FFPE tissue might either bind to the active site of the polymerase, which is responsible for binding the template DNA, or to the site which binds to the dNTPs.[30]

In the present study, HPV 16 infection was found in 3 cases characterized by moderately differentiated SCC representing 5.0% (n = 3/58), two of them were keratinized SCC. Additionally, 3.4% cases (2/58) were keratinized well-differentiated SCC. Late tumors stage has been reported in unscreened populations, however if these risk populations are regularly screened, the mortality might be reduced. Furthermore, it has been well reported that regular screening of HPV using Pap smear reduce HPV incidence among women worldwide,[31] hence it would also be effective in a low resource country like Sudan. Cervical cancer screening methods such as Pap smear has saved multitudes of lives in developed countries. However, this is not the case in developing countries, because of the nature of society and low level of cervical cancer awareness.[32] Additionally, the HPV16 infection was associated with moderately differentiated SCC including keratinized SCC. In addition, the keratinized well-differentiated SCC cases were associated with advanced stages, which can be attributed to the lack of public awareness regarding cervical cancer screening programs.

One of the limitations of the present study is the small sample size, as well as, screening for HPV 16 only because of the limited resources. The long-term studies with large sample size are required further to identify types of HR HPV to help in introducing of vaccines.


 > Conclusion Top


The prevalence of HPV types 16 was higher in cervical SCC in Sudanese women but low in comparison to other African countries and globally. In this study, high rate of infection with HPV 16 was observed at age >70 years representing (20%) of malignant cases, while high rate of infection observed in age group 61–70 representing (15%) in control. Contrary to our expectations, no statistically significant association was found between HPV16 infection and cervical SCC. The baseline information about the HR-HPV infection among Sudanese women will be helpful for policy makers in making an informed decision regarding introduction and implementation of HPV vaccination in Sudan. Finally, further study with large sample size is required to get conclusive results on the situation of HR-HPV infection, study on HPV16 variants are recommended to determine which variant associate with the disease and also E6 gene in HPV16 should be sequenced in Sudanese population in order to identify new SNPs could be associated with SCC. In addition, the cervical cancer-screening program can also be benefit from the adequate knowledge and practice of physicians on cervical cancer in Sudan.

Acknowledgements

We would like to thank Elhassan Elray'ah, and Abdalla Khalaf for their technical help in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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