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Table of Contents
ORIGINAL ARTICLE
Year : 2015  |  Volume : 2  |  Issue : 2  |  Page : 92-99

Detection of HPV and its association with different known risk factors for neoplastic cervical lesions


1 Demonstrator, Department of Pathology, Fatima Jinnah Medical College, Lahore, Pakistan
2 Professor of Pathology, Department of Pathology, Fatima Jinnah Medical College, Lahore, Pakistan
3 Professor of Pathology, Department of Pathology, Gujran, Wala Medical College, Gujran Wala, Pakistan
4 Professor of Medicine, Fatima Jinnah Medical College, Lahore, Pakistan
5 Research Student, Department of Molecular Cell Biology, National University of Science and Technology, Islamabad, Pakistan
6 Associate Professor, Department of Molecular Cell Biology, University of Health Science, Lahore, Pakistan
7 Professor and HOD, Department of Molecular Cell Biology, National University of Science and Technology, Islamabad, Pakistan
8 Department of International Research Scholar Epidemiology and Preventive Medicine, Graduate School of Medicine and Dental Science, Kagoshima University, Kagoshima, Japan

Date of Web Publication5-Jul-2017

Correspondence Address:
Mulazim Hussain Bukhari
Professor of Pathology, Department of Pathology, Fatima Jinnah Medical College, Lahore
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.5530/ami.2015.3.9

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  Abstract 


Objective: Cervical carcinoma is one of the most common diseases in our setup studies show that it is preceded by precursor lesions. It has been suggested that persistent infections with human papillomavirus (HPV) is the major risk factor in the development of this invasive cervical neoplasia. The rationale of this study was, whether detection may contribute to the identification as a major risk factor in cervical neoplastic lesions. Study Design: Experimental study. Methods: 102 cases were selected after screening 1000 specimens through Papanicolaou stains of cervical cytology and histopathology for detection of HPV and its subtype PCR. Data for risk factors were collected by a questionnaire and association of HPV was seen with Positive PCR results. Patient demographics including their age, sexual partners, marital status, socioeconomic condition, contraceptive and screening history were evaluated to determine whether subsidiary risk factors are associated with HPV and the development of cervical lesions among Pakistani women. Major Outcome: 85% cases of cervical carcinoma were associated with high risk HPV infection. Results: 46/102 (45%) cases were low grade squamous cell intraepithelial lesions(L-SILs),twenty two (21.5%) cases were high grade squamous cell intraepithelial lesions (H-SILs), 14(13.7%) cases were squamous cell carcinomas(SCC), 6 (5.8%) cases showed features of adenocarcinoma, 10(9.8%) cases showed cytology of atypical squamous cells of undetermined significance(ASCUS) and 4(3.9%) cases were of atypical glandular cells of undetermined significance(AGUS). Out of 102 cases, 88/102(86.27%) were positive for HPV and among them 32/88 (36%) cases were of HPV-16 and 56/88(64%) cases of HPV-18. There was strong association of HPV positivity with young age, early marriages, poor socioeconomic condition, abortions, multiparity and smoking but there was no association with multiple marriages. Conclusion: Frequency of HPV-18 was greater than HPV-16 in cervical neoplastic lesions and was strongly associated with certain known risk factors for cervical carcinoma.

Keywords: Papanicolaou smear, Squamous Cell Intra Epithelial Lesion, Polymerase chain reaction, Human Papilloma Virus


How to cite this article:
Saba K, Bukhari MH, Khaleel E, Imam SF, Zainab M, Siddiqa A, Ahmad G, Qadri I, Anwar M. Detection of HPV and its association with different known risk factors for neoplastic cervical lesions. Acta Med Int 2015;2:92-9

How to cite this URL:
Saba K, Bukhari MH, Khaleel E, Imam SF, Zainab M, Siddiqa A, Ahmad G, Qadri I, Anwar M. Detection of HPV and its association with different known risk factors for neoplastic cervical lesions. Acta Med Int [serial online] 2015 [cited 2019 Aug 17];2:92-9. Available from: http://www.actamedicainternational.com/text.asp?2015/2/2/92/209661




  Introduction Top


Cervical pathology represents the most frequent cause of mortality and morbidity with regard to female genital diseases but is also an important health problem. According to a practice bulletin (2002) of American Congress of Obstetricians and Gynecologists, cervical carcinoma is estimated to be the second most common cause of worldwide cancer death in females.[1],[2] It is also a leading cause of death in middle aged females in developing countries causing a three-quarter of the global burden from this dreadful disease.[3],[4],[5],[6],[7] Cervical carcinoma is becoming a burden in Pakistan and has become the fourth most common cancer among Pakistani females after breast, head and neck cancer.[8],[9] The incidence is rising, according Bhurgri,Y, Cancer cervix ranked sixth in the 1995-97 period the age standardized incidence rate (ASR) world and crude incidence rate (CIR) per 100,000 were 6.81 and 3.22. It reached the fifth ranking in the 1998-2002 period with an ASR and CIR of 7.5 and 4.0 per 100,000 and now cervical cancer has become the third leading cause of cancer death amongst women in Pakistan.[10],[11]

Although the incidence of cervical cancer in Pakistan is lower than that in various western countries, yet the mortality is higher. This higher rate of mortality is attributed to the late presentation of cervical cancers in Pakistan.[12]

In the past decade, a strong etiologic association between infection with high-risk HPV types and development of cervical cancer has been established, and vaccines targeting HPV 16 and 18 have been shown to prevent persistent HPV infections in clinical trials.[2],[13] For the effective screening method of cervical lesions, the Papanicolaou cervical cytology is used to detect premalignant and malignant conditions. It is included in the screening program for evaluation of all patients with abnormal cervical smears in developed countries. This test is adopted because it is a simple and cost-effective technique for the detection of early changes in the cervical epithelium. Unfortunately, many developing countries lack the ability to carry out widespread Pap screening.[14],[15],[16],[17]

According to Centers for Disease Control and Prevention, Cervical cancer mortality in the United States has decreased over the last five decades by 70%, largely as a result of the introduction of the Papanicolaou (Pap) test.[18]

The study was conducted to detect HPV by PCR and to see any association of different known risk factors for neoplastic (premalignant and malignant) cervical Lesions.


  Materials and Methods Top


Study Design

This study was conducted in two phases. In one phase a cross sectional study was conducted on 1000 females to screen neoplastic lesions of the cervix. (The details of the study are specified elsewhere). In second phase experimental study was conducted on the lesions selected from phases one.

Study Universe and Study Population

Sample size was calculated according to the prevalence of HPV in Pakistan and 102 cases were selected out of 1000 Papanicolaou (Pap) smears of cervical cytology, who attended the Gynecology Clinics of Lady Wellingdon Hospital and Lady Aitchison Hospital Lahore, Pakistan, from January 2008 to June 2009.

Criteria of Study Population

Patients coming to Lady Wellingdon Hospital and Lady Aitchison Hospital Lahore from all areas of Pakistan.

Data Collection

Cases of premalignant and malignant cervical lesions were selected according to the Bethesda System 2001.[19] These cases of cervical lesions were followed for biopsy (punch biopsy, conization and hysterectomies in 29, 34 and 39 patients respectively).

Patient's Interviews

The study was conducted according to the standardized protocol of the International Agency for Research on Cancer (IARC) HPV Prevalence Surveys.[20] All participants signed an informed consent form (Urdu and English) and were administered a questionnaire. Ethical approval of ethical principles for medical research for human's subjects for the study was received from the Review board of ethical committee of the University, Lahore.

Inclusion Criteria

  1. Cases suspicious for malignancy on Pap smear by Bethesda System 2001.
  2. All malignant cases on Pap smears by Bethesda System 2001.
  3. Females age range 20-70 years.


Exclusion Criteria

  1. Virgin females
  2. Infective smears.


Selection Criteria for Control

20 biopsies from normal hysterectomies with normal cervix. The biopsies were randomly selected from Lady Aitchison and Lady Weligdon hospitals

Methodology for Epidemiological Components and Data Collection

This research project was planned to study the epidemiology of various types of gynecological lesions (infective, premalignant and malignant) cancer among population of Pakistan. A descriptive study based on passive surveillance system was planned. The data of patients were collected from the Lady Aitchison and Lady Weligdon Hospital two major teaching Hospitals of Lahore. To meet the objective of this study we took data from the patients coming to these hospitals for any gynecological problem. (From January 2008 to June 2009). All the information were collected through a specially designed questionnaire. Information of the patients was collected from their respective registered files. The questionnaire included information on demographic characteristics (like name, age, gender, residence and city, occupation, marital status), date of presentation, diagnosis and type of lesion. The information of those patients was collected whose diagnosis was confirmed through cytopathology and histopathological reports of the hospital laboratory. All the data and information was collected by researcher herself to minimize the bias in the study.

Analytical Procedures

Pap smear samples were taken from those patients who were not menstruating and were 10-20 days after first day of menstrual cycle. They had not douched or used tampons or vaginal medication for at least 24 hours before the Pap test was done. Cases were analyzed for HPV by PCR. Twenty normal cervical biopsies (operated for some other reasons) were included as control

Diagnostic Criteria

The smears were fixed immediately with methanol and stained with Papanicolaou stain. A qualified Cytopathologist examined the pap stained slides which were then were confirmed on H&E by a Histopathologist. Their observations were compared independently without informing the reporting consultants to enhance the reproducibility. All the smears were classified according the Bethesda method 2001. Normal and infective smears were excluded from this study[21]. Biopsies of these samples were obtained and confirmed by H&E stain

Use of PCR for Detection of HPV

There were 102 specimens which were screened through cervical smearing through Papanicolaou stain and histopathological examination by H&E stained and 20 control cases (cervical biopsied which were histopathologically negative for cervicitis, dysplasia and malignancy) were followed for detection of HPV by PCR. 20 to 60 μm thick sections from each formalin-fixed paraffin-embedded tissue samples were prepared. The samples were added with 1 ml of xylene, and then with 1 ml of ethanol. After centrifugation, the pellet was dried and resuspended in digestion buffer (100mM Tris HCl, 0.05% tween 20 pH=7.5) containing 200 μg of Proteinase K (Invitrogen Corp., USA) and incubated for 24 hours at 56°C. The solution was heated at 90°C for 10 min and centrifuged at 10,000 rpm for 2 min. The supernatant was then purified by using Qiagen DNA purification kit. HPV amplification with GP5+/GP6+ primers was performed in a reaction mixture that contained 5 μl of template DNA, 200 μM dNTP, 0.5 μM of each primer and 1.0 U Taq DNA polymerase in a total volume of 25 μl of reaction buffer (50 mM KCl, 20 mM Tris-Cl, pH 8.3). The amplification result of a beta globin fragment with a set of PCO3/PCO4 primers was used as internal positive control.[22] The PCR program was as previously reported: initial denaturation at 94°C for 4 min; subsequent 35 cycles at 94°C for 30 sec, 54°C for 40 sec and 72°C for 30 sec; final extension at 72°C for 5 min. For Genotyping, the type specific primers were used i.e. TS16 and TS18. The PCR program for HPV type 16 was as follows: initial denaturation at 94°C for 4 min; subsequent 35 cycles at 94°C for 30 sec, 54°C for 40 sec and 72°C for 30 sec; final extension at 72°C for 5 min. The PCR program for HPV type 18 was: initial denaturation at 94°C for 4 min; subsequent 35 cycles at 94°C for 30 sec, 58°C for 40 sec and 72°C for 30 sec; final extension at 72°C for 5 min.

All reactions were carried out in Eppendorf master cycler in 20 μl reaction volume. Master mixtures were prepared containing, 200 μM dNTPs (each), 1 μM forward and reverse primers (each) and 1 unit of Taq DNA polymerase (Fermentas). Master mixture was pipetted in 0.2 ml PCR tubes (Thermo scientific) and 100-200 ng of template DNA was used for each reaction. For the amplification 100 ng template was used. Positive and negative controls were used to control the specificity and reliability of the PCR. The reactions were ended with a final extension of 7 minutes at 72°C and kept on hold at 4°C until tubes were taken out of the cycler.

Agarose gel electrophoresis was carried out to analyze the amplified DNA samples. 1.5 % (w/v) agarose gel was prepared by melting 0.6 gram of agarose in 40 ml of 1 X TBE buffer (0.89 M Tris-Borate, 0.032 M EDTA, pH 8.3) in microwave oven for two minutes. 6μl ethidium Bromide solution (10 mg/ml) was added to stain DNA. Amplified DNA samples were mixed with loading dye (0.25 % bromo-phenol blue prepared in 40 % Glycerol solution) and loaded into the wells. Electrophoresis was performed at 90 volts for 20 minutes in 1X TBE running buffer. After electrophoresis amplified product was detected by placing the gel on UV Transilluminator (Biometra, Germany).

Statistical Analysis

Data was entered and analyzed through SPSS version 16 by researcher herself. Quantitative variable like age was presented in form of mean ± SD with respect to type of cancer and year. The qualitative variable like gender, type of cancer etc were presented in form of frequency tables and appropriate charts.

Simple percentage of the positive lesions was calculated and compared with histopathology reports. The accuracy rate, sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, and false negative rate were calculated by definition. The chi-square test was performed for comparative analysis and the results were considered statistically significant at p-values <0.05. Fisher exact test was used to determine the statistical significance of the difference.


  Results Top


There were 102/1000 (10.2%) neoplastic cases diagnosed initially from Pap stained cervical smears and then by H&E through biopsies. The age ranged between 20 and 70 years with mean age of 47.43±12.21 years. The peak frequency of pre-malignant and malignant cervical lesions (62/102; 60.8%) were seen in postmenopausal females but HPV infection was more common in premenopausal women (38/40; 95% versus 50/62; 80%) [Table 1],[Table 2].
Table 1: Cytological findings in Papanicolaou stained cervical smears of 1000 cases

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Table 2: Association of HPV with known risk factors of cervical carcinoma

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On cytological examination of Pap smears, there were 10 (9.8%), 4 (3.9%), 46 (45%) and 22 (21.8%) cases of ASCUS, AGUS, LSILs and HSILs respectively while 14(13.7%) cases were of Squamous Cell Carcinoma and 6/102 cases (5.8%) of adenocarcinoma. [Table 1] and [Figure 1]. Histological examination revealed 10 benign, 72 premalignant and 20 malignant cases [Figure 1],[Figure 2],[Figure 3]A,B [Table 3].
Figure 1: Photomicrographs (Pap stain 40x) of a: High grade Squamous intraepithelial lesion (HSIL showing parabasal type cells, moderate to severe hyperchromasia, High N:C ratio,nuclear membrane irregular and Coarse chromatin, b: Histopathology of this case showing dysplasia grade III and c: PCR positive for HPV 18.

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Figure 2: a: Photomicrograph of Pap smear of squamous cell carcinoma large cell type (non keratinizing) showing Syncytial aggregates of cells, round to polygonal cells, ill defined cell borders, Irregular nuclei, Coarsely granular chromatin and Dirty background, cellular debris, b: Histopathology of this case of Poorly differentiated SCC and c: showing positive HPV-16

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Figure 3: Photomicrographs (Pap stain 40x) of a: Squamous carcinoma (keratinizing ), showing Dirty hemorrhagic background (Tumor diathesis), marked variation in cell size and shape,elongated cells,moderate to marked hyperchromasia and high N:C ratio. b: Histopathology of Keratinizing SCC. c: This case was positive for HPV-16 and 18.

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Table 3: Association of HPV from different Cervical Lesions detected by Cervical Smears and Histopathology

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Out of 102 cases, 88/102 (86.27%) were positive for HPV and among them 32/88 (36%) cases were of HPV-16 and 56/88(64%) cases of HPV-18. There was strong association of HPV positivity with young age, early marriages, poor socioeconomic condition, abortions, multiparity and smoking but there was no association with multiple marriages. Out of 10 benign cases, HPV was positive in 7 cases (HPV 18 in 2 and HPV 16 in 5 cases) while out of 72 premalignant cases, there were 64 positive cases of HPV (40 cases were positive for HPV 18 and 24 cases for HPV 16). Out of 20 malignant cases, 17 cases were positive for HPV and among them 14 cases were of HPV-18 and 3 cases of HPV-16 [Table 3],[Table 4],[Table 5], [Figure 1],[Figure 2],[Figure 3]C.
Table 4: Comparison of HPV with compared to normal biopsies

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Table 5: Relative risk with 95% CI as compared to control

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The HPV infection was seen in 38/40 (95%) females in reproductive ages and 50/62 (80%) cases in postmenopausal ages. 50/57 (97%) females gave history of early marriage, and 67(97%) cases were multiparous. HPV infection was seen in females with history of multiple abortions [Table 2].


  Discussion Top


It is important to have knowledge about the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer for the introduction of prophylactic vaccines program. The worldwide genotype distribution in patients with invasive cervical cancer provides the comprehensive data that high-risk HPV-DNA-based screening tests and protocols should focus on HPV types 16, 18, and 45 which are the most prevalent infections worldwide.[23] In Pakistan, like other developing countries, cytology screening by Pap-test is an important procedure used to control cervical malignancy; however organized screening programs are rare. In spite of having these facilities in our clinical centers, the incidence of invasive cervical carcinomas still remains high, especially in low socioeconomic patients. The failure of cytological testing is likely due to a number of factors, like poor infrastructure, lack of trained health professionals and cytotechnicians, absence of organized community-based-screening-programs and inadequate follow-up of abnormal smears.

In our study, out of 102 positive cases, 14 women (13.7%) had clear cut Squamous Cell Carcinoma (invasive) while 6 cases revealed morphology of an adenocarcinoma. 10 (9.8%), 4 (3.9%), 46 (45%) and 22 (21.8%) cases of ASCUS, AGUS, LSIL and HSIL respectively.

Epidemiological studies have clearly shown a causal association between tobacco exposure and cervical cancer.[24],[25],[26] but in our study smoking was not a common finding. Malignancy of the cervix is strongly associated with sexual behavior, and the risk of acquiring cervical cancer and its precursor lesions increases with the number of sex partners, early age at first intercourse, increasing parity, and cigarette smoking. Low dietary intake of vitamin A and long-term use of oral contraceptives have also been suspected of elevating the risk of SIL and cervical cancer. Multiparity (women having more than 2 children) and early age at first intercourse were common in our study and were seen in 67% and 55.8% of the cases of these cervical lesions. The findings are consistent with Burkett et al and Kjaer et al, who observed positive correlations of all these risk factors with cervical neoplasia.[27],[28],[29] An interesting association was seen for HPV and some risk factors in our community. Age, socioeconomic status, early marriage, abortions and parity was directly associated with frequency of HPV in neoplastic cervical lesions. [Table 2]

The overall frequency was 88 (86%). 56/88 (64%) cases were positive for HPV 18 while 32/88 (36%) cases were positive for HPV-16. Other subtypes could not be identified since the kit used in the study had probes of 16&18 only. Four cases showed co- infections with both HPV 18 and HPV 16.

Only a few studies have reported HPV genotypes in cervical cancer patients in some other regions of Pakistan. In a study, Yousuf et al (2010) detected HPV in nine (18%) out of fifty paraffin embedded tissues of squamous cell carcinoma of cervix. Out of the 9 cases that were positive, five (55.6%) were infected with HPV-16.[30]

In another study by Khan et al (2007)[31] HPV was detected in 98% cases and HPV 16 was positive in 93% cases while only one case was positive for HPV-18. In a similar study by Anwar et al (1991), HPV-16&18 were detected only in 17% of the non-neoplastic specimens and in 69% of cervical cancers from Pakistan.[32]

The present study indicated that HPV-18 is very common among all positive samples but there could be other causes for squamous cell carcinoma in women tested or probably a different subtype of HPV might be prevalent in Pakistani population, therefore our findings are different from the above three studies in Pakistan. The reason could be geographical distribution of HPV as the present study was conducted at Lahore while all other studies were conducted at Karachi.

Only 7/20 (35%) cases showed positivity for HPV in control biopsies as compared to 88/102 (92%) diseased cases in malignant cases, therefore the difference was highly significant from the malignant biopsies (Relative risk≈2.5, Odd ration≈11 and p=0.0000056). The infection was more common in SCC as compared to ADC which is statistically highly significant (P=0.0020) [Table 4].

The frequency is different from Japanese data, where HPV positivity has been reported as 68% positive for HPV.[32] The present study findings are also inconsistent from the study of Bulk et al (2006), who found that HPV18 is mainly a risk factor for the development of adenocarcinoma whereas HPV16 is associated with both SCC and adenocarcinoma.[33] When the findings of the present study were compared to the neighboring country India, the results were almost similar and the only difference was in genotypes. They found 87.8% high-risk HPV types in (HPV 16 in 66.7% and HPV 18 in 19.4%) cervical squamous cell carcinomas.[34]

It has been seen in a recent study that, the prevalence of HPV in the general population is 2.8%, with no evidence of higher HPV prevalence in young women. The positivity of HPV was associated with women's lifetime number of sexual partners, but particularly with the age difference between spouses and other husbands' characteristics, such as extramarital sexual relationships and regular absence from home.[35]

Association of HPV was also studied with certain known risk factors for cervical carcinoma and it was found that there was strong association of HPV positivity with young age, early marriages, poor socioeconomic condition, abortions, multiparity and smoking but there was no association with multiple marriages. Our findings are consistent with others for some co factors like age, socioeconomic status, education, early marriage, parity, and smoking but not for multiple sexual partners (multiple marriages)[36] Studied have shown that HPV may interact with certain co factors like smoking and it may induce impaired antibody response in HPV16/18-infected young women.[37],[38] It has also been seen that high HPV concentrations combined with smoking significantly raise risks of cervical cancer.[39]

Age is also an important co factor for HPV infection. In our study, the HPV infection was more common in females below 49 years in a reproductive period of life as compared to postmenopausal life. In another study it has been seen that HPV infection was more common in young age group and in females with younger age at first sexual experience and first pregnancy, and more pregnancies.[36]


  Conclusion Top


Neoplastic lesions of cervix are not uncommon in Pakistan. The conventional Pap test is still the most commonly used modality for screening, diagnosing and surveillance of cervical lesions. HPV -16 and 18 were the aetiogical risk factor for development of this cervical cancer, triggered with some additional co-factors that may promote the oncogenic potential of HPV infection. HPV 18 was more frequent as compared to HPV-16. There was strong association of HPV with certain known risk factors for cervical carcinoma like young age, poor socioeconomic condition, early marriage and multiparity.

Article Focus

  1. Detection of HPV from cervical tumors in Pakistan
  2. Association of risk factors with HPV
  3. Hypothesis: HPV may be responsible for causing of cervical carcinoma and may be associated with certain other known factors as a co factor.


Recommendations

  1. All married females above 30 years should be screed for HPV by Pap smears.
  2. Prevention of HPV includes, the same recommendations as for prevention of other sexually transmitted diseases like monogamous relationships, use of condoms, limiting the number of sexual partners, early marriage and adaption of regular screening program.
  3. All possible routes of transmission (contact with infected urogenital secretions from sharing towels or bathing together, vertical transmission etc) should be avoided to prevent HPV infection other than sexual transmission
  4. Vaccination programs should introduced.


Strength and Limitations of study

  1. Small data, could not conduct cohort study due to budget constraints and economic problems.
  2. The main limitation were limited numbers of samples (102), small size of control, population was randomly selected and was not well defined and electrophoresis results of amplicons were not confirmed by specific hybridization because we used already published PCR primers sequences for GP5/GP6.
  3. Strength of this study is prevalence of High Risk HPV in our sample.


List of Abbreviations

SCP: Squamous Cell Papilloma, SCD: Squamous Cell Dysplasia, HPV: Human Papilloma Virus, SCC: Squamous cell carcinoma, ADC: Adenocarcinoma. IHC: Immunohistochemistry, PCR: polymerase chain Reaction, WDSCC: Well differentiated Squamous cell carcinoma, MDSCC: Moderately Differentiated Squamous Cell carcinoma, PDSCC; Poorly differentiated Squamous cell carcinoma.

Competing Interest

The authors declare that they have no competing interests.

Funding Statement

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors'.


  Authors Contribution Top


MBH: Conceived and Designed the study, MA: Performed analysis on HPV- R-PCR, MZ: Performed Genotyping of HPV, GA: Supervised PCR, IQ: Supervised PCR, FA: Clinical data analysis, AA: analysis of data by IHC, AH; Biostatistics.


  Acknowledgement Top


The authors are grateful to Professor Muhammad Munir (Chairman Department of Pathology) who provided moral support. They are also grateful to laboratory staff at King Edward Medical University and NUST Islamabad, Pakistan for their cooperation during the research work.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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