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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 54  |  Issue : 3  |  Page : 498-501
 

Prevalence of human papillomavirus in oral squamous cell carcinoma: A rural teaching hospital-based cross-sectional study


1 Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka, India
2 Department of Otorhinolaryngology and Head and Neck Surgery, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka, India

Date of Web Publication24-May-2018

Correspondence Address:
Dr. Sharath Balakrishna
Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijc.IJC_272_17

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 » Abstract 


BACKGROUND: Human papillomavirus (HPV) is a well-established oncogenic agent in the pathogenesis of cervical carcinoma. Its role in the oncogenesis of tumors such as oral squamous cell carcinoma (OSCC) is not clear. Globally, approximately 3% of OSCCs are positive for HPV. Studies conducted in India indicate its prevalence from as low as 0% to as high as 74%. However, a recent Indian study on leukoplakia failed to find any evidence of HPV involvement. This motivated us to reexamine the HPV status in OSCC. AIM: To evaluate the prevalence of HPV in OSCC. SETTINGS AND DESIGN: This was a rural teaching hospital-based cross-sectional study. SUBJECTS AND METHODS: Sixty histopathologically confirmed samples of OSCC were used for the study. Genomic DNA was isolated from frozen, surgically-resected specimens. HPV positivity was tested by polymerase chain reaction-based method using GP5+/6+ primers in the L1 consensus region of the viral genome. RESULTS: None of the samples were HPV positive. CONCLUSIONS: Results of this study indicate that the association between HPV and OSCC may be overestimated. Hence, multicentric studies covering diverse geographical and socioeconomic groups are needed to delineate the profile of HPV infectivity and OSCC in the Indian subcontinent.


Keywords: Human papillomavirus, India, oral squamous cell carcinoma, prevalence


How to cite this article:
Rajesh D, Mohiyuddin S M, Kutty A V, Balakrishna S. Prevalence of human papillomavirus in oral squamous cell carcinoma: A rural teaching hospital-based cross-sectional study. Indian J Cancer 2017;54:498-501

How to cite this URL:
Rajesh D, Mohiyuddin S M, Kutty A V, Balakrishna S. Prevalence of human papillomavirus in oral squamous cell carcinoma: A rural teaching hospital-based cross-sectional study. Indian J Cancer [serial online] 2017 [cited 2019 Dec 11];54:498-501. Available from: http://www.indianjcancer.com/text.asp?2017/54/3/498/233138





 » Introduction Top


Human papillomavirus (HPV) is a DNA virus that plays an important role in the pathogenesis of cervical carcinoma. The virus produces two proteins, namely E6 and E7, that bind and inactivate host cell tumor-suppressor genes p53 and Rb, respectively. The latter is crucial for the tight regulation of cell cycle. The strong association between HPV and cervical carcinoma motivated the development of HPV vaccine. More than 100 different strains of HPV have been identified, of which approximately 15 are high-risk strains with respect to oncogenicity. Globally, HPV 16 and 18 are the predominant high-risk strains seen in 70% of cervical carcinoma.[1]

The role of HPV in the development of head and neck cancers is not clear primarily because its prevalence is not nearly 100% as in cervical carcinoma. HPV incidence is not uniform in all head and neck cancers. Its global incidence in oral cancers is approximately 3%, whereas that of oropharyngeal cancers is approximately 12%.[2],[3] This site-based difference is attributed to sexual practices and addictions. The profile reported in India is, however, not consistent with the global statistics. In India, the incidence of HPV in oral cancers ranges from 0 to 74%, and 26 to 70% in case of oropharyngeal cancers.[4],[5],[6],[7],[8],[9] However, a recent study from Lucknow, India, found no evidence of HPV in leukoplakia.[10] This indicates that the prevalence of HPV in oral cancers may not be uniform across the country and may vary significantly in terms of demographic factors such as geographical region, socioeconomic category, and personal habits.

Knowledge of HPV profile in oral cancers is important to understand the epidemiology and oncogenetic mechanisms as these factors affect not only disease-prevention strategies but also treatment involving chemoradiotherapy. We conducted this study in a rural teaching hospital to evaluate the presence of HPV in oral squamous cell carcinoma (OSCC) using polymerase chain reaction (PCR) test in the L1 consensus region.


 » Subjects and Methods Top


Sample collection

Tumor specimens were obtained from 60 histopathologically confirmed OSCC patients of the Department of Otorhinolaryngology, R. L. Jalappa Hospital and Research Centre, Kolar, Karnataka. The study was approved by the University Ethics Committee. Demographic details such as gender, age, and personal habits (tobacco use, smoking, and alcohol) were obtained proforma at the time of admission. Clinical and histopathological details were collected from patients' medical records. Staging was done according to tumor, node, and metastasis staging system for OSCC.

DNA isolation

Resected tumor specimens were collected in saline and stored at −20°C until processing. Tissue homogenate of the specimen was used for the genomic DNA isolation using a previously published method with the following modification.[11] Tissue sample was minced and placed in a microcentrifuge tube to which 270 μl of 20% sodium dodecyl sulfate and 30 μl of 20 mg proteinase K were added. Contents of the tube with 3 ml of cell lysis buffer solution were incubated at 37°C until complete digestion of the tissue. After the tissue digestion, 500 μl 5M NaCl and 4 ml of isopropyl alcohol was used for DNA precipitation. Then, the resultant DNA was washed thrice with 70% ethanol, dried, and Tris–ethylenediaminetetraacetic acid buffer was added. Tissue homogenate was used as the source instead of peripheral blood. Quantity and purity of the preparation was estimated by ultraviolet spectroscopy.

Polymerase chain reaction

HPV DNA was amplified with GP5+/6+ primers [Table 1].[12] The 20 μl reaction mixture included 1X assay buffer, 100 ng genomic DNA, 0.2 mM dNTP, 1 picomole of each primer, 1.5 mM MgCl2, and 1-unit Taq DNA polymerase (Bangalore Genei, India). PCR program comprised an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 2 min, and extension at 72°C for 1.5 min, as well as a final extension 72°C for 5 min. Each PCR run included a negative control (sterile water in place of DNA) and a positive control (1:1 mix of genomic DNA of HPV 18-positive cervical carcinoma and SiHa cell line). SiHa cell line was obtained from the National Centre for Cell Science, Pune. Primers for HCP5 g ene were included as internal control.[13] PCR products were analyzed on 2% agarose gel.
Table 1: Primer sequences used for polymerase chain reaction

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 » Results Top


The demographic profile of the patients and the histopathological profile of the samples are summarized in [Table 2]. Patient's age ranged from 21 to 80 years and the mean age was 53.7 ± 12.7 years. Majority of the patients were female (75%) and were from lower socioeconomic class. All patients had one or more addictions to carcinogens (consumption of betel nut, chewable tobacco, gutka, smoking, or alcohol) with the mean duration of adverse habits being 20 ± 5 years. All patients were habituated to chewing betel tobacco and 3.3% of the patients were habituated to tobacco chewing, gutka, and smoking followed by alcohol (1.6%). Approximately 10% of the patients had type 2 diabetes. Burning sensation (86%) and trismus (13%) were the main complaints. Majority of the patients were of Stage IV (62%), 25% were Stage III, and 13% were Stage II. Squamous cell carcinoma was found to be well differentiated in 78% of the patients, moderately differentiated in 18%, and poorly differentiated in 3% patients. Mean duration of symptoms was 6 ± 4 months. Lesion size was 2–4 cm 2 in a majority of the patients (83%). A total of 17% of patients had lesion size > 4 cm 2. Buccal mucosa was the most commonly involved site (70%); the other sites included lower alveolus, tongue, retromolar trigone, and floor of mouth.
Table 2: Demographic and clinicopathological parameters in oral squamous cell carcinoma patients included in the study

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We tested a total of 60 DNA samples and failed to find a HPV-positive reaction in any. Approximately, 20% of the samples were randomly chosen and retested. The result of the second test was 100% concordant with the first test. Several measures were included in the assay design to ensure that the observed results were true and not false negative. First, a positive control comprising 1:1 mix of genomic DNA of HPV 18-positive cervical carcinoma and SiHa cell line was used during every run. SiHa (ATCC No. HTB35) is an immortalized cell line derived from cervical carcinoma. DNA of HPV 16 is integrated into the genomic DNA of SiHa cells. Second, an internal control was included to ensure that the negative results did not arise due to sample deterioration. Primers for HCP5 gene whose PCR amplicon is distinct from that of the test amplicon were included as internal controls [Figure 1]. Results were scored only after confirming that the positive control sample and the internal control added to each sample produced the required PCR amplicon. Furthermore, in an independent experiment, 10% of the samples were supplemented with the positive control DNA and analyzed by PCR. Supplemented samples showed positive result whereas unsupplemented samples were negative. This experiment confirmed that the samples did not harbor any substance or sequence that was inhibitory to the amplification by GP5+/6+ primers.
Figure 1: Representative agarose gel image showing polymerase chain reaction assay for human papillomavirus GP5+/GP6+. Lane 1: 100 bp ladder, Lane 2: Human papillomavirus positive control (with GP5+/GP6+ primer set), Lane 3: Human papillomavirus positive control (with GP5+/GP6+ primer set + internal control), Lane 4: Oral squamous cell carcinoma sample

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We used primers for GP5+/6+ sequences in the L1 consensus region of the viral DNA. This ensures that the test is generic and not strain-specific. Some previous studies used only primers specific for HPV 16/18. A negative result in such a test only rules out common high-risk strains and not the presence of rarer strains. The use of GP5+/6+ primers ensured that samples used in the study were completely HPV-negative.


 » Discussion Top


To our knowledge, this is the first study to report total absence of HPV in the DNA of OSCC from South India. A similar scenario was previously noticed in studies from India (Gujarat), Bangladesh, the Netherlands, and China.[4],[11],[14],[15] Survey of HPV status in cancer cases has assumed significance in the recent past owing to the increased responsiveness of HPV-positive cases to chemoradiotherapy. A number of clinical trials are presently being conducted to evaluate radiation dose reduction in HPV-positive patients and to ameliorate some of the adverse side effects.[16] Furthermore, results from the recent studies indicate that HPV vaccine, which is primarily targeted against cervical carcinoma, may have multisite benefit.[17] These likely benefits warrant a systematic assessment of HPV status in common cancers at various centers so that suitable preventive and therapeutic options can be delivered to the patients.

Among head and neck cancers, oral cancers are more common than oropharyngeal cancers. The frequency of oral cancers in our center is 30%.[18] Thus, understanding the molecular profile of oral cancer is important. Previous survey of HPV status in oral cancers in India reported a frequency ranging from 0 to 74%.[4],[5],[6],[7],[8],[9] This is exceptionally high compared to the global incidence of 3%. The global incidence of HPV in oropharyngeal cancers is 12%; thus, the global ratio of oral-to-oropharyngeal cancers is 1:4.[2],[3] Incidence of HPV in oropharyngeal cancers in India ranges from 26 to 39%.[1] Not only this number is 2- to 3-folds higher than the global incidence but also almost equal to the incidence in oral cancers. This indicates that the profile of HPV is uniquely different in Indian head and neck cancer patients, or the available data are incomplete. The list of Indian studies given in [Table 3] encourages us to assume that the need of data from diverse centers is probably the cause for the discrepancy.[4],[5],[6],[7],[8],[9]
Table 3: List of Indian studies on the prevalence of human papillomavirus in oral cancer

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The demographic profile given in [Table 1] indicates that tobacco consumption continues to be a major risk factor in our study population, which is mostly rural.


 » Conclusions Top


The results of this study highlight that screening studies at local and regional centers provide better insight into the risk profile rather than large studies based on few centers. Data from such studies will enable tailoring preventive and therapeutic strategies according to the local needs.

This study was funded by Sri Devaraj Urs Academy of Higher Education and Research, Tamaka, Kolar.

Financial support and sponsorship

This study was funded by Sri Devaraj Urs Academy of Higher Education and Research, Tamaka, Kolar.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

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Shukla S, Bharti AC, Mahata S, Hussain S, Kumar R, Hedau S, et al. Infection of human papillomaviruses in cancers of different human organ sites. Indian J Med Res 2009;130:222-33.  Back to cited text no. 1
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2.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.  Back to cited text no. 2
    
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Parkin DM, Bray F. Chapter 2: The burden of HPV-related cancers. Vaccine 2006;24 Suppl 3:S3/11-25.  Back to cited text no. 3
    
4.
Patel KR, Vajaria BN, Begum R, Desai A, Patel JB, Shah FD, et al. Prevalence of high-risk human papillomavirus type 16 and 18 in oral and cervical cancers in population from Gujarat, West India. J Oral Pathol Med 2014;43:293-7.  Back to cited text no. 4
    
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Balaram P, Nalinakumari KR, Abraham E, Balan A, Hareendran NK, Bernard HU, et al. Human papillomaviruses in 91 oral cancers from Indian betel quid chewers – High prevalence and multiplicity of infections. Int J Cancer 1995;61:450-4.  Back to cited text no. 5
    
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Elango KJ, Suresh A, Erode EM, Subhadradevi L, Ravindran HK, Iyer SK, et al. Role of human papilloma virus in oral tongue squamous cell carcinoma. Asian Pac J Cancer Prev 2011;12:889-96.  Back to cited text no. 6
    
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Nagpal JK, Patnaik S, Das BR. Prevalence of high-risk human papilloma virus types and its association with P53 codon 72 polymorphism in tobacco addicted oral squamous cell carcinoma (OSCC) patients of Eastern India. Int J Cancer 2002;97:649-53.  Back to cited text no. 7
    
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Barwad A, Sood S, Gupta N, Rajwanshi A, Panda N, Srinivasan R. Human papilloma virus associated head and neck cancer: A PCR based study. Diagn Cytopathol 2012;40:893-7.  Back to cited text no. 8
    
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Koppikar P, deVilliers EM, Mulherkar R. Identification of human papillomaviruses in tumors of the oral cavity in an Indian community. Int J Cancer 2005;113:946-50.  Back to cited text no. 9
    
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Bhargava A, Shakeel M, Srivastava AN, Raza TS, Rizvi S, Varshney P. Role of human papilloma virus in oral leukoplakia. Indian J Cancer 2016;53:206-9.  Back to cited text no. 10
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Akhter M, Ali L, Hassan Z, Khan I. Association of human papilloma virus infection and oral squamous cell carcinoma in Bangladesh. J Health Popul Nutr 2013;31:65-9.  Back to cited text no. 11
    
12.
Pezeshkpoor F, Jafarian AH, Ghazvini K, Yazdanpanah MJ, Sadeghian A, Esmaili H, et al. An association of human papillomaviruses low risk and high risk subtypes with skin tag. Iran J Basic Med Sci 2012;15:840-4.  Back to cited text no. 12
    
13.
Nasi M, Riva A, Borghi V, D'Amico R, Del Giovane C, Casoli C, et al. Novel genetic association of TNF-α-238 and PDCD1-7209 polymorphisms with long-term non-progressive HIV-1 infection. Int J Infect Dis 2013;17:e845-50.  Back to cited text no. 13
    
14.
Siebers TJ, Merkx MA, Slootweg PJ, Melchers WJ, van Cleef P, de Wilde PC. No high-risk HPV detected in SCC of the oral tongue in the absolute absence of tobacco and alcohol – A case study of seven patients. Oral Maxillofac Surg 2008;12:185-8.  Back to cited text no. 14
    
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Chen XJ, Sun K, Jiang WW. Absence of high-risk HPV 16 and 18 in Chinese patients with oral squamous cell carcinoma and oral potentially malignant disorders. Virol J 2016;13:81.  Back to cited text no. 15
    
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Kimple RJ, Harari PM. Is radiation dose reduction the right answer for HPV-positive head and neck cancer? Oral Oncol 2014;50:560-4.  Back to cited text no. 16
    
17.
Kiatpongsan S, Campos NG, Kim JJ. Potential benefits of second-generation human papillomavirus vaccines. PLoS One 2012;7:e48426.  Back to cited text no. 17
    
18.
Kalyani R, Das S, Bindra Singh MS, Kumar H. Cancer profile in the Department of Pathology of Sri Devaraj Urs Medical College, Kolar: A ten years study. Indian J Cancer 2010;47:160-5.  Back to cited text no. 18
[PUBMED]  [Full text]  


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    Tables

  [Table 1], [Table 2], [Table 3]



 

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