BUJOD theases-1 2012 septa 31-45

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Original Research DETECTION OF HUMAN PAPILLOMAVIRUS 18 AND 16 IN SQUAMOUS CELL CARCINOMA OF ORAL CAVITY OF VADODARA DISTRICT. A PRELIMINARY STUDY Authors: Vandana Shah*, Shreyash Shah**, Deepak Pateel***, Vikas Bhakhar**** ABSTRACT: BACKGROUND: Over the last 20 years, there has been increasing awareness of a subset of squamous cell carcinomas of the head and neck (HNSCC), i.e. Human Papilloma Virus (HPV) -positive HNSCC. HPV, the causal agent of cervical cancer, appears to be involved in the etiology of cancer of the oral cavity and oropharynx. However, the reported prevalence of HPV in Oral Cavity in Gujarati population does not exist. METHODS: DNA was extracted from all the 30 samples considered for the study and viral genome was examined by PCR/DNA sequencing. HPV-positive samples were analyzed separately for the high-risk types HPV 16 and 18. RESULTS: HPV 16 and 18 were detected in 11 (36.66%) and 15 (50%) of the 30 samples included for the present study. CONCLUSION: The positive rates of HPV 16 and 18 types were significantly higher in OSCC samples. Keywords: HPV 16, HPV 18, SCC, PCR INTRODUCTION: Oral cancer is a heterogeneous group of cancers arising from different parts of the oral cavity, with different predisposing factors, prevalence, and treatment outcomes. It is the sixth most common cancer reported globally.[1] Oral cancer remains one of the most common forms of cancer in the Indian subcontinent and in other parts of Asia [2]. There is a significant difference in the incidence of oral cancer in different regions of the world. The age-adjusted rates of oral cancer vary from over 20 per 100,000 population in India, to 10 per 100,000 in the U.S., [3] and less than 2 per 100,000 in the Middle East. HNSCC has a 75% overall 5-year survival rate if [4] detected early . Despite advances in detection and treatments over recent decades, most patients present with metastatic disease at the time of ADDRESS FOR CORRESSPONDENCE: Dr Vandana Shah Department of Oral and Maxillofacial Pathology, K.M. Shah Dental college and Hospital, SVU Campus, Piparia, Waghodia, Vadodara - 391760 E Mail: [email protected]

diagnosis, reducing the overall 5-year survival rate to 35% .[5] It is well known that there is a strong association between gene, environment and cancer. Several factors are involved in oral carcinogenesis, such as age, gender, ethnicity, lifestyle, genetic background, status of health and exposure to one or more oncogenic factors [6]. In several epidemiologic studies, tobacco smoking and alcohol consumption have been well documented as major risk factors for oral cancer, with attributable fractions of approximately 90%. However, 15-20% of HNC have [7, 8] no known tobacco or alcohol exposure . Thus, other agents, such as viruses, are being investigated. Today it is well established that the infection with specific types of HPV can cause cervical cancer. More than 95% of cervical cancer biopsies contain high[9] risk HPV genomes . The involvement of human papillomavirus (HPV) in head and neck carcinogenesis was first suggested by Syrjanen in 1983 [10]. Since Syrjanen's first proposal regarding the involvement of HPV in head and neck cancer,

* Professor and HOD, *** Reader, ****P.G. Student Department of Oral and Maxillofacial Pathology, K.M. Shah Dental college and Hospital, Vadodara. **Senior Lecturer Department of Oral and Maxillofacial Pathology, Vyas Dental College and Hospital, Jodhpur, Rajasthan

BHAVNAGAR UNIVERSITY'S JOURNAL OF DENTISTRY

Vol. 2 Issue-3 Sept. 2012

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BUJOD several other studies have demonstrated HPV in oral cavity, pharyngeal and laryngeal SCC. To date over 100 human papilomavirus (HPV) genotypes have been identified. In the oral cavity, 24 HPV genotypes (HPV-1, 2, 3, 4, 6, 7, 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 45, 52, 55, 57, 59, 69, 72 and 73) in benign lesions and 12 HPV genotypes (HPV-2, 3, 6, 11, 13, 16, 18, 31, 33, 35, 52 and 57) in malignant 11 lesions have been identified . During the past three decades, data supporting human papilloma virus (HPV) as a causative agent in the development and progression of head and neck cancer, particularly that of oropharynx has accumulated [12-15]. Different types of HPV have been detected in oral cancers, which are mainly HPV16 and HPV18 with [16, 17, 18] noticeable detection rates from 8% to 64% . The primary objective of the present case control study was to determine the prevalence of HPV16 and 18 in oral squamous cell carcinoma. It is the first cross-sectional survey conducted in Vadodara District as per our knowledge. Our data will provide novel information, still missing from Gujarat, about HPV occurrence in the oral cavity in Guajarati population.

A/TS18-B, subtype-specific primers for HPV subtypes 16 and 18, respectively and PC03/PC04 for β-globin sequences for the given samples, along with the length of respective PCR amplimers and the targeted gene. Amplified PCR products were run on 2% agarose gel and stained with Ethidium Bromide. The PCR products were identified on the basis of their predicted fragment size (Fig 1).

RESULTS: Table1: Clinical parameters of the Oral Squamous Cell Carcinoma lesions PARAMETER

Material and Methods

ORAL

CELL CARCINOMA (n)

30 cases of histologically confirmed cases of Oral Squamous cell carcinoma were considered in the present study. (Table 1). All specimens were taken from formalin fixed, paraffin-embedded tissues from files of the Oral Pathology and microbiology department, K. M. Shah Dental College and Hospital, Vadodara, Gujarat. The present study was evaluated and approved by Institutional Ethics Committee.

Total no of cases

30

Age, mean (years)

50

Formalin fixed tissue embedded in paraffin wax blocks were obtained from the archives and two sections of 4-5 microns were prepared. One Section was stained with routine Hematoxylin and Eosin stain. Slides were examined by 3 examiners to confirm the histological diagnosis. DNA was extracted from the paraffin blocks by using standard criteria as per the manufacturer's protocol. After DNA extraction the samples were analyzed by PCR using four sets of primers, i.e., GP5/GP6, general primers for HPV; TS16-A/TS16-B and TS18-

Tobacco habits:


SQUAMOUS

Gender: a. Male

22

b. Female

08

a. Smokers

25

b. Chewers

22

Most common anatomic sites: a. Tongue

12

b. Cheek

17

c. Both

1


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Table-2: Prevalence of HPV 16 and 18 among Oral SCC patients

Table-3: Correlation between HPV DNA sequence and Site S ite o f lesio n

H PV 16+

H PV 16-

H PV 18+

H PV 18-

T o n gu e

8 (6 6 .6 7 % )

4 (3 3 .3 3 % )

8 (6 6 .6 7 % )

4 (3 3 .3 3 % )

C h eek

1 2 (7 0 .5 9 % )

5 (2 9 .4 1 % )

1 2 (7 0 .5 9 % )

5 (2 9 .4 1 % )

Bo t h

1 (1 0 0 % )

0

1 (1 0 0 % )

0

T o ta l

21

9

21

9

The products were electophoresed on 2% agarose gel and stained with eth idium bromide. Lane N:negative control, lane P: positive control, lanes 14 HPV (general primer) positive tumor samples, lanes 5-6 HPV 16 positive tumor samples, lanes 7-8 HPV 18 positive tumor samples. Discussion: Oral cancer is a multifactorial disease, with smoking and regular alcohol consumption being the main etiological agents involved in oral carcinogenesis [19]. However, these habits do not always explain the development of these types of neoplasm [20, 21]. Over the last few years, HPV has called the attention of research as a possible etiological agent of oral cancer [22, 23, 24, 25]. This virus is strongly associated with cancer of the uterine cervix and is detected in almost 100% of cases of this neoplasm, but its role in oral [21,24,26,27,28]. carcinogenesis is still inconclusive In the present study, we analyzed paraffinembedded tissue samples of OSCC for HPV detection using a highly sensitive method- PCR. The HPV16 DNA was detected in 11 (36.37%) and HPV18 DNA was detected in 15 (50%) out of 30 examined specimens. (Table 2) This percentage is similar to data reported [24, 29] elsewhere . Previous studies have provided


evidence of an association of both HPV 16 and 18 and Oral Squamous Cell Carcinomas. In some studies HPV 18 was more prevalent and in few others HPV 16 [21, 22, 24, 26, 29, 30, 31]. HPV prevalence in OSCC tissue samples has varied from 0% to 100%. Discrepant results can be explained by the different diagnostic methods employed, distinct epidemiological characteristics of the various patient groups and the difficulty in performing comparable studies of OSCC in different head and neck sites.[19] Previous reports show a tendency for higher HPV prevalence in male patients (Miller CS & White DK. 1996), besides some researchers have found no association between HPV and gender (Gillis on ML et al 2000; Paz IB et al. 1997). Why male are more HPV positive is not clear, it may be due to that male are more exposed to HPV transmission risk factors. There was no significant difference between HPV 16 and 18 sites and the SCC samples (Table 3). This finding is in line with other studies, where no correlation was found between HPV positivity and [32] site. The predominance of the HPV high-risk types, HPV 18 and 16, in OSCC in our study provides further evidence for its etiologic importance. It is likely that


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BUJOD HPV plays a role in oral carcinogenesis, though only in a small subset of cases. Other aspects such as HPV load and persistence in infected cells of the oral mucosa need to be analyzed in more detail. Progress in the understanding of the significance of HPV in head and neck cancer can be made only with multidisciplinary efforts, as is evident from the research history of cervical cancer.

8.

9.

Conclusion: Our preliminary study shows a high prevalence of both HPV 16 and 18 among SCC patients. These finding should trigger additional research to further instigate and investigate the role of HPV in OSCC patients especially in of Guajarati population. References: 1. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol 2001; 2:533-543. 2. Moore SR, Johnson NW, Pierce AM, Wilson DF. The epidemiology of mouth cancer: a review of global incidence. Oral Dis 2000 Mar; 6(2):6574. 3. Sankaranarayan R, Masuyer E, Swaminathan R, Ferley J, Whelan S; Head and neck cancer: a global perspective on epidemiology and prognosis. Anticancer Res 18:4779-86, 1998) 4. Silveira NJ, Varuzza L, Machado-Lima A, Lauretto MS, Pinheiro DG, Rodrigues RV, Severino P, Nobrega FG, Silva WA, Jr., de BPCA, Tajara EH. Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries. BMC Med Genomics 2008;1:56 5. Chin D, Boyle GM, Williams RM, Ferguson K, Pandeya N, Pedley J, Campbell CM, Theile DR, Parsons PG, Coman WB. Novel markers for poor prognosis in head and neck cancer. Int J Cancer 2005; 113:789-797. 6. Llewellyn CD, Johnson NW, Warnakulasuriya KA: Risk factors for oral cancer in newly diagnosed patients aged 45 years and younger: a case-control study in southern England. J Oral Pathol Med 2004, 33:525-32. 7. Castellsague X, Quintana MJ, Martinez MC, Nieto A, Sanchez MJ, Juan A, et al: The role of type of tobacco and type of alcoholic beverage in oral carcinogenesis. Int J Cancer 2004, 108:741-9.


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Source of Support :

NIL

Conflict of Interest :

NOT DECLARED

Date of Submission :

07-07-2012

Review Completed :

02-08-2012


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