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1 ASSESSMENT OF HUMAN PAPILLOMA VIRUS SUBTYPES BY POLYMERASE CHAIN REACTION AND THEIR IMPACT ON THE DEGREE OF DYSPLASIA IN ORAL LEUKOPLAKIA Submitted on: XXXX Dr. N. Kannan, Dr Teja Srinivas, Dr. Rakesh Kumar Manne, Dr. P. V. Sarath, Dr. Swapna Accepted on: XXXX For Correspondence Dreedevi, Dr. K. V. Suneel ID: Narayana Dental College and Hospital, Nellore Submitted on: January 2016 Accepted on: January 2016 For Correspondence ID: Medrech ISSN No Original Research Article Abstract Aim: The study was intended to determine the prevalence of HPV subtypes in patients with oral leukoplakia & assess the possible correlation between HPV and dysplastic changes in oral leukoplakia. Materials and Methods: Research was carried out in 25 patients with oral leukoplakia. HPV DNA was determined in incisional biopsy specimens by PCR and HPV subtypes by direct DNA sequencing. Results: Statistical analysis was done using the SPSS version HPV DNA was detected in 15 patients (60%) out of 25 leukoplakia patients. HPV 16 and 18 were detected in 8 patients (32%), HPV 18 was detected in 6 patients (24%) and HPV16 alone was detected in 1patient (4%). 10 patients (40%) were negative for HPV DNA. There was a statistical significant correlation between HPV DNA and degree of dysplasia (p value 0.037). Conclusion: An increased prevalence of HPV infection was found in oral leukoplakia; It was observed that HPV might play a crucial role in malignant transformation of potentially malignant lesion like oral leukoplakia. Keywords: human papilloma virus, leukoplakia, dysplasia, polymerase chain reaction, HPV16, HPV18, Toluidine blue staining Introduction A precancerous lesion is a morphologically altered tissue in which cancer is more likely to occur than in its apparently normal counterpart. The most common precancerous lesion of the oral cavity is leukoplakia. The term leukoplakia should be used to recognize white plaques of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer. Leukoplakia is a clinical term with no specific histological features. It may show features like hyperkeratosis, acanthosis suggestive of epithelial dysplasia. It has a variable behavioural and a variable rate of malignant transformation. 1 18

2 Tobacco usage is the most widely accepted etiological agent for the development of oral leukoplakia. However, we do come across patients with no history of tobacco habits. In such patients, viruses like human papilloma virus are hypothesized as risk factors for the development of leukoplakia and oral cancers. Oral potentially malignant disorders maybe suspected to harbour several subtypes of HPV, like 6, 11, 16, and The aim of the present study was to assess the prevalence of HPV subtypes in patients with oral leukoplakia & evaluate the presence of any correlation between HPV and dysplastic changes in oral leukoplakia. Materials and Methods The study was conducted on 25 patients with varying stages of oral leukoplakia who were selected from the outpatient section of the department of oral medicine & radiology. Prior approval from the institutional ethical committee was obtained before undertaking the study. Written informed consent was obtained as per the study protocol. The study included only those patients who were diagnosed clinically Results & Observation as having oral leukoplakia. Patients with other potentially malignant lesions occurring concomitantly were excluded from the study group Detailed clinical examination of the oral cavity was done. Toluidine blue staining was done with 1% toluidine blue solution for clinical assessment of degree of dysplasia & for selection of the biopsy site. Clinical investigations like complete hemogram, blood sugar level, screening for HIV and HBs Ag were done prior to the biopsy procedure. Incisional biopsy was done with local anaesthesia under aseptic conditions. The biopsy specimen was divided into two parts and one part was evaluated for histological grading of dysplasia by oral pathologist into mild dysplasia, moderate dysplasia or severe dysplasia based on classification by Warnakulasuriya et al. 4 and the other part was subjected to Polymerase Chain Reaction analysis using methodology described by Giovannelli L et al. 5 for detecting the HPV subtypes. Table 1: Correlation between types of Leukoplakia & HPV Subspecies HPV HOMOGENOUS SPECKLED TOTAL Subspecies LEUKOPLAKIA LEUKOPLAKIA HPV HPV HPV 16, NO HPV Chi-Square Tests Value df p-value Pearson Chi- Square Not Sig Table 2: Correlation between HPV subspecies & T. Blue Staining Based Staging of dysplasia 19

3 HPV Severe Moderate Mild No Stain Total Subspecies Dysplasia Dysplasia Dysplasia HPV HPV HPV 16, NO HPV Chi-Square Tests Value df p-value Pearson Chi- Square Not Sig table 3: Correlation between HPV subspecies & Histopathological staging of dysplasia Histopathological Grading of Dysplasia HPV Mild Moderate Severe C.A. in Situ Total Subspecies HPV HPV HPV 16, NO HPV Chi-Square Tests Value df p-value Pearson Chi-. Square Sig Out of 25 patients with leukoplakia, 3 patients (12%) were females and 22 patients (88%) were males. 21 patients(84%) were clinically diagnosed as having homogenous leukoplakia and 4(16%) were having speckled leukoplakia. 10 patients (40%) showed positive staining with toluidine blue, doubtful staining was observed in 13 patients (52%) and negative staining was observed in 2 patients (8%). Histopathologically, 6 patients (24%) showed mild dysplasia, 12 patients (48%) showed moderate dysplasia, 6 patients (24%) showed severe dysplasia and 1 patient (4%) showed histological features of carcinoma-in-situ. Only HPV16 was detected in 1 patient (4%) with homogenous leukoplakia, HPV 18 was detected in 24% of the leukoplakia patients including 5 patients with homogenous leukoplakia and 1 patient with speckled leukoplakia, both HPV 16 and 18 were detected in 32% patients including 5 patients with homogenous leukoplakia and 3 patients with speckled leukoplakia. 10 leukoplakia patients (40%) were negative for HPV DNA. All the female leukoplakia patients were positive for HPV DNA. HPV 16 was detected in 1 male patient. HPV 18 was detected in 5 males and 1 female. Both HPV 16 and 18 were detected in 6 males and 2 females. HPV DNA was not detected in 10 males. HPV DNA was not detected in any patients with negative toluidine blue staining. HPV 16 alone was detected in 1 patient with positive TB staining. HPV 18 20

4 was detected in 2 patients with positive staining and 4 patients with doubtful staining. Both HPV 16 and 18 were detected in 6 patients with positive staining and 2 patients with doubtful staining. HPV 16 alone was detected in only one patient who had carcinoma in situ. HPV 18 alone was detected in 4 patients with moderate dysplasia and 2 patients with severe dysplasia. Both HPV 16 and 18 were detected in 4 patients with moderate dysplasia and 4 patients with severe dysplasia. HPV DNA was not detected in any of the cases of mild dysplasia nor in the 4 cases of moderate dysplasia. Statistical analysis was done using the SPSS version The data has been considered to be a non-parametric data so non parametric test chi-square test had been applied. p value <0.05 was taken as having statistical significance. Our study showed statistical significant correlation between HPV DNA and degree of dysplasia (p value 0.037). Other parameters (TB staining, clinical variants of leukoplakia, gender) showed some mild changes which were not statistically significant. Discussion It has been proved that HPV may play a role in the development of chromosomal mutations. The E6 and E7 proteins of HPV 16 have been shown to bind and inactivate two important negative cell cycle regulators, the p53 and retinoblastoma proteins (RB), respectively. In response to chromosomal damage, p53 turns off the cell cycle and stimulates expression of DNA repair proteins, thereby minimizing the likelihood that chromosomal mutations will accumulate in response to a cellular insult. Inactivation of p53 by the HPV E6 protein would therefore negate its protective effect and lead to chromosomal instability. The most important epidemiologic risk factor for OSCC, tobacco use, has mutagenic capability and could potentiate the development of OSCC alone or in conjunction with HPV by contributing to chromosomal instability. 6 In the present study HPV DNA was detected in 60% of leukoplakia lesions similar to findings of Loning.TH et al. who found HPV in 58% of leukoplakia lesions 7. Palefsky JM, Silverman Jr.et al. 8 in their study detected HPV 16 DNA in 89% of proliferative verrucous leukoplakia lesions by PCR. Other subtypes of HPV were not included in their study. In the present study the prevalence of HPV 16 was (6.6%), which included homogenous and speckled leukoplakia lesions. Rajaram Gopalakrishnan, Christopher M et al. in their study detected HPV DNA in 25% of proliferative verrucous leukoplakia lesions by PCR 9. They also detected mutated and wild-type p53 expression. Giuseppina Campisi et al. in their study detected HPV DNA in 17.6% of oral leukoplakia lesions. They detected HPV-18 more frequently followed by HPV 16, HPV 33, HPV 31 and HPV 6. In the present study both HPV 16 AND 18 were detected more frequently (53%) followed by HPV 18 (40%) and HPV 16 (6%). Acay R, Rezende N et al. in their study detected HPV DNA in 24% of patients with leukoplakia by using in situ hybridization with signal amplification (CSA-ISH) technique 6. They found that HPV types 16 and 18 were the most prevalent, and types 6 and 11 were only found in groups of mild or no dysplasia. These results were consistent with the present study which detected HPV 16 and 18 as more prevalent subtypes. Szarka K, Tar I et al. reported HPV prevalence as around 40.9% by PCR. Most frequently detected subtype was HPV 16, followed by HPV 18 and HPV However 21

5 in the present study both HPV 16 and 18 were detected frequently. The present study found no statistically significant correlation between HPV and TB staining (p = 0.083). The present study shows statistically significant correlation of HPV with degree of dysplasia p = Conclusion The present study confirmed a positive correlation between HPV and dysplastic changes in leukoplakia. However, it failed to demonstrate a relationship between HPV infection and TB staining. Taking into account the oncogenic potential of HPV, the presence of oral HPV infection should be determined in all variants of leukoplakia, along with an adequate longterm follow-up of lesions found to be HPVpositive. It is known that HPV alone are not capable of mediating malignant transformation of oral lesions but they can enhance the effect of other carcinogens or agents provoking dysplasia. Our results suggest that the debate on the etiological role of HPVs is probably worth extending to include oral potentially malignant disorders such as leukoplakia. Further studies with larger sample size, are necessary to elucidate the relation between HPV and the development of premalignant and malignant lesions of the oral cavity. References 1. Axe ll T, Pindborg JJ, Smith CJ, van der Waal International Collaborative Group on Oral White Lesions. Oral white lesions with special reference to precancerous and tobacco-related lesions: conclusions of an international symposium held in Uppsala, Sweden, May 18 21, J Oral Pathol Med 1996; 25: Giuseppina Campis, Lucia Giovannelli et al. HPV DNA in clinically different variants of oral leukoplakia and lichen planus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 98: Gokul Sridharan, Akhil A Shankar. Toluidine Blue; A Review of its chemistry and clinical utility. Journal of Oral and Maxillofacial Pathology. Vol.16, Issue 2, May-Aug S. Warnakulasuriya, J. Rebel et al. Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med (2008) 37: Giovannelli L, Campisi G, Lama A, Giambalvo O, Osborn J, Margiotta V, et al. Human papillomavirus DNA in oral mucosal lesions. J Infect Dis 2002; 185: Acay R, Rezende N, Fontes A, Aburad A. Human papillomavirus as a risk factor in oral carcinogenesis: a study using in situ hybridization with signal amplification. Oral Microbiology Immunology 2008: 23: Th. Loning, P. Reichart et al. Occurrence of papilloinavirus structural antigens in oral papillomas and leukoplakias. Journal of Oral Pathology 1984: 13: Palefsky JM, Silverman Jr et al. Association between proliferative verrucous leukoplakia and infection with human papillomavirus type 16. J Oral Pathol Med 1995; 24: Rajaram Gopalakrishnan, Christopher M. et al. Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and Squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997, 83:

6 Figure 1 Speckled Leukoplakia before Toluidine Blue Staining Figure 2 Speckled Leukoplakia with Toluidine Blue Stain 23

7 Figure 3 PCR positive for HPV 16 Figure 4 PCR positive for HPV 16 24

8 Figure 5 PCR positive for HPV 16 Figure 6 PCR positive for HPV 18 25

9 Figure 7 PCR positive for HPV 18 Figure 8 PCR positive for HPV 18 26

10 Fig 9 Microphotograph showing Mild Dysplasia Fig 10 Microphotograph showing Moderate Dysplasia 27

11 Fig 11 Microphotograph showing Severe Dysplasia Fig 12 Microphotograph showing C.A. in Situ 28