DOI:http://dx.doi.org/10.7314/APJCP.2015.16.16.7077 Do Human Papilloma Viruses Play Any Role in Oral Squamous Cell Carcinoma in North Indians? RESEARCH ARTICLE Do Human Papilloma Viruses Play Any Role in Oral Squamous Cell Carcinoma in North Indians? Vineeta Singh1,3, Nuzhat Husain1*, Naseem Akhtar2, Vijay Kumar2, Shikha Tewari1, Sridhar Mishra1, Sanjeev Misra2, M.Y. Khan3 Abstract Background: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy among males in India. While tobacco and alcohol are main aetiological factors, human papilloma virus (HPV) presence has surprisingly increased in head and neck Squamous Cell Carcinoma (HNSCC) in the past two decade but its frequency in OSCCS is still uncertain. We aim to explore the frequency of HPV and its major genotypes in North Indian patients and their association with clinicopathological and histopathological features and p16 expression pattern. Materials and Methods: The study group comprised 250 histologically proven cases of OSCC. HPV was detected by real time PCR in tumor biopsy specimens and confirmed by conventional PCR with PGMY09/ PGMY11 primers. Genotyping for high-risk types 16/ 18 was conducted by type specific PCR. p16 expression was assessed by immunohistochemsitry. Results: HPV presence was confirmed in 23/250 (9.2%) OSCC cases, of which 30.4% had HPV 16 infection, 17.4%were positive for HPV 18 and 26.1% had co-infections. HPV presence was significantly associated with male gender (p=0.02) and habit of pan masala chewing (p=0.01). HPV positive cases also had a history of tobacco consumption in 91.3% cases. p16 over expression was observed in 39.1% of HPV positive cases but this was not significantly different from negative cases (p=0.54). Conclusions: The frequency of HPV in OSCC is low in North-India and majority of cases are associated with a tobacco habit. It appears that tobacco shows a confounding effect in HPV positive cases and use of p16 protein as a reliable marker to assess the potential etiological role of HPV in OSCC in our population is not suggested. Keywords: Oral squamous cell carcinoma - human papilloma virus - p16 - head & neck squamous cell carcinoma. Asian Pac J Cancer Prev, 16 (16), 7077-7084 Introduction Oral squamous cell carcinoma (OSCC) is one of the most prevalent malignancy in India with approximately 83,000 new cases and more than 46,000 deaths occur yearly (Bray et al., 2013). It ranks number one in terms of incidence among men and third among women (Byakodi et al., 2012). According to Indian Council of Medical Research there is a sharp increase in the number of oral cancer cases by 2020 is expected. In India 16% population smoke tobacco, 20% chew tobacco/pan masala and 30% either smoke or chew tobacco (Rani et al., 2003) and these are the well established risk factors for OSCC. The relative risk for OSCC among tobacco and alcohol abusers is 20 times that of non-smokers and non-drinkers (Hashibe et al., 2009). An increased involvement of human papilloma virus (HPV) in the Head &Neck Squamous Cell Carcinoma (HNSCC) has been reported in past 10 years (Chaturvedi et al., 2008) but its presence is not as consistent in it as in cervical cancer and therefore its actual prevalence is still vague in HNSCC and need the more solemn attention of researchers in this direction. HPV is a DNA virus that presents tropism for epithelial cells, causing infection of the skin and mucous membrane. Its presence is more allied to oropharyngeal cancer for instance about 40-80% of oropharyngeal cancers are caused by HPV in USA, whereas in Europe the proportion varies from around 90% in Sweden to less than 20% in communities with the highest rates of tobacco use (Marur et al., 2010). The etiologic role of HPV in HNSCC sites other than oropharynx is still controversial. In India a wide variation in HPV associated OSCC has been reported, for instance in Western India only 15% OSCC patients showed association with HPV, 33.6% cases in Eastern India while its prevalence is surprisingly higher (70.6 %) in South Indian population (Balaram et al., 1995; D’Costa et al., 1998; Priya et al., 2005; Alok et al., 2006; Chaudhary et al., 2013). These variations might be due 1 Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, 2Department of Surgical Oncology, King George’s Medical University, 3Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow, UP, India *For correspondence: drnuzhathusain@hotmail.com Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 7077 Vineeta Singh et al to sensitivity of the employed technique, the sample size, the state of conservation of the clinical specimens, and epidemiological factors of the studied population. Apart from having a different epidemiology and aetiology, the HPV-positive HNSCC are different clinicopathologically and showed distinct histopathology. These are usually poorly differentiated, nonkeratinizing and have a basaloid appearance (Gillison et al., 2000). Furthermore, patients with HPV-positive HNSCC in general tend to be younger at time of diagnosis (Smith et al., 2004) and generally have a better survival and favorable prognosis compared to the HPV-negative patients (Hafkamp et al., 2008; Lassen et al., 2009). HPV positive oral cancer also represents a distinct molecular phenotype with a unique mechanism of tumorigenesis, independent of the mutagenic effect of tobacco and alcohol. A characteristic over expression of p16, tumor suppressor protein, has been observed in HPV positive HNSCC. Two viral onco-proteins E6 and E7 cause inactivation of p53 and retinoblastoma (Rb) respectively, this event leads further activation of CDKN2A gene and increased expression of p16 has occurred. Thus, p16 overexpression is an indicator of an aberrant expression of viral oncogenes and considered as a surrogate biomarker for HPV presence (Klussmann et al., 2009; Ahmed et al., 2012). Therefore, the present study aims to explore the frequency of HPV association in OSCC, distribution of its major high risk types and correlation with other risk factors in North Indian population. Materials and Methods Study Population: A total of 250 clinically and histologically proven cases of OSCC were included prospectively in the study between Oct 2013 to Jan 2015. Tissue biopsies were collected from department of Surgical Oncology, King George’s Medical University (KGMU) Lucknow after obtaining approval from the Institutional Ethics Committee and written informed consent from the patients. Sample Collection: Tissue biopsies were collected in 10% buffered formalin at RT for histopathological diagnosis and in 1X phosphate buffer saline (PBS, pH 7.4) and stored at -80°C for molecular analysis. The demographic and clinical details of the patients were recorded on the standard questionnaire. DNA Extraction: The DNA from biopsy tissues was isolated by commercially available Genomic DNA Mini kit (Invitrogen, USA) as per manufacturer’s instructions. The extracted DNA was eluted in 80μl of the elution buffer (provided in the kit) and kept at -80°C until further use. All the DNA samples were qualitatively confirmed on 0.8% agarose gel electrophoresis and the concentration and purity was checked by DS-11 spectrophotometer (Denovix, USA) at 260/280 nm wavelengths. HPV detection: HPV detection was performed by using the following methodology Real Time PCR based methodology for HPV detection: Presence of HPV in OSCC samples were detected by Real 7078 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 time PCR (Biorad CFX 96TM) using 13 HIGH RISK HPV REAL TIME PCR KIT (Hybribio Limited, China) as per manufacturer’s instructions. Briefly, each PCR reaction contained 17.5µl of PCR master mix, 0.5µl of Taq DNA polymerase and 2µl of DNA. The PCR amplification protocol was as follows- initial denaturation at 95°C for 10min and 45 cycles of - denaturation for 3min at 95°C, annealing for 60sec at 60°C, and extension at 72°C for 20sec. The amplification was followed by a 5sec final extension step at 38°C. Presence or absence of HPV DNA was confirmed by the Ct values obtained for the sample. Conventional PCR based methodology: Further validation of HPV was done using PGMY09/11 primers as reported by P.E.Gravitt et al; 2000, designed to amplify a 450bp HPV L1 gene fragment. This region is used because it is highly conserved between different HPV types but has sufficient variation for the identification of each one. Cervical cancer cases which were confirmed previously for HPV presence were used as positive control. PCR amplification was carried out in a volume of 20μl containing 50ng/μl of genomic DNA, 4mM of MgCl2 (Invitrogen, USA), 200µM of each dNTPs. Concentration of PGMY09/11 primers was reduced from 10pmol as previously used to 5pmol, also the final concentration of AmpliTaq DNA polymerase (Invitrogen, USA) was reduced from 7.5U/µl to 5U/µl. Amplification was performed in thermal cycler (S100TM, Biorad, USA) under the following conditions: initial denaturation at 95ºC for 9min, followed by 35 cycles of - 95ºC for 1min (denaturation), primer annealing at 55°C for 1min, extension at 72ºC for 1min and a final extension at 72ºC for 5min. Positive and negative controls were run simultaneously. HPV genotyping: Samples that were positive for HPV presence were proceeded further for high risk HPV 16 & 18 genotyping. HPV genotyping was done with the previously described type specific primers for HPV 16 and 18 (Sharma et al., 2005). PCR amplification was carried out in a 20μl reaction volume containing 50ng/μl of genomic DNA, 10pmol each of forward and reverse primer, 2mM MgCl2 (Invitrogen, USA), 200µM of each dNTPs, 0.5U/µl of AmpliTaq DNA polymerase (Invitrogen, USA) along with 10X (NH4)2SO4 buffer (Invitrogen, USA). Amplification was performed in thermal cycler (S100TM, Biorad, USA) under the following PCR conditions: An initial denaturation at 94ºC for 10min, followed by 35 cycles of 94ºC for 1min (cycle denaturation), primer annealing at 52°C for 1min for HPV 16 & 62°C for 1min for HPV 18, extension at 72ºC for 1min and a final extension of 72ºC for 5min. PCR products were confirmed for their respective amplicon size on 2% agarose gel electrophoresis and visualized by UV-transillumination (Gel Doc XR+ , Biorad, USA). Expression of p16 by Immunohistochemsitry (IHC): Paraffin-embedded tissues were sectioned, 4μm, using a microtome (Leica, Germany), and transferred to tissue bond-coated slides (Biocare, USA). After overnight incubation in a 60°C dry oven, paraffin-embedded sections were deparaffinized in xylene and rehydrated through graded ethanol series 100%, 70% and 50%. Endogenous peroxidase activity was blocked with 3% DOI:http://dx.doi.org/10.7314/APJCP.2015.16.16.7077 Do Human Papilloma Viruses Play Any Role in Oral Squamous Cell Carcinoma in North Indians? hydrogen peroxide in methanol for 30min. Antigen retrieval was done by placing the slides in Tris-EDTA buffer (pH 9.0) in Pascal. These sections after cooling to room temperature (RT) were incubated with p16 primary antibody (Biogenex) at RT for one hour, followed by treatment with polymer based secondary antibody kit with DAB (DAKO, Denmark). Positive reactions were visualized using diaminobenzidine, DAB (1:50). Sections were finally counter-stained with 0.1% hematoxylin. The positive cells expressing the p16 positivity were assessed for cytoplasmic as well as nuclear staining at higher magnification.A tumor was recorded positive if more than 10% of tumor cells showed immunoreactivity (Names et al., 2006). Data analysis: The diagnostic criteria used for the detection of HPV considered only those OSCC cases that were positive by at least two methods described above (Real time PCR, PCR, 16 PCR & 18 PCR). The real time PCR data was analyzed using Bio-Rad CFX Manager software v3.0. The results are presented in mean±SD and percentages. The Chi-square test was used to compare the dichotomous/categorical variables and unpaired t-test was used to compare the continuous variables. The univariate binary logistic regression was carried out to find the strength of associations. The odds ratio with its confidence interval was calculated. The p-value<0.05 was considered significant. Kaplan-Meier survival curve was made and survival time was compared by using Log rank test. All the analysis was carried out by using SPSS 16.0 version (Chicago, Inc., USA). Figure 1. Detection of HPV by real time and conventional PCR. (A) Amplification Plot by Real Time PCR, (B) 2% Agarose gel with amplified product of HPV (450bp) by PGMY09/11 primers-M: 100bp DNA ladder, L1: Positive control, L2: Negative control, L3-L7: HPV in cases. (C) 2% Agarose gel with amplified product of HPV 16 (223bp) M: 100bp DNA ladder, L7: Positive control, L6: Negative control, L1-L5: HPV 16 in cases. (D) 2% Agarose gel with amplified product of HPV 18 (217bp) M: 100bp DNA ladder, L12: Positive control, L13: Negative control, L1-L11: Samples Results The study encompasses 250 histologically proven cases of OSCC. Out of these HPV presence was confirmed in 23(9.2%) cases (Figure 1A&B) according to diagnostic criteria predefined in data analysis. Table 1 summarizes the clinical, histopathological, demographic and behavioral characteristics of patients in HPV positive and negative cases. The mean age of HPV positive patients was 47.17 while HPV negative cases had mean age 47.69, but this difference was statistically insignificant. HPV presence associated significantly with male gender [p=0.02*,OR (95%CI) =0.34 (0.13-0.83)]. Buccal Mucosa was the most frequent site (52.2%) in patients. Most of HPV positive cases were well differentiated SCC (60.9%), eight cases were moderately differentiated or keratinized while only 4.3% cases showed a basaloid morphology (Figure 2A). HPV positivity did not find to be associated with age, marital status, domicile, sub-site, stage, tumor grade, nodal status and outcome. In HPV positive cases 91.3% had taken tobacco in any form while only 8.7% patients had no history of any risk factor. History of multiple risk factors was present in 13.00% cases. HPV positivity significantly associated with habit of pan masala chewing [p=0.01*,OR (95%CI) = 0.32 (0.13-0.79)]. HPV subtypes in OSCC: Out of 23 HPV positive cases 30.4% cases had HPV 16 infection while 17.4% were positive for HPV 18. Six cases (26.1%) co-expressed Figure 2. Microphotograph showing. (A) Basaloid morphology, (B) p16 expression in HPV positive cases (DAB x 200 x digital magnification) Figure 3. Kaplan-Meier survival curve of patients. (A) According to HPV status, (B) According to HPV16 status, (C) According to HPV18 status. Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 7079 Vineeta Singh et al Table 1. Association of HPV with Clinicopathological, Histopathological, Demographic and Behavioral Characteristics in OSCC Patients HPV Characteristics Positive Negative (n=23) (n=227) OR 95%CI p -value1 No. % No. % <30 2 8.7 7 3.1 2.57 0.39-16.86 0.32 30-40 5 21.7 73 32.2 0.616 0.15-2.43 0.49 41-50 7 30.4 69 30.4 0.91 0.25-3.32 0.89 51-60 5 21.7 42 18.5 1.07 0.26-4.29 0.92 >60 4 17.4 36 15.9 1 Ref. Male 14 60.9 186 81.9 0.34 0.13-0.83 Female 9 39.1 41 18.1 1 Ref. Married 23 100 223 98.2 Unmarried 0 0 4 1.8 NA NA Age in years Gender 0.02* Marital status Residence Rural 16 69.6 159 70 0.97 0.38-2.48 Urban 7 30.4 68 30 1 Ref. 0.96 BM 12 52.2 115 50.7 0.99 0.35-2.77 FM 0 0 4 1.8 - - LA 4 17.4 40 17.6 0.95 0.25-3.58 0.94 Palate/Upper alveolus 0 0 6 2.6 - - 0.58 Subsite RMT 1 4.3 5 2.2 1.9 0.18-19.06 Tongue 6 26.1 57 25.1 1 Ref. 0.08 Site Left 16 69.6 116 51.1 2.18 0.86-5.51 Right 7 30.4 111 48.9 1 Ref. 0.09 Stage I 1 4.3 21 9.3 0.47 0.05-3.88 0.48 II 4 17.4 45 19.8 0.88 0.26-2.93 0.84 III 7 30.4 51 22.5 1.37 0.50-3.74 0.53 IV 11 47.8 110 48.5 1 Ref. Yes 12 52.2 110 48.5 1.16 0.49-2.73 No 11 47.8 117 51.5 1 Ref. Yes 8 34.8 141 62.1 0.32 0.13-0.79 No 15 65.2 86 37.9 1 Ref. Yes 19 82.6 171 75.3 1.55 0.50-4.76 No 4 17.4 56 24.7 1 Ref. Yes 7 30.4 66 29.1 1.06 0.42-2.71 No 16 69.6 161 70.9 1 Ref. Smoking 0.73 Pan masala 0.01* Tobacco 0.43 Alcohal Multiple habit 7080 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 0.89 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.16.7077 Do Human Papilloma Viruses Play Any Role in Oral Squamous Cell Carcinoma in North Indians? Present 3 13 30 13.2 0.98 0.27-3.51 Absent 20 87 197 86.8 1 Ref. Any habit 21 91.3 212 93.4 0.74 0.15-3.47 No habit 2 8.7 15 6.6 1 Ref. 0.98 No habit 0.7 Treatment ABS 0 0 3 1.3 - CT 9 39.1 62 27.3 1.52 0.30-7.62 0.6 CT/RT 3 13 34 15 0.92 0.14-6.01 0.93 CT/SX/RT 2 8.7 31 13.7 0.67 0.08-5.19 0.7 SX 7 30.4 76 33.5 0.96 0.18-5.01 0.96 SX/RT 2 8.7 21 9.3 1 Ref. WD 14 60.9 129 56.8 0.65 0.07-5.80 0.7 MD 8 34.8 92 40.5 0.52 0.05-4.88 0.56 PD 1 4.3 6 2.6 1 Ref. Large 22 95.7 226 99.6 0.09 0.01-1.61 Basaloid 1 4.3 1 0.4 1 Ref. Present 15 65.2 132 58.1 1.34 0.55-3.31 Absent 8 34.8 95 41.9 1 Ref. Recurrence 1 9.1 9 7 1.33 0.15-11.61 No recurrence 10 90.9 120 93 1 Ref. Dead 7 30.4 58 25.6 1.27 0.49-3.25 Alive 16 69.6 169 74.4 1 Ref. Positive 9 39.1 7 30.4 1.46 0.43-4.9 Negative 14 60.9 16 69.6 1 Ref. Differentiation Cellular morphology 0.1 Node 0.51 Recurrence (n=140) 0.79 Survival 0.61 p16 Protein expression (n=23) 0.54 1 p value <0.05 by Binary logistic regression method.; Abbreviations: HPV- Human Papilloma Virus, OR-Odds Ratio, BM-Buccal Mucosa, FMfloor of Mouth, LA-Lower Alveolus, RMT-Retro Moral trigone, SX = Surgery, CT=Chemotherapy, CT/RT=Chemotherapy and Radiotherapy both, SX/RT=Surgery and Radiotherapy both, CT/SX/RT=Chemotherapy, Surgery and Radiotherapy, WD- Well Differentiated, MD- Moderately Differentiated, PD- Poorly Differentiated Table 2. HPV Subtypes in OSCC Patients HPV Result HPV positive cases HPV negative cases HPV Types HPV16 only HPV18 only HPV16 and 18 co-infection Negative for HPV 16 /18 or suspected subtypes other than 16/18 No. of Patient % 23 227 9.2 90.8 7 4 6 6 30.4 17.4 26.1 26.1 Abbreviation: HPV- Human Papilloma Virus DNA of both HPV 16 and 18 subtypes and 26.1% cases were negative for both 16 and 18 or supposed to had HPV subtypes other than 16/18 (Table 2, Figure 1C& D). Correlation of HPV 16 and 18 with clinicopathological variables Association of HPV type 16 & 18 with all clinical, demographic & behavioral profile of patients were evaluated but it was found to be shown no association with any of these (data not shown). Correlation of HPV with Survival of Patients Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 7081 Vineeta Singh et al Table 3. Association of Survival of Patients with HPV Presence and Its Subtypes. HPV Status HPVa Positive Negative Total cases HPV16b Positive Negative HPV18c Positive Negative Median Survival in Month 16.5 12.9 15.6 14 12 13 14 Log Rank test ap=0.62, bp=0.68, cp=0.72 (Insignificant); Abbreviation: HPV- Human Papilloma Virus According to Log rank test median survival of HPV positive patients was better (16.5) compared to HPV negative patients (12.9) but the difference was not significant (p=0.62) (Figure 3A, B & C). The survival status of patients in the study is shown in Table 3. Correlation of HPV with p16 protein expression p16 expression was evaluated in all HPV positive cases and equal number of HPV negative cases (n=23) that were selected randomly. Diffuse nuclear staining with some cytoplasmic positivity for p16 protein was seen in 39.13% HPV positive patients and 30.43% HPV negative patients (Table 1, Figure 2B). p16 over expression was not associated with the presence of HPV ( p=0.54). Discussion HPV has been identified as a prime suspect in the etiology of HNSCC due to their morphological similarities with genital epithelia and their ability to transform and immortalize oral keratinocytes (Termine et al., 2008). Speculation on the role of HPV in the etio-pathogenesis of oral carcinoma has been voiced worldwide.We have explored the frequency and major genotypes of HPV in 250 histologically confirmed cases of OSCC in NorthIndian population. The overall prevalence of HPV associated OSCC at worldwide level varies from 0-30%.In North America 5.9% positivity was reported by Mark W. Lingen., 2013, in Canada 4% by Jarry Machado et al., 2010, 27.5% in China by Li-Li Gan et al., 2014 and only 3% in Bangladesh by Mahmuda Akhter et al., 2013. Most Indian studies have reported prevalence of HPV associated OSCC between 15-37.9% (Priya et al., 2005; Alok et al., 2006; Chaudhary et al., 2013) with an exception in southern India where higher prevalence of HPV (70.6%) was reported (Kulkarni et al., 2011). Our study is in concordance with previous studies and shows that HPV was associated in 9.2% of OSCC cases with HPV 16 showing higher (30.4%) prevalence than HPV 18 (17.47%). In one fourth of the cases it is not single subtype which was present, but the co-infection of 16/18. Tobacco and betel nut chewing habits were the major etiological factors involved in our study population. 7082 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 In contrast to oropharyngeal cancer, oral cancer and other HNSCC occasionally harbor HPV (Dayyani et al., 2010; Machado et al., 2010). Its presence is strongly associated with oropharynx, most notably in tonsils and base of tongue (El-Mofty, 2003; Gillison et al., 2004). This might be due to the fact that these tumors may constitute an etiologically different subgroup within head and neck tumors. HPV might play a role in progression of premalignant lesions to advanced cancer form (Miller et al., 2001) but evidences in this direction are not sufficient. Low prevalence of HPV in oral cavity carcinoma was reported by Jerry Machado et al., 2010. Luca Scapoli et al., 2008 also found a very low prevalence of HPV in oral cavity (2%) and demonstrated no significant correlation of HPV in OSCC. Association of HPV with other factors is also controversial worldwide, for instance Luciano MarquesSilva et al., 2012 reported no association of HPV positivity with age. To the contrary Abdul Samad Gichki et al., 2012 reported higher HPV incidence between 20-59 years of age. Jerry Machado et al., 2010 found no significant association between HPV presence and smoking, alcohol status, tumor differentiation, stage and survival. Our study is in concordance with previous reports of no significant association of HPV positivity with age, marital status, demographic profile, tumor stage, grade and site of tumor. HPV in our case series was significantly associated with male gender (p=0.02) this is might be due to the fact that HNSCC is more common in males compared to females because of the presence of traditional risk factors here. Earlier studies report that majority of HPV related carcinomas of the oropharynx are nonkeratinizing squamous cell carcinoma (NKSCC) with a characteristic basaloid cellular morphology and these tumors were found to be more responsive to treatment with a favorable patient outcome and good prognosis. But HPV positive OSCC, unlike oropharynx, do not exhibit distinct morphology and have poor prognosis (El-Mofty et al., 2014). Our study is in agreement with studies where HPV positive cases did not demonstrate basaloid morphology (p=0.10) and we also found no significant differences in survival outcome (p=0.62) between HPV positive and negative cases. It is assumed that carcinogenic potential of HPV increases with viral integration to host genome (Spence et al., 2005). Smoking induces DNA damage which may favor the integration of HPV to human genome at these sites (Luo et al., 2005) thus enhancing the oncogenic potential of virus. In our study majority of HPV positive cases (91.3%) had one or multiple of the tobacco related habits like tobacco and pan masala chewing/smoking. We have observed significant relationship of HPV with pan masala chewing (0.01), a habit peculiar to the Indian subcontinent. The possibility of this may be the local injury caused to buccal mucosa during tobacco/pan masala chewing creates atmosphere for virus to infect easily. The limitation of the present study was that we were unable to evaluate the oncogenic expressions of HPV E6/E7 in our case series due to scarce sample amount. Furthermore, due to lack of relevant information regarding behavioral history of patients, the route of transmission of virus is not recognized here. The FDA approved DOI:http://dx.doi.org/10.7314/APJCP.2015.16.16.7077 Do Human Papilloma Viruses Play Any Role in Oral Squamous Cell Carcinoma in North Indians? by p16INK4a immunostaining. Asian Pac J Cancer Prev, hybrid capture method was not implemented in our cases. 13, 6083-6. However we have utilized both in-house conventional AK Chaudhary, S Pandya, M Singh, et al (2013). Identification PCR assay and a kit based real time PCR analysis to of high-risk human papillomavirus-16 and -18 infections confirm HPV presence. Further sub typing for 16 and by multiplex PCR and their expression in oral submucous 18 also show independent positive results. Hence we fibrosis and oral squamous cell carcinoma. Head Neck feel confident about the presence of HPV in our cases. Oncol, 5, 4. Despite of some limitations our data is contributing Alok Mishra, Alok C Bharti, Prishla Varghese, et al (2006). needful information regarding HPV presence in OSCC of Differential expression and activation of NF-jB family North Indian population that will be useful for the future proteins during oral carcinogenesis: Role oh high risk human papilloma infection. Int J Cancer, 119, 2840-50. treatment implications. Anil K Chaturvedi, Eric A Engels, William F Anderson, et al HPV positive cancers usually show over-expression (2008). Incidence trends for human papillomavirus -related of p16, while the loss of the p16 expression by deletion, and -unrelated oral squamous cell carcinomas in the united hypermethylation or mutation is common in tobacco states. J Clin Oncol, 26, 612-19. related cancers. Therefore, p16 up regulation is an Balaram P, Nalinakumari KR, Abraham E, et al (1995). Human indication of expression of viral oncogenes and we can papilloma virus in 91 oral Indian betel quid chewers: High expect the presence of HPV (Tran et al., 2007; Vidal et al., prevalence and multiplicity of infections. Int J Cancer, 61, 2008; Klussmann et al., 2009; Ahmed et al., 2012), but its 450-4. relevance for the site of HNSCC other than oropharynx Byakodi R, Byakodi S, Hiremath S, et al (2012). Oral cancer in India: an epidemiologic and clinical review. J Community is ambiguous. In our cases presence of HPV was not Health, 37, 316-9. significantly associated with p16 expression and all p16 Dayyani F, Etzel CJ, Liu M, et al (2010). Meta- analysis of the positive cases had history of tobacco consumption. It impact of human papillomavirus (HPV) on cancer risk and is possible that tobacco related oncogenic pathway cooverall survival in head and neck squamous cell carcinomas existed with HPV related events in our cases. Hence, (HNSCC). Head Neck Oncol, 2, 15. p16 expression was not found to be a reliable marker D’Costa J, Saranath D, Dedhia P, et al (1998). Detection of HPVfor HPV presence in our population. Our findings are 16 genome in human oral cancers and potentially malignant in concordance with Pradit Rushatamukayanunt et al., lesions from India. Oral Oncol, 34, 413-20. 2014 who also could not relate p16 expression for HPV D’Souza, A R Kreimer, R Viscidi, et al (2007). Case-control study of human papillomavirus and oropharyngeal cancer. infection in OSCC. New Engl J Med, 356, 1944-56. In conclusion, our findings illustrate that 9.2% OSCC El-Mofty SK, Lu DW (2003). Prevalence of human papillomavirus cases harbor HPV in North Indian population which type 16 DNA in squamous cell carcinoma of the palatine is slightly lower than that observed in previous Indian tonsil, and not the oral cavity, in young patients: A distinct studies and we report tobacco as a major risk factor in clinicopathologic and molecular disease entity. Am J Surg both HPV negative as well as positive cases. Therefore the Pathol, 27, 1463-70. independent role of HPV in the causation of oral cancer Elaine M Smith, Justine M Ritchie, Kurt F Summersgill, et al is difficult to evaluate in our case series due to the strong (2004). Age, sexual behavior and human papillomavirus confounding influence of tobacco. We also find that p16 infection in oral cavity and oropharyngeal cancers. Int J.Cancer, 108, 766-72. expression is not a reliable marker in the oral cavity to Freddie Bray, Jian-Song Ren, Eric Masuyer, et al (2013). Global assess the potential etiologic role of HPV. Further studies estimates of cancer prevalence for 27 sites in the adult on larger and well defined population are needed to population in 2008. Int J Cancer, 132, 1133-45. elucidate the role of HPV induced oral oncogenesis and Gillison ML (2004). Human papillomavirus-associated head co-carcinogenesis pathways need to be explored. and neck cancer is a distinct epidemiologic, clinical, and molecular entity. Semin Oncol, 31, 744-54. Acknowledgements Hafkamp HC, Manni JJ, Haesevoets A, et al (2008). Marked Differences in Survival Rate between Smokers and The authors wish to thank all those who have Nonsmokers with HPV 16-Associated Tonsillar Carcinomas. Int J Cancer, 122, 2656-64. cooperated in the study. Present work was funded by the JC De Vicente, LMJ Gutierrez, AH Zapatero, et al (2004). 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