Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India

Background Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc. Methods A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR). Results The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit. Conclusions Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.


Results
The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr-HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit.
to degradation of p53 through its interaction with the E3 ubiquitin ligase E6AP [31,32]. The active E7 degrades the retinoblastoma tumor suppressor protein (pRb) due to which transcription factor E2F is stimulated and results in overexpression of p16 INK4A, a cyclin-dependent kinase inhibitor [31,32]. By evading the above mentioned master guardians of cell cycle, HPV genes take control over the cellular proliferation that leads to uncontrolled cell division.
The North Eastern (NE) region of India has high age adjusted incidence rate (AAR) per 1, 00,000 persons for HNCs in India [33]. Among all population based cancer registries (PBCR), the Kamrup Urban district (KUD) of NE region of India was ranked second (AAR-156. 3) in females and fourth (AAR-185.2) in males for all cancer sites incidence. The AAR for leading HNC sites in KUD PBCR is as follows: tongue 9.4 (male), 3.2 (female); mouth 7.7 (male), 7.6 (females); hypo pharynx 7.7 (male) and 7.6 (females); larynx 8.2(male). Tobacco related cancers (TRC) accounted for >40% but <50% in males whereas in females it was >30% but <40% of all HNC cases in KUD PBCR [33]. The population of NE region represents unique ethnicity, distinctive life style and food habits which can play important role in the complex interplay of environmental and genetic factors that may be associated with high incidence of HNC in this region. There is lack of sufficient data on the hr-HPV status in HNC cases of NE India. The aim of this study was to investigate the prevalence of hr-HPV infection and their association with betal quid chewing, smoking, tobacco, alcohol consumption and clinico-pathological characteristics of patients. In the present study, E6 nested multiplex PCR method was used for the sensitive and type-specific detection of HPV infections based on the amplification of the viral E6/E7 oncogene as described previously [28,34]. The Hybrid Capture 2 (HC2) test, which is FDA-USA approved and WHO recommended for detection of HPV in clinical specimens of cervical intraepithelial lesions (CINs) have been applied for the first time for hr-HPV detection in HNC specimens of NE India.

Patients Characteristics
A total of 106 HNC patients were enrolled in this study (male 73; female 33). Tumor tissue specimens were obtained from the Head and Neck oncology surgery unit of Dr. Bhubaneswar Borooah Cancer Institute (BBCI), Regional Cancer Center, Guwahati, India. The histopathologically confirmed HNC cases were enrolled during the period of October, 2011 to September, 2013. The demographic and lifestyle information were collected using a pre-designed questionnaire through personal interview. The information collected about demographic variables included age, gender, ethnicity, education, socio-economic status, life style factors of alcohol, cigarette smoking, betel nut chewing and other food habits. The American Joint Committee on Cancer's TNM staging was used for staging and diagnosis. The patients were informed about the study and written consent was obtained prior to collection of the specimens. This study was approved by the institutional ethics committee of Dr. Bhubaneswar Borooah Cancer Institute (BBCI), Guwahati, Assam (India).

Genomic DNA isolation from tumor tissue
Tissue biopsy specimens were collected in 1mL volume of phosphate buffered saline (PBS) and the genomic DNA was extracted from the homogenized tissue by using QIAamp DNA mini kit (Qiagen, Germany) following the manufacturer's instructions. The quantity and quality of the isolated genomic DNA was confirmed by Nano-spectrophotometer (Bio-photometer, Eppendorf, Germany) and agarose gel electrophoresis respectively.

High -risk HPV DNA detection by Hybrid Capture 2 (HC2) Assay
Genomic DNA isolated from biopsy samples of the patients were suspended in the specimen transport medium (STM) (Qiagen, Germany) and the presence of HPV was detected by HC2 High-Risk HPV DNA Test™ kit (Qiagen, Germany) as per the instructions of the manufacturer. In the patient STM samples, chemiluminescent detection of the 13 most common hr-HPV types (16,18,31,33,35,39,45,51,52,56,58,59, and 68) was performed using automated DML 2000 Luminometer system (Digene, USA). Light signals were measured as relative light units (RLUs) with light intensity indicating the presence or absence of target DNA in the tested specimen. The mean RLU value for high risk HPV calibrator of positive control samples was considered as positive cutoff (CO) value. Patients specimen with RLU/CO value ratio 1 with high-risk HPV probe were considered "positive" for any of the hr-HPV types (16,18,31,33,35,39,45,51,52,56,58,59 and 68). The absence of hr-HPV DNA below the detection limit was denoted in sample with RLU/ CO <1. When RLU = CO it confers presence of approximately 5000 virus copies in the specimen. Two confirmed cases of cervical cancer with hr-HPV positivity were also used in each test run and carrier DNA as negative control [35,36]. The test reports were generated as per the format of the Hybrid Capture TM software ver. 2.0. All the samples were tested twice and samples with hr-HPV positive outcome in both test run were only scored as hr-HPV positive.

E6 nested multiplex PCR (NMPCR) for HPV genotyping
The samples tested positive for hr-HPV DNA in the HC2 assay were further analysed for HPV genotyping. The nested E6 PCR reaction 1 st round was done in 20 μL volume using 25-100 ng genomic DNA, 1X Maxima Hot Start PCR buffer, 1.5-2.0 mM MgCl 2 (Thermo Scientific, USA), 250 nM of each primers (Metabion, Germany), 250 μM of each dNTPs and 0.5 U Maxima Hot Start Taq DNA Polymerase (Thermo Scientific, USA). The thermal cycling condition for 1 st round of E6 PCR was as: Denaturation-95°C-5 min, 38 amplification cycles with Denaturation-95°C-30 sec, Annealing-55°C-45 sec, Extension-72°C-90 sec, and final Extension-72°C for 7 min. The 1 st round E6 PCR product (630bp) was used as a template (1 μL) for the 2 nd round of nested reaction for HPV genotyping using multiplex set of HPV type specific primers. The reaction volume and constituents were similar as mentioned above. The 2 nd round nested PCR condition for HPV genotyping was similar to 1 st round with changes only in Annealing-55°C-40 sec and Extension-72°C-45 sec [34]. All PCR products were visualized in 2% agarose gel (Amresco, USA) stained with ethidium bromide (Amresco, USA) in Gel Doc XR™ system (Bio-Rad, USA). The hr-HPV positive cervical squamous cell carcinoma samples DNA was used as positive control in each PCR run. The primer sequences are described in Table 1.

Statistical Analysis
The data was represented as mean ± SD or in frequencies (%). The χ² test and Fisher exact test were employed to identify the association between clinico-pathological characteristics and hr-HPV status. Univariate analysis was done by calculating the odds ratios (ORs) along with 95% confidence intervals (CIs) and P 0.05 was considered to be the significance level. Epi-info Version 6 software was employed for the statistical analysis

Characteristics of the Patient
In the study population (n = 106), 68.86% of patients (n = 73) were males and 31.13% were (n = 33) females. Patients' age ranged from 25 to 80 years (Mean = 53.41 years). More than three fourths (80.18%) of the patients were from rural areas, rest (19.56%) were from semi urban and urban areas. Non-vegetarian dietary habit was reported by >92% of the patients ( Table 2). The study population was grouped into either of 'hr-HPV positive' (n = 33) or 'HPV negative' (n = 73) based on the presence of hr-HPV DNA. Demographic characteristics of the patients and their dietary habits are mentioned in Table 2. The demographic factors were not significantly associated with hr-HPV infection (p>0.05). Out of the 106 cases, the anatomical sites among clinico-pathological characteristics of patients were of oral cavity 81.13% (n = 86), oropharynx 4.71% (n = 5), hypopharynx 2.83% (n = 3), larynx 8.49% (n = 9) in (Table 3). Percentages of patients diagnosed with advanced stage HNCs were stage III 24.52% (n = 26), IVA- Table 1. HPV type-specific nested PCR Primer Sequences.

hr-HPV detection and its association with Demographic and Clinico-Pathological characteristics
The high risk HPV DNA was detected by nested multiplex PCR (NMPCR) in 31.13% (n = 33) and hybrid capture in 24.52% (n = 26) of the HNC patients. The sensitivity and negative positive predictively was found to be 100% each; the specificity and positive predictive value (PPV) was observed as 91.19% and 78.78% respectively. This test classified 93.29% of the cases correctly and the area under the curve was found as 89.50% (p<0.001) with 95%CI (81.30% -97.80%). To confirm the biologically active HPVs, p16 expression was analyzed in both HPV positive and HPV negative cases, and over expression of p16 was found in HPV positive cases which is an indirect indicator for the presence of HPV (data not shown). In HC2 tested samples, mean RLU value was in the range of 99-3466, and the mean positive cut off (CO) value for hr-HPV was 347 (Qiagen, Germany) as per the instruction. The High risk HPV positive cases were genotyped for HPV subtypes using E6 nested multiplex PCR. The primer details used for the E6 nested multiplex PCR [29] are mentioned in the  Table 2. The alcohol drinker samples have shown to bear hr-HPV infection compared to non-drinkers. To look out for the synergistic or complementary effect between tobacco and alcohol habits, we observed significant association with hr-HPV positivity (OR = 3.09; 95% CI. 1.00-9.65, p = 0.02), but when cases of smokers and alcohol habits were together analyzed and calculated we found that there was no significant association with hr-HPV positivity. The betel nut chewers also had shown more hr-HPV infection. Among hr-HPV positive cases (n = 33), 39.40% (n = 13) patients were tobacco non-chewers and 60.60% (n = 20) were tobacco chewers. Non-smokers group 69.69% (n = 23) have more likelihood of hr-HPV infection compared to smokers 30.31% (10). Patients residing in rural areas have more hr-HPV positivity 87.88% (n = 29) compared to semi urban 9.09% (n = 3) and urban 3.03% (1%) area residents. There was no significant association found between hr-HPV  (Table 2). Among hr-HPV positive cases (n = 33), oral cavity 72.72% (n = 24), larynx 6.06% (n = 2), hypopharynx 6.06% (n = 2) and oropharynx 15.15% (n = 5) had hr-HPV DNA (Table 3). There was significant association between hr-HPV infection and oropharyngeal tumor site (p = 0.01). Majority of the hr-HPV positive cases 90.90%, (30 of 33) were presented with advanced stage III, IVA or IVB disease. Histologically, 90.90% hr-HPV positive cases were squamous cell carcinoma, 6.06% were verrucous carcinoma and 3.03% were other carcinomas (adenoid cystic carcinoma, spindle cell malignant) ( Table 3). There was no significant association of TNM tumor stage, histology grade and morphology with hr-HPV positivity (p>0.05). When HNC cases were analyzed with lifestyle factors, alcohol consumption was found to be significantly associated with hr-HPV infection (p<0.001) ( Table 2). Next we analysed hr-HPV status in oral cavity cancers alone (Table 4) and compared with smoking, tobacco, alcohol, betel nut use and found that there was significant association of hr-HPV positivity and alcohol consumption (OR = 2.98; 95% CI. 0.95-9.43, p = 0.03). To find the synergistic or complementary effect of tobacco and alcohol habits, from univariate analysis it was found that there is significant association of hr-HPV positivity with tobacco and alcohol habits(OR = 2.94; 95% CI. 0.95-8.88, p = 0.05) ( Table 4). But when analyzed the case of smokers and alcohol habits together, no significant association was found with hr-HPV positivity.

Discussion
The Human papilloma virus (HPV) has been identified as an etiological factor in subset of head and neck cancer especially oropharyngeal cancer [37]. Most of the HPV positive associated oropharyngeal carcinoma patients were reported to be younger in age and mostly had no history of tobacco/alcohol use [38]. Some research groups have advocated that HPV positive and HPV negative HNC cases should be considered as two distinct clinical, pathological entities and the treatment regimens need to be devised accordingly [37][38][39]. The tumor hypoxia fraction and proliferation abilities were significantly decreased in HPV positive tumor cells after irradiation in comparison to HPV negative cells [40]. It was demonstrated that HPV positive locally advanced HNC patients have shown significantly higher pathological complete response as compared to HPV negative cases undergoing chemotherapy regimen [41]. Numerous studies have reported HPV DNA detection in HNSCC with varying rates, the detection techniques included in situ hybridization and southern blot hybridization (lower sensitivity methods) or PCR (high sensitivity methods) [42]. Moreover, methods used for sampling and storage also vary. The lack of universally acceptable standardized and clinically relevant procedure for HPV detection has raised concerns about their application in routine clinical setting. Our study has, for the first time assessed the applicability of HC2 assay in clinical specimens of HNC cases from NE India. We propose that Hybrid Capture 2 (HC2) assay, which is approved by Food and Drug Administration (FDA, USA), for hr-HPV detection in clinical cervical specimens, can also be used for hr-HPV detection in HNC specimens. Due to semi-quantitative nature of HC2 assay it may have application for prognostic assessment of hr-HPV in HNC patients.
We used E6 nested multiplex PCR (NMPCR) for HPV genotyping, and found HPV-16 in 81.81% (n = 27) and HPV-18 in 18.18% (n = 6) of the hr-HPV positive (n = 33) cases, which is 31.13% of HNC patients (33/106). Our study data is supported by previous studies from India which had found HPV DNA in the range of 22% to 73% in oral cancer cases [43][44][45]. Among (n = 33) hr-HPV cases, we found (n = 27) HPV 16 in which 70.37%, 14.82%, 7.41%, 7.41% and (n = 7) HPV-18 in 83.33%, 16.66%, 0%, and 0% of oral cavity, oropharyngeal, laryngeal and hypopharyngeal cancer patients respectively. Our study shows higher HPV-16, 18 infection rate compared to earlier studies from India [43][44][45][46]. We found that oropharyngeal cancer site compared to other tumor sites has shown significant risk for high-risk HPV infection. Our data is supported by earlier study, in which it was found to have association of oropharyngeal cancer with high-risk HPV infection [47]. Epidemiology of HPV positive oropharyngeal squamous cell carcinoma (OPSCC) are distinct from HPV negative ones and are characterized by younger age and strongly associated with sexual behavior. HPV associated oropharyngeal cancer (HPV-OPC) is growing in incidence and has distinct clinical, pathological, molecular and epidemiologic features [38]. The difference in our observations with other studies can be attributed to different methodologies, ethnic variation, tumor sites etc [48]. Our data show that betel nut chewing, smoking may not have significant association with hr-HPV infection. The hr-HPV positivity in the HNC cases was small which may confound the role of betel nut, smoking as risk factors for HPV infection. It was demonstrated by Ang et al (2010) that HPV positive oropharyngeal cancer progression may not be strongly associated with tobacco-smoking [47]. But other studies have shown tobacco association with HPV infection [49]. We observed in our data that tobacco chewers have significantly higher hr-HPV infection, posing tobacco chewing as a risk factor for hr-HPV infection. The concentration of nicotine and its exposure duration to the cell may alter the antigen mediated signaling pathways [50]. Alteration in the antigen mediated signaling events in the immune cells may be the mechanism by which nicotine or other constituents of cigarette inhibit the response of cell for bacterial/ viral infection [50]. We suggest that tobacco associated carcinogens may induce alterations in genetic events which may lead to molecular changes, making the individual susceptible to hr-HPV infection.
In the present study hr-HPV infection was found to be significantly associated with alcohol consumption in HNC cases. Oh et al (2014) recently demonstrated that hr-HPV load and alcohol consumption may have synergistic effect on hr-HPV infection and its persistence in cervical cancer [51]. Another recent study has shown that alcohol intake was associated with significantly increased risks for HPV infection in men [52]. This supports our observation that alcohol intake, may act as a risk factor for HPV infection.
Ethanol, a major constituent of alcoholic beverages, is oxidized to acetaldehyde by alcohol dehydrogenases (ADH). Acetaldehyde is metabolized to acetate by aldehyde dehydrogenases (ALDH). Poor oral hygiene and smoking can change the oral bacterial flora which can lead to increased acetaldehyde production [53]. In animal models, acetaldehyde has been proved as carcinogenic and mutagenic [54]. Genetic factors such as ADH/ALDH2 polymorphism, lack of ALDH2 or low levels of expression of ALDH2 and alcohol drinking habits were reported to be associated with higher risk of head and neck cancer [55,56].
Alcohol can also modify innate immune response in dose dependent manner which can result in altered inflammatory response to infection [57]. Since immune response plays major role in HPV infection, alcohol consumption may help in evading the immune response for HPV infection. Systemic immunity components such as cytokine serum level have been found to be increased in persistent HPV infection cases [58].
Our study provides evidence about increase in vulnerability for hr-HPV infection in tobacco chewers and alcohol drinkers mostly in oral cavity cancer patients. Our observation is well supported by previous HNC studies from India and worldwide [14]. From the total number of cases when tobacco chewing and smoking habits were analyzed with HPV, the associations were not significant (Table 2), as major proportion of population were HPV negative and HPV positive cases were less. But when the chewing habits were taken along HPV positive cases alone, significant association was found (Table 4) proving that tobacco chewing and alcohol consumption act as risk factors for HPV infection. More number of oropharyngeal cancers in our study might have brought a better assessment of alcohol and tobacco association with hr-HPV in these cancers. We focused primarily on hr-HPV DNA detection by employing newer methodology such as HC2 assay and E6-NMPCR which are less used in HNC. The alcohol drinking and tobacco chewing/smoking are widespread habits in North-East Indian population. We feel that mass sensitization through screening cum awareness programmes about HNC risk factors can help in reducing the HNC incidence in the NE population in future.
We conclude from our present study data that hr-HPV infection may be more prevalent in tobacco chewers, alcohol drinkers and they act as risk factors for HPV infection in HNC cases of North-East India. Hybrid Capture 2 (HC2) assay due to its high negative predictive value, specificity and sensitivity can be applied for hr-HPV detection in HNC clinical specimens.