Prevalence and trends of human papillomavirus in oropharyngeal cancer in a predominantly north Indian population

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1 ORIGINAL ARTICLE Prevalence and trends of human papillomavirus in oropharyngeal cancer in a predominantly north Indian population Ankur Bahl, MD, 1 Pankaj Kumar, MSc, 2 Lalit Dar, MD, 2 Bidhu K. Mohanti, MD, 3 Atul Sharma, MD, 1 * Alok Thakar, MS, 4 Venkat Karthikeyan, MS, 4 Kapil Sikka, MS, 4 Chiromamit Singh, MS, 4 Kamala Poo, MS, 4 Jaini Lodha, MS 4 1 Department of Medical Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, All India Institute of Medical Sciences, New Delhi, India, 2 Department of Microbiology, All India Institute of Medical Sciences, All India Institute of Medical Sciences, New Delhi, India, 3 Department of Radiation Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, All India Institute of Medical Sciences, New Delhi, India, 4 Department of Otorhinolaryngology, All India Institute of Medical Sciences, New Delhi, India. Accepted 8 February 2013 Published online 1 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. Human papillomavirus (HPV) prevalence in oropharyngeal squamous cell cancer (SCC) remains variable and studies have estimated that up to 60% may be positive. Methods. One hundred five treatment-naïve oropharyngeal SCC patients were included. HPV genotyping was done by consensus polymerase chain reaction (PCR) and reverse line-blot hybridization assay. HPV prevalence was co-related with sex, age, tobacco consumption, alcohol use, and high-risk sexual behavior. Results. HPV prevalence was 22.8%. No significant associations were seen between tobacco or alcohol consumption with HPV status. The mean number of lifetime sexual partners and indulgence in high-risk sexual behavior was significantly more in patients who are HPV positive. There were no significant associations between the 2 groups with respect to the stage of the tumor. Conclusion. Results of this study confirms that patients who are HPV positive are younger, and with high-risk sexual behavior. We did not find any impact of smoking and alcohol consumption on HPV status. VC 2013 Wiley Periodicals, Inc. Head Neck 36: , 2014 KEY WORDS: human papillomavirus, prevalence, trends, oropharyngeal cancer, Indian patients INTRODUCTION Every year, 650,000 patients worldwide are diagnosed with head and neck squamous cell cancer (HNSCC) and approximately 350,000 die of this disease. 1 In India, approximately 56,000 new cases are diagnosed per year, which is probably one of the highest incidence rates in the world, accounting for around 20% of all the cancers. 2,3 oropharyngeal SCC (squamous cell carcinoma) comprises approximately a third of head and neck cancer in India. 4 Tobacco chewing, cigarette smoking, and alcohol consumption are the major etiological factors of HNSCC. These 3 factors are synergistic and dose dependent. 5,6 The median age for various oropharyngeal cancers worldwide is 64 years, whereas in India, it is 57 years. 4,7 However, a subgroup of the population develops oropharyngeal cancer at a younger age than this median and without having been exposed to these risk factors, suggesting other etiological factors, such as genetic predisposition, diet, and viral agents particularly human papillomavirus (HPV) infection. *Corresponding author: A. Sharma, Additional Professor of Medical Oncology, Department of Medical Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi , India. atul1@hotmail.com HPV is frequently present in humans, but only a small subset develops cancer. The HPV family contains over 70 different types that can be classified based on their ability to induce cancer. Mucosal and genital HPV, consisting of about 30 types, have been divided into low-risk types (HPV 6, 11, 42, 43, and 44) and high-risk types (HPV 16, 18, 31, 33, 35, 45, 51, 52, and 56), according to their presence in malignant lesions of the cervix. 8 The transforming ability of high-risk HPV seems to depend primarily on deregulated expression of E6 and E7 HPV oncoproteins. The viral E6 and E7 genes of the high-risk types are preferentially retained and expressed in the tumors, where they inactivate the p53 and Rb tumor suppressor proteins, respectively. 9 A lot of interest has been generated regarding the role of HPV infection in oropharyngeal SCC. In the oropharynx, particularly the tonsils and the base of the tongue have been associated with HPV infection and transformation. This may be because of easy access to infection and an embryonic development similar to that of the cervix (ie, from endoderm). Tonsils contain deep invaginations in the mucosal surface that favor capture and processing of antigens. 10 Over the last couple of decades, there has been a change in the epidemiologic pattern of HNSCC. A study from the United States has reported that the incidence of oral cavity cancers has decreased, whereas oropharyngeal SCC incidence has increased. This has been observed specially in the younger population. 11 Surveillance, Epidemiology, and HEAD & NECK DOI /HED APRIL

2 ANKUR ET AL. TABLE 1. Selected studies for the association of human papillomavirus in oropharyngeal cancer. Author, year, and reference number Country Incidence Tachezy, Czech Republic 62% Pintos, Canada 43% D Souza, United States 50% Hansson, Sweden 36% Mizumachi, Japan 33% Li, Hong Kong 29% End Results data showed an increase in the annual incidence of base of tongue and tonsillar cancers by 2.1% and 3.9%, respectively, from 1973 to 2001 among white individuals between 20 to 44 years of age, whereas incidence at other sites declined. 12 An increased incidence of sexual behavior associated with viral transmission indirectly demonstrated by a concurrent increase in herpes simplex seroprevalence through this period may explain this change in demographics. A recent systematic review from 60 studies reported that the overall HPV prevalence in HNSCC was 25.9% and was significantly higher in oropharyngeal cancer (35.6%). 13 Similar to the trends seen in the United States, increased incidence of HPV infection in oropharyngeal SCC has been seen in other parts of world (Table 1) HPV infection is mainly diagnosed by molecular methods because reliable serological tools are not available and culture of the virus is not possible. 20 Light microscopy, electron microscopy, and immunohistochemistry techniques have resulted in inconsistent or irreproducible findings. The use of DNA hybridization revolutionized the detection of HPV DNA types in benign and malignant lesions. Hybridization techniques (Southern blot, dot blot, and in situ hybridization) have been used to identify specific viral genetic sequences in cells and tissues. Southern blot has been considered the gold standard for typing HPV genotypes. Although it requires about 10 ng of DNA, 0.1 copy of HPV DNA per cell can be detected with a homologous probe. 21 Southern blotting is labor-intensive and complex, and there is significant inter-laboratory variation in detection and typing. 22 Polymerase chain reaction (PCR), a method of amplifying target sequences from a DNA specimen, provides a higher degree of sensitivity and specificity than traditional hybridization methodologies. 23 PCR requires relatively small amounts of target DNA (range, ng). 24 Consequently, the technique can be applied to DNA extracted from cell suspensions or from formalin-fixed paraffin-embedded tumor tissues. Although more and more reports of HPV-positive oropharyngeal cancer are emerging from different parts of the world, there is no published Indian data on the association of HPV and oropharyngeal cancer. The purpose of this study was to determine the prevalence of HPV positivity in oropharyngeal SCC in a predominantly north Indian population. This study also assesses the differences according to sex, age, tobacco, or alcohol use and highrisk sexual behavior of the patients between HPV-positive and HPV-negative oropharyngeal cancer sites. MATERIALS AND METHODS This prospective study was carried out at the All India Institute of Medical Sciences by the departments of otorhinolaryngology, medical oncology, microbiology, and radiation oncology from May 2010 through June One hundred five newly diagnosed, biopsy proven oropharyngeal SCC patients attending the head and neck cancer clinic were enrolled in this study. Exclusion criteria included patients with malignancy other than oropharyngeal cancer, second primary cancer, or those who had taken treatment for cancer before coming to our institution. Informed written consent was taken from the patients. Ethical clearance was obtained from the institutional review board. Clinical examination and investigation All the patients were reviewed and examined by a multidisciplinary team of otorhinolaryngologists, medical oncologists, and radiation oncologists. Information about demographic characteristics, oral hygiene, medical history, family history of cancer, lifetime sexual behavior, lifetime history of smoking, tobacco chewing, and alcohol use was obtained and entered in a pre-planned proforma. Light alcohol drinking was defined as consumption of less than 2 drinks per day in men and less than 1 drink in women, whereas heavy drinking was defined as consumption of more than 2 drinks per day in men and more than 1 drink per day in women. 25 Staging was done according to the American Joint Committee on Cancer 7th Edition Guidelines. Sample storage, processing, and human papillomavirus testing One part of the oropharyngeal biopsy was sent for histopathological examination and the other part was immediately transferred to the virology laboratory in normal saline and preserved in phosphate buffer saline at 70 C until further processing. HPV testing was carried out using the linear array HPV Genotyping Test kit (Roche Molecular System, Branchburg, NJ). In brief, 150 ll of the sample was digested with 15 ll of 10 X digestion buffer (containing 700 ll of20mm Tris-HCl-1 mm ethylene diaminetetraacetic acid (TE) buffer, 100 ll 10% Tween-20, and 200 ll of 20 mg/ml proteinase K) at 65 C for 1 hour, followed by heat inactivation at 95 C for 10 minutes. The DNA was precipitated with ethanol and ammonium acetate at 20 C overnight. After centrifugation at 13,000 rpm for 30 minutes at 4 C for pelleting the DNA, the pellet was dried, resuspended in 50 ll of TE, and stored at 20 C until amplification for HPV testing. The specimen DNA was amplified using PGMY 09/ 11 HPV-specific primers that amplify the 450-bp fragment of L1 open reading frame of genital HPV. A human b-globin target was coamplified with HPV consensus primers to determine adequacy of the specimen. The PCR products were denatured and hybridized to type-specific HPV probes immobilized as a linear array on strips. Positive and negative controls were included in every PCR run. Positive hybridization was detected by color precipitation at the probe site and the type determined by matching with a reference overlay. For analysis purposes, 506 HEAD & NECK DOI /HED APRIL 2014

3 PREVALENCE OF HPV IN OROPHARYNGEAL CANCER IN INDIAN POPULATION TABLE 2. Site-specific trends among human papillomavirus infection. Site No. of patients HPV positive, No. (%) HPV 16, No. (%) HPV 18, No. (%) Other strain, No. (%) p value Base of tongue (18.6) 12 (16) 1 (1.3) 1 (1.3)*.086 Tonsil (40) 7 (28) 2 (8) 1 (4) Soft palate 5 0 (0) 0 (0) 0 (0) 0 (0) Abbreviation: HPV, human papillomavirus. * HPV strain: 33. HPV strain: 31. samples were considered sufficient for HPV determination if the b-globin probe was detected. All b-globin negative samples were excluded from further analysis. HPV types were grouped into high-risk and low-risk types as follows: high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, and its subtype IS039) and low-risk (6, 11, 40, 42, 54, 55, 57, 61, 62, 64, 71, 72, 83, 84, and 89). 8 Statistical methods Chi-square and Fisher s exact test were used to examine the association of HPV detection with sex, age, primary site location, stage, TNM classification, sexual behavior, smoking, and alcohol usage history. SPSS software version 16 was used for the analysis. RESULTS During the study period, 105 patients with oropharyngeal SCC were enrolled. The group consisted of 91 men (86%) and 14 women (14%). The site-specific distribution of oropharyngeal SCC is given in Table 2. Of the 105 patients, 24 (22.8%) had detectable HPV DNA. Nearly 19% (14 of 75) of base of tongue and 40% (10 of 25) of tonsillar carcinomas were positive for various strains of HPV (Table 2). HPV positivity was seen more commonly in tonsillar carcinoma than in other subsites of the oropharynx. Among HPV-positive samples, HPV 16 strain was the most common (79%) followed by HPV 18 (12%) and others (9%). Three of the 24 patients who were HPV positive had multiple strains. The demographic characteristics of HPVpositive and patients who are HPV negative are given in Table 3. The median age of the patients in the study population was 55 years (range, years). The median age of the patients who were HPV positive was 8 years younger than the HPV-negative group (HPV positive, 49 years vs HPV negative, 57 years; p ¼.002). When patients were stratified into young (<55 years) and old (>55 years) based upon the median age of the study group, HPV was more likely to be detected in the younger patient group (Table 3; p ¼.003). Patients living in the urban areas were more prone to being infected with HPV as compared to those living in rural areas (66% vs 40%; p ¼.02). However, the use of alcohol and smoking habits were not different between HPV positive and negative groups, with p values of.18 and.86, respectively. Contrary to this, consumption of smokeless tobacco was more profound and common in patients who are HPV positive (p ¼.028). Patients in the HPV-positive group had a higher number of mean lifetime sexual partners (1.6 vs 1.3; p ¼.049) and indulged in high-risk sexual behavior (oral and anal sex) as compared to patients who are HPV negative (58% vs 21%, respectively; p ¼.001). There was no difference of the median age at first sexual activity between the 2 groups. None of the patients in both the groups reported homosexual and bisexual tendencies. Approximately a third of the patients in both the study groups had poor dental and oral hygiene. Ninetyfour percent of the study population had stage III or IV disease. We found no significant difference between the 2 study groups in relation to tumor (T) classification, nodal (N) classification, or the overall stage of the tumor at the time of presentation. Similarly, no difference in tumor TABLE 3. Demographic characteristics of patients who are human papillomavirus positive and patients who are human papillomavirus negative. Total no. of patients (105) No. (%) HPV positive (24) HPV negative (81) p value Sex.515 Male 91 (86) 22 (91) 69 (85) Female 14 (14) 2 (9) 12 (14) Male:female ratio 6.5:1 11:1 5.7:1.09 Age <55 48 (46) 17 (70) 31 (38).003 >55 57 (54) 7 (30) 50 (62) Living Rural 59 (56) 8 (34) 48 (60).02 Urban 46 (44) 16 (66) 33 (40) Smoker Never 13 (12) 4 (17) 9 (11).86 Current 37 (35) 8 (33) 29 (36) Former 55 (53) 12 (50) 43 (53) Smokeless tobacco 18 (17) 8 (33) 10 (12).028 Alcohol Never 87 (83) 17 (71) 70 (86).18 Light 7 (6) 2 (8) 6 (9) Heavy 11 (11) 5 (21) 5 (4) Median age at first sexual activity Mean number of lifetime sexual partners High-risk sexual behaviors 31 (29) 14 (58) 17 (21).001 Frequency of high-risk sexual behavior Never 74 (70) 10 (42) 64 (80).003 Rarely 16 (15) 7 (29) 9 (11) Frequently 15 (15) 7 (29) 8 (9) Abbreviation: HPV, human papillomavirus. HEAD & NECK DOI /HED APRIL

4 ANKUR ET AL. TABLE 4. Clinical and pathologic characteristics of patients who are human papillomavirus positive and patients who are human papillomavirus negative. Variables Total no. of patients (105) No. (%) HPV positive (24) HPV negative (81) p value Oral hygiene Good 14 (13) 4 (17) 10 (12).81 Satisfactory 56 (53) 13 (54) 43 (53) Poor 35 (34) 7 (29) 28 (35) Dental hygiene Good 13 (12) 4 (17) 9 (11).72 Satisfactory 54 (51) 15 (63) 41 (51) Poor 38 (37) 5 (20) 31 (38) T classification T 1 6 (5.5) 0 (0) 6 (7).13 T 2 16 (15) 3 (12) 13 (16) T 3 53 (50) 17 (70) 36 (44) T 4 30 (29.5) 4 (18) 26 (33) N classification N 0 30 (29) 5 (21) 25 (31).42 N 1 25 (24) 4 (16) 21 (27) N 2a 15 (14) 4 (16) 11 (14) N 2b 4 (3) 2 (9) 2 (2) N 2c 14 (13) 3 (13) 11 (13) N 3 17 (17) 6 (25) 11 (13) Stage I 2 (2) 0 (0) 2 (2).54 II 5 (4) 2 (8) 3 (3) III 24 (23) 4 (17) 20 (25) IV 74 (71) 18 (75) 56 (70) Grade Well differentiated 0 (0) 0 (0) 0 (0).62 Moderately 95 (91) 20 (84) 75 (92) Poorly 3 (2) 1 (4) 2 (3) Not known 7 (7) 3 (12) 4 (5) grade was appreciable between the 2 groups on histopathological examination (Table 4). DISCUSSION Overall, 22.8% of the patients with oropharyngeal SCC in our study tested positive for HPV. HPV 16 and HPV 18 accounted for 79% and 12%, respectively, of the total positive cases. HPV 16 and HPV 18 infection in patients with HNSCC could vary by site and also by geography. Nishioka et al, 24 while studying oral cavity cancers and hyperplasia in the Greek population, reported a prevalence of 44% and 22% for HPV 18 and HPV 16, respectively. In a study of 91 oral cancers in a south Indian population, Balaram et al 26 reported an HPV 16 and HPV 18 detection rates of 42% and 47%, respectively. Both studies also showed a different HPV 16 and HPV 18 type distribution compared with our study in which HPV 16 positivity was more prevalent than HPV 18. As India is a large country, a possible explanation for this may be regional geographic differences in HPV prevalence and distribution between the studied populations. Kreimer et al 23 conducted a systematic meta-analysis to review the available literature in the field and ascertain the worldwide prevalence of HPV in HNSCC. They analyzed 60 eligible studies using PCR detection from 26 countries which included 5046 cases of SCCs of head and neck; 2642 oral cancers, 969 oropharyngeal cancers, and 1435 laryngeal cancers. HPV prevalence was 35.6% in oropharyngeal cancers, 23.5% in oral cancers, and 24.0% in laryngeal cancers. The overall prevalence of HPV in HNSCC was estimated at 26%. Similar to our study, HPV 16 was the most common subtype in all types of HPV-positive cancers in 86.7% of oropharyngeal, 68.2% of oral, and 69.2% of laryngeal cancers. HPV 18 was next most common in 1% of oropharyngeal, 8.0% of oral, and 3.9% of laryngeal cancers. The low HPV positivity rate in the present study could probably be explained by the relatively elderly study population (median age ¼ 55 years), the small size of the study group (n ¼ 105) and unequal sex distribution (women ¼ 14, men ¼ 91). Various studies have included oral, oropharyngeal, and hypopharyngeal primary sites in the study population. In the present study, only malignant lesions of the oropharynx were included. Similar to our study, various other studies have shown higher frequency of HPV positivity in cancers arising from the tonsils followed by the base of the tongue. 11,27 29 A 40% yield of HPV positivity in tonsillar cancer in our study is consistent with 21% to 100% yield of HPV 16, HPV 18, or HPV 33 in literature, strongly supporting this anatomic preference of HPV Our patients with HPV detectable in their tumors were more likely to be less than 55 years of age compared with those who were negative for the virus. Similar to the study by Ringstrom et al, 36 our patients who were HPV positive were 8 years younger than the patients who were HPV negative. Sisk et al 37 reported the risk for HPV positivity to be 1.25 greater among the younger population. In light of the increasing incidence of tonsillar cancers in young adults, HPV might represent an important etiologic factor in these patients. 38 Tobacco smoking and alcohol drinking are 2 major etiological factors involved in HNSCC development. In our study, 88% of the participants were smokers. It has been postulated that the usual high-risk behavior, like smoking and alcohol, are not as important in HPV-related HNSCC as in non HPV-related cancers, and the carcinogenesis process in the former differs from the latter. Various studies have shown that HPV-related carcinomas were found more often in those who had lower alcohol and/or tobacco exposure. 6,39,40 In the study by Smith et al, 41 they found that patients were at higher risk of detection with HPV types if they were low to moderate smokers compared to heavy smokers. Snijders et al 42 concurred that HPV status was not correlated with smoking or alcohol use in their study on 63 HNSCC tumors. D Souza et al 16 also found that exposure to HPV increased the association with oropharyngeal cancer regardless of tobacco smoking or alcohol use. They showed that the odds of developing oropharyngeal cancer increased in patients who are HPV negative who were heavy users of alcohol and smoking but did not increase in patients who were HPV positive. The present study found an insignificant difference between patients who were HPV positive and HPV negative as far as prevalence of smoking and alcohol consumption was concerned. Eighty-eight percent of the study population was smokers. The prevalence of 508 HEAD & NECK DOI /HED APRIL 2014

5 PREVALENCE OF HPV IN OROPHARYNGEAL CANCER IN INDIAN POPULATION smoking in the HPV positive and negative groups was 83% and 89%, respectively. This insignificant difference may be attributed to the already high prevalence of smoking in our study population. A vast majority of the Indian patients with HNSCC are also chronic tobacco chewers. However, as for smokers, not all these patients proceed to develop cancer and there could be various other predisposing factors, which are commonly associated with oropharyngeal SCC. It remains to be established how betel nut, tobacco chewing, HPV, and oropharyngeal SCC are interrelated. It is likely that repeated trauma to the oral cavity because of betel quid chewing is an important etiologic factor in India, as it not only stimulates mitosis (for wound healing) but also exposes germinal basal cells to infection by HPV. 26 In the present study, 17% of the study group were tobacco chewers. Consumption of smokeless tobacco was significantly higher in the HPV-positive group. Koppiker et al 43 also concluded that HPV infection was more prevalent in patients with the tobacco chewing habit. In our study, 91% of the HPV-positive tumors occurred in men. HNSCC, as such, is more common in the male population and it was a matter of chance that only 14% of the study population was female. Previous researchers have found either no association between HPV presence and sex. 30,41,44,45 Chaturvedi et al, 11 in their analysis of the Surveillance, Epidemiology, and End Results data of oropharyngeal SCCs from 1973 to 2004, also found that HPV-related tumors were more likely to be seen in men as compared to HPV-unrelated cancers. The small sample size and fewer numbers of female patients may limit our study ability of sex differences in HPV infection. Fifty-six percent of our study population was from rural areas. However, 66% of the patients who were HPV positive lived in the urban areas compared to 40% in the HPV-negative group (p ¼.02). Debanth et al 46 have also reported this association of high HPV positivity with urban residence. The probable reasons given for this association were lifestyle (late marriage, individual preference to remain unmarried, more sexual partners, and living in places apart from their families for the purpose of work, particularly in cases of drivers, businessmen, and laborers) that led to greater sexual activity. All the study subjects were married. There was no significant difference between the 2 groups in the age of initiation of sexual activity. However, similar to other published series, patients who were HPV positive were more involved in high-risk sexual behavior (orogenital and oroanal contact), had a greater number of lifetime sexual partners, and had significantly higher indulgence in highrisk sexual habits. In the population-based case control study conducted by Stephen et al 47 decreasing age at first intercourse, increasing number of sex partners, and a history of genital warts were associated with HPV positivity. Smith et al 41 found an increased risk of HPV detection with more sex partners (more than 4) in patients with HNSCC compared to HPV-negative controls. D Souza et al 16 in their case control study of HPV and oropharyngeal cancers, found an association with increasing number of vaginal sex and oral sex partners. Although a detailed sexual history was elicited from each subject in the study, taboos on this subject in India cannot be ruled out. Various investigators have tried to find the correlation between HPV status and tumor characteristics; like grades of differentiation, stage at presentation, and lymph node status. The results have been conflicting. Smith et al 41 found that over three quarters of HPV-positive cases (as against only about half of patients who are HPV negative) were identified with advanced stage of disease (III or IV), and that HPV positives were more than twice as likely to show involvement of lymph nodes. The presence of oncogenic HPV types was not significantly different between well differentiated and moderately differentiated cancers (13% vs 17%; p ¼.5). Lindel et al, 28 in their prospective study of 99 patients with oropharyngeal SCC, found no correlation between T or N classification, grade of differentiation, and HPV positivity. The findings of Sedaghat et al 48 among 75 patients diagnosed with oropharyngeal SCC who had been treated from 2000 to 2005 at the Greater Baltimore Medical Center were similar. No difference in T classification, N classification, and grades of differentiation were observed between the 2 groups in this study. The majority of our patients presented with stage III or IV disease. 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