|Year : 2016 | Volume
| Issue : 3 | Page : 387-393
Human papillomavirus/p16 positive head and neck cancer in India: Prevalence, clinical impact, and influence of tobacco use
V Murthy1, M Swain2, T Teni3, S Pawar3, P Kalkar3, A Patil4, A Chande3, S Ghonge1, SG Laskar2, T Gupta1, A Budrukkar2, J Agrawal2
1 Department of Radiation Oncology, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, Maharashtra, India
2 Department of Radiation Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India
3 Department of Teni Lab, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, Maharashtra, India
4 Department of Pathology, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, Maharashtra, India
|Date of Web Publication||24-Feb-2017|
Department of Radiation Oncology, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
BACKGROUND: Limited data are available on the prevalence and prognostic significance of human papillomavirus (HPV) in squamous cell carcinoma of head and neck (SCCHN) in the Indian population. AIM: The present study aimed to determine the prevalence of HPV and p16 in an Indian cohort of SCCHN and assess their correlation and influence of tobacco use on patient outcomes. MATERIALS AND METHODS: The p16 and HPV status of 170 patients of SCCHN treated with curative chemoradiotherapy was determined using immunohistochemistry and polymerase chain reaction, respectively, and further correlated with their demographic characteristics. In addition, genotyping of HPV-positive samples was performed. Survival outcomes were analyzed and compared for both p16 positive (p16 +ve) and p16 negative (p16 −ve) population. The influence of tobacco use on outcomes was assessed. RESULTS: p16 expression was observed in 20% (34/170) cases whereas HPV positivity was detected in 39.4% (67/170) of SCCHN patients with HPV16 being the most common (91%) subtype. About 73.5% patients were p16 +ve among the tobacco users in this cohort (83.5%). Interestingly, p16 positivity was significantly associated with nonusers of tobacco (P = 0.02) and younger females (P = 0.06). The p16 +ve and p16 −ve groups did not exhibit a significant difference in the 5-year cause-specific survival (CSS) (79% vs. 72.2%), disease-free survival (DFS) (78.3% vs. 68.3%, P = 0.5), and locoregional control (LRC) (82.2% vs. 71.5%, P = 0.4). However, the outcome analyses in tobacco nonusers revealed a definite large improvement in CSS (P = 0.08) and a trend toward improvement in DFS (P = 0.15) and LRC (P = 0.11) in the p16 +ve versus the p16 −ve groups. CONCLUSION: The low prevalence of p16 positivity (20%) and dual HPV and p16 positivity (38.8%) in the studied Indian cohort indicates the low utility of p16 as a surrogate for HPV in the background of high tobacco burden. The outcomes are largely improved in a small subset of SCCHN cases comprising p16 +ve tobacco nonusers.
Keywords: Head and neck cancer, human papillomavirus, outcome, p16, tobacco nonusers
|How to cite this article:|
Murthy V, Swain M, Teni T, Pawar S, Kalkar P, Patil A, Chande A, Ghonge S, Laskar S, Gupta T, Budrukkar A, Agrawal J. Human papillomavirus/p16 positive head and neck cancer in India: Prevalence, clinical impact, and influence of tobacco use. Indian J Cancer 2016;53:387-93
|How to cite this URL:|
Murthy V, Swain M, Teni T, Pawar S, Kalkar P, Patil A, Chande A, Ghonge S, Laskar S, Gupta T, Budrukkar A, Agrawal J. Human papillomavirus/p16 positive head and neck cancer in India: Prevalence, clinical impact, and influence of tobacco use. Indian J Cancer [serial online] 2016 [cited 2017 Mar 26];53:387-93. Available from: http://www.indianjcancer.com/text.asp?2016/53/3/387/200668
| » Introduction|| |
Squamous cell carcinoma of head and neck (SCCHN) is a major public health problem in the developing countries. Tobacco and alcohol act synergistically and are the two most important etiological factors responsible for about 75% of SCCHN. Studies have reported changing trends in the incidence of SCCHN in the United States and Europe with oropharyngeal cancer (OPC) showing an increasing trend, particularly in younger age group and in the absence of the above risk factors. This shift in epidemiology of OPC has been attributed to the infection by human papillomavirus (HPV).
Presence of HPV in tumor tissue does not imply its causative role, rather the transformative pathways lead to the development of cancer. Expression of the viral oncoproteins E6 and E7 causes degradation and functional inactivation of p53 and retinoblastoma (Rb) proteins, respectively. The E7-induced downregulation of Rb leads to the overexpression of p16 and helps to detect the oncogenic HPV. p16 has been identified as a reliable surrogate marker of HPV infection.,
HPV positive (HPV +ve) SCCHN has unique tumor biology, risk factor profile, clinical and demographic characteristics, and outcome.,,,,,,, Meta-analysis of 24 retrospective studies showed that patients with HPV +ve SCCHN have a lower risk of dying than those with HPV-negative (HPV −ve) cancers. A prospective Phase II study by Fakhry et al. (Eastern Cooperative Oncology Group 2399) and secondary analysis from different studies supports the evidence of better prognosis and overall survival (OAS) of HPV +ve oropharyngeal carcinoma.,,,,
Although there are reasonably robust data on the prevalence and outcome in HPV +ve SCCHN, especially OPCs from Europe and the US, published literature from India is sparse where the use of tobacco is extensive. The aim of this study was to evaluate the clinicodemographic characteristics, prevalence of HPV infection, p16 status, and its impact on survival in HPV +ve SCCHN in the Indian study population. We also aimed to study the influence on tobacco use in this cohort of patients.
| » Materials and Methods|| |
This is an analysis of patients of SCC of oropharynx, hypopharynx, and larynx whose clinical and follow-up information was available in the prospectively maintained database in the department and formalin-fixed, paraffin-embedded (FFPE) blocks were retrievable. The study was approved by Tata Memorial Centre - Advanced Centre for Treatment, Research and Education in Cancer Institutional Ethics Committee. It included the patients with nonmetastatic SCCHN of tumor stage T1–T3 and nodal stage N0–N2 who were treated in institutional prospective trials and had good follow-up information. They received standard radiotherapy or combined chemoradiotherapy depending on the stage of the disease. Radiotherapy was with conventional technique or intensity-modulated radiation therapy to an effective dose of 70 Gy using standard immobilization, planning, and on-treatment care. Most patients received weekly cisplatin-based chemotherapy (30 mg/m 2) with standard premedication and hydration.
p16 expression by immunohistochemistry staining
Expression of p16 protein in 5 µM FFPE sections on silane-coated slides was evaluated using CINtec Histology kit (Roche MTM Laboratories AG, Germany). After epitope retrieval, sections were incubated with prediluted primary mouse antihuman p16INK4a antibody for 30 min followed by chromogenic substrate diaminobenzidine and counterstaining with hematoxylin (S D Fine Chem. Ltd., Mumbai, India). p16 expression in all immunostained slides was evaluated and scored independently by two of the investigators. Differences in interpretation were reviewed jointly to obtain a consensus. Immunostaining in tumors was quantified based on the intensity score of nuclear (N) and cytoplasmic (C) staining (none = 0, weak = 1, moderate = 2, and strong = 3) and also the percentage of cells stained in nuclear and cytoplasmic staining calculated by obtaining the mean of C plus N (0% = 0, 1%–10% = 1, 11%–50% = 2, 51%–80% = 3, and 81%–100% = 4). The total score for each stained slide was determined by multiplying the intensity score with the positivity score, the minimum score being 0 and a maximum score being 12. A score of 0–3 was classified as negative staining and score of 4–12 indicated positive p16INK4a expression.
Human papillomavirus polymerase chain reaction and genotyping
Genomic DNA extraction from archival FFPE tissues was performed using a heat treatment protocol. Presence of HPV in the DNA extracted from FFPE tissues was checked using INNO-LiPA HPV Genotyping Kit (Innogenetics, Belgium). Polymerase chain reaction (PCR) was prepared as per manufacturer's recommendation. PCR products were subjected to a line probe assay to identify different HPV genotypes. This assay is based on the principle of reverse line hybridization in which 28 different genotype-specific HPV probes are immobilized as parallel lines on nitrocellulose membrane strips. Each PCR-amplified product was hybridized on a single typing strip. Results were interpreted manually by aligning the provided reading card to ascertain the position of the bands. Appearance of purple band at a specific position was considered positive and samples were scored positive for specific HPV type/types in accordance with the interpretation chart provided with the kit.
Descriptive statistics were used for frequency counts and for the prevalence of HPV and p16. Fisher's exact test and Chi-square test were used to determine the association of p16 with various clinicodemographic characteristics. For survival analysis and locoregional control (LRC), the time frame was taken from the date of start of radiotherapy. Events were defined for OAS (death from any cause), cause-specific survival (CSS, death attributed to disease), disease-free survival (DFS, failure of treatment, i.e., unsalvageable residual disease, locoregional, and/or distant failure), and LRC (locoregional failure or un-salvageable residual disease). Kaplan–Meier method was used to determine the time to event and comparison was made between p16 positive (p16 +ve) and p16 negative (p16 –ve) group using log-rank test. SPSS software version 20 Statistical Package for the Social Sciences (SPSS) software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp) was used for statistical analysis. Results were considered statistically significant if the P < 0.05.
| » Results|| |
Paraffin blocks of 205 patients could be retrieved of which blocks of 170 (82.9%) patients were evaluable for HPV genotyping and p16 immunohistochemistry (IHC) expression. Paraffin blocks of 35 (17.1%) patients were unusable due to improper storage, scanty tissue material, or absence of tumor tissue in the sampled material.
Demographics and prevalence
Majority of the patients were from Western India. Although being a tertiary referral center, this cohort includes patients from various parts of the country [Figure 1]. The median age of the study population was 54 years (range: 31–74 years) with male predominance (88.8%). Oropharynx was the most common site constituting 58.8% of all cases. The p16 positivity was seen in 20% (n = 34) of cases. The clinicodemographic characteristics of the study population with respect to p16 status are given in [Table 1]. Overall, 83.5% of patients were tobacco users and of p16 +ve patients, 73.5% were users of tobacco. The p16 positivity was significantly associated with females (P = 0.06) and nonusers of tobacco (P = 0.02). The prevalence by subsite was no different for oropharynx, larynx, and hypopharynx. The p16 +ve patients show a trend toward having small tumor size and were node positive (N+) compared to p16 −ve cases.
|Figure 1: Distribution of the study population (map is representative only): The regional distribution of the study population in India with predominance from Western India|
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|Table 1: Demographic characteristics of the study population with relation to p16 status|
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HPV positivity was detected in 39.4% of the patients with HPV16 being the most common subtype (91%). The HPV genotype with the site-wise distribution is given in [Table 2]. Seven cases were positive for multiple strains. Four cases were positive for type 31, 33, and 40 along with type 16 and 18 of which three cases were additionally positive for type 52, 53, and 58 and two cases were positive for type 39, 68, and 73 without the presence of HPV16 and one case was positive for both HPV 16 and 31.
Human papillomavirus and p16 correlation
The use of p16 IHC revealed 20% (34/170) SCCHN were positive in contrast to 39.4% (67/170) HPV DNA positivity. Nearly 76.5% (26/34) p16 +ve cases were also HPV +ve while only 38.8% HPV +ve cases were p16 +ve. About 7.8% cases were p16 +ve in the absence of HPV infection [Table 3].
|Table 3: Correlation of human papillomavirus and p16 status: Showing number (percentage) of p16 positive and p16 negative cases of total HPV DNA detected|
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Survival analysis was done for all the patients who completed planned treatment (95.7%) and was compared on the basis of p16 positivity status. Median follow-up of surviving patients was 43.9 months (range: 1.8–149.0 months). The 3- and 5-year OAS, CSS, DFS, and LRC were compared between p16 +ve and p16 −ve groups [Table 4]. There was no statistically significant difference in the outcomes between the groups although a trend for better CSS, DFS, and LRC was seen in p16 +ve group at 3 and 5 years [Figure 2].
|Table 4: Comparison of median survival, 3- and 5-year outcomes in p16 positive and p16 negative patients|
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|Figure 2: Survival and p16 status: Kaplan–Meier curve comparing overall survival, cause-specific survival, disease-free survival, and locoregional control in p16 positive and p16 negative patients shows no difference between two groups|
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Tobacco use and relation with outcomes
The impact of tobacco use on OAS, CSS, DFS, and LRC in p16 +ve group was analyzed separately for tobacco users and nonusers [Figure 3] and [Figure 4]. There was a definite improvement in OAS and CSS among p16 +ve tobacco nonusers which reached close to statistical significance (P = 0.08). Furthermore, a definite trend toward the improvement in DFS and LRC was observed in p16 +ve nonusers of tobacco although it did not reach statistical significance (P = 0.15 and 0.11). No difference in survival was observed in patients using tobacco.
|Figure 3: Overall survival and cause-specific survival in p16 positive tobacco user and tobacco nonuser: Kaplan–Meier curve comparing overall survival and cause-specific survival in p16 positive user and nonusers of tobacco shows trend toward better survival in nonusers of tobacco|
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|Figure 4: Disease-free survival and locoregional control in p16 positive tobacco user and tobacco nonuser: Kaplan–Meir curve comparing disease-free survival and locoregional control in p16 positive user and nonusers of tobacco shows trend toward benefit in nonusers of tobacco|
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| » Discussion|| |
This is one of the largest studies from India to report the prevalence of p16 and HPV in SCCHN with predominance of patients from Western India and the first attempt to provide data on clinicodemographic characteristics and survival outcomes of SCCHN based on the tumor p16 expression.
In this study, the overall prevalence of p16 positivity and p16 positivity in oropharyngeal carcinoma is in agreement with the study from North India in which 22.8% of OPCs were HPV +ve. It may however be noted that Bahl et al. determined the HPV DNA status without reporting the p16 positivity which is a surrogate for transcriptionally active HPV infection. In a recent study by Bhosale et al., 427 patients of SCCHN were analyzed for p16 expression, which showed a very low p16 and HPV positivity (9/427 and 7/427, respectively). There are small series of patients with oral cancers showing a prevalence of HPV ranging from 17% to 50% in India.,,, Studies from the West show a higher p16 positivity rate of about 43%–66%, especially in oropharyngeal carcinoma.,,, A recent systematic review and meta-analysis by Mehanna et al. showed an overall HPV prevalence of 47.7% and 21.8% in oropharyngeal and non-OPCs, respectively. While the prevalence in oropharyngeal site is lower here, p16 positivity in nonoropharyngeal sites (hypopharynx and -larynx) was 20% and fits with the Western data. There are no previously reported data on HPV prevalence in carcinoma hypopharynx and larynx from India.
A typical patient from the West with HPV +ve SCCHN is a young male with the median age varying from 54 to 57 years.,, Analysis of demographic characteristics of the present cohort showed that p16 +ve tumors are more likely to be detected in a younger female with a median age of 53 years. This may be explained by the differences in the pattern of tobacco use in females, patterns of sexual behavior in India compared to the US as p16 positivity is high likely to be detected among the nonsmokers and those with high-risk sexual behaviors., The overall prevalence of smoking is 18.1% with 14.5% in women compared to 20.1% in men in the US, whereas in India it is 2.9% among females compared to 24.3% in males.
While it is generally believed that a classical HPV +ve patient has a small tumor size with large, multiple cystic nodes,,,,, there are conflicting data about the correlation of HPV/p16 status and tumor characteristics.,,,, In this study, although the p16 +ve patients did show a trend toward having small tumor size and node positivity, it did not reach statistical significance, due to the small number of p16 +ve tumors.
The high prevalence of tobacco use (83.5%) in this study is supported by a large series of OPC from India with prevalence of tobacco use of 80.5%. In this study, despite the higher prevalence of tobacco use in both p16 +ve and p16 −ve groups (73.5% and 86%, respectively), nonusers of tobacco were more likely to be positive (P = 0.02). Karpathiou et al., in a study of 120 patients, have shown that p16 positivity was significantly associated with nonsmoking. Although HPV +ve SCCHN is common in nonsmokers and nondrinkers, the degree of synergism of HPV infection with the classical risk factors is not clear, and data exist for both synergistic  and additional effect.
Based on the above, it may be inferred that a typical patient of p16 +ve SCCHN in the present study is a young female, nonuser of tobacco, possibly with a small tumor and positive node of either oropharynx (tonsil) or nonoropharyngeal site.
p16 specifically identifies transcriptionally active HPV in the tumors. A number of studies have shown the correlation between HPV and p16 status in SCCHN.,, In this study, although HPV positivity was seen in 39.4%, 38.8% of the HPV infection were transcriptionally active. The importance of p16 positivity lies in the fact only p16 +ve HPV +ve tumors are biologically and clinically relevant. During analysis, we classified the tumors into three subgroups based on both HPV and p16 status (Class I: HPV −ve/p16 –ve; n = 90, Class II: HPV +ve/p16 –ve; n = 41, Class III: HPV +ve/p16 +ve; n = 24), but survival analysis was not meaningful due to relatively small number of p16 +ve patients overall. Weinberger et al. classified tumors into the above subgroups and showed that Class III patients have better outcomes, and mere presence of HPV does not have a significant prognostic value. This group also had the highest viral load to suggest being the causal factor in SCCHN. Although the usefulness of p16 alone is proven, Junor et al. reported the importance of HPV +ve/p16 −ve (Class II) group as a separate clinical entity having better survival with the use of chemotherapy. The discordant HPV +ve/p16 −ve group may represent three possible scenarios: (a) HPV is present as a commensal without any role in pathogenesis, (b) HPV is active but tobacco-induced promoter hypermethylation of p16 gene silences the function and prevents overexpression of p16, and (c) HPV is active but through activation of other Rb-induced pathways rather than p16 pathway. The significance of discordant HPV/p16 remains unresolved and needs further study.
We did not find any statistically significant difference in survival outcome between HPV/p16 +ve and p16 −ve patients. At first this seemed surprising, but on a detailed search of the literature, we found that a number of studies have reported either no difference or even worst outcome in HPV +ve tumors.,,, A site-specific meta-analysis showed that the survival outcomes were improved in HPV +ve oropharyngeal tumors, but not in nonoropharyngeal sites. On subset analysis of our data (not shown), there was no difference in survival in nonoropharyngeal subsites based on p16 status in the present cohort. A large majority (73.5%) of p16 +ve patients were tobacco users in this cohort. The lack of definite survival advantage among the p16 +ve patients in our study population may be due to the predominant activation of multiple known and unknown tobacco-related pathways of tumorigenesis. The high tobacco use even in the HPV +ve patients may have masked the prognostic significance of p16 positivity in the present cohort. On analysis of outcomes in p16 +ve tobacco users and nonusers separately, a clear, large, and clinically significant survival advantage was observed in p16 +ve nonusers of tobacco. There was no difference (clinical and statistical) in survival among the p16 +ve and p16 −ve users of tobacco.
The strengths of this study are that it is the largest study of p16 prevalence in SCCHN from India with clinical outcome data. The HPV status was confirmed by both IHC and DNA PCR, and p16 prevalence is reported in nonoral/nonoropharyngeal sites. This may be taken as the benchmark for the future studies on the prevalence and outcomes in HPV +ve SCCHN in India and South Asian region. The limitations of this study include lack of data on the high-risk sexual behavior and its relation with the p16 prevalence. Although the data on the tobacco use were available, the quantity, duration, and the quitting status were not recorded. About 17.1% blocks were not usable as blocks were old and may have suffered from storage and archival artifacts. We have also not reported the E6/E7 status which is the ultimate proof for active HPV infection.
| » Conclusion|| |
The prevalence of p16 positivity is low (20%), and tobacco use is high even in p16 +ve SCCHN in the study cohort compared to the West. In this setting, no difference in outcome was found in p16 +ve SCCHN. However, a large and clinically significant difference in survival was observed in p16 +ve nonusers of tobacco. p16 positivity becomes less reliable as a surrogate of HPV infection in the presence of high tobacco burden. This may have implication in doing p16 routinely in clinical practice in the country and implication while considering for treatment de-intensification strategies.
We thank Mrs. Sadhana Kannan for her guidance in the statistical analysis of the data.
Financial support and sponsorship
The study was supported by the Department of Biotechnology (Grant Number: BT/PR15004/MED/30/585/2010).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]