|Year : 2022 | Volume
| Issue : 3 | Page : 72-77
Incidence and characteristics of human papillomavirus-positive oropharyngeal cancers by p16 expression
Murugaiyan Nagarajan, Ramesh Banu, Ananthakrishnan Radha, Sasikala Saranya
Department of Oncology, Valavadi Narayanasamy Cancer Centre, G. Kuppuswamy Naidu Memorial Hospital, Coimbatore, Tamil Nadu, India
|Date of Submission||30-Apr-2021|
|Date of Decision||07-Dec-2022|
|Date of Acceptance||12-Dec-2022|
|Date of Web Publication||21-Dec-2022|
Department of Oncology, Valavadi Narayanasamy Cancer Centre, G. Kuppuswamy Naidu Memorial Hospital, Coimbatore - 641 037, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Head-and-neck cancers are one of the most common cancers in the Indian subcontinent. The rising incidence of human papillomavirus (HPV), especially in oropharyngeal cancers is likely to increase the burden by many folds. Hence, we decided to study the incidence of HPV in oropharyngeal carcinoma and its characteristics. Materials and Methods: Patients with primary squamous cell carcinoma (SCC) of the oropharynx registered in our hospital between September 2018 and July 2020 were included in the study after obtaining informed consent. A total of 60 patients were included in the study. The evaluation of HPV status was done by immunohistochemistry for p16 expression. Results: The median age of presentation was 60 years (range: 38–85 years). Fifty-four patients were male and six patients were female. The incidence of HPV in oropharyngeal carcinoma was 21.7% (n = 13). There was no difference seen when we compare HPV-positive patients with HPV-negative patients as well as with the entire study population for the demographic characteristics such as age (P = 0.569), gender (P = 0.754), smoker (P = 0.368), history of alcohol consumption (P = 0.558), and history of tobacco chewing (P = 0.781). We did not find any association between HPV-positive and HPV-negative patients with anatomical subsites (P = 0.369), tumor stage (P = 0.397), and nodal stage (P = 0.592). HPV-positive oropharyngeal SCC (OPSCC) patients presented more at early stage as compared to HPV-negative patients (P = 0.005). HPV-positive patients had higher incidence of histological poor differentiation than HPV-negative patients (P = 0.024). Conclusion: The study highlighted the incidence of HPV (21.7%) among OPSCC patients using p16 expression. HPV-positive patients have propensity for early stage of presentation and histological poor differentiation. The demographic characteristics and anatomical subsites of OPSCC had no impact on HPV status.
Keywords: Human papillomavirus, incidence, oropharyngeal carcinoma, p16 expression
|How to cite this article:|
Nagarajan M, Banu R, Radha A, Saranya S. Incidence and characteristics of human papillomavirus-positive oropharyngeal cancers by p16 expression. Oncol J India 2022;6:72-7
|How to cite this URL:|
Nagarajan M, Banu R, Radha A, Saranya S. Incidence and characteristics of human papillomavirus-positive oropharyngeal cancers by p16 expression. Oncol J India [serial online] 2022 [cited 2023 Jun 6];6:72-7. Available from: https://www.ojionline.org/text.asp?2022/6/3/72/364562
| Introduction|| |
During the past few decades, a significant increase in oropharyngeal cancers was found despite the decline in tobacco addiction. These divergent incidence patterns led to postulations of various theories where human papillomavirus (HPV) infection was considered to be an additional risk which was substantiated with subsequent molecular studies in several countries and reported dramatic increase in proportion of HPV-positive oropharyngeal carcinomas. Chaturvedi et al. in a study reported 22.5% increase in the incidence of HPV-positive oropharyngeal cancer in the United States from 1988 to 2004 instead of 50% decline in HPV-negative cancers.
Thus, this study aims to analyze the incidence of HPV in oropharyngeal cancers using p16 as a surrogate marker of HPV infection in head-and-neck squamous cell carcinomas (HNSCCs) and its characteristics.
| Materials and Methods|| |
This observational prospective study was conducted in patients with oropharyngeal cancers registered in our center between September 2018 and July 2020. All newly diagnosed primary cancer of the oropharynx (Any T and Any N) and those who expressed their willingness to participate in the study were included in the analysis.
This study was approved by the Institutional Ethics Committee via letter number A2018/IEC/015.
Pretreatment workup includes general physical examination, ear–nose–throat examination, laryngopharyngoscopy, routine blood counts, liver and renal function tests, and viral markers. Imaging such as contrast-enhanced computed tomography scan of the neck and chest X-ray was done. Biopsy from the lesion was taken and was subjected to histopathological confirmation and grading of the lesion. Patients were staged according to the tumor, node, metastasis (TNM) staging system of the American Joint Committee on Cancer manual 8th edition considering the HPV (p16) status.
Evaluation of human papillomavirus status
The biopsy sampled tissue was subjected to immunohistochemistry (IHC) for p16 expression is a surrogate marker for HPV. IHC staining was carried out with ready-to-use mouse monoclonal antibody against p16(INK4A) on the Ventana BenchMark ULTRA (Ventana Medical Systems, Tucson, USA). The standard protocol included deparaffinization, rehydration, retrieval, quenching, and incubation with primary antibody, and further treatment with polydetector diaminobenzidine system (Ultra view Universal DAB detection kit, Ventana Medical System), and the slides were counterstained with hematoxylin and results interpreted using light microscopy. IHC expression for p16 was reported to be positive if there was diffuse nuclear and cytoplasmic staining in more than 70% of tumor cells [Figure 1].
|Figure 1: Photomicrograph of tumor section showing p16(INK4A) overexpression (×40)|
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Descriptive analysis was carried out by mean and standard deviation for quantitative variables, frequency, and proportion for categorical variables. All quantitative variables were checked for normal distribution within each category of explanatory variable using visual inspection of histograms and normality Q–Q plots. The Shapiro–Wilk test was also conducted to assess normal distribution. Categorical outcomes were compared between study groups using the Chi-square test/Fisher's exact test. The key outcome variable considered for the analysis was the incidence of HPV and P < 0.05 was considered statistically significant. IBM SPSS version 22 (Armonk, NY: IBM Corp) was used for statistical analysis.
| Results|| |
A total of 60 oropharyngeal cancer patients were included in the study which consisted of 54 males and six females. The basic characteristics of the study population are depicted in [Table 1]. The median age of presentation was 60 years (range 38–85 years). According to exposure of tobacco/alcohol, 78.3% of patients were smokers, 53.3% of patients had history of alcohol consumption, and 20% had history of tobacco chewing. The site-specific distribution of oropharyngeal squamous cell carcinoma (OPSCC) is depicted in [Table 2]. The posterior one-third of the tongue (n = 29) is the most common site for OPSCC followed by the tonsil (n = 18). The majority of patients were Stage III/IV OPSCC (n = 42), whereas only 18 patients were having Stage I/II disease [Table 2].
|Table 2: Comparison of demographic characteristics of human papillomavirus-positive and -negative oropharyngeal cancers|
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On IHC examination of biopsy tissue sample, p16 expression was detected in 13 patients showing HPV prevalence of 21.7% in oropharyngeal cancers and the rest 47 were HPV negative.
The comparison of demographic characteristics of both HPV-positive and HPV-negative patients is depicted in [Table 2]. HPV-positive patients consist of 12 men and one woman. The median age of presentation was 59 years (range 51–78) in HPV-positive groups which was only 1 year younger than that of the HPV-negative group (60 years). When we characterize the age group into ≤60 years and >60 years, we did not find an association between HPV-positive and HPV-negative groups with age of presentation (P = 0.569). Moreover, we did not find any difference in between HPV-positive and HPV-negative groups for gender character (P = 0.754), smoker (P = 0.368), history of alcohol consumption (P = 0.558), and tobacco chewer (P = 0.781).
The comparison of tumor characteristics between HPV-positive and HPV-negative patients is depicted in [Table 3]. Seven out of 29 (24.1%) posterior one-third of the tongue and 5 out of 18 (27.8%) tonsillar carcinomas were HPV positive. According to site-specific distribution, there was no significant difference seen between HPV-positive and HPV-negative groups (P > 0.05). Most of the patients in our study presented with T3 disease at the time of diagnosis (61.5% in HPV positive). The most common nodal presentation in the study population was N2 (69.2% in the HPV-positive group) [Table 3].
|Table 3: Tumor characteristics of human papillomavirus-positive and -negative oropharyngeal cancers|
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Early stage of presentation was found more in the HPV-positive group than in the HPV-negative group (61.5% vs. 21.3%: P =0.005). Regarding the grade of tumor, histological poor differentiation was observed more in HPV-positive patients than HPV-negative patients (46.1% vs. 23.4%: P =0.024) [Table 3].
| Discussion|| |
This study highlighted the incidence of HPV among patients with oropharyngeal carcinoma diagnosed at our center and compared the patient characteristics between HPV-positive and HPV-negative groups.
According to GLOBOCAN 2018 data, HNSCC belonging to sites such as the lip, oral cavity, larynx, oropharynx, and hypopharynx account for 2%–4% of cancers worldwide with an age-standardized incidence rate of oropharyngeal cancers being 1.1/100,000 population more for men than women (1.8 vs. 0.4). Gupta et al. studied the global epidemiology of HNSCC which showed that some parts of India had an increase in the incidence of oral and pharyngeal cancers. A dramatic rise in the incidence of oropharyngeal cancers was seen in both genders. This was attributed to the persistent increased use of tobacco and its products and the prevalence of high-risk HPV genotypes in this region. The study also projected that there will be an alarming steady increase in the incidence of pharyngeal cancers by around 60% in underdeveloped and developing countries like India by 2030.
HPV testings on a molecular level are based on the presence of HPV DNA, mRNA transcripts, or translated proteins (E6/E7 protein, p16) in paraffin-embedded tissue. HPV is detected either by HPV DNA and mRNA through polymerase chain reaction (PCR)/in situ hybridization (ISH) or by detecting p16 expression by IHC., To assay for biologic activity of HPV in tumors, analysis of E6 and E7 expression by detecting E6 and E7 mRNA by reverse transcription followed by real-time PCR is also often performed. HPV screening is often conducted by ISH in many centers, and at times p16 immunohistochemical analysis is used as a substitute to assay for biologically active HPV as it is cost-effective and can be done in laboratories with minimal equipment. The sensitivity of HPV mRNA ISH, HPV DNA ISH, and HPV DNA PCR were 93.4%, 86.3%, and 83.5%, respectively. The corresponding specificity was 92.4%, 95.3%, and 89.1%, respectively. On taking all the above tests into consideration, there are no perfectly sensitive and specific tests to detect biologically active HPV in tumor specimens. ISH and PCR are sensitive but may not correlate with actual HPV DNA cellular integration. They are expensive and performed in a reference/experimental laboratory. IHC staining of p16 has gained broad acceptance as a surrogate marker of HPV infection in HNSCCs and its correlation has been consistently established in many of the studies.
As per the American Society for Clinical Pathology and College of American Pathologists' opinion diffuse cytoplasmic and nuclear staining in >70% of tumor cells must be the criteria for p16 staining to be stated as positive.
In view of the proven prognostic implications of HPV in OPSCC, IHC for p16 has been incorporated a new staging system for HPV-positive OPSCC into the 8th edition of the AJCC by the International Collaboration for Oropharyngeal Cancer Network for Staging.
In our study, HPV was detected in patients with oropharyngeal carcinoma by IHC for p16. On evaluation, 21.7% of the patient population tested HPV positive for p16 and 78.3% tested negative for p16. Our data are comparable to the study done by Bahl et al. which reported an incidence of 22.8% HPV positivity among OPSCC in an Indian cohort. Jalouli et al. in a study on 155 oral squamous cell carcinoma (SCC) patients from eight different countries from different ethnic groups and continents and with different socioeconomic backgrounds demonstrated HPV prevalence of 35%. In India, there are some series of head-and-neck SCC patients showing a HPV detection rate of 31% in the Indian community.
The age-specific prevalence rates of HNSCC were highest in patients belonging to the 50–59 years age group and also observed that 56% of patients with oropharyngeal cancer were among the age group 51–60 years. Various studies from west also have reported that patient with HPV-positive HNSCC were seen in young males with the median age varying from 54 to 60 years.,, In this study, we did not find the difference in age of presentation between HPV-positive and HPV-negative patients (P = 0.569), and the possible explanation for no difference in age of presentation toward HPV positivity is due to the small sample size. HNSCC including oropharyngeal cancers is common in males when compared to females supporting our findings.
HPV positivity is common in nonsmokers and nondrinkers in HNSCC. However, the degree of synergism of HPV infection with the classical risk factors is not clear, and data exist for both synergistic and additional effects. In this study, history of smoking, alcohol consumption, and tobacco chewing are noncontributary for HPV status. Bahl et al. a study on 105 OPSCC patients did not detect any association of smoking or alcohol consumption on HPV status. The majority of them were current smokers in both groups. The high prevalence of tobacco use in this study has been supported by a large series of oropharyngeal carcinoma from India with 80.5% prevalence of tobacco use.
According to previously published literature, HPV prevalence varies for anatomical subsites in oropharyngeal cancers., Jiang et al. reported that the prevalence of HPV in OPSCC was seen more in base of the tongue (80%) followed by the tonsil (63%), whereas Dahlstrom et al. reported a prevalence of HPV among 54.6% of tonsillar OPSCC followed by 44.1% of base of the tongue. In our study, we find HPV positivity in 38.5% of tonsil primary and 53.8% of posterior one-third of tongue primary.
Previously published studies have shown that classical HPV-positive patient has a small tumor size with large multiple metastatic cystic nodes., There is no robust data in correlation with HPV/p16 status and tumor characteristics., HPV-positive patients normally have an advanced stage of presentation but with a lower death rate. In our study, HPV-positive patients present more at early-stage disease (Stage I/II) when compared to that of HPV-negative patients (P = 0.005), whereas Bahl et al. in an Indian setting find no association of HPV status with respect to the stage of presentation for OPSCC patients. In this study, although the p16-positive patients did show a trend toward having small tumor size and node positivity, it did not reach statistical significance. It could be due to the small number of p16-positive tumors and high prevalence of smoking in them might have nullified the effect of HPV as explained by Liu et al.
HPV-positive patients have a correlation with poorly keratinizing tumor histology. We found that HPV-positive patients have higher incidence of histological poor differentiation than HPV-negative patients. Our data suggested a possible influence of the viral oncogenes on tumor cell differentiation. Klussmann et al. also reported a significant correlation between the HPV status with histological differentiation supporting our data. Liu et al. in a study reported Grade 2 tumor as the most common histologic grade for HPV-negative groups, whereas Grade 3 histology is more seen in HPV-positive patients.
Oropharyngeal cancers are predominantly locoregional diseases; therefore, achieving locoregional control is essential in survival. Chemoradiation is the most popular adopted modality of treatment in oropharyngeal cancers irrespective of HPV status. Studies have established that HPV-associated tumors have a higher rate of response to radiation or chemoradiation as they have higher intrinsic sensitivity to radiation and better radiosensitization with chemotherapy resulting in better locoregional control.
Indeed, there has been a recent focus for dose de-escalation for HPV-positive tumors of the head and neck assuming that they are been overtreated., However, our study highlights only the epidemiological profile of HPV-positive OPSCC and does not take into account the treatment part.
HPV-related OPSCC has been recently recognized as their own entity besides HPV-negative OPSCC with respect to the new TNM staging criteria incorporated in the AJCC 8th edition in 2018.
This study has some limitations. First of all, this is a single-center study with small sample size. The treatment part and outcomes are salient in this study and need to be addressed. The low incidence of HPV in India hindering comparison studies on par with HPV-negative oropharyngeal cancers.
| Conclusion|| |
Nowadays, HPV testing for oropharyngeal cancer is mandatory. This study highlighted the incidence of HPV (21.7%) among patients with oropharyngeal carcinoma. P16 can be used as a surrogate marker for HPV testing. Since HPV-positive oropharyngeal cancer has a prognostic implication and future studies are directed toward de-escalation treatment strategies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Canto MT, Devesa SS. Oral cavity and pharynx cancer incidence rates in the United States, 1975-1998. Oral Oncol 2002;38:610-7.
Laskar SG, Swain M. HPV positive oropharyngeal cancer and treatment deintensification: How pertinent is it? J Cancer Res Ther 2015;11:6-9.
Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, et al.
Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J Clin Oncol 2011;29:4294-301.
Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, et al.
Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer 2019;144:1941-53.
Gupta B, Johnson NW, Kumar N. Global epidemiology of head and neck cancers: A continuing challenge. Oncology 2016;91:13-23.
van Houten VM, Snijders PJ, van den Brekel MW, Kummer JA, Meijer CJ, van Leeuwen B, et al.
Biological evidence that human papillomaviruses are etiologically involved in a subgroup of head and neck squamous cell carcinomas. Int J Cancer 2001;93:232-5.
Halec G, Holzinger D, Schmitt M, Flechtenmacher C, Dyckhoff G, Lloveras B, et al.
Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma. Br J Cancer 2013;109:172-83.
Randén-Brady R, Carpén T, Jouhi L, Syrjänen S, Haglund C, Tarkkanen J, et al. In situ
hybridization for high-risk HPV E6/E7 mRNA is a superior method for detecting transcriptionally active HPV in oropharyngeal cancer. Hum Pathol 2019;90:97-105.
Golusiński P, Pazdrowski J, Szewczyk M, Misiołek M, Pietruszewska W, Klatka J, et al.
Is immunohistochemical evaluation of p16 in oropharyngeal cancer enough to predict the HPV positivity? Rep Pract Oncol Radiother 2017;22:237-42.
Zhan KY, Eskander A, Kang SY, Old MO, Ozer E, Agrawal AA, et al.
Appraisal of the AJCC 8th
edition pathologic staging modifications for HPV-positive oropharyngeal cancer, a study of the national cancer data base. Oral Oncol 2017;73:152-9.
Bahl A, Kumar P, Dar L, Mohanti BK, Sharma A, Thakar A, et al.
Prevalence and trends of human papillomavirus in oropharyngeal cancer in a predominantly north Indian population. Head Neck 2014;36:505-10.
Jalouli J, Jalouli MM, Sapkota D, Ibrahim SO, Larsson PA, Sand L. Human papilloma virus, herpes simplex virus and epstein barr virus in oral squamous cell carcinoma from eight different countries. Anticancer Res 2012;32:571-80.
Koppikar P, deVilliers EM, Mulherkar R. Identification of human papillomaviruses in tumors of the oral cavity in an Indian community. Int J Cancer 2005;113:946-50.
Mehrotra R, Singh M, Gupta RK, Singh M, Kapoor AK. Trends of prevalence and pathological spectrum of head and neck cancers in North India. Indian J Cancer 2005;42:89-93.
] [Full text]
Klussmann JP, Weissenborn SJ, Wieland U, Dries V, Kolligs J, Jungehuelsing M, et al.
Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas. Cancer 2001;92:2875-84.
Bhattacharya N, Roy A, Roy B, Roychoudhury S, Panda CK. MYC gene amplification reveals clinical association with head and neck squamous cell carcinoma in Indian patients. J Oral Pathol Med 2009;38:759-63.
Gaur DS, Kishore S, Harsh M, Kusum A, Bansal R. Pattern of cancers amongst patients attending Himalayan institute of medical sciences, Dehradun. Indian J Pathol Microbiol 2006;49:193-8.
Gandini S, Botteri E, Iodice S, Boniol M, Lowenfels AB, Maisonneuve P, et al.
Tobacco smoking and cancer: A meta-analysis. Int J Cancer 2008;122:155-64.
Agarwal JP, Mallick I, Bhutani R, Ghosh-Laskar S, Gupta T, Budrukkar A, et al.
Prognostic factors in oropharyngeal cancer – Analysis of 627 cases receiving definitive radiotherapy. Acta Oncol 2009;48:1026-33.
Jiang R, Ekshyyan O, Moore-Medlin T, Rong X, Nathan S, Gu X, et al.
Association between human papilloma virus/Epstein-Barr virus coinfection and oral carcinogenesis. J Oral Pathol Med 2015;44:28-36.
Dahlstrom KR, Bell D, Hanby D, Li G, Wang LE, Wei Q, et al.
Socioeconomic characteristics of patients with oropharyngeal carcinoma according to tumor HPV status, patient smoking status, and sexual behavior. Oral Oncol 2015;51:832-8.
Gillison ML, Broutian T, Pickard RK, Tong ZY, Xiao W, Kahle L, et al.
Prevalence of oral HPV infection in the United States, 2009-2010. JAMA 2012;307:693-703.
Weinberger PM, Merkley MA, Khichi SS, Lee JR, Psyrri A, Jackson LL, et al.
Human papillomavirus-active head and neck cancer and ethnic health disparities. Laryngoscope 2010;120:1531-7.
Lassen P, Eriksen JG, Krogdahl A, Therkildsen MH, Ulhøi BP, Overgaard M, et al.
The influence of HPV-associated p16-expression on accelerated fractionated radiotherapy in head and neck cancer: Evaluation of the randomised DAHANCA 6 and 7 trial. Radiother Oncol 2011;100:49-55.
Goldenberg D, Begum S, Westra WH, Khan Z, Sciubba J, Pai SI, et al.
Cystic lymph node metastasis in patients with head and neck cancer: An HPV-associated phenomenon. Head Neck 2008;30:898-903.
Liu C, Talmor G, Low GM, Wang TV, Mann DS, Sinha UK, et al.
How does smoking change the clinicopathological characteristics of human papillomavirus-positive oropharyngeal squamous cell carcinoma? One medical Center experience. Clin Med Insights Ear Nose Throat 2018;11:1179550618792248.
Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tân PF, et al.
Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010;363:24-35.
Choi KH, Song JH, Kim YS, Moon SH, Lee J, Oh YT, et al.
Survey of radiation field and dose in human papillomavirus-positive oropharyngeal cancer: Is de-escalation actually applied in clinical practice? Radiat Oncol J 2021;39:174-83.
Gabani P, Lin AJ, Barnes J, Oppelt P, Adkins DR, Rich JT, et al.
Radiation therapy dose de-escalation compared to standard dose radiation therapy in definitive treatment of HPV-positive oropharyngeal squamous cell carcinoma. Radiother Oncol 2019;134:81-8.
[Table 1], [Table 2], [Table 3]