Introduction

According to GLOBOCON 2020, head and neck cancer (HNC) poses a significant health burden, ranking as the seventh-most common malignancy globally.

In addition to traditional risk factors such as tobacco and alcohol consumption, emerging factors like genetic predisposition, dietary habits, and human papillomavirus (HPV) infection are gaining recognition as distinct contributors to HNC development. Epidemiological investigations in Western cohorts have illuminated a distinct rise in HPV-positive HNC cases, particularly in oropharyngeal squamous cell carcinoma. Among the plethora of HPV genotypes, HPV-16 and -18 are predominantly implicated in malignant alterations, with HPV-16 alone responsible for over 90% of HNSCC cases.1 Persistent HPV infection triggers the overexpression of tumor suppressor protein p16, a biomarker often used for clinical detection of biologically active HPV infection. Moreover, the revised eighth American Joint Committee on Cancer/Union for International Cancer Control classification advocates for p16 as an HPV surrogate marker in Oropharyngeal Cancer (OPC) due to its correlation with enhanced survival rates.2

Against this backdrop, the present study aims to investigate the association of high-risk HPV-16, HPV-18, and p16 expression with clinicopathological parameters, risk behaviors, and survival outcomes in HNC among the western Indian population.

Materials and Methods

Results

Association of HPV-16 Infection and p16 Expression with Overall Survival in HNC Patients

The relationship between HPV-16 infection, p16 expression, and overall survival (OS) in HNC patients was investigated in this study. A follow-up period of 18 months yielded complete follow-up details for 55 out of the initial 92 HNC patients, forming the cohort for OS analysis. Kaplan–Meier survival analysis was conducted to assess OS in these 55 patients, stratified by HPV-16 infection and p16 expression status.

The log-rank test was employed to compare survival curves. It was observed that the OS of HNC patients having HPV-16 infection was comparatively lower than the patients who did not have HPV-16 infection. However, HPV-16 infection did not emerge as a significant prognostic factor for OS in HNC patients (log rank = 0.963, df = 1, p = 0.326; Fig. 1A).

Close

Fig. 1: Kaplan–Meier curves with log-rank analysis of overall survival. (A) Association of HPV-16 infection with overall survival. (B) Association of p16 expression with overall survival. (C) Association of combined HPV-16 and p16 expression with overall survival.

Export to PPT

Similarly, p16 expression in HNC patients did not demonstrate prognostic capability for OS (log rank = 0.043, df = 1, p = 0.835; (Fig. 1B). Furthermore, the combined analysis of HPV-16 infection and p16 expression status did not reveal any significant association with OS (log rank = 0.244, df = 1, p = 0.622; Fig. 1C).

Discussion

Increasingly, HPV infection is acknowledged as a significant contributor to carcinogenesis in various human cancers. High-risk HPV strains, notably HPV-16 and HPV-18, are implicated in the development of cancers across anatomical squamous tissue sites, including the head and neck regions. Many studies across the globe have reported that HPV prevalence exhibits considerable variation worldwide, ranging from 15 to 49%. This discrepancy is evident between developed and developing nations, with a larger proportion of HPV-positive HNC cases observed in developed regions. Moreover, Indian studies highlight wide geographical diversity in HPV prevalence, spanning from 0 to 74%.34

In the present study, HPV-16 infection was detected in 13% of HNC patients, while 87% patients tested negative for HPV-16 infection. Conversely, no instances of HPV-18 infection were identified among the enrolled HNC patients. These findings are consistent with previous studies conducted in North, West, and Central India, indicating similar patterns of HPV-16 and HPV-18 prevalence. However, it is noteworthy that HPV-18 exhibits higher prevalence rates in southern India.14 Notably, current results strongly suggest that HPV-18 may not play a significant role in oral carcinogenesis in the studied population.

The current study showed that only 14% of HNC patients exhibited positive p16 expression, while 86% displayed negative p16 expression. However, this discrepancy diverges from findings in prior research that consistently highlighted the association between overexpression of p16 and HPV infection. Consequently, immunohistochemical expression of p16 is widely considered as a surrogate marker for oncogenic HPV infection.5

Furthermore, in the present study, all cases positive for HPV-16 had oropharyngeal cancer, while no cases of HPV-16 infection were detected in oral cavity cancers. These findings align with previous research indicating a significantly lower prevalence of HPV infection in oral cavity cancers compared with oropharyngeal cancer. Thus, the oral cavity appears to be relatively less susceptible to HPV carcinogenicity, although the underlying reasons for this anatomical site's differing vulnerability remain unresolved.6 Additionally, in the present study, a trend of increased HPV-16 expression was observed in patients with cancer in the base of tongue in relation to patients with cancer in other clinical site subtypes (p = 0.063).

Statistically significant association was observed between positive HPV-16 infection and metastasis involvement, as all cases with HPV-16 infection had presence of metastasis (p < 0.001). Moreover, findings of the present study indicated that although not significant, HPV-16 infection tended to be more prevalent among younger age groups (≤59 years). This observation is consistent with a prior study,1 which reported that HPV-16 positivity was more common in oral squamous cell carcinoma cases among younger individuals aged 21 to 44 years.

Additionally, all cases in this study exhibited advanced stages of HNC, with the majority reporting a history of prolonged and intense tobacco exposure, a recognized risk behavior. Further, it was observed that a majority of HNC patients with history of tobacco exposure exhibited positive HPV-16 and p16 expression. These findings contradict a previous study, which reported a significant overexpression of p16 in patients with HNSCC who did not have a history of tobacco exposure, particularly those who chewed paan.5 Similarly, a population study in Brazil identified a co-occurrence of risk factors, namely tobacco and alcohol, alongside a low frequency of HPV DNA, leading to the conclusion that HPV may not play a significant role in the development of oral cavity squamous cell carcinoma.6 Hence, future studies may necessitate robust strategies to explore HPV infection while considering major confounding factors.

Moreover, the current study did not reveal any significant correlations between p16 expression and clinicopathological factors or risk behaviors. This is consistent with prior research findings where no notable associations were observed between p16 expression and variables such as gender, age, tumor differentiation, lymph node involvement, or survival outcomes.7 Similarly, there was no significant relationship observed between p16 expression and tumor site, which is in line with earlier studies that also found no statistical differences in p16 expression across various tumor sites.58

This study found that HPV-16 infection was not a significant predictor of OS in HNC patients. These results contradict the findings of prior studies in Western countries, where it was reported that patients with HPV-positive cancer exhibited better OS and progression-free survival compared with patients with HPV-negative cancer.910 Nevertheless, our findings indicated that there was no significant correlation between p16 expression and OS, consistent with previous research findings.511 The data presented in this study indicate that there is no significant correlation between HPV-16 infection and p16 expression. Additionally, a prior study has reported for prognostic evaluation, and p16 alone appears to be adequate, while direct detection of HPV-DNA should be incorporated for therapy stratification.10

Summarizing the findings from present study, a small number of HNC cases, primarily localized in the oropharyngeal region, attributed to HPV-16 infection. The expression of the p16 protein did not serve as an accurate surrogate marker for HPV-16 infection in HNC. Furthermore, HPV-16 DNA was detected in some HNC cases that were negative for p16 expression. Given the limited number of HPV-16 DNA-positive cases in our study, further investigation is warranted to explore the presence of HPV DNA and the expression of E6 and E7 mRNA, irrespective of p16 expression, using a larger cohort. It is important to note that our study had limitations, such as the utilization of samples from FFPE blocks instead of fresh tissue. Additionally, our cohort comprised tumors from various sites of HNC (oral cavity and oropharyngeal sites) treated with diverse regimens, which could potentially obscure significant findings.

Conclusion

In view of outcomes discussed, diverse observations related to HPV infection and p16 expression as compared with studies from Western countries may be attributed to high prevalence of tobacco exposure in the studied HNC patients from western India. This emphasizes the importance of understanding the associations between HPV-16, HPV-18, and p16 expression with clinicopathological parameters, risk behaviors, and survival outcomes among HNC patients. Furthermore, it may be suggested that comprehensive multi-institutional efforts are mandatory to gain a better understanding of oral carcinogenesis and factors that affect its clinical relevance in terms of staging and treatment strategies.