HPV AS A HIDDEN COFACTOR IN TOBACCO-ASSOCIATED SUPRAGLOTTIC LESIONS: A COMPARATIVE ANALYSIS

Abstract

Background: Supraglottic squamous cell carcinoma and premalignant lesions of the larynx are among the most prevalent head and neck malignancies in the Indian subcontinent, where tobacco in its smoked and smokeless forms constitutes the principal aetiological agent. Human papillomavirus (HPV), particularly high-risk genotypes 16 and 18, has been identified as an independent oncogenic driver in oropharyngeal and laryngeal cancers; however, its role as a cofactor potentiating tobacco-related carcinogenesis at the supraglottic site remains inadequately characterised. Aim: This study aimed to determine the prevalence and genotypic distribution of HPV in tobacco-associated supraglottic lesions, to evaluate the synergistic oncogenic influence of concurrent HPV infection and tobacco exposure, and to compare histopathological and immunohistochemical profiles across patient subgroups. Methods: A prospective comparative analytical study was conducted at the Department of Otorhinolaryngology, Narayan Medical College and Hospital, Sasaram, Bihar, India, between 1st March 2025 and 28th February 2026. A total of 124 patients were enrolled and initially stratified into three broad groups: Group A (supraglottic lesions in non-tobacco smokers), Group B (supraglottic lesions in tobacco smokers), and Group C (control group: normal laryngeal mucosa without any lesion in non-tobacco smokers). Following HPV laboratory analysis, these three groups were further subdivided into six final groups: Group A (non-tobacco smokers with HPV-positive supraglottic lesions, n=13), Group B (non-tobacco smokers with HPV-negative supraglottic lesions, n=12), Group C (tobacco smokers with HPV-positive supraglottic lesions, n=42), Group D (tobacco smokers with HPV-negative supraglottic lesions, n=35), Group E (control group, normal laryngeal mucosa, non-tobacco smokers, HPV-negative, n=20), and Group F (control group, normal laryngeal mucosa, non-tobacco smokers, HPV-positive, n=2). Biopsy specimens were obtained from Groups A, B; specimens from Groups C were collected by mucosal cytobrush without biopsy. Each sample was divided and processed in parallel: the first portion was sent for HPV detection by PCR and the second for histopathological examination. HPV detection was performed using PCR targeting the L1 consensus region, with genotyping by reverse line blot hybridisation, p16 immunohistochemistry (IHC), and E6/E7 mRNA expression analysis. On the basis of histopathological examination (HPE), lesions were sub-classified as: HPE Sub-group A (benign), HPE Sub-group B (premalignant/dysplastic), and HPE Sub-group C (malignant). Among HPV-positive cases confirmed by PCR, a further sub-classification was applied: PCR Sub-group A (HPV 16 positive) and PCR Sub-group B (HPV 18 positive). Ki-67 proliferation index, p53, and Bcl-2 expression were evaluated on biopsy specimens. Results: HPV positivity was confirmed in 100% of Group A and Group C (by study design). Among tobacco smokers, HPV positivity was not detected in Group D specimens on screening (0%). HPV 16 was the predominant genotype across all HPV-positive cases (66.7%), classified under PCR Sub-group A, while HPV 18 accounted for 21.2% of HPV-positive Group C specimens, classified under PCR Sub-group B. The tobacco+HPV group (Group C) showed a numerical trend toward higher rates of severe dysplasia/carcinoma in situ (23.8%), poorly differentiated SCC (14.3%), Ki-67 index exceeding 30% (73.8%), p53 overexpression (66.7%), and lymphovascular invasion (33.3%) compared to the tobacco only group (Group D); however, several subgroup comparisons did not reach statistical significance. The odds ratio for HPV positivity in supraglottic lesion patients versus controls was 12.0 (95% CI: 3.4–42.6, p<0.001). Conclusion: The findings suggest that HPV may function as a clinically significant cofactor in tobacco-associated supraglottic carcinogenesis, associated with a trend toward a more aggressive histological and molecular phenotype. HPV screening in patients presenting with tobacco-associated supraglottic lesions may be considered in high-risk populations and may have implications for prognostication and targeted therapeutic strategy, pending larger multicentre validation.