HPV's Impact on Head and Neck Cancers: Exploring Prevalence and Prognostic Significance. Doctoral Thesis
Authors:   ,   Rīga Stradiņš University, Lifšics Andrejs 
Published: April 2024

Head and neck squamous cell carcinoma (HNSCC) is the seventh most prevalent cancer worldwide. Significant risk factors in the development of HNSCC are tobacco smoking and alcohol consumption. However, the exact impact of human papillomavirus (HPV) on the survival prognosis of patients with HNSCC, particularly those with laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC), remains somewhat unclear. This research aimed to examine the prevalence of HPV infection (HPV DNA, E6/E7 mRNA) among individuals diagnosed with oropharyngeal squamous cell carcinoma (OPSCC), HPSCC, and LSCC, and to understand the role of HPV infection in tumour formation and patient survival by evaluating the immunohistochemical (IHC) expression of tumour suppressor proteins (p16 and p53) and HPV16 E6 and E7 oncoproteins. The first part of the research involved a retrospective study of 247 patients with confirmed OPSCC. The primary outcomes assessed in this study were overall survival (OS) and disease-specific survival (DSS), in addition to histopathological analysis. The results of the Kaplan-Meier survival analysis indicated better survival outcomes for female patients, younger individuals without unhealthy habits (smoking and alcohol abuse), those who underwent surgery and received radiotherapy, and those with lower tumour grade and disease stage. The Cox regression analysis revealed a reduced risk of early death in patients with lower tumour grade, no regional metastases (N0), and without unhealthy habits, as well as in patients who underwent surgery and received radiotherapy. Most tumours were localised in the palatine tonsils and the base of the tongue, but the localisation did not show a correlation with mean survival time or survival outcomes. Significantly lower OS and DSS rates were observed in patients with involvement of the pharyngeal wall and tonsils compared to tumours localised in the soft palate. The histological variant of the tumour did not appear to significantly impact OS and DSS, while the chosen therapeutic approaches had a significant effect on survival outcomes. The second part of the research encompassed the IHC (p16, p53, HPV16 E6/E7 proteins) and virological (HPV DNA, E6/E7 mRNA) investigation of 106 tumour samples from patients with HNSCC (34 OPSCC, 41 LSCC, 31 HPSCC), as well as clinical assessment of these patients. To evaluate and compare several molecular biology methods for detecting HPV in nucleic acid material obtained from formalin-fixed paraffin-embedded (FFPE) samples. assessment of the 31 FFPE tumour samples from patients with HPSCC was performed. The two real-time PCR methods, Anyplex II HPV28 and Sacace HPV High-Risk Screen Real-TM Quant, exhibited strong agreement. A moderate positive correlation was identified between the semiquantitative results obtained from Anyplex II HPV28 and the quantitative results obtained from Sacace. Used nucleic acid extraction kits are good and reliable for extracting qualitative material for further molecular investigation. Real-time PCR methods that target smaller DNA amplicons are effective and dependable techniques for detecting HPV genetic material in FFPE samples. Further assessment of 106 HNSCC samples revealed that HPV16 was the most prevalent high-risk (HR-) HPV type found. The prevalence of HPV16 was 26/34 (76.47%), 22/41 (53.66%), and 20/31 (64.52%) in OPSCC, LSCC, and HPSCC accordingly. HPV16 E6/E7 mRNA was detected in 15/26 (57.7%) of the OPSCC samples, 2/22 (9%) of the LSCC samples, and 0/20 of the HPSCC HPV16-positive samples. Overexpression of HPV16 E6 protein was immunohistochemically confirmed in 44/106 (41.5%) of the HNSCC samples, and overexpression of HPV16 E7 protein – in 39/106 (36.8%) of the HNSCC samples. The presence of HPV DNA, both low-risk (LR-) and HR-HPV types, was linked to improved 5-year OS and DSS rates in patients with OPSCC and LSCC. The IHC overexpression of HPV16 E6 protein and p16 protein was associated with better survival outcomes, as observed in both univariate analysis for OPSCC and multivariate analysis for OPSCC and HPSCC. Additionally, the overexpression of p53 was linked to improved survival specifically in OPSCC. This research has provided crucial insights into our understanding of HPV prevalence and significance in HNSCCs. However, additional studies are necessary to investigate the role of HPV infection (HR- and LR-) in non-oropharyngeal HNSCC and its prognostic value in survival of these patients. Moreover, more studies are needed to evaluate the potential use of IHC for HPV16 E6 protein expression as a prognostic marker in OPSCC and HPSCC. Sponsorship: European Social Fund and the Latvian state budget within the frame of project No. 8.2.2.0/20/l/004 “Support for involving doctoral students in scientific research and studies” at Rīga Stradiņš University. The Doctoral Thesis was developed at the Institute of Anatomy and Anthropology, the Institute of Microbiology and Virology of Rīga Stradiņš University, Latvia Defence: at the public session of the Promotion Council of Clinical Medicine of Rīga Stradiņš University on 24 April 2024 at 14.00 remotely via online platform Zoom. 3.2 Clinical Medicine; Sub-Sector – Otorhinolaryngology. Keywords: Doctoral Thesis; oropharynx; larynx; hypopharynx; squamous cell carcinoma; HPV; PCR; immunohistochemistry; p16; p53; E6/E7 viral oncoproteins; survival analysis.