Development and validation of new biomarkers for the early diagnosis and personalised management of patients with HPVrelated head and neck cancers

The escalating global incidence of Human Papillomavirus (HPV) related Head and Neck Squamous Cell Carcinoma (HPV-related HNSCC) highlights the urgent need for improved diagnostic and surveillance tools, especially given the absence of screening programs. Liquid biopsies, such as blood and saliva, are a good source of circulating tumour DNA, which contains tumour-specific genetic information, and are promising for HPV- related HNSCC screening programs and post-treatment surveillance. Unfortunately, the lack of a standardised workflow from the pre-analytical to analytical stages for the use of liquid biopsy in HPV testing for HNSCC has hampered consistent and reliable diagnostic outcomes. This thesis aimed to address these critical gaps by contributing to the technical development and validation of the OncoPredict HPV Quantitative Typing assay. Its primary objectives included assessing the assay’s ability to detect and quantify in blood and saliva samples from patients with HPV-related HNSCC, evaluating its accuracy for quantifying HPV 16 viral loads against digital droplet PCR (ddPCR), and assessing its clinical performance for post-treatment monitoring of disease recurrence or relapse among oropharyngeal squamous cell carcinoma (OPSCC) patients. Additionally, the thesis aimed to evaluate the molecular target CCR5 for reliable normalisation of viral load in saliva samples. The work involved a clinical, prospective, multicentre, non-profit study (IDENTIFY study), 74 patients with newly diagnosed HNSCC were enrolled, with 67 patients (51/67 (76.1%) had OPSCC) included in the final analysis. Blood and saliva samples were collected at baseline and during longitudinal follow-up. The OncoPredict HPV QT assay demonstrated high analytical accuracy and sensitivity, showing moderate to strong concordance with p16 immunohistochemistry (p16 IHC), a widely accepted surrogate marker, achieving Cohen's kappa values of 0.65 for plasma and 0.60 for saliva, and a combined value of 0.77. This concordance between the OncoPredict HPV QT assay and p16 IHC suggests its utility as a complementary test, as relying solely on p16 IHC can lead to false positives and false negatives, making it insufficient for HPV status determination. The assay’s agreement with ddPCR for HPV 16 viral loads quantification, its ability to detect and quantify all 12 high-risk HPV genotypes, and its cost-effectiveness with shorter turnaround time and standard input sample volume position it as a standardised tool for quantitative viral load assessment. Furthermore, longitudinal monitoring using the OncoPredict HPV QT assay exhibited potential for detecting cancer recurrence weeks to months earlier than conventional clinical imaging, with persistent or increasing HPV DNA levels serving as strong indicators of relapse. In conclusion, the validation of the OncoPredict HPV QT assay confirms its reliability and potential as a valuable tool for HPV DNA detection and quantification in HPV-related OPSCC patients. Its robust analytical performance, strong concordance with p16 IHC, and high agreement with ddPCR affirm its utility in advancing HPV diagnostics. The integration of this validated, commercially available and standardised CE-marked assay into a comprehensive liquid biopsy strategy offers an accessible tool to facilitate the development of screening programs, advance early detection, personalise management, and enhance continuous posttreatment surveillance of this challenging disease, ultimately improving patient care.