Development of a rapid qualitative and quantitative system for immunophenotyping and early differential detection of precancerous and cancerous oral lesions.

Oral squamous cell carcinoma (OSCC) represents the most common histological subtype of oral cancer and continues to show an increasing global incidence. Despite advances in oncological management, prognosis remains poor in advanced stages. The current diagnostic gold standard, surgical biopsy, while effective, presents several limitations related to its invasiveness, restricted sampling capacity, and unsuitability for repeated or longitudinal assessments. These factors highlight the need for alternative, minimally invasive diagnostic strategies capable of providing reliable molecular information and facilitating early detection and disease monitoring. The PhD project is based on evaluating, developing and optimizing a cytobrush-based sampling technique analyzed through the Stark Oral Screening® in vitro diagnostic assay combined with the FemtoHunter® phenotyper device. Cytolo-salivary samples are obtained both from the tumor site and from adjacent clinically healthy mucosa in patients affected by histologically confirmed OSCC. Six molecular biomarkers EGFR, p53, Ki67, B7-H6, PD-L1, and HLA-E are quantified using a chemiluminescent ELISA-based analytical method. The results demonstrate a consistent overexpression of all six biomarkers in tumor tissue compared with the surrounding healthy mucosa. The analysis of the biomarkers performed through the FemtoHunter® , confirmed excellent diagnostic accuracy, showing that this non-invasive multimarker approach can effectively discriminate between malignant and normal tissues. These findings indicate that cytobrush-based biomarker profiling, integrated with chemiluminescent ELISA technology, provides diagnostic performance comparable to that of conventional biopsy while eliminating the associated invasiveness. The inclusion of immune checkpoint–related biomarkers, such as PD-L1, HLA-E, and B7-H6, alongside classical oncogenic markers, offers a broader and more comprehensive diagnostic perspective by capturing both tumor proliferation and immune evasion signatures. Overall, this project supports the use of cytobrush-based biomarker analysis as a reliable, accurate, and patient-side method for OSCC detection and follow-up. The strong diagnostic performance, combined with ease of sampling and excellent patient compliance, underscores the clinical potential of this approach. Compared to previous pilot investigations, the expanded dataset obtained in this work provides robust confirmation of diagnostic validity and highlights the feasibility of integrating this multimarker strategy into future clinical workflows for early diagnosis and longitudinal monitoring of oral cancer.