Diagnostic accuracy of combinatorial mRNA biomarkers for non-invasive detection and therapy monitoring of oral and oropharyngeal SCC

BACKGROUND: Oral squamous cell carcinoma (OSCC) is increasingly common, with over 380,000 new cases annually. Despite its high incidence (6.0 per 100,000 males; 2.3 per 100,000 females) and poor prognosis, no molecular biomarkers exist for early detection. Non-invasive sampling could improve diagnosis and patient outcomes. METHODS: This pilot study used RNA sequencing to identify significantly upregulated mRNA targets in swab samples from oral and oropharyngeal SCC patients and healthy probands. After filtering, four potential biomarkers were further validated in 79 samples using RT-qPCR. CombiROC analysis assessed diagnostic performance. Additional RT-qPCR on tumour and normal tissues and fluorescence staining in FFPE tumour sections evaluated expression at mRNA and protein levels. RESULTS: A panel of three markers (c-JUN, SFN, HSP90AB1) showed high diagnostic accuracy: 92.3% specificity, 92.3% sensitivity, and AUC of 0.91. Fluorescence staining confirmed significantly higher protein expression in tumour tissues, supporting RNA findings. The panel showed stronger diagnostic performance in men than in women. CONCLUSION: This study presents a promising non-invasive biomarker panel for oral and oropharyngeal SCC detection. Further validation in larger cohorts is needed to confirm diagnostic value and clarify sex specificity. The approach is adaptable to other tumour types and sample materials, supporting molecular diagnostics. The workflow shows all project steps from sampling to marker localisation in FFPE tissue. In the discovery phase, RNA sequencing was used to find potential mRNA markers in swab samples. Via strict filtering, markers for RT-qPCR were selected and verified in a bigger cohort in the validation phase. Selected markers were then used to do combinatorial analysis to improve the specificity and sensitivity compared to that of the single markers. Finally, markers were verified and localised in primary oral and oropharyngeal SCC FFPE tissue. Created with BioRender.com.