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Background: Oral cancer is often surrounded by epithelium that clinically appears normal but harbours genetic aberrations, including pre-cancerous changes, a phenomenon known as field cancerization. In patients with squamous cell carcinoma of the oral cavity (SCCOC), it is crucial to study not only the tumour itself but also the clinically normal tissue that remains post-therapeutically. Additionally, liquid biopsy approaches, such as the analysis of plasma samples, have emerged as promising minimally invasive methods for detecting cancer-related alterations. The aim of this doctoral study is to characterize the clinically normal tissue and plasma in patients with SCCOC and to establish a panel of changes that may contribute to early detection, diagnosis or prognosis.

Materials & Methods: Microarray gene expression data of healthy tongue tissue, tumour and clinically normal tongue contralateral to tumour (NTCT) from patients with SCC of the oral tongue (SCCOT) were analysed. Reverse transcription quantitative PCR and immunohistochemistry were performed to validate microarray data and investigate protein expression, respectively. Data from whole exome sequencing and RNA sequencing were investigated to identify correlations between copy number variation and differential gene expression in paired tumour and NTCT samples. Finally, proteomics data based on the Olink explore 3072 platform were examined to compare plasma protein levels between healthy controls and patients with SCCOC. The prognostic impact of cancerrelated alterations was assessed using Kaplan-Meier and Cox regression survival analysis.

Results: Focusing on transporter associated with antigen processing 1 (TAP1) and TAP2, two key factors in antigen presentation and immune evasion, our microarray data showed that TAP1 mRNA levels increased progressively from healthy controls to NTCT to tumour, whereas TAP2 mRNA levels were upregulated only in tumours. Notably, higher TAP1 mRNA levels in NTCT were associated with worse survival outcomes, while TAP1 levels in tumours provided no prognostic information. Immunohistochemistry confirmed elevated TAP protein expression in tumours. Similarly, TAP protein levels in tumours had no overall impact on survival but exhibited sex-specific associations. Further comprehensive analysis of microarray data revealed upregulation of apoptosis-related genes in NTCT. A positive correlation between copy number and mRNA levels was identified for the pro-apoptotic tumour suppressor Zinc Finger Protein 395 (ZNF395). Finally, plasma proteomics analysis revealed decreased levels of Secreted Frizzled Related Protein 4 (SFRP4) in SCCOC patients, with lower SFRP4 levels associated with worse survival outcomes.

Conclusions: We provide further evidence that NTCT harbours genetic aberrations, is susceptible to malignant transformations, and contains biomarkers that may aid in early detection and prognosis. Plasma protein analysis, meanwhile, revealed systemic alterations with prognostic significance. Taken together, our findings demonstrate that molecular profiling of NTCT and plasma could improve our understanding of tumorigenesis and enhance early detection, risk stratification, and personalized surveillance strategies for SCCOC patients.