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Background: The incidence of squamous cell carcinoma of the oral tongue (SCCOT) among young adults is increasing in several regions of the world. Age-dependent differences in the biology of SCCOT have been suspected.

Methods: We used the Olink Explore 3072 high-throughput platform to comprehensively quantify plasma proteins in 24 young (≤ 40 years of age) and 50 old (> 50 years of age) individuals. Eight young and 20 old individuals were diagnosed with SCCOT, four young and nine old individuals with SCC at other oral subsites (SCCOO), and the remaining 12 young and 21 old individuals were healthy controls. Dimension reduction analysis, differential expression analysis, and functional enrichment analysis were performed to characterize young patient-specific biological signatures.

Results: Plasma levels of 2923 proteins were obtained. Principal component analysis indicated age-related expression patterns. Comparing young patients to young controls/old patients/old controls, differential abundance analysis showed that increases in protein levels of Peroxiredoxin 2 (PRDX2) and C-C motif chemokine ligand 26 (CCL26) and a decrease in Kallikrein related peptidase 4 (KLK4) were young patient-specific. Reactome pathway enrichment analysis identified “Cellular response to chemical stress,” “Detoxification of reactive oxygen species” and “Cellular responses to stimuli” as the top altered pathways in young patients with SCCOT.

Conclusions: Abnormal cellular stress and aberrant immune regulation could thus be linked to cancer development in young patients. The unique plasma proteomic signature observed in young patients with SCCOT suggests that they constitute a specific group with distinct underlying pathophysiological processes.

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.

PURPOSE: The field cancerization concept indicates the presence of pre-cancerous changes in clinically normal tissue surrounding the tumor. In squamous cell carcinoma of the oral tongue (SCCOT) which is infrequently linked to human papillomavirus infection, we have previously reported that clinically normal tongue contralateral to tumor (NTCT) is molecularly abnormal. Here, combining our transcriptomic and genomic data, we aimed to investigate the contribution of molecular changes in NTCT to cancer development.

METHODS: Microarray gene expression data of 14 healthy controls, 23 NTCT and 29 SCCOT samples were investigated to characterize transcriptional profiles in NTCT. Whole exome sequencing and RNA-sequencing data of paired NTCT and tumor samples from 15 SCCOT patients were used to study correlation between copy number variation and differential gene expression.

RESULTS: Using supervised multivariate partial least squares discriminant analysis, a total of 61 mRNAs that distinguish NTCT from healthy tongue were selected. Functional enrichment analysis of the 22 upregulated genes showed increased "positive regulation of nitrogen compound metabolic process" in NTCT. All 12 genes involved in this process have roles in apoptosis (anti- and/or pro-apoptotic). Compared to healthy controls, Zinc Finger Protein 395 (ZNF395), a pro-apoptotic tumor suppressor located on chromosome 8p, was the only gene showing increased mRNA level in NTCT whereas decreased in SCCOT. Given the frequent loss of chromosome 8p in SCCOT, the impact of ZNF395 copy number variation on gene expression was further examined, revealing a positive correlation between copy number and mRNA level (correlation coefficient = 0.572, p < 0.001).

CONCLUSION: NTCT is susceptible to malignant transformation, where tissue homeostasis is maintained at least partly through regulation of apoptosis. Loss of the pro-apoptotic gene ZNF395 could thus initiate cancer development.

Transporter associated with antigen processing 1 (TAP1) and TAP2 serve pivotal roles in adaptive immunity. Tumor cells often show reduced antigen presentation on their surface as one mechanism to escape immune recognition. Whether downregulation of TAPs is a common mechanism of tumor immune evasion in squamous cell carcinoma of the oral tongue (SCCOT) is unclear. In the present study, samples from 78 patients with SCCOT and 17 patients with benign hyperplastic tongue lesions were analyzed for TAP1 and TAP2 expression by immunohistochemistry. The percentage of positive cells and staining intensity were scored. Associations with clinicopathological variables and survival outcome were also investigated. The results demonstrated that TAP1 and TAP2 levels were highly associated with each other in individual samples and were upregulated in SCCOT compared with benign lesions (P<0.001). The proportion of TAP1‐ or TAP2‐positive tumor cells was >80% in all but two of the tumors, whereas 25.6 and 23.0% of the tumors showed weak intensity of TAP1 and TAP2, respectively. There were no significant associations with clinicopathological variables or survival outcomes between TAP‐intermediate/strong and TAP‐weak tumors. However, in patients <70 years old and with early stage SCCOT, male patients had better outcomes than female patients (log‐rank P<0.05), and the best outcome was observed in male patients with intermediate/strong TAP expression. In conclusion, loss of TAP was not a frequent event in SCCOT and stronger TAP expression in male patients was associated with improved survival, providing further evidence for sex‐specific immune modulation in cancer.

Oral cancers are surrounded by epithelium that histologically might seem normal, but genetically has aberrations. In patients with squamous cell carcinoma of the oral tongue (SCCOT), it is therefore important to study not only the tumor but also the clinically tumor-free contralateral tongue tissue that remains in the patient after treatment to map changes of prognostic and/or diagnostic value. The transporter associated with antigen processing (TAP) dimer is a key factor in the process of activating cytotoxic T cells. By downregulating the expression of TAP, tumor cells can escape cytotoxic T cell recognition. Biopsies from tumor and clinically tumor-free contralateral tongue tissue in 21 patients with SCCOT were analyzed together with tongue biopsies from 14 healthy individuals, which served as the control group. Dividing patients into TAP1-high and TAP1-low groups according to the median TAP1 level in tumor-free samples showed that patients with lower TAP1 mRNA levels in tumor-free samples had better overall (p = 0.003) and disease-free survival (p = 0.002). The results showing that TAP1 levels in tumor-free tongue tissue contralateral to the SCCOT correlate with survival is an important contribution to early diagnosis and follow up of SCCOT.