Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

Abstract

Background: Despite its low sensitivity (approximately 30%), squamous cell carcinoma (SCC) antigen is commonly utilized as a serum tumor marker for oral SCC (OSCC) in clinical settings. The objective of this research was to identify novel biomarkers for OSCC.

Methods: Initially, we performed microarray analysis to evaluate the gene expression signatures of primary OSCC and normal oral mucosal tissues. Our findings showed the C-X-C motif chemokine ligand 13 (CXCL13) to be a promising novel biomarker as it was consistently overexpressed in primary OSCC tissues, a conclusion corroborated by polymerase chain reaction results. Subsequently, we measured serum CXCL13 levels in 125 patients with OSCC using a sandwich enzyme-linked immunosorbent assay and compared the results with those of 29 healthy individuals.

Results: Remarkably, the levels of serum CXCL13 were consistently elevated in patients with OSCC, and the high expression of serum CXCL13 was notably associated with tumor size and neck lymph node metastasis. Patients with advanced OSCC with high-serum CXCL13 levels exhibited poor prognosis regarding both overall and disease-free survival. Finally, spatial transcriptome analysis revealed CXCL13 and CD8 expressions within tumor area clusters but not in adjacent normal areas, suggesting specific overexpression of CXCL13 in primary OSCC tissues.

Conclusion: These findings imply that serum CXCL13 holds diagnostic and prognostic value, showing promise as a novel biomarker for OSCC.

Keywords: C‐X‐C motif chemokine ligand 13; biomarker; oral squamous cell carcinoma; prognosis.

FIGURE 1

Expression of CXCL13 mRNA in primary OSCC tissues by RT‐qPCR. (A) CXCL13 expression was higher in tumor tissues compared to adjacent normal tissues in all cases. (B) Significant upregulation of CXCL13 mRNA was observed in tumor tissues. **p < 0.01 compared to adjacent normal tissues.

FIGURE 2

Serum levels of CXCL13 protein in patients with OSCC. (A) The average serum CXCL13 protein in 125 patients with OSCC was 72.8 ρg/mL. (B) The average serum CXCL13 protein in healthy individuals was 33.5 ρg/mL. (C) The reference value determined by the ROC curve for maximizing the TPF/FPF ratio was 53.0 ρg/mL. FPF, false‐positive fraction; TPF, true‐positive fraction.

FIGURE 3

Serum CXCL13 and clinicopathological factors in patients with OSCC. (A) Serum CXCL13 protein levels in T classification. The serum CXCL13 proteins increased in accordance with primary tumor size. The serum CXCL13 levels in T1 were significantly higher than in T3 and T4. (B) The serum CXCL13 proteins in recurrence. The serum levels of CXCL13 proteins in patients with recurrence were significantly elevated. (C) Patients with recurrence were classified into primary recurrence, neck LNM, and distant metastasis (including multiple responses). The serum levels of CXCL13 proteins in neck LNM were significantly elevated. *p < 0.05, **p < 0.01.

FIGURE 4

Serum CXCL13 and prognosis in patients with OSCC. A comparison of overall survival (OS) and disease‐free survival (DFS) in patients with OSCC was made among the two groups, which were classified based on the median serum CXCL13 level using the Kaplan–Meier method with a log‐rank test. High expression of serum CXCL13 indicated poor prognosis in both OS (A, p = 0.044) and DFS (B, p = 0.018) across all stages. In Stage I/II, the groups had no significant difference in OS (C, p = 0.696) and DFS (D, p = 0.435). In Stage III/IV, both OS (E, p = 0.020) and DFS (F, p = 0.009) were significantly poorer in patients with high CXCL13 levels.

FIGURE 5

Visium spatial transcriptome analysis using two cases of primary OSCC tissues. (A) Case 1: Primary tongue SCC tissues were classified into eight clusters based on the gene expression profile. Among these, the tumor area comprised four clusters (Cluster 1, 3, 5, and 6). CXCL13 expression was upregulated in three of four clusters within the tumor area. CD8 expression was detected in all tumor clusters. (B) Case 2: In Case 2, primary tongue SCC tissues were classified into seven clusters. The tumor area consisted of four clusters (Clusters 1, 2, 3, and 6). The expression of CXCL13 and CD8 was upregulated in three of four clusters within the tumor area. CD4 expression was detected only in Cluster 2 in the tumor area but not in Case 1.