Abstract / Summary
Nasopharyngeal carcinoma (NPC) is highly prevalent in Southeast Asia, including Indonesia, with most patients presenting at advanced clinical stages. Reliable, minimally invasive circulating biomarkers for monitoring treatment response are lacking. This study aimed to identify candidate blood-based transcriptomic biomarkers for advanced-stage NPC through integrative bioinformatic analysis and to evaluate the expression dynamics of a prioritised candidate gene, TSPAN32, in peripheral blood of patients undergoing chemoradiation. Differentially expressed genes (DEGs) were identified from the GEO microarray dataset GSE53819 (18 advanced-stage NPC tissues vs. 18 non-cancerous nasopharyngeal tissues) using GEO2R. Candidate DEGs were prioritised through protein–protein interaction (PPI) network analysis (STRING confidence ≥ 0.9), functional enrichment analysis (GO via Enrichr; KEGG pathway via ShinyGO v0.82), and survival analysis (Kaplan–Meier). Overlapping DEGs were identified against a previously published blood-based NPC transcriptomic dataset with 11 early-stage NPC patients, 11 advanced-stage NPC patients, and 11 healthy donors. Analysis of GSE53819 identified 943 upregulated and 1503 downregulated DEGs (|log2FC| ≥ 1; Padj < 0.05). Functional enrichment of upregulated DEGs was dominated by inflammatory response and cytokine activity pathways, while downregulated DEGs were enriched in B-cell activation and immunoglobulin receptor binding. Protein–protein interaction network construction (STRING confidence ≥ 0.9) and hub gene identification using the MCC algorithm identified six candidate genes among the downregulated DEGs: ADRA2A, SELP, TSPAN32, PEAR1, P2RX1, and DGKG. Kaplan-Meier survival analysis demonstrated that lower expression of SELP (HR = 0.57; p = 0.00053), TSPAN32 (HR = 0.56; p = 0.00025), and P2RX1 (HR = 0.48; p = 1.2 × 10−5) was significantly associated with shorter overall survival in head and neck carcinoma, supporting TSPAN32 as the primary candidate for clinical validation. RT-qPCR examination demonstrated that TSPAN32 transcript abundance in peripheral blood was significantly upregulated following chemoradiation in nine of ten patients (mean Log2FC = 2.08 ± SD 1.09, 95% CI 1.29–2.86; one-sample t-test t(9) = 6.01, p = 0.0002; Wilcoxon signed-rank test p = 0.002). Primer specificity for TSPAN32 and ACTB was confirmed by melt-curve analysis and in silico validation (Primer-BLAST, OligoAnalyzer, UCSC In-Silico PCR). RBC transfusion status did not significantly modulate the magnitude of TSPAN32 expression change (independent-samples t-test, t = 1.86, p = 0.101). This integrative study identifies TSPAN32 as a candidate circulating biomarker responsive to chemoradiation in advanced-stage NPC. These exploratory findings require confirmation in larger, prospective, longitudinal cohorts before clinical translation.