晚期鼻咽癌放化疗患者外周血中TSPAN32可检测性作为潜在循环生物标志物的初步试点评估

文献信息

中文摘要

背景： 鼻咽癌（NPC）在东南亚（包括印度尼西亚）高度流行，大多数患者就诊时已处于晚期临床阶段。目前缺乏可靠、微创的循环生物标志物用于监测治疗反应。本研究旨在通过整合生物信息学分析，识别用于晚期NPC的候选血液转录组生物标志物，并评估优先候选基因TSPAN32在接受放化疗患者外周血中的表达动态。
方法： 使用GEO2R从GEO微阵列数据集GSE53819（18例晚期NPC组织 vs. 18例非癌鼻咽组织）中识别差异表达基因（DEGs）。通过蛋白质-蛋白质相互作用（PPI）网络分析（STRING置信度 ≥ 0.9）、功能富集分析（通过Enrichr进行GO分析；通过ShinyGO v0.82进行KEGG通路分析）和生存分析（Kaplan-Meier）对候选DEGs进行优先级排序。与先前发表的血液NPC转录组数据集（包含11例早期NPC患者、11例晚期NPC患者和11例健康供者）进行重叠DEGs识别。
结果： 对GSE53819的分析识别出943个上调DEGs和1503个下调DEGs（|log2FC| ≥ 1；Padj < 0.05）。上调DEGs的功能富集以炎症反应和细胞因子活性通路为主，而下调DEGs富集于B细胞活化和免疫球蛋白受体结合。通过STRING置信度 ≥ 0.9构建PPI网络并使用MCC算法识别枢纽基因，在下调DEGs中识别出六个候选基因：ADRA2A、SELP、TSPAN32、PEAR1、P2RX1和DGKG。Kaplan-Meier生存分析表明，SELP（HR = 0.57；p = 0.00053）、TSPAN32（HR = 0.56；p = 0.00025）和P2RX1（HR = 0.48；p = 1.2 × 10-5）的低表达与头颈癌较短的总生存期显著相关，支持TSPAN32作为临床验证的主要候选基因。RT-qPCR检测表明，十例患者中有九例在放化疗后外周血中TSPAN32转录本丰度显著上调（平均Log2FC = 2.08 ± SD 1.09，95% CI 1.29-2.86；单样本t检验 t(9) = 6.01，p = 0.0002；Wilcoxon符号秩检验 p = 0.002）。通过熔解曲线分析和计算机验证（Primer-BLAST、OligoAnalyzer、UCSC In-Silico PCR）确认了TSPAN32和ACTB的引物特异性。红细胞输注状态未显著调节TSPAN32表达变化的幅度（独立样本t检验，t = 1.86，p = 0.101）。
结论： 这项整合性研究确定TSPAN32作为晚期NPC中对放化疗有反应的候选循环生物标志物。这些探索性发现需要在更大规模的前瞻性纵向队列中确认，才能进行临床转化。

英文摘要

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.