DataSheet3_Potential diagnostic of lymph node metastasis and prognostic values of TM4SFs in papillary thyroid carcinoma patients.ZIP

Background: Although the prognosis of papillary thyroid carcinoma (PTC) is relatively good, it causes around 41,000 deaths per year, which is likely related to recurrence and metastasis. Lymph node metastasis (LNM) is an important indicator of PTC recurrence and transmembrane 4 superfamily (TM4SF) proteins regulate metastasis by modulating cell adhesion, migration, tissue differentiation, and tumor invasion. However, the diagnostic and prognostic values of TM4SF in PTC remain unclear.
Methods: This study aimed to identify TM4SF genes with predictive value for LNM and prognostic value in PTC using bioinformatic analysis. We screened the differentially expressed genes (DEGs) of the TM4SF family in PTC using data from TCGA, constructed a PPI network using STRING, and evaluated the predictive role of TM4SF1 in LNM via a binary logistic regression analysis and ROC curve. We assessed the association between TM4SF1 expression and DNA methylation, and determined the functional and mechanistic role of TM4SF1 in promoting LNM via GSEA, KEGG, and GO. We estimated the relationship between each TM4SF gene and overall survival (OS, estimated by Kaplan-Meier analysis) in patients with PTC and established a predictive model of prognostic indicators using a LASSO penalized Cox analysis to identify hub genes. Finally, we explored the correlation between TM4SFs and TMB/MSI.
Results: We identified 21 DEGs from the 41 TM4SFs between N0 (without LNM) and N1 (with LNM) patients, with TM4SF1, TM4SF4, UPK1B, and CD151 being highly expressed in the N1 group; several DEGs were observed in the TNM, T, and N cancer stages. The “integrins and other cell-surface receptors” pathway was the most significantly enriched functional category related to LNM and TM4SFs. TM4SF1 was identified as an indicator of LNM (AUC= 0.702). High levels of TM4SF1 might be related to Wnt/β-catenin pathway and epithelial–mesenchymal transition (EMT) process in PTC. The higher expression of TM4SF1 was also related to DNA promoter hypomethylation. CD9, TM4SF4, TSPAN2, and TSPAN16 were associated with OS in PTC patients and TSPAN2 has great potential to become a prognostic marker of PTC progression. For the prognostic model, the riskscore = (-0.0058)*CD82+(-0.4994)*+(0.1584)*TSPAN11+(1.7597)*TSPAN19+(0.2694)*TSPAN2 (lambda.min = 0.0149). The AUCs for 3-year, 5-year, and 10-year OS were 0.81, 0.851, and 0.804. TSPAN18, TSPAN31, and TSPAN32 were associated with both TMB and MSI in PTC patients.
Conclusion: Our findings identified TM4SF1 as a potential diagnostic marker of LNM and TSPAN2 as a prognostic factor for patients with PTC. Our study provides a novel strategy to assess prognosis and predict effective treatments in PTC.

背景：尽管乳头状甲状腺癌（papillary thyroid carcinoma, PTC）的预后相对良好，但每年仍造成约41000例死亡，该死亡情况大概率与肿瘤复发及转移相关。淋巴结转移（lymph node metastasis, LNM）是PTC复发的重要预测指标，而四次跨膜蛋白超家族（transmembrane 4 superfamily, TM4SF）可通过调控细胞黏附、迁移、组织分化及肿瘤侵袭过程参与转移调控。但目前TM4SF在PTC中的诊断及预后价值仍未明确。
方法：本研究旨在通过生物信息学分析，筛选在PTC中对LNM具有预测价值且具备预后价值的TM4SF家族基因。我们借助癌症基因组图谱（The Cancer Genome Atlas, TCGA）的数据集，筛选PTC组织中TM4SF家族的差异表达基因（differentially expressed genes, DEGs）；通过STRING数据库构建蛋白质相互作用（Protein-Protein Interaction, PPI）网络；采用二元logistic回归分析与受试者工作特征（Receiver Operating Characteristic, ROC）曲线，评估TM4SF1在LNM预测中的作用。我们分析了TM4SF1表达与DNA甲基化的关联，并通过基因集富集分析（Gene Set Enrichment Analysis, GSEA）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）及基因本体（Gene Ontology, GO）富集分析，明确TM4SF1促进LNM的功能与机制。我们通过Kaplan-Meier分析，评估PTC患者中每个TM4SF基因与总生存期（overall survival, OS）的相关性；并采用LASSO惩罚Cox回归分析构建预后预测模型，以筛选枢纽基因。最后，我们探究了TM4SF家族基因与肿瘤突变负荷（Tumor Mutational Burden, TMB）、微卫星不稳定性（Microsatellite Instability, MSI）的相关性。
结果：我们在41个TM4SF家族基因中，筛选出N0组（无LNM）与N1组（伴LNM）PTC患者之间的21个DEGs；其中TM4SF1、TM4SF4、UPK1B及CD151在N1组中呈高表达。在TNM分期、T分期及N分期中均检测到部分DEGs的差异表达。与LNM及TM4SF家族相关的最显著富集功能通路为"integrins and other cell-surface receptors"通路。TM4SF1被证实可作为LNM的预测指标（AUC=0.702）。PTC组织中TM4SF1的高表达可能与Wnt/β-连环蛋白通路及上皮间质转化（epithelial–mesenchymal transition, EMT）过程相关。TM4SF1的高表达同时与DNA启动子低甲基化存在关联。CD9、TM4SF4、TSPAN2及TSPAN16与PTC患者的OS显著相关，其中TSPAN2具备成为PTC进展预后标志物的巨大潜力。本研究构建的预后预测模型风险评分公式为：风险评分 = (-0.0058)*CD82 + (-0.4994)* + (0.1584)*TSPAN11 + (1.7597)*TSPAN19 + (0.2694)*TSPAN2（lambda.min=0.0149）。该模型对PTC患者3年、5年及10年OS的AUC分别为0.81、0.851及0.804。TSPAN18、TSPAN31及TSPAN32与PTC患者的TMB及MSI均存在相关性。
结论：本研究证实TM4SF1可作为LNM的潜在诊断标志物，TSPAN2可作为PTC患者的预后危险因素。本研究为PTC的预后评估及有效治疗预测提供了全新的策略。