Two molecular subgroups predict most recurrences in advanced laryngeal squamous cell carcinoma

The aim of this study was to test whether intratumoral heterogeneity explains inconsistent reports on VEGF-A as a prognostic marker in laryngeal squamous cell carcinoma (LSCC) and to identify expression phenotypes (mRNA/miRNA) associated with recurrence. In a prospective cohort of 60 T3/T4 LSCC patients undergoing primary laryngectomy, we sampled four regions per case (tumor surface, tumor depth, peritumoral mucosa ≤1 cm, paired distant normal mucosa). mRNA levels of HIF-1α/2α/3α, VEGF-A, VEGFR1/2, and ETS-1 were quantified by RT-qPCR; cohort-matched miRNA profiling (microarray with RT-qPCR validation) was integrated. Elevated VEGF-A at tumor depth predicted recurrence (log-rank p = 0.0001), while surface VEGF-A had no prognostic value (p = 0.170). Pairwise testing confirmed higher VEGF-A at depth versus surface (Wilcoxon p = 0.026). A subgroup with depth VEGF-A RQ > 2 and HIF-1α RQ < 2 showed a 64% recurrence rate. An independent subgroup defined by high miR-93-5p/miR-144-3p/miR-210-3p expression also had significantly worse outcomes. The two subgroups were non-overlapping in most patients and accounted for 76% of recurrences. The prognostic relevance of VEGF-A in LSCC is region-dependent, with clinically meaningful value confined to tumor depth. Together with a high-risk three-miRNA signature, these findings delineate two molecular subgroups capturing most recurrences and may inform sampling strategies and risk stratification.本研究旨在验证肿瘤内异质性是否可解释喉鳞状细胞癌（LSCC）中血管内皮生长因子A（VEGF-A）作为预后标志物的相关研究结果不一致的问题，并鉴定与肿瘤复发相关的表达表型（mRNA/微小RNA（miRNA））。 本研究纳入60例接受首次喉切除术的T3/T4期喉鳞状细胞癌患者作为前瞻性队列，每例患者采集4个区域的样本：肿瘤表面、肿瘤深部、瘤周黏膜≤1cm处以及配对的远端正常黏膜。采用逆转录实时定量聚合酶链反应（RT-qPCR）定量检测缺氧诱导因子-1α/2α/3α（HIF-1α/2α/3α）、VEGF-A、血管内皮生长因子受体1/2（VEGFR1/2）及ETS-1的mRNA水平；整合了队列匹配的miRNA表达谱分析数据（经RT-qPCR验证的微阵列芯片数据）。 结果显示，肿瘤深部的VEGF-A高表达可预测肿瘤复发（对数秩检验p=0.0001），而肿瘤表面的VEGF-A表达则无预后价值（p=0.170）。两两比较证实，肿瘤深部的VEGF-A表达水平显著高于肿瘤表面（Wilcoxon检验p=0.026）。其中，肿瘤深部VEGF-A相对定量值（RQ）>2且HIF-1α RQ<2的患者亚组，复发率达64%。此外，以高表达miR-93-5p/miR-144-3p/miR-210-3p定义的独立患者亚组，其预后也显著更差。这两个亚组在大多数患者中无重叠，且覆盖了76%的复发病例。 本研究证实，VEGF-A在喉鳞状细胞癌中的预后相关性具有区域依赖性，其临床有效价值仅局限于肿瘤深部区域。结合高危三miRNA特征标志物，本研究明确了两个可覆盖绝大多数复发案例的分子亚组，可为临床采样策略及风险分层提供参考依据。