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.