Data from Two Molecular Subgroups Predict Most Recurrences in Advanced Laryngeal Squamous Cell Carcinoma

Abstract 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 patients with T3/T4 LSCC undergoing primary laryngectomy, we sampled four regions per case (tumor surface, tumor depth, peritumoral mucosa ≤1 cm, and paired distant normal mucosa). mRNA levels of hypoxia-inducible factor (HIF) 1α/2α/3α, VEGF-A, VEGFR1/2, and ETS-1 were quantified by qRT-PCR; cohort-matched miRNA profiling (microarray with qRT-PCR validation) was integrated. Elevated VEGF-A at tumor depth predicted recurrence (log-rank P = 0.0001), whereas 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 relative quantification (RQ) > 2 and HIF1α 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. Significance: We demonstrate that VEGF-A has prognostic value in laryngeal carcinoma only when assessed at tumor depth. Alongside a distinct high-risk miRNA signature, we define two molecular subgroups accounting for most recurrences, highlighting the clinical importance of spatial heterogeneity in biomarker evaluation.