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 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.

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.