# Circulating miR-99a-5p and miR-1246 as Diagnostic and Stage-Associated Biomarkers in Laryngeal Squamous Cell Carcinoma

## TL;DR

This study identifies miR-99a-5p as a potential blood-based diagnostic marker for laryngeal cancer and miR-1246 as a marker for cancer stage.

## Contribution

The study provides new evidence on the diagnostic and staging potential of miR-99a-5p and miR-1246 in laryngeal squamous cell carcinoma.

## Key findings

- miR-99a-5p showed significantly lower relative expression in cancer patients compared to controls.
- miR-1246 expression was lower in advanced-stage cancer patients compared to early-stage patients.
- miR-99a-5p had strong diagnostic performance with high sensitivity and specificity.

## Abstract

Background and Objectives: Circulating microRNAs may provide minimally invasive biomarkers for laryngeal squamous cell carcinoma (LSCC), but clinically interpretable data for miR-99a-5p and miR-1246 remain limited. We compared circulating levels of these two miRNAs between LSCC patients and controls and explored stage-associated differences within the cancer cohort. Methods: This single-center case–control study was conducted in Timișoara, Romania. Circulating miRNAs were quantified by RT-qPCR. Expression was summarized as ΔCt [Ct(target) − Ct(miR-16)] and as the relative expression (2−ΔΔCt) using the control group as a calibrator. Group comparisons used Mann–Whitney U tests, associations used Spearman correlation, and the diagnostic performance was assessed by ROC analysis and multivariable logistic regression. Results: Fourteen controls were compared with cancer patients with available miRNA measurements (miR-99a-5p, n = 53; miR-1246, n = 49). miR-99a-5p showed significantly higher ΔCt values in cancer patients than in the controls (5.308 [IQR 4.139–6.864] vs. 3.184 [2.142–3.708], p < 0.001), corresponding to a lower relative expression (fold-change 0.200 [0.068–0.449], p < 0.001). miR-1246 did not differ significantly between cancer and controls (p = 0.09). Within the cancer cohort, advanced-stage disease showed a lower relative miR-1246 expression than early-stage disease (ΔCt 5.820 [4.502–6.972] vs. 4.233 [3.109–5.372], p = 0.01; fold-change 0.363 vs. 1.091, p = 0.01), while miR-99a-5p showed a non-significant difference in the same direction (p = 0.052). miR-99a-5p discriminated cancer patients from the controls with an AUC of 0.842 (95% CI 0.744–0.931), sensitivity of 77.4%, and specificity of 92.9% at ΔCt = 4.018. In multivariable analysis, ΔCt(miR-99a-5p) remained independently associated with cancer status (OR 1.89, 95% CI 1.19–3.00; p = 0.007). Conclusions: Circulating miR-99a-5p showed the strongest diagnostic signal in LSCC, whereas miR-1246 appeared more informative for stage-associated biological stratification.