miRquad: first-in-class dPCR multiplex TaqMan™ Advanced RUO assay for microRNA detection in head and neck cancer

Background: Despite remarkable therapeutic progress, cancer resistance remains one of the major challenges in oncology, often resulting in disease relapse and poor patient outcomes. Resistance arises from multiple genetic and non-genetic mechanisms, ultimately limiting the effectiveness of chemo- and targeted therapies. Within the RNA family, microRNAs (miRNAs) regulate core biological processes and have been recognized also as critical contributors of tumor resistance and therapy failure. Being pivotal, they have been increasingly exploited as biomarkers in various settings. Although in silico analyses facilitate miRNAs identification, PCR-based approaches remain essential to validate their expression. Currently, a plethora of well-established methods exist but multiplex detection from the same input have been only rarely explored. Methods: We present miRquad, the first-in-class digital PCR (dPCR) TaqMan™ multiplex RUO assay for miRNA detection in head and neck (HNC) cancers. Based on a patented prognostic signature including miR-21-5p, miR-96-5p, miR-21-3p and miR-429, the assay enables simultaneous miRNA analysis via qPCR and dPCR across multiple clinically relevant sample types. Results: We designed and optimized miRquad using both synthetic controls and retrospective patient-derived tissues, sera and saliva. A multicentre ring study was conducted to evaluate assay reliability across different platforms, demonstrating strong correlation with commercial singleplexes, broad applicability, and cost-effectiveness. Finally, we provide evidence for its potential clinical application in different HNC settings, testing miRquad on tumoral and peritumoral tissues, sera and saliva samples collected throughout patient follow up. Conclusions : The assay overcomes common challenges associated with multiple miRNAs detection, particularly in liquid biopsy samples, and provides robust and accurate detection, demonstrating potential for real-time patient monitoring and prognostication in HNC.