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

## TL;DR

The paper introduces miRquad, a new digital PCR assay for detecting microRNAs in head and neck cancer, which could improve patient monitoring and prognosis.

## Contribution

miRquad is the first multiplex dPCR TaqMan assay for miRNA detection in head and neck cancer, enabling simultaneous analysis from multiple sample types.

## Key findings

- miRquad demonstrated strong correlation with commercial singleplex assays and broad applicability across sample types.
- The assay reduces turnaround time and cost while enabling accurate miRNA detection in liquid biopsy samples.
- miRquad shows potential for real-time patient monitoring and predicting disease outcomes in head and neck cancer.

## Abstract

Cancer resistance is one of the major challenges in oncology, often resulting in disease relapse and poor patient outcomes. 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 are 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, single-target methods exist but multiplex detection from the same input have been only rarely explored.

We present miRquad, the first-in-class digital PCR (dPCR) TaqMan™ multiplex clinical research 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 would enable simultaneous miRNA analysis via qPCR and dPCR on multiple clinically relevant sample types.

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, reduced turnaround time (TAT) and cost-effectiveness. Finally, we provide evidence for its potential clinical application to predict disease outcome in HNC, testing miRquad on tumoral and peritumoral tissues, sera and saliva samples collected throughout patient follow up.

The assay overcomes common challenges associated with multiple miRNAs detection, particularly in liquid biopsy samples (e.g., multiple pipetting issues, increased consumption of sample for multiple assessment, extended TAT for complete profiling) and provides robust and accurate detection, demonstrating potential for real-time patient monitoring and prognostication in HNC.

The online version contains supplementary material available at 10.1186/s13046-025-03590-6.