Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma

Background: Circulating miRNAs (cmiRNAs) are emerging as valuable non-invasive biomarkers for monitoring disease progression and therapeutic response in cancer. Their stability in biological fluids, tissue-specific expression, and functional roles in tumor biology make them particularly suitable for liquid biopsy approaches. However, challenges related to quantification accuracy and assay standardization have limited their clinical translation. Digital PCR (dPCR) offers a highly sensitive and reproducible solution for absolute quantification of low-abundance transcripts, addressing many of these limitations. Methods: We developed and analytically validated the first duplex dPCR assay for the simultaneous detection of miR-4488 and miR-579-3p in serum samples from patients with BRAF-mutant metastatic melanoma receiving MAPK inhibitor therapy. These two cmiRNAs were previously identified by our group as biomarkers predictive of treatment response. Using fluorescently labelled probes, both targets were co-amplified in a single reaction. The assay was tested for analytical performance, including comparison with singleplex formats and quantitative Real-Time PCR (qRT-PCR). We then applied the duplex assay to assess the prognostic potential of the expression ratio between the two miRNAs, termed miRatio, at baseline and over treatment timepoints. Results: The duplex assay maintained analytical performance comparable to singleplex reactions while reducing sample and reagent use. Compared to qRT-PCR, dPCR showed superior sensitivity, particularly for detecting low-abundance miRNAs like miR-4488. miRatio effectively predicts disease outcome when measured at baseline prior to MAPKi therapy and exhibits dynamic changes during treatment, supporting its potential as a longitudinal biomarker. ROC analysis demonstrated strong prognostic value, with improved accuracy over previous qRT-PCR-based evaluations. Conclusions: This study highlights duplex dPCR as a robust, sensitive, and scalable technology for circulating miRNA quantification in liquid biopsy applications. By enabling absolute and simultaneous detection of miR-4488 and miR-579-3p, the assay provides a technically advanced platform for real-time monitoring in metastatic melanoma. While miRatio remains a promising biomarker, the key innovation of this work is the development of a duplex assay suitable for clinical implementation in precision oncology.