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. 2025 Aug 13;23(1):904.
doi: 10.1186/s12967-025-06941-1.

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

Alex De Gregorio  1 , Andrea Sacconi  2 , Chiara Mandoj  3 , Fabio Valenti  4 , Valentina De Pascale  4 , Gabriele Madonna  5 , Mariaelena Capone  5 , Teresa Meinardi  5 , Francesca Guerresi  5 , Irene Terrenato  2 , Giovanni Cigliana  3 , Michelangelo Russillo  6 , Virginia Ferraresi  6 , Maurizio Fanciulli  1 , Giovanni Blandino  4 , Paolo Antonio Ascierto  5 , Gennaro Ciliberto  7 , Rita Mancini  8 , Luigi Fattore  9
Affiliations

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

Alex De Gregorio et al. J Transl Med. .

Abstract

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.

Keywords: Circulating MiRNAs; Digital PCR; Duplex assay; Liquid biopsy; Metastatic melanoma.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The study was approved by the Ethics Committees of both participating institutions: the IRCCS “G. Pascale Foundation” (Approval Numbers DSC/1504, June 11, 2014 and DSC/2893, April 11, 2015) and the IRCCS Regina Elena National Cancer Institute-IFO (Approval Number 8393, July 23, 2017), in accordance with the Declaration of Helsinki. Inclusion criteria specified patients aged ≥ 18 years with histologically confirmed, locally advanced or metastatic melanoma (Stage IIIB-IIIC or IV according to the American Joint Committee on Cancer, 7th Edition), eligible for MAPK-targeted therapy, and capable of providing informed consent. Samples from healthy donors were collected with the approval of the IRCCS Regina Elena National Cancer Institute (Rome, Italy) institutional ethics committee (Approval Number 7182, May 24, 2019). Written informed consent was obtained from all participants before inclusion in the study. Consent for publication: All the authors agreed for publication of this research. Competing interests: All the authors declare no conflict of interest with the exceptions of P.A.A. P.A.A. has/had a consultant/advisory role for Bristol MyersSquibb, Roche-Genentech, Merck Sharp & Dohme, Novartis, Merck Serono, Pierre-Fabre, AstraZeneca, Sun Pharma, Sanofi, Idera, Sandoz, Immunocore, 4SC, Italfarmaco, Nektar, Boehringer-Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Pfizer, Oncosec, Nouscom, Lunaphore, Seagen, iTeos, Medicenna, Bio-Al Health, ValoTX, Replimmune. He also received research funding from Bristol Myers Squibb, Roche-Genentech, Pfizer, Sanofi. Travel support by Pfizer. The funders of this study had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Fig. 1
Fig. 1
Comparison of circulating miR-4488 and miR-579-3p levels in healthy donors’ serum using qRT-PCR and dPCR. Quantification of miR-579-3p (panels A and B) and miR-4488 (panels C and D) in serum samples from healthy donors was performed using qRT-PCR (A, C) and dPCR (B, D). qRT-PCR results are shown as relative expression values (2−Ct), while dPCR provides absolute quantification, expressed as copies per microliter
Fig. 2
Fig. 2
Comparison of circulating miR-4488 and miR-579-3p levels in melanoma patients’ serum using qRT-PCR and dPCR. Quantification of miR-579-3p (panels A and B) and miR-4488 (panels C and D) in serum samples from melanoma patients using qRT-PCR (A, C) and dPCR (B, D). qRT-PCR results are expressed as relative levels (2−Ct), while dPCR provides absolute quantification in copies per microliter
Fig. 3
Fig. 3
Comparison of singleplex and duplex dPCR assays for circulating miR-4488 and miR-579-3p quantification. Expression levels of miR-4488 (A) and miR-579-3p (C) measured by singleplex and duplex dPCR across 40 serum samples. Correlation between the two formats is shown for miR-4488 (B) and miR-579-3p (D) using Pearson’s correlation coefficient. Panel E compares miRatio (miR-4488/miR-579-3p) values from both approaches, with corresponding correlation in (F). All expression levels are reported as copies per microliter. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test; ns = not significant
Fig. 4
Fig. 4
Prognostic and diagnostic value of miRatio in melanoma patients. Panel A shows baseline serum miRatio levels in DC and PD to targeted therapy, with significantly higher values observed in PD. Panel B presents the ROC curve evaluating the predictive performance of baseline miRatio in classifying treatment response. Panel C displays Kaplan–Meier survival curves stratifying patients by high or low baseline miRatio, demonstrating its prognostic relevance. KM curve generated using the 2.79 threshold calculated by ROC curve in panel (B). Panels D and E show longitudinal changes in miRatio levels in melanoma patients: panel D displays values at the first on-treatment timepoint (T1), while panel (E) reports levels at later timepoints corresponding to disease progression or sustained response (T2). Panel F compares miRatio values between healthy donors and melanoma patients, revealing significant upregulation in the latter group. Panel G presents the ROC curve assessing the diagnostic accuracy of miRatio in distinguishing melanoma patients from healthy controls. Statistical significance was evaluated using the Mann–Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001
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