Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma
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 suit...
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2025-08-01
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| Online Access: | https://doi.org/10.1186/s12967-025-06941-1 |
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| author | Alex De Gregorio Andrea Sacconi Chiara Mandoj Fabio Valenti Valentina De Pascale Gabriele Madonna Mariaelena Capone Teresa Meinardi Francesca Guerresi Irene Terrenato Giovanni Cigliana Michelangelo Russillo Virginia Ferraresi Maurizio Fanciulli Giovanni Blandino Paolo Antonio Ascierto Gennaro Ciliberto Rita Mancini Luigi Fattore |
| author_facet | Alex De Gregorio Andrea Sacconi Chiara Mandoj Fabio Valenti Valentina De Pascale Gabriele Madonna Mariaelena Capone Teresa Meinardi Francesca Guerresi Irene Terrenato Giovanni Cigliana Michelangelo Russillo Virginia Ferraresi Maurizio Fanciulli Giovanni Blandino Paolo Antonio Ascierto Gennaro Ciliberto Rita Mancini Luigi Fattore |
| author_sort | Alex De Gregorio |
| collection | DOAJ |
| description | 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. Graphical abstract |
| format | Article |
| id | doaj-art-a86f91b0306e4a35a5fadb268b219e9a |
| institution | Kabale University |
| issn | 1479-5876 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | BMC |
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| series | Journal of Translational Medicine |
| spelling | doaj-art-a86f91b0306e4a35a5fadb268b219e9a2025-08-20T03:43:02ZengBMCJournal of Translational Medicine1479-58762025-08-0123111310.1186/s12967-025-06941-1Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanomaAlex De Gregorio0Andrea Sacconi1Chiara Mandoj2Fabio Valenti3Valentina De Pascale4Gabriele Madonna5Mariaelena Capone6Teresa Meinardi7Francesca Guerresi8Irene Terrenato9Giovanni Cigliana10Michelangelo Russillo11Virginia Ferraresi12Maurizio Fanciulli13Giovanni Blandino14Paolo Antonio Ascierto15Gennaro Ciliberto16Rita Mancini17Luigi Fattore18SAFU Laboratory, Department of Research, Advanced Diagnostics and Technological Innovation, Translational Research Area, IRCCS Regina Elena National Cancer InstituteUOSD Clinical Trial Center, Biostatistics and Bioinformatics, IRCCS Regina Elena National Cancer InstituteClinical Pathology Unit and Cancer Biobank, IRCCS Regina Elena National Cancer InstituteOncogenomic and Epigenetic Unit, Department of Diagnostic Research and Technological Innovation, IRCCS Regina Elena National Cancer InstituteOncogenomic and Epigenetic Unit, Department of Diagnostic Research and Technological Innovation, IRCCS Regina Elena National Cancer InstituteUnit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. PascaleUnit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. PascaleUnit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. PascaleUnit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. PascaleUOSD Clinical Trial Center, Biostatistics and Bioinformatics, IRCCS Regina Elena National Cancer InstituteClinical Pathology Unit and Cancer Biobank, IRCCS Regina Elena National Cancer InstituteSarcomas and Rare Tumors Departmental Unit, IRCCS Istituto Nazionale Tumori Regina ElenaSarcomas and Rare Tumors Departmental Unit, IRCCS Istituto Nazionale Tumori Regina ElenaSAFU Laboratory, Department of Research, Advanced Diagnostics and Technological Innovation, Translational Research Area, IRCCS Regina Elena National Cancer InstituteOncogenomic and Epigenetic Unit, Department of Diagnostic Research and Technological Innovation, IRCCS Regina Elena National Cancer InstituteUnit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. PascaleIRCCS Regina Elena National Cancer InstituteDepartment of Clinical and Molecular Medicine, University of Rome “Sapienza”Department of Life Health Sciences and Health Professions, Link Campus UniversityAbstract 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. Graphical abstracthttps://doi.org/10.1186/s12967-025-06941-1Digital PCRDuplex assayLiquid biopsyCirculating MiRNAsMetastatic melanoma |
| spellingShingle | Alex De Gregorio Andrea Sacconi Chiara Mandoj Fabio Valenti Valentina De Pascale Gabriele Madonna Mariaelena Capone Teresa Meinardi Francesca Guerresi Irene Terrenato Giovanni Cigliana Michelangelo Russillo Virginia Ferraresi Maurizio Fanciulli Giovanni Blandino Paolo Antonio Ascierto Gennaro Ciliberto Rita Mancini Luigi Fattore Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma Journal of Translational Medicine Digital PCR Duplex assay Liquid biopsy Circulating MiRNAs Metastatic melanoma |
| title | Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma |
| title_full | Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma |
| title_fullStr | Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma |
| title_full_unstemmed | Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma |
| title_short | Development of an innovative duplex digital PCR assay for circulating MiRNA ratio quantification in metastatic melanoma |
| title_sort | development of an innovative duplex digital pcr assay for circulating mirna ratio quantification in metastatic melanoma |
| topic | Digital PCR Duplex assay Liquid biopsy Circulating MiRNAs Metastatic melanoma |
| url | https://doi.org/10.1186/s12967-025-06941-1 |
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