Cross-method comparison for BRAF p.V600 mutation cfDNA testing in Melanoma: BRAFI study

<h2>ABSTRACT</h2><h3>Background</h3> BRAF p.V600 mutation is the most frequent molecular driver alteration in melanoma. Detection of BRAF mutations in circulating-free DNA (cfDNA) reflects the shedding of tumor DNA and offers a potential non-invasive biomarker for disease monitoring and prognosis. However, the lack of standardized methodologies and inter-assay variability hinders its clinical implementation. <h3>Methods</h3> The sensitivity, agreement and concordance of seven <i>BRAF</i> mutation detection assays were assessed across four laboratories. <i>BRAF</i> p.V600 mutation in pretreatment plasma samples was analyzed in 51 patients diagnosed with advanced stage melanoma using two digital PCR-based assays (droplet digital PCR -ddPCR- Bio-Rad and microfluidic digital PCR -Absolute Q, ThermoFisher Scientific-), three RT-PCR based assays (Idylla®, Cobas®, PNA-Q-PCR) and two NGS based assays (Oncomine™ Pan-Cancer Cell-Free Assay and Illumina Platforms). <h3>Results</h3> digital PCR-based assays and Cobas® exhibited the highest sensitivity (51.0%), followed by NGS Illumina® (45.1%), Oncomine NGS / PNA-Q-PCR (43.1%) and Idylla® (37.2%). Results of different techniques showed a moderate to strong agreement, except for the comparison of Cobas with Idylla that was poor (Kappa=0.57). There was near-perfect agreement on detection of <i>BRAF</i> mutation between both NGS platforms and the NGS Illumina® with PNA-Q-PCR (Kappa = 0.92). Concordance of the quantitative results in terms of mutant allele frequency was near-perfect between NGS Illumina and ddPCR Bio-Rad assays (ICC = 0.99). <h3>Conclusions</h3> Our study demonstrates substantial agreement among multiple cfDNA <i>BRAF</i> mutation detection assays, particularly between NGS and digital PCR assays. These findings support the potential utility of different techniques for BRAF testing in cfDNA.

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Journal
EJC Skin Cancer
Published
2025-01-01
DOI
https://doi.org/10.1016/j.ejcskn.2025.100738
Primary Topic
Cancer Genomics and Diagnostics
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article
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article

Cross-method comparison for BRAF p.V600 mutation cfDNA testing in Melanoma: BRAFI study

Ana Arance, Silvia Calabuig‐Fariñas, Joselyn Valarezo, José Luís Zamora Manzano et al.
EJC Skin Cancer
Cancer Genomics and Diagnostics
article

Cross-method comparison for BRAF p.V600 mutation cfDNA testing in Melanoma: BRAFI study

Ana Arance, Silvia Calabuig‐Fariñas, Joselyn Valarezo, José Luís Zamora Manzano, Juan Francisco Rodríguez-Moreno, Rafael Rosell, Susana Muñoz, Eloísa Jantus‐Lewintre, Enrique Espinosa, Teresa Puértolas, Miguel Ángel Molina‐Vila, Clara Mayo de las Casas, Eva Muñoz‐Couselo, Alfonso Berrocal, Miguel‐Ángel Berciano‐Guerrero, María González‐Cao, María Quindós-Varela, Sebastián Ortiz Reina, Guillermo Crespo, Pablo Miguel, Joan Anton Puig‐Butillé, Pablo Cerezuela, Ana Drozdowskyj, Almudena García Castaño
article en

Abstract

<h2>ABSTRACT</h2><h3>Background</h3> BRAF p.V600 mutation is the most frequent molecular driver alteration in melanoma. Detection of BRAF mutations in circulating-free DNA (cfDNA) reflects the shedding of tumor DNA and offers a potential non-invasive biomarker for disease monitoring and prognosis. However, the lack of standardized methodologies and inter-assay variability hinders its clinical implementation. <h3>Methods</h3> The sensitivity, agreement and concordance of seven <i>BRAF</i> mutation detection assays were assessed across four laboratories. <i>BRAF</i> p.V600 mutation in pretreatment plasma samples was analyzed in 51 patients diagnosed with advanced stage melanoma using two digital PCR-based assays (droplet digital PCR -ddPCR- Bio-Rad and microfluidic digital PCR -Absolute Q, ThermoFisher Scientific-), three RT-PCR based assays (Idylla®, Cobas®, PNA-Q-PCR) and two NGS based assays (Oncomine™ Pan-Cancer Cell-Free Assay and Illumina Platforms). <h3>Results</h3> digital PCR-based assays and Cobas® exhibited the highest sensitivity (51.0%), followed by NGS Illumina® (45.1%), Oncomine NGS / PNA-Q-PCR (43.1%) and Idylla® (37.2%). Results of different techniques showed a moderate to strong agreement, except for the comparison of Cobas with Idylla that was poor (Kappa=0.57). There was near-perfect agreement on detection of <i>BRAF</i> mutation between both NGS platforms and the NGS Illumina® with PNA-Q-PCR (Kappa = 0.92). Concordance of the quantitative results in terms of mutant allele frequency was near-perfect between NGS Illumina and ddPCR Bio-Rad assays (ICC = 0.99). <h3>Conclusions</h3> Our study demonstrates substantial agreement among multiple cfDNA <i>BRAF</i> mutation detection assays, particularly between NGS and digital PCR assays. These findings support the potential utility of different techniques for BRAF testing in cfDNA.

EJC Skin CancerVol. 3
Universidad San Pablo CEU (ES), Hospital Universitario La Paz (ES), Hospital General Universitario Santa María del Rosell (ES), Instituto Oncológico Dr. Rosell (ES), Hospital Regional Universitario de Málaga (ES), Hospital San Pedro de Alcántara (ES), Hospital Clínic de Barcelona (ES), Hospital Universitario de Burgos (ES), Agbar (Spain) (ES), Vall d'Hebron Hospital Universitari (ES), Hospital General Universitario De Valencia (ES), Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (ES), Fundación General (ES), Complexo Hospitalario Universitario A Coruña (ES), Santa Lucía University General Hospital (ES), Hospital Universitario Virgen de la Arrixaca (ES), Hospital Universitari Germans Trias i Pujol (ES), Centro de Investigación Biomédica en Red de Cáncer (ES), Centro de Investigacion Principe Felipe (ES)
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