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Updated: Jul 6, 2025
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Resistance to BRAF inhibition explored through single circulating tumour cell molecular profiling in BRAF-mutant
Laura Mezquita1,2, Marianne Oulhen3,4, Agathe Aberlenc3,4
1Gustave Roussy, Université Paris-Saclay, Department of Medicine, F-94805, Villejuif, France.
Background:
Resistance mechanisms to combination therapy with dabrafenib plus trametinib remain poorly understood in patients with BRAFV600E-mutant advanced non-small-cell lung cancer (NSCLC). We examined resistance to BRAF inhibition by single CTC sequencing in BRAFV600E-mutant NSCLC.
Methods:
CTCs and cfDNA were examined in seven BRAFV600E-mutant NSCLC patients at failure to treatment. Matched tumour tissue was available for four patients. Single CTCs were isolated by fluorescence-activated cell sorting following enrichment and immunofluorescence (Hoechst 33342/CD45/pan-cytokeratins) and sequenced for mutation and copy number-alteration (CNA) analyses.
Results:
BRAFV600E was found in 4/4 tumour biopsies and 5/7 cfDNA samples. CTC mutations were mostly found in MAPK-independent pathways and only 1/26 CTCs were BRAFV600E mutated. CTC profiles encompassed the majority of matched tumour biopsy CNAs but 72.5% to 84.5% of CTC CNAs were exclusive to CTCs. Extensive diversity, involving MAPK, MAPK-related, cell cycle, DNA repair and immune response pathways, was observed in CTCs and missed by analyses on tumour biopsies and cfDNA. Driver alterations in clinically relevant genes were recurrent in CTCs.
Conclusions:
Resistance was not driven by BRAFV600E-mutant CTCs. Extensive tumour genomic heterogeneity was found in CTCs compared to tumour biopsies and cfDNA at failure to BRAF inhibition, in BRAFV600E-mutant NSCLC, including relevant alterations that may represent potential treatment opportunities.
Insights
Mechanisms of resistance to BRAF inhibition in non-small-cell lung cancer (NSCLC) are complex. Circulating tumor cells (CTCs) reveal extensive genomic heterogeneity, offering potential new treatment targets.
Area of Science:
- Oncology
- Genomics
- Cancer Biology
Background:
- Resistance to BRAF/MEK inhibitor combinations (dabrafenib/trametinib) in BRAF V600E-mutant non-small-cell lung cancer (NSCLC) is not fully understood.
- Understanding resistance mechanisms is crucial for improving treatment outcomes in advanced NSCLC.
Purpose of the Study:
- To investigate resistance mechanisms to BRAF inhibition in BRAF V600E-mutant NSCLC using single circulating tumor cell (CTC) sequencing.
- To characterize the genomic landscape of CTCs at treatment failure.
Main Methods:
- Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) were analyzed from seven BRAF V600E-mutant NSCLC patients at treatment failure.
- Single CTCs were isolated and sequenced for mutation and copy number alteration (CNA) analyses.
- Matched tumor tissue was available for four patients.
Main Results:
- BRAF V600E mutations were detected in tumor biopsies and cfDNA, but rarely in CTCs.
- CTCs exhibited extensive genomic diversity, including alterations in MAPK-independent pathways, cell cycle, DNA repair, and immune response pathways, which were missed by tumor biopsy and cfDNA analysis.
- CTC profiles showed significant copy number alterations (CNAs) not present in matched tumor biopsies.
Conclusions:
- Resistance to BRAF inhibition in BRAF V600E-mutant NSCLC is not driven by BRAF V600E-mutant CTCs.
- CTCs reveal substantial tumor genomic heterogeneity at treatment failure, offering potential therapeutic targets beyond BRAF V600E.
- Single CTC sequencing provides a more comprehensive view of tumor evolution and resistance mechanisms compared to tumor biopsies and cfDNA.
