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. 2024 Mar;130(4):682-693.
doi: 10.1038/s41416-023-02535-0. Epub 2024 Jan 4.

Resistance to BRAF inhibition explored through single circulating tumour cell molecular profiling in BRAF-mutant non-small-cell lung cancer

Laura Mezquita #  1   2 , Marianne Oulhen #  3   4 , Agathe Aberlenc  3   4 , Marc Deloger  5 , Mihaela Aldea  1 , Aurélie Honore  6 , Yann Lecluse  7 , Karen Howarth  8 , Luc Friboulet  4 , Benjamin Besse  1 , David Planchard  1 , Françoise Farace  9   10
Affiliations

Resistance to BRAF inhibition explored through single circulating tumour cell molecular profiling in BRAF-mutant non-small-cell lung cancer

Laura Mezquita et al. Br J Cancer. 2024 Mar.

Abstract

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.

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Conflict of interest statement

LM: Sponsored Research: Amgen, Bristol-Myers Squibb, Boehringer Ingelheim. Consulting, advisory role: Roche Diagnostics, Takeda, Roche. Lectures and educational activities: Bristol-Myers Squibb, Tecnofarma, Roche. Travel, Accommodations, Expenses: Bristol-Myers Squibb, Roche. Mentorship program with key opinion leaders: funded by AstraZeneca. BB: Sponsored Research at Gustave Roussy Cancer Center Abbvie, Amgen, AstraZeneca, Biogen, Blueprint Medicines, BMS, Celgene, Eli Lilly, GSK, Ignyta, IPSEN, Merck KGaA, MSD, Nektar, Onxeo, Pfizer, Pharma Mar, Sanofi, Spectrum Pharmaceuticals, Takeda, Tiziana Pharma. DP: Consulting, advisory role or lectures: AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Daiichi Sankyo, Eli Lilly, Merck, Novartis, Pfizer, prIME Oncology, Peer. CME, Roche. Honoraria: AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Eli Lilly, Merck, Novartis, Pfizer, prIME Oncology, Peer CME, Roche. Clinical trials research: AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Eli Lilly, Merck, Novartis, Pfizer, Roche, Medimmune, Sanofi-Aventis, Taiho Pharma, Novocure, Daiichi Sankyo. Travel, Accommodations, Expenses: AstraZeneca, Roche, Novartis, prIME Oncology, Pfizer. The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Timelines of treatments, tumour biopsies and blood sample collection for cfDNA and CTC analysis.
CtDNA monitoring data is also provided. Only cfDNA mutations with a VAF threshold ≥ 0.25% are presented, except for BRAFV600E mutation from patient P4 (VAF, 0.06875%).
Fig. 2
Fig. 2. Mutational profiles of CTCs, matched tumour biopsies and cfDNA at combined dabrafenib plus trametinib therapy failure.
Variants in red are known or predicted as drivers according to Cancer Genome Interpreter.
Fig. 3
Fig. 3. Low-pass whole-genome CNA profiles and ploidy of CTCs and matched tumour biopsies at combined dabrafenib plus trametinib therapy failure.
a CNA profiles of CTCs, corresponding germline DNA and leucocyte controls, and matched tumour biopsies from patient P3. Gains are shown in red, losses in blue. b Comparative CNA analysis of CTCs and matched tumour biopsies from patients P1, P2, P3 and P6. Numbers of total CNAs detected in each patient are mentioned in parentheses. c Ploidy level determined for each single CTC (black dots) and tumour biopsy (white dots) samples. *CTC samples that show a flat diploid CNA profile but harbour mutations.
Fig. 4
Fig. 4. Heatmap of selected CNA and SNV oncogenic drivers in CTCs, matched tumour biopsies and cfDNA at combined dabrafenib plus trametinib therapy failure, according to their clinical relevance and/or presence in ≥5 patients.
Altered genes are attributed to pathways. The four main pathways are sorted from the most altered to the least altered. The number of altered genes per pathway is shown in parentheses. CNA driver function (activating or loss of function) is mentioned in the “Role” column. Frequencies of CNAs and driver SNVs in the 57 samples (53 CTCs and 4 tumour biopsies) and the 64 samples (53 CTCs, 4 tumour biopsies and 7 cfDNA), respectively, are provided. Red and blue colours represent gains and losses, respectively.
Fig. 5
Fig. 5. Shared recurrent driver alterations detected in the seven patients.
Numbers of recurrent CNA and SNV driver alterations in RTK/RAS/PI3K, cell cycle, DNA repair and immune response pathways are presented.
See this image and copyright information in PMC

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