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

British Journal of Cancer ARTICLE www.nature.com/bjc OPEN Molecular Diagnostics Resistance to BRAF inhibition explored through single circulating tumour cell molecular profiling in BRAF-mutant nonsmall-cell lung cancer Laura Mezquita1,2,9, Marianne Oulhen3,4,9, Agathe Aberlenc3,4, Marc Deloger5, Mihaela Aldea 1, Aurélie Honore6, Yann Lecluse7, ✉ Karen Howarth8, Luc Friboulet 4, Benjamin Besse1, David Planchard1,10 and Françoise Farace 3,4,10 © The Author(s) 2024 1234567890();,: 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 MAPKindependent 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. British Journal of Cancer; https://doi.org/10.1038/s41416-023-02535-0 INTRODUCTION Lung cancer is the most common cause of cancer-related death worldwide, owing to its metastatic spread at the time of diagnosis [1]. The molecular characterisation of Non-Small-Cell Lung Cancer (NSCLC) and discovery of oncogene driver alterations have revolutionised the therapeutic landscape of NSCLC. Molecularly targeted therapy using tyrosine kinase inhibitors (TKIs) has led to major clinical improvement in about 25% of patients with NSCLC harbouring epidermal growth factor receptor (EGFR) activating alterations, anaplastic lymphoma kinase (ALK) gene or c-ros oncogene 1 (ROS1) fusions [2]. More recently, BRAF mutations—responsible for the constitutive activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway—have emerged as a novel molecular target in around 2% of NSCLC patients [3, 4]. Initial studies demonstrated the clinical activity of selective inhibitors dabrafenib or vemurafenib as single agents in previously treated patients with BRAFV600E-mutant NSCLC, observed in 50% of patients with a BRAF mutation [5, 6]. Similar to melanoma, superior efficacy of combined BRAF and MEK inhibition compared to BRAF inhibitor monotherapy was observed in BRAFV600E-mutant NSCLC. The combination of dabrafenib and MEK inhibitor trametinib produced substantial antitumor activity (ORR, 66.7%) with durable responses (median PFS, 10.2 months) in previously treated BRAFV600E-mutant NSCLC patients [5]. Moreover, significant clinical improvement of this combination therapy over both single-agent dabrafenib and conventional chemotherapy was observed in untreated BRAFV600E NSCLC [6]. These studies have led to the European Medicines Agency and US Food and Drug Administration 1 Gustave Roussy, Université Paris-Saclay, Department of Medicine, F-94805 Villejuif, France. 2Medical Oncology Department, Hospital Clinic of Barcelona, Laboratory of Translational Genomics and Targeted Therapies in Solid Tumors, IDIBAPS, Barcelona, Spain. 3Gustave Roussy, Université Paris-Saclay, “Rare Circulating Cells” Translational Platform, CNRS UMS3655—INSERM US23 AMMICA, F-94805 Villejuif, France. 4INSERM, U981 “Identification of Molecular Predictors and new Targets for Cancer Treatment”, F-94805 Villejuif, France. 5Gustave Roussy, Université Paris-Saclay, Bioinformatics Platform, CNRS UMS3655—INSERM US23 AMMICA, F-94805 Villejuif, France. 6Gustave Roussy, Université Paris-Saclay, Genomic Platform, CNRS UMS3655—INSERM US23 AMMICA, F-94805 Villejuif, France. 7Gustave Roussy, Université Paris-Saclay, “Flow cytometry and Imaging” Platform, CNRS UMS3655—INSERM US23AMMICA, F-94805 Villejuif, France. 8Inivata Ltd, Babraham Research Park, Cambridge, UK. 9These authors contributed equally: Laura Mezquita, Marianne Oulhen. 10These authors jointly supervised this work: David Planchard, Françoise Farace. ✉email: Received: 2 July 2023 Revised: 24 November 2023 Accepted: 30 November 2023 L. Mezquita et al. 2 approval of dabrafenib-trametinib combination for the treatment of BRAFV600E-mutant NSCLC and its recent recommendation as upfront and standard of care treatment in this malignancy. Nevertheless, in spite of high objective response rates, acquired resistance to targeted therapy inevitably develops, leading to disease progression in patients with BRAFV600E-mutant NSCLC. Knowledge about resistance mechanisms to BRAF inhibition results mainly from studies conducted in metastatic melanoma. Very limited data are available for NSCLC so far. Unlike EGFR or ALK, acquired resistance mutations within the BRAF gene remain to be elucidated. In melanoma, it has been proposed that the development of secondary resistance mechanisms can be due to (1) ERK reactivation through the MAPK pathway, (2) bypass signalling tracks leading to constitutive activation of alternative oncogenic pathways, (3) other unknown mechanisms [7–9]. Reactivation of ERK upstream or downstream of BRAF kinase constitutes the main secondary resistance mechanism to BRAF inhibition in metastatic melanoma. Activation of bypass pathways such as PI3K-AKT represents another critical mechanism of acquired resistance in melanoma. In NSCLC, mechanisms of ERK reactivation mainly involved BRAF variants, BRAF gene amplification or secondary mutations in other genes of the MAPK/ERK signalling pathway such as NRAS/KRAS or MEK mutations, leading to BRAF-independent reactivation of ERK signalling [10]. Mechanisms of secondary resistance to dual inhibition of BRAF and MEK are more complex but in most cases also involve the reactivation of MAPK pathway and ERK signalling as observed for single-agent resistance [11–14]. Genomic studies of primary tumours and metastases have unravelled the complex and heterogeneous molecular landscape of NSCLC and its implication in response to therapy. Liquid biopsy components such as circulating tumour cells (CTCs) and cell-free (cf) DNA are likely r (...truncated)