Circulating tumor cell copy-number heterogeneity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors

Marianne Oulhen,Benjamin Besse,Genny Buson,Charles Naltet,Nicolò Manaresi,Marianna Garonzi,Laura Mezquita,Tala Tayoun,Agathe Aberlenc,Patrycja Pawlikowska,Yann Lecluse,Pernelle Lavaud,David Planchard,Françoise Farace,Claudio Forcato

doi:10.1038/s41698-021-00203-1

Abstract

Gatekeeper mutations are identified in only 50% of the cases at resistance to Anaplastic Lymphoma Kinase (ALK)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in ALK-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six ALK-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1st and 3rd ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK+/cytokeratins-, 46 (56%) ALK-/cytokeratins+ and 4 (5%) ALK+/cytokeratins+. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial ALK-negative CTCs may drive resistance through activation of bypass signaling pathways, while ALK-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.

Highlights

Targeted therapies have produced substantial clinical benefits in about 20% of non-small cell lung cancer (NSCLC) patients, including a 4% subgroup harboring the anaplastic lymphoma kinase (ALK) gene rearrangements[1]
We have identified Circulating tumor cells (CTCs) harboring a high level of numerical chromosomal instability (CIN) in ROS1rearranged NSCLC22 and reported the predictive value of CTCs with aberrant ALK copy number for crizotinib efficacy[23]
Six ALK-rearranged NSCLC patients (P41, P43, P45, P46, P49, and P50) with CTC counts ≥10 CTCs/20 mL blood at resistance to ALKTKI were analyzed in this study

Summary

Introduction

Targeted therapies have produced substantial clinical benefits in about 20% of non-small cell lung cancer (NSCLC) patients, including a 4% subgroup harboring the anaplastic lymphoma kinase (ALK) gene rearrangements[1]. For patients relapsing on crizotinib, more potent and selective secondgeneration ALK inhibitors such as ceritinib, alectinib and brigatinib, have produced substantial clinical benefits, re-inducing responses to treatment in the majority of crizotinib-resistant patients[3,4,5]. Mutations in ALK kinase domain emerging on treatment with second-generation ALK inhibitors are in most cases targetable by the third-generation TKI lorlatinib[1,6].

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Results

Conclusion