Abstract 3654: Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in ALK-rearranged non-small-cell lung cancer (NSCLC)
Authors: Pailler Emma , Faugeroux Vincent , Oulhen Marianne , Forcato Claudio , Laporte Mélanie , Lecluse Yann , Lacroix Ludovic , NgoCamus Maud , Nicotra Claudio , Remon Jordi , Mezquita Laura , Planchard David , Soria Jean-Charles , Manaresi Nicolò , Besse Benjamin , Farace Françoise 
Published: August 2018

Abstract
Background
Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. We recently reported how CTCs with abnormal ALK FISH-patterns monitored on treatment can stratify patients at risk of early-resistance to crizotinib (Pailler et al., Cancer Res, 2017). ALK-rearranged NSCLC patients treated with crizotinib develop resistance which manifest by the upregulation of bypass signaling pathways in two-thirds of cases. Here, we evaluated whether individual CTCs could identify resistance mutations to crizotinib and represent tumor diversity compared to single-site tumor-biopsies.
Methods
Individual CTCs were isolated from seven ALK-rearranged patients at resistance to crizotinib. Matched tumor-biopsies were available for three. Two isolation strategies were used: (i) ISET-filtration, immunofluorescence (Hoescht/CD45/epithelial-markers/mesenchymal-marker), scanning, laser-microdissection; (ii) RosetteSep, immunofluorescence (Hoescht/CD45/ALK/cytokeratins), cell-sorting. A process including Ampli1 whole-genome amplification, quality controls, multiplex PCR with two panels (Ampli1 CHPCustomBeta cancer panel developed by Silicon Biosystems and a home-made panel targeting all ALK mutations) and Ion Torrent next-generation sequencing (NGS) was established. Ten to 17 pools with 1-5 CTCs and one CD45+ cell pool were analyzed for each patient. A bioinformatic workflow was developed including determination of allele drop-out (ADO), false-positive rate (FPR) and positive predicted value (PPV).
Results
By comparing germline variants present in constitutional and CD45+ pool DNA, we calculated for each sample the statistics of mean ADO, FPR and PPV which were respectively 19%, 2.6x10-4 and 69%. When variants were present into two pools, the PPV and FPR were 96% and 2.39 10-5 respectively. In the three patients with an available tumor-biopsy, a limited number of shared mutations between CTC and matched tumor-biopsies were identified. In contrast, CTC-private mutations (exclusively present in CTCs and not in matched biopsies) present in at least two CTC pools were identified including in FLT3, SMAD4, KIT, PI3KCA, EGFR, KRAS, PDGFRA genes among which several were COSMIC mutations. A higher degree of mutational diversity was observed in CTCs compared to tumor-biopsies.
Conclusion
Using a rigorously qualified workflow, we reported for the first time the identification of resistance mutations in individually isolated CTCs of ALK-rearranged patients. In accordance with published series from tumor-biopsies, our CTC results highlighted the role of bypass signaling pathways in resistance to crizotinib. Our study provides important perspectives on using CTCs for precision medicine and informing on tumor heterogeneity in NSCLC patients.
Citation Format: Emma Pailler, Vincent Faugeroux, Marianne Oulhen, Claudio Forcato, Mélanie Laporte, Yann Lecluse, Ludovic Lacroix, Maud NgoCamus, Claudio Nicotra, Jordi Remon, Laura Mezquita, David Planchard, Jean-Charles Soria, Nicolò Manaresi, Benjamin Besse, Françoise Farace. Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in ALK-rearranged non-small-cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3654.