Acquired Resistance Mutations to ALK Inhibitors Identified by Single Circulating Tumor Cell Sequencing inALK-Rearranged Non–Small-Cell Lung Cancer

Abstract

Purpose: Patients with anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) inevitably develop resistance to ALK inhibitors. New diagnostic strategies are needed to assess resistance mechanisms and provide patients with the most effective therapy. We asked whether single circulating tumor cell (CTC) sequencing can inform on resistance mutations to ALK inhibitors and underlying tumor heterogeneity in ALK-rearranged NSCLC. Experimental Design: Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib (n ¼ 14) or lorlatinib (n ¼ 3). Three strategies including filter laser-capture microdissection, fluorescence activated cell sorting, and the DEPArray were used. One hundred twenty-six CTC pools and 56 single CTCs were isolated and sequenced. Hotspot regions over 48 cancer-related genes and 14 ALK mutations were examined to identify ALK-independent and ALK-dependent resistance mechanisms. Results: Multiple mutations in various genes in ALKindependent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS (EGFR, KRAS, BRAF genes) and TP53 pathways were recurrently mutated. In one lorlatinib-resistant patient, two single CTCs out of 12 harbored ALK compound mutations. CTC-1 harbored the ALK G1202R/F1174C compound mutation virtually similar to ALK G1202R/F1174L present in the corresponding tumor biopsy. CTC-10 harbored a second ALK G1202R/T1151M compound mutation not detected in the tumor biopsy. By copy-number analysis, CTC-1 and the tumor biopsy had similar profiles, whereas CTC-10 harbored multiple copynumber alterations and whole-genome duplication. Conclusions: Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in ALK-rearranged patients. Single CTC sequencing may be a unique tool to assess heterogeneous resistance mechanisms and help clinicians for treatment personalization and resistance options to ALK-targeted therapies.

The Use of Three-Dimensional DNA Fluorescent In Situ Hybridization (3D DNA FISH) for the Detection of Anaplastic Lymphoma Kinase (ALK) in Non-Small Cell Lung Cancer (NSCLC) Circulating Tumor Cells

“…A number of studies have reported on the presence of ALK-translocated CTCs in blood taken from NSCLC patients [12,14,15,17,[21][22][23] with recent findings from Pailler et al, 2019, sequencing single CTCs that revealed the heterogeneity and resistance mutations in ALK-rearranged patients. The study by Pailler et al, 2019, demonstrated that various genes involved in the RTK-KRAS and TP53 pathways were found in patients with crizotinib resistance [24]. Therefore, the ability to assess the mutational changes in real time and over the course of therapy is of critical importance.…”

Epidermal Growth Factor Receptor (EGFR)-Mutated Non-Small-Cell Lung Cancer (NSCLC)

“…Molecular characterization of CTCs has revealed that genomic changes of the tumor can be readily assessed via CTCs. This was recently highlighted by a number of studies identifying translocations/mutations in anaplastic lymphoma kinase (ALK) and EGFR in CTCs [ 74 , 75 , 76 , 77 , 78 ]. For EGFR mutations, the sensitivity of the assay increased with higher numbers of detected CTCs with concordance between EGFR mutation status in the tumor and CTC samples [ 77 ].…”

Genomic Instability in Circulating Tumor Cells

“…A number of studies have reported the presence of ALK-translocated CTCs in blood from NSCLC patients [ 80 , 147 , 148 ]. Provencio et al, 2017, used CTCs for the dynamic monitoring of NSCLC patients with ALK rearrangement, as an example [ 80 ].…”

“…Provencio et al, 2017, used CTCs for the dynamic monitoring of NSCLC patients with ALK rearrangement, as an example [ 80 ]. Recently, Pailler et al (2019) demonstrated that many genes involved in the RTK- KRAS and TP53 pathways were found in patients with crizotinib resistance, showing the clinical utility of CTCs to identify therapeutic resistance mutations in ALK-rearranged patients and for treatment decisions [ 148 ].…”