Acquired Resistance Mutations to ALK Inhibitors Identified by Single Circulating Tumor Cell Sequencing in <i>ALK</i>-Rearranged Non-Small-Cell Lung Cancer.

Abstract

Patients with anaplastic lymphoma kinase (<i>ALK</i>)-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 <i>ALK</i>-rearranged NSCLC. Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib (<i>n</i> = 14) or lorlatinib (<i>n</i> = 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 <i>ALK</i>-independent and <i>ALK</i>-dependent resistance mechanisms. Multiple mutations in various genes in ALK-independent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS (<i>EGFR, KRAS, BRAF</i> genes) and TP53 pathways were recurrently mutated. In one lorlatinib-resistant patient, two single CTCs out of 12 harbored <i>ALK</i> compound mutations. CTC-1 harbored the <i>ALK</i> ^{G1202R/F1174C} compound mutation virtually similar to <i>ALK</i> ^{G1202R/F1174L} present in the corresponding tumor biopsy. CTC-10 harbored a second <i>ALK</i> ^{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 copy-number alterations and whole-genome duplication. Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in <i>ALK</i>-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.