Purpose: Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary ALK mutations. Here, we investigated utility of plasma genotyping for identifying ALK resistance mutations at relapse on next-generation ALK TKIs. Experimental Design: We analyzed 106 plasma specimens from 84 patients with advanced ALK-positive lung cancer treated with second-and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify ALK mutations. Results: By genotyping plasma, we detected an ALK mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of ALK mutations (67% vs. 63%), but plasma specimens were more likely to harbor !2 ALK mutations (24% vs. 2%, P ¼ 0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an ALK mutation in 22 (76%), including 14 (48%) with !2 ALK mutations. The most frequent combinations of ALK mutations were G1202R/L1196M and D1203N/1171N. Detection of !2 ALK mutations was significantly more common in patients relapsing on lorlatinib compared with second-generation ALK TKIs (48% vs. 23%, P ¼ 0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that eight (53%) acquired !1 new ALK mutations on lorlatinib. Conclusions: ALK resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of ALK resistance mutations leading to treatment-refractory compound ALK mutations.

Background: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. Methods: We performed fluorescence in-situ hybridization and/or next-generation sequencing on 207 post-treatment tissue (n=101) or plasma (n=106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and two patients with MET-driven resistance. Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (p=0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition re-sensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may select for MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Driver mutations alter cells from normal to cancer through several evolutionary epochs: premalignancy, early malignancy, subclonal diversification, metastasis and resistance to therapy. Later stages of disease can be explored through analyzing multiple samples collected longitudinally, on or between successive treatments, and finally at time of autopsy. It is also possible to study earlier stages of cancer development through probabilistic reconstruction of developmental trajectories based on mutational information preserved in the genome. Here we present a suite of tools, called Phylogic N-Dimensional with Timing (PhylogicNDT), that statistically model phylogenetic and evolutionary trajectories based on mutation and copy-number data representing samples taken at single or multiple time points. PhylogicNDT can be used to infer: (i) the order of clonal driver events (including in pre-cancerous stages); (ii) subclonal populations of cells and their phylogenetic relationships; and (iii) cell population dynamics. We demonstrate the use of PhylogicNDT by applying it to whole-exome and whole-genome data of 498 lung adenocarcinoma samples (434 previously available and 64 of newly generated data). We identify significantly different progression trajectories across subtypes of lung adenocarcinoma (EGFR mutant, KRAS mutant, fusion-driven and EGFR/KRAS wild type cancers). In addition, we study the progression of fusiondriven lung cancer in 21 patients by analyzing samples from multiple timepoints during treatment with 1st and next generation tyrosine kinase inhibitors. We characterize their subclonal diversification, dynamics, selection, and changes in mutational signatures and neoantigen load. This methodology will enable a systematic study of tumour initiation, progression and resistance across cancer types and therapies.

Introduction:The current standard initial therapy for advanced ALK receptor tyrosine kinase (ALK)-positive NSCLC is a second-generation ALK tyrosine kinase inhibitor (TKI) such as alectinib. The optimal next-line therapy after failure of a second-generation ALK TKI remains to be established; however, standard options include the thirdgeneration ALK TKI lorlatinib or platinum/pemetrexedbased chemotherapy. The efficacy of platinum/ pemetrexed-based chemotherapy has not been evaluated in cases that are refractory to second-generation TKIs.Methods: This was a retrospective study performed at three institutions. Patients were eligible if they had advanced ALK-positive NSCLC refractory to one or more second-generation ALK TKI(s) and had received platinum/ pemetrexed-based chemotherapy.Results: Among 58 patients eligible for this study, 37 had scans evaluable for response with measurable disease at baseline. The confirmed objective response rate to platinum/pemetrexed-based chemotherapy was 29.7% (11 of 37 patients; 95% confidence interval [CI]: 15.9% -47.0%), with median duration of response of 6.4 months (95% CI: 1.6 monthsnot reached). The median progression-free survival for the entire cohort was 4.3 months (95% CI: 2.9 -5.8 months). Progression-free survival was longer in patients who received platinum/pemetrexed in combination with an ALK TKI compared to those who received platinum/pemetrexed alone (6.8 months vs. 3.2 months, respectively; hazard ratio ¼ 0.33; p ¼ 0.025).Conclusions: Platinum/pemetrexed-based chemotherapy shows modest efficacy in ALK-positive NSCLC after failure of *Corresponding author.

Introduction: Circulating tumor DNA analysis is an emerging genotyping strategy that can identify tumorspecific genetic alterations in plasma including mutations and rearrangements. Detection of ROS1 fusions in plasma requires genotyping approaches that cover multiple breakpoints and target a variety of fusion partners. Compared to other molecular subsets of NSCLC, experience with detecting ROS1 genetic alterations in plasma is limited. Methods: To describe the spectrum of ROS1 fusions in NSCLC and determine sensitivity for detecting ROS1 fusions in plasma, we queried the Guardant Health plasma dataset and an institutional tissue database and compared plasma findings to tissue results. In addition, we used the Guar-dant360 NGS assay to detect potential genetic mediators of resistance in plasma from patients with ROS1-positive NSCLC who were relapsing on crizotinib. Results: We detected seven distinct fusion partners in plasma, most of which (n ¼ 6 of 7) were also represented in the tissue dataset. Fusions pairing CD74 with ROS1 predominated in both cohorts (plasma: n ¼ 35 of 56, 63%; tissue: n ¼ 26 of 52, 50%). There was 100% concordance between the specific tissue-and plasmadetected ROS1 fusion for seven patients genotyped with both methods. Sensitivity for detecting ROS1 fusions in plasma at relapse on ROS1-directed therapy was 50%. Six (33%) of 18 post-crizotinib plasma specimens harbored ROS1 kinase domain mutations, five of which were ROS1 G2032R. Two (11%) post-crizotinib plasma specimens had genetic alterations (n ¼ 1 each BRAF V600E and PIK3CA E545K) potentially associated with ROS1independent signaling. Conclusions: Plasma genotyping captures the spectrum of ROS1 fusions observed in tissue. Plasma genotyping is a promising approach to detecting mutations that drive resistance to ROS1-directed therapies.