Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer

Abstract

Introduction: Despite initial effectiveness of ALK receptor tyrosine kinase inhibitors (TKIs) in patients with ALK+ NSCLC, therapeutic resistance will ultimately develop. Serial tracking of genetic alterations detected in circulating tumor DNA (ctDNA) can be an informative strategy to identify response and resistance. This study evaluated the utility of analyzing ctDNA as a function of response to ensartinib, a potent second-generation ALK TKI.

Methods: Pre-treatment plasma was collected from 76 patients with ALK+ NSCLC who were ALK TKI-naive or had received prior ALK TKI, and analyzed for specific genetic alterations. Longitudinal plasma samples were analyzed from a subset (n = 11) of patients. Analysis of pre-treatment tumor biopsy specimens from 22 patients was compared with plasma.

Results: Disease-associated genetic alterations were detected in 74% (56 of 76) of patients, the most common being EML4-ALK. Concordance of ALK fusion between plasma and tissue was 91% (20 of 22 blood and tissue samples). Twenty-four ALK kinase domain mutations were detected in 15 patients, all had previously received an ALK TKI; G1269A was the most prevalent (4 of 24). Patients with a detectable EML4-ALK variant 1 (V1) fusion had improved response (9 of 17 patients; 53%) to ensartinib compared to patients with EML4-ALK V3 fusion (one of seven patients; 14%). Serial changes in ALK alterations were observed during therapy.

Conclusions: Clinical utility of ctDNA was shown, both at pre-treatment by identifying a potential subgroup of ALK+ NSCLC patients who may derive more benefit from ensartinib and longitudinally by tracking resistance. Prospective application of this technology may translate to improved outcomes for NSCLC patients treated with ALK TKIs.

Keywords: ALK receptor tyrosine kinase; Circulating tumor DNA; Liquid biopsies; NSCLC; Next-generation sequencing.

Conflict of interest statement

Conflict of Interests: All other authors do not have any conflicts to disclose.

Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Detection of molecular alterations in plasma from patients with ALK+ NSCLC enrolled in the eXALT2 trial.

(A) Summary of all mutations identified by individual patient at the beginning of treatment with ensartinib. Alterations are color-coded per the figure legend below the image. The red or brown line around a particular mutation (or lack thereof) means that the mutation is either confirmed or not confirmed in the tissue, respectively. The mutation frequencies for each gene are graphed in the right panel, with the denominator equal to total number of patients (i.e., 76). Boxes with yellow and green alternating strips represent a gene for which both an SNV (yellow) and an indel (green) were detected. KRAS, NRAS, NTRK1, RET, and ROS1 were included in the NGS panel; however, no alterations in these genes were detected. ALK, anaplastic lymphoma kinase gene; MET, mesenchymal-epithelial transition gene; MAP2K, mitogen-activated protein kinase 2 gene; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene; TP53, tumor protein p53 gene. (B) Frequency of various ALK fusions detected in the plasma at the time of study entrance. “EML4-ALK Other” includes three patients with EML4-ALK fusions that had breakpoints in regions that could not be mapped to a specific variant. An ALK fusion of any kind was not detected in six patients. Note: frequencies in the bar graphs are expressed based upon the total number of patients (n=76). (C) Frequency and distribution of ALK kinase domain mutations detected across the study population. Orange bars represent samples from patients who had received prior crizotinib. Blue bars represent samples from patients who had received crizotinib and at least one second-generation ALK inhibitor. Frequencies are expressed based upon the total number of patients that received post-crizotinib (n=37) or received post-crizotinib and a second-generation ALK inhibitor (n=23).

Figure 2.. Longitudinal assessment of molecular alterations detected in plasma during ensartinib treatment.

Graphs illustrate the change in allelic fraction of ALK and TP53 alterations for patient #34 (A), patient #74 (B), and patient #39 (C) during treatment with ensartinib. The time of radiographic response and progression are annotated below the x-axis. An x-axis point with a missing marker designates that plasma was not analyzed at that time point. The duration of each treatment cycle is 28 days.

Figure 3.. Efficacy of ensartinib against ALK kinase domain mutations.

BA/F3 cells expressing the indicated ALK kinase domain mutations within the context of EML4-ALK variant 1 (A) or EML4-ALK variant 3 (B) were treated with increasing concentrations of crizotinib, ceritinib, ensartinib, alectinib, brigatinib, and lorlatinib (0–10 µmol/L) for 72 hours. Cell titer blue assays were performed to assess cell viability. Experiments were performed with six replicates per drug concentration and repeated three times. IC50 values with 95% confidence intervals were generated with the data from the dose response curves using GraphPad Prism 7. All variants were tested three independent times, except for V1 E1210K, V3 D1203N, and V3 E1210K, which were tested two independent times.