De novo ALK kinase domain mutations are uncommon in kinase inhibitor-naïve ALK rearranged lung cancers

Abstract

Introduction: Anaplastic lymphoma kinase (ALK) rearranged lung adenocarcinomas are responsive to the multitargeted ALK inhibitor crizotinib. One of the common mechanisms of resistance to crizotinib is the acquisition of ALK kinase domain mutations. However, the presence of ALK mutations in crizotinib-naïve tumors has not been widely reported and it is unclear if de novo ALK mutations affect the response to crizotinib.

Methods: We analyzed preclinical models of ALK rearranged lung cancers that were sensitive/resistant to ALK inhibitors, probed our institutional and other lung cancer databases for tumors with ALK kinase domain mutations, and evaluated tumor response to crizotinib.

Results: ALK rearranged cell lines with ALK kinase domain mutations were heterogeneously less inhibited by increasing concentrations of crizotinib than cells driven solely by EML4-ALK fusions. Previous ALK rearranged lung cancer cohorts did not report ALK kinase mutations in inhibitor-naïve tumors. We identified one TKI-naïve ALK rearranged tumor with an ALK kinase domain mutation: ALK-S1206F (mutations at ALK-S1206 shifted crizotinib inhibitory curves only minimally in preclinical models). The never smoker whose tumor harbored de novo EML4-ALK-E5;A20+ALK-S1206F only achieved a 4-month radiographic response to crizotinib 250mg twice daily.

Conclusions: Combining data from our and prior cohorts, ALK kinase domain mutations were uncommon events (<3% of cases) in ALK inhibitor-naïve ALK rearranged lung adenocarcinomas but their effect on intrinsic resistance to ALK inhibitors should be better evaluated.

Keywords: ALK; Adenocarcinoma; Crizotinib; Kinase domain; Lung cancer; Mutation.

Figure 1. Preclinical models of acquired resistance to crizotinib in ALK rearranged lung cancers

A. Dose-inhibition curves for crizotinib using H3122, H3122 CR_A and H3122 CR1, with 50% inhibitory concentration (IC₅₀) using nanomolar (nM) concentrations indicated. B. Western blot results showing the intracellular signaling effects of crizotinib 1000nM and certinib 100nM after 6 hours of exposure to H3122 cells, with inhibition of phosphorylated (p) levels of each protein indicating drug activity. The same intracellular signaling is shown for H3122 CR_A and H3122 CR1 cells grown in the presence of continuous 1000nM of crizotinib. C. Dose-inhibition curves for ceritinib using H3122, H3122 CR_A and H3122 CR1, with IC₅₀ concentrations indicated. D. Dose-inhibition curves for ceritinib in the presence of afatinib 100nM using H3122, H3122 CR_A and H3122 CR1, with IC₅₀ concentrations indicated.

Figure 2. ALK kinase domain mutations in different NSCLC cohorts

A. Frequencies of ALK genomic changes and graphic representation of the ALK protein with ALK mutations identified in the TCGA 2014 lung adenocarcinoma cohort indicated. B. ALK rearrangements and ALK kinase domain mutations identified in the BIDMC NSCLC tumor-pair cohort. C. Tabulated ALK rearranged NSCLC cohort and percentage (%) of ALK kinase domain mutations in TKI-naïve and crizotinib-resistant biopsies. References to the original publication of the cohort are indicated in parenthesis. * We assumed that crizotinib-resistant samples with wild-type (WT) ALK kinase domain would also have lacked a mutation in the TKI-naïve setting and we excluded cases that had ALK kinase mutations in the crizotinib-resistant setting but were not analyzed in the TKI-naïve biopsy.

Figure 3. ALK kinase domain mutants and EML4-ALK+ALK-S1206F

A. Crizotinib 50% inhibitory concentration (IC₅₀) fold changes induced by different ALK kinase mutants in the background of EML4-ALK-E13;A20, as reported by Friboulet et al (reference 10), tabulated with possible clinical response to achievable concentrations of crizotinib 250mg twice daily. B. Computed tomography (CT) to exemplify response to crizotinib in the patient whose lung adenocarcinoma harbored de novo EML4-ALK-E5;A20+ALK-S1206F. Shown are pre-crizotinib (baseline), month 1 and month 2 of crizotinib 250mg twice daily representative images. The white arrows highlight sites of tumor burden in the thorax and liver.