Multiple Acquired Resistance Mutations of the ALK Tyrosine Kinase Domain after Sequential Use of ALK Inhibitors

Currently, it is reasonable that for oncogene-addicted cancer, including anaplastic lymphoma receptor tyrosine kinase gene (ALK)-rearranged NSCLC, targeted therapies are administered in a linear manner. Wide arrays of acquired ALK mutations offer differential sensitivities to the various newly developed second-generation anaplastic lymphoma kinase (ALK) inhibitors.1Heuckmann J.M. Holzel M. Sos M.L. et al.ALK mutations conferring differential resistance to structurally diverse ALK inhibitors.Clin Cancer Res. 2011; 17: 7394-7401Crossref PubMed Scopus (157) Google Scholar, 2Sullivan I. Planchard D. Treatment modalities for advanced ALK-rearranged non-small-cell lung cancer.Future Oncol. 2016; 12: 945-961Crossref PubMed Scopus (15) Google Scholar Choosing different ALK inhibitors might result in the emergence of new resistance mutations; however, the evolution process of tumor clones is not well studied. Herein, we report a patient with ALK-rearranged advanced NSCLC in whom multiple point mutations in the ALK kinase domain developed after treatment with sequential ALK inhibitors. We focused on the evolution of acquired resistance mutations in the ALK kinase domain. Stage IIIa NSCLC adenocarcinoma was diagnosed in a 48-year-old Asian man. Endobronchial biopsy revealed adenocarcinoma positive for thyroid transcription factor 1 and ALK (2+) on immunohistochemistry. He began receiving sequential ALK-targeted therapy in the order crizotinib, belizatinib, and ceritinib while his tumor progressed with brain, left adrenal gland, spinal, and spleen metastases. The patient received crizotinib, belizatinib, and ceritinib for 9, 4, and 6 months, respectively. The patient achieved partial response after initiation of all three of these ALK inhibitors. However, the disease inevitably progressed. The patient died 40 months after diagnosis of his cancer (Fig. 1). Genetic analysis of treatment-naive bronchoscopic biopsy specimens revealed no mutations in the ALK kinase domain. Reverse-transcriptase polymerase chain reaction of RNA extracted from malignant pleural effusion after disease progression despite crizotinib treatment, revealed echinoderm microtubule associated protein like 4 gene (EML4)-ALK fusion variant 1 and a single point mutation, L1196M, in the ALK kinase domain. Continuous genotyping of tumor cells isolated from pleural effusion after treatment with crizotinib and belizatinib revealed that the resistance panel had shifted to G1269A and F1174L double point mutations. The final pleural effusion analysis after disease progression despite ceritinib therapy but before death showed one more new point mutation, D1203N, together with the two previous G1269A and F1174L mutations. (Table 1).Table 1List of Acquired Resistance Mutations in Our PatientNucleotide AlterationAmino Acid MutationDomain in ALKALK Inhibitor SensitivityCrizotinibCeritinibAlectinib3586C→AL1196MGatekeeper mutationRSS3806G→CG1269A–1 to the DFG activation loopRSSaIn cell lines and in vivo models.3522C→GF1174LEnd of αC helixRRS3607G→AD1203NBetween β5 strand and αD helix?RaIn cell lines and in vivo models.?ALK, anaplastic lymphoma kinase; R, resistant; S, sensitive; DFG, Asp-Phe-Gly motif.a In cell lines and in vivo models. Open table in a new tab ALK, anaplastic lymphoma kinase; R, resistant; S, sensitive; DFG, Asp-Phe-Gly motif. We examined the sensitivity of PE-3314 cells carrying the three mutations that were derived from patient’s malignant pleural effusion by using methyl thiazolyl tetrazolium assay and confirmed their high-resistance nature (Fig. 2). Our patient received three different ALK inhibitors during treatment. The tumor developed multiple point mutations in the ALK kinase domain in a stepwise manner under selective pressure of sequential ALK inhibitors. Although an increasing number of targeted therapies have expanded the therapeutic options, rapid antineoplastic adaptation remains a treatment obstacle. The findings of our case demonstrate that tumor survival under targeted therapy is a cancer evolutionary process. Three basic components interplay dynamically in cancer evolution: mutation variability, selection, and genetic drift.3Lipinski K.A. Barber L.J. Davies M.N. Ashenden M. Sottoriva A. Gerlinger M. Cancer evolution and the limits of predictability in precision cancer medicine.Trends Cancer. 2016; 2: 49-63Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Genetic drift is a stochastic process and hard to predict. However, the heavy disease burden of our patient might have accelerated mutation generation. The sequential ALK inhibitors were the determining factors in Darwinian selection and governed cancer evolution in our patient. Therefore, understanding the evolution of resistance-driving cancer genotypes is crucial for precision medicine. The sequence of ALK inhibitors chosen might determine the surviving tumor clones. Gainor el al. recently reported that sequential ALK inhibitors may predispose a patient to development of compound mutations.4Gainor J.F. Dardaei L. Yoda S. et al.Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in ALK-rearranged lung cancer.Cancer Discov. 2016; 6: 1118-1133Crossref PubMed Scopus (758) Google Scholar The similar findings in our case indicate that compound mutations might be an increasingly common issue and exhibit high resistance. As we cannot predict the dynamic cancer evolutionary process, repeat tumor genetic analysis for ALK-rearranged NSCLC before alteration of treatment strategies is realistic and should be performed in current clinical scenarios. We thank Dr. Chao-Hua Chiou (Taipei Veterans General Hospital, Taipei, Republic of China) for providing the ALK TKI–sensitive cell line (A33/R10).