Case Report
Secondary Mutations at I1171 in the ALK Gene Confer Resistance to Both Crizotinib and Alectinib Gouji Toyokawa, MD, PhD, Fumihiko Hirai, MD, PhD, Eiko Inamasu, MD, Tsukihisa Yoshida, MD, Kaname Nosaki, MD, Tomoyoshi Takenaka, MD, PhD, Masafumi Yamaguchi, MD, PhD, Takashi Seto, MD, PhD, Mitsuhiro Takenoyama, MD, PhD, and Yukito Ichinose, MD, PhD
T
he first case was a 27-year-old female nonsmoker who was diagnosed with advanced adenocarcinoma with bone and brain metastases. A FISH study of a biopsy sample of the left clavicle detected ALK rearrangement, and the patient was confirmed to harbor variant 2 of the EML4-ALK fusion gene by reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing (Figure 1A). After the failure of first-line chemotherapy with cisplatin plus pemetrexed (Figure 1B), the patient was enrolled in a phase III study comparing crizotinib with standard chemotherapy in patients with ALK-rearranged non-small-cell lung cancer (NSCLC), and was then randomized to crizotinib. Although the patient experienced a partial response (Figure 1C), a relapse of brain metastases was observed approximately 8 months after the administration of crizotinib, and stereotactic radiotherapy was administered. The patient continued to be treated with crizotinib beyond the progression of the brain metastases, and pleural effusion became evident 1 year and 6 months after the first administration of crizotinib, and gradually expanded (Figure 1D). A rebiopsy of the pleural effusion showed a previously unidentified secondary mutation of the ALK gene at codon 1171 (I1171T; Figure 1E). The second case was a 49-year-old female nonsmoker with locally advanced NSCLC, which was first treated with chemoradiation using cisplatin plus vinorelbine (radiation dose: 60 Gy in total). Approximately 7 months after the start of chemoradiation, positron emission tomography/computed tomography (PET/CT) showed multiple skin, hepatic and bone metastases, and variant 3 of the EML4-ALK fusion was identified by fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and RT-PCR of a biopsied skin lesion (Figure 2A). Second-line chemotherapy with pemetrexed led to a partial response, but the patient experienced disease failure after eight cycles (Figure 2B). Thereafter, the patient was enrolled in a phase I study investigating alectinib, and the agent induced tumor reduction in the primary lesion Department of Thoracic Oncology, National Kyushu Cancer Center, Fukuoka, Japan Disclosure: The authors declare no conflict of interest. Address for correspondence: Gouji Toyokawa, Department of Thoracic Oncology, National Kyushu Cancer Center, 3-1-1 Notame, Minami-ku, Fukuoka 811-1395, Japan. E-mail: [email protected] DOI: 10.1097/JTO.0000000000000358 Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/14/0912-e86
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and hepatic metastases, as shown in Figure 2C. However, a relapse of the primary lesion and hepatic metastases was seen approximately 7 months after the administration of alectinib (Figure 2D), and a rebiopsy of the hepatic lesion showed a secondary mutation of the ALK gene at codon 1171 (I1171N; Figure 2E). In both cases, bypass track resistance through the activation of an alternative driver oncogene, such as EGFR or KRAS, was not identified. Various mechanisms which confer resistance to crizotinib have been identified, including secondary mutations,
FIGURE 1. Direct sequencing by capillary electrophoresis of a biopsy sample obtained from the left clavicle before crizotinib treatment using an ABI PRISM 310 Genetic Analyzer (A). Computed tomography scans showed metastatic mediastinal lymph nodes before (B) and approximately 7 months after the administration of crizotinib (C). There was expansion of the pleural effusion with no relapse of the mediastinal lymph nodes (D). Direct sequencing of a biopsy sample of pleural effusion after a relapse on crizotinib identified a secondary mutation of the ALK gene at codon 1171 (I1171T) (E).
Journal of Thoracic Oncology ® • Volume 9, Number 12, December 2014
Journal of Thoracic Oncology ® • Volume 9, Number 12, December 2014
Case report
FIGURE 2. Direct sequencing by capillary electrophoresis of a biopsy sample obtained from the skin metastasis before the administration of alectinib using an ABI PRISM 310 Genetic Analyzer (A). Computed tomography scans showing the primary lesion in the right lower lobe and hepatic metastases before (B) and approximately 4 months after (C) the administration of alectinib. A computed tomography scan indicating the relapse of the primary lesion and hepatic metastases approximately 7 months after the administration of alectinib (D). Direct sequencing of a biopsy sample obtained from the hepatic metastasis after a relapse on alectinib showing a secondary mutation of the ALK gene at codon 1171 (I1171N) (E).
a copy number gain of the ALK gene, the activation of other oncogenes and so on.1 With regard to the secondary mutations of the ALK gene, different mutations have been observed to confer resistance to crizotinib and second-generation ALK inhibitors, such as ceritinib and alectinib (i.e., those conferring resistance to crizotinib include 1152Tins, L1152R, C1156Y, F1174C/L, L1196M, G1202R, D1203N, S1206Y, and G1269A; G1202R and F1174C/V confer resistance to ceritinib; G1202R confers resistance to alectinib, whereas mutations at codon 1174 do not confer resistance to alectinib).1–6 Although an in vitro mutagenesis screen identified I1171T in the ALK gene, mutations at codon 1171, which is located in the vicinity of the kinase DFG (Asp-PheGly) motif of the activation loop, have yet to be identified in patients with ALK-rearranged NSCLC who have acquired resistance to ALK inhibitors.7 In addition, in vitro analyses by Friboulet et al2 and Ceccon et al8 showed that I1171T and I1171N confer resistance to crizotinib. In addition to the novel finding of mutations at I1171 in ALK-rearranged patients, it is intriguing that mutations at I1171 confer resistance to both crizotinib and alectinib, which is a representative second-generation ALK inhibitor. It remains unclear why these mutations confer resistance to both of these agents; however, further basic and clinical analyses are warranted to investigate the biological and clinical significance of mutations at I1171.
Acknowledgments The authors thank Brian Quinn for providing critical comments on the manuscript. REFERENCES 1. Camidge DR, Doebele RC. Treating ALK-positive lung cancer–early successes and future challenges. Nat Rev Clin Oncol 2012;9:268–277. 2. Friboulet L, Li N, Katayama R, et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov 2014;4:662–673. 3. Choi YL, Soda M, Yamashita Y, et al; ALK Lung Cancer Study Group. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010;363:1734–1739. 4. Ignatius Ou SH, Azada M, Hsiang DJ, et al. Next-generation sequencing reveals a Novel NSCLC ALK F1174V mutation and confirms ALK G1202R mutation confers high-level resistance to alectinib (CH5424802/ RO5424802) in ALK-rearranged NSCLC patients who progressed on crizotinib. J Thorac Oncol 2014;9:549–553. 5. Kodama T, Tsukaguchi T, Yoshida M, et al. Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance. Cancer Lett 2014;351:215–221. 6. Sakamoto H, Tsukaguchi T, Hiroshima S, et al. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell 2011;19:679–690. 7. Zhang S, Wang F, Keats J, et al. Crizotinib-resistant mutants of EML4ALK identified through an accelerated mutagenesis screen. Chem Biol Drug Des 2011;78:999–1005. 8. Ceccon M, Mologni L, Bisson W, Scapozza L, Gambacorti-Passerini C. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors. Mol Cancer Res 2013;11:122–132.
Copyright © 2014 by the International Association for the Study of Lung Cancer
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Secondary Mutations at I1171 in the ALK Gene Confer Resistance to Both Crizotinib and Alectinib Gouji Toyokawa, MD, PhD, Fumihiko Hirai, MD, PhD, Eiko Inamasu, MD, Tsukihisa Yoshida, MD, Kaname Nosaki, MD, Tomoyoshi Takenaka, MD, PhD, Masafumi Yamaguchi, MD, PhD, Takashi Seto, MD, PhD, Mitsuhiro Takenoyama, MD, PhD, and Yukito Ichinose, MD, PhD
T
he first case was a 27-year-old female nonsmoker who was diagnosed with advanced adenocarcinoma with bone and brain metastases. A FISH study of a biopsy sample of the left clavicle detected ALK rearrangement, and the patient was confirmed to harbor variant 2 of the EML4-ALK fusion gene by reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing (Figure 1A). After the failure of first-line chemotherapy with cisplatin plus pemetrexed (Figure 1B), the patient was enrolled in a phase III study comparing crizotinib with standard chemotherapy in patients with ALK-rearranged non-small-cell lung cancer (NSCLC), and was then randomized to crizotinib. Although the patient experienced a partial response (Figure 1C), a relapse of brain metastases was observed approximately 8 months after the administration of crizotinib, and stereotactic radiotherapy was administered. The patient continued to be treated with crizotinib beyond the progression of the brain metastases, and pleural effusion became evident 1 year and 6 months after the first administration of crizotinib, and gradually expanded (Figure 1D). A rebiopsy of the pleural effusion showed a previously unidentified secondary mutation of the ALK gene at codon 1171 (I1171T; Figure 1E). The second case was a 49-year-old female nonsmoker with locally advanced NSCLC, which was first treated with chemoradiation using cisplatin plus vinorelbine (radiation dose: 60 Gy in total). Approximately 7 months after the start of chemoradiation, positron emission tomography/computed tomography (PET/CT) showed multiple skin, hepatic and bone metastases, and variant 3 of the EML4-ALK fusion was identified by fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and RT-PCR of a biopsied skin lesion (Figure 2A). Second-line chemotherapy with pemetrexed led to a partial response, but the patient experienced disease failure after eight cycles (Figure 2B). Thereafter, the patient was enrolled in a phase I study investigating alectinib, and the agent induced tumor reduction in the primary lesion Department of Thoracic Oncology, National Kyushu Cancer Center, Fukuoka, Japan Disclosure: The authors declare no conflict of interest. Address for correspondence: Gouji Toyokawa, Department of Thoracic Oncology, National Kyushu Cancer Center, 3-1-1 Notame, Minami-ku, Fukuoka 811-1395, Japan. E-mail: [email protected] DOI: 10.1097/JTO.0000000000000358 Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/14/0912-e86
e86
and hepatic metastases, as shown in Figure 2C. However, a relapse of the primary lesion and hepatic metastases was seen approximately 7 months after the administration of alectinib (Figure 2D), and a rebiopsy of the hepatic lesion showed a secondary mutation of the ALK gene at codon 1171 (I1171N; Figure 2E). In both cases, bypass track resistance through the activation of an alternative driver oncogene, such as EGFR or KRAS, was not identified. Various mechanisms which confer resistance to crizotinib have been identified, including secondary mutations,
FIGURE 1. Direct sequencing by capillary electrophoresis of a biopsy sample obtained from the left clavicle before crizotinib treatment using an ABI PRISM 310 Genetic Analyzer (A). Computed tomography scans showed metastatic mediastinal lymph nodes before (B) and approximately 7 months after the administration of crizotinib (C). There was expansion of the pleural effusion with no relapse of the mediastinal lymph nodes (D). Direct sequencing of a biopsy sample of pleural effusion after a relapse on crizotinib identified a secondary mutation of the ALK gene at codon 1171 (I1171T) (E).
Journal of Thoracic Oncology ® • Volume 9, Number 12, December 2014
Journal of Thoracic Oncology ® • Volume 9, Number 12, December 2014
Case report
FIGURE 2. Direct sequencing by capillary electrophoresis of a biopsy sample obtained from the skin metastasis before the administration of alectinib using an ABI PRISM 310 Genetic Analyzer (A). Computed tomography scans showing the primary lesion in the right lower lobe and hepatic metastases before (B) and approximately 4 months after (C) the administration of alectinib. A computed tomography scan indicating the relapse of the primary lesion and hepatic metastases approximately 7 months after the administration of alectinib (D). Direct sequencing of a biopsy sample obtained from the hepatic metastasis after a relapse on alectinib showing a secondary mutation of the ALK gene at codon 1171 (I1171N) (E).
a copy number gain of the ALK gene, the activation of other oncogenes and so on.1 With regard to the secondary mutations of the ALK gene, different mutations have been observed to confer resistance to crizotinib and second-generation ALK inhibitors, such as ceritinib and alectinib (i.e., those conferring resistance to crizotinib include 1152Tins, L1152R, C1156Y, F1174C/L, L1196M, G1202R, D1203N, S1206Y, and G1269A; G1202R and F1174C/V confer resistance to ceritinib; G1202R confers resistance to alectinib, whereas mutations at codon 1174 do not confer resistance to alectinib).1–6 Although an in vitro mutagenesis screen identified I1171T in the ALK gene, mutations at codon 1171, which is located in the vicinity of the kinase DFG (Asp-PheGly) motif of the activation loop, have yet to be identified in patients with ALK-rearranged NSCLC who have acquired resistance to ALK inhibitors.7 In addition, in vitro analyses by Friboulet et al2 and Ceccon et al8 showed that I1171T and I1171N confer resistance to crizotinib. In addition to the novel finding of mutations at I1171 in ALK-rearranged patients, it is intriguing that mutations at I1171 confer resistance to both crizotinib and alectinib, which is a representative second-generation ALK inhibitor. It remains unclear why these mutations confer resistance to both of these agents; however, further basic and clinical analyses are warranted to investigate the biological and clinical significance of mutations at I1171.
Acknowledgments The authors thank Brian Quinn for providing critical comments on the manuscript. REFERENCES 1. Camidge DR, Doebele RC. Treating ALK-positive lung cancer–early successes and future challenges. Nat Rev Clin Oncol 2012;9:268–277. 2. Friboulet L, Li N, Katayama R, et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov 2014;4:662–673. 3. Choi YL, Soda M, Yamashita Y, et al; ALK Lung Cancer Study Group. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010;363:1734–1739. 4. Ignatius Ou SH, Azada M, Hsiang DJ, et al. Next-generation sequencing reveals a Novel NSCLC ALK F1174V mutation and confirms ALK G1202R mutation confers high-level resistance to alectinib (CH5424802/ RO5424802) in ALK-rearranged NSCLC patients who progressed on crizotinib. J Thorac Oncol 2014;9:549–553. 5. Kodama T, Tsukaguchi T, Yoshida M, et al. Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance. Cancer Lett 2014;351:215–221. 6. Sakamoto H, Tsukaguchi T, Hiroshima S, et al. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell 2011;19:679–690. 7. Zhang S, Wang F, Keats J, et al. Crizotinib-resistant mutants of EML4ALK identified through an accelerated mutagenesis screen. Chem Biol Drug Des 2011;78:999–1005. 8. Ceccon M, Mologni L, Bisson W, Scapozza L, Gambacorti-Passerini C. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors. Mol Cancer Res 2013;11:122–132.
Copyright © 2014 by the International Association for the Study of Lung Cancer
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