Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Choi YL, Soda M, Yamashita Y, et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 21;363:1734-9.
Choi et al. 1 Supplementary Methods DNA sequencing Oligo(dT)-primed cdnas were generated from specimen RNAs extracted with the use of the EZ1 system (Qiagen) and were subjected to PCR with the primers ALK-TK-F (5 -TACAACCCCAACTACTGCTTTGCT-3 ) and ALK-TK-R1 (5 -AGGCACTTTCTCTTCCTCTTCCAC-3 ) (see Figure 2 in Supplementary Appendix). The PCR products corresponding to the kinase domain of ALK were then fragmented, and sequenced with an Illumina Genome Analyzer II (GAII) for 76 bases from both ends by the multiplexed paired-end sequencing system (Illumina). Raw read data were quality-filtered on the basis of the presence of the PCR primer sequences and a Q value of 2 for all bases. The filter-passed reads were then aligned to the ALK cdna sequence with the use of the Bowtie algorithm (http://bowtie-bio.sourceforge.net/index.shtml). For capillary sequencing with a 313xl Genetic Analyzer (Applied Biosystems), PCR products were prepared from cdnas with the combination of the EA-F-g-S (5 -CCACACCTGGGAAAGGACCTAAAG-3 ) and ALK-TK-R2 (5 -CCTCCAAATACTGACAGCCACAGG-3 ) primers (see Figure 2 in Supplementary Appendix). The described studies were approved by the ethics committees of Jichi Medical University and Osaka General Medical Center, and written informed consent was obtained from the enrolled subjects. Properties of mutant EML4-ALK A cdna encoding FLAG epitope tagged EML4-ALK variant 1 was inserted into the pmx-irescd8 retroviral vector 1 for simultaneous expression of FLAG-tagged EML4-ALK and mouse CD8. Nucleotide changes corresponding to the C1156Y or L1196M mutations of ALK were introduced into the plasmid individually for expression of
Choi et al. 2 EML4-ALK(C1156Y) or EML4-ALK(L1196M), respectively. Recombinant retroviruses based on these plasmids were generated as described previously 1 and were used to infect the mouse interleukin-3 dependent cell line BA/F3 (RIKEN Bio Resource Center). The resulting CD8-positive cells were purified with the use of a minimacs cell separation column and magnetic beads conjugated with antibodies to CD8 (both from Miltenyi Biotec). Crizotinib was obtained from Selleck, and another ALK-inhibitor, a 2,4-pyrimidinediamine derivative (PDD), was synthesized by Astellas Pharma. For examination of the tyrosine phosphorylation of EML4-ALK, BA/F3 cells expressing the fusion protein were exposed to ALK inhibitors for 15 h, after which EML4-ALK was immunoprecipitated from cell lysates with antibodies to FLAG (Eastman Kodak) and subjected to immunoblot analysis with antibodies to Tyr 164 -phosphorylated ALK (Cell Signaling Technology). An in vitro kinase assay was performed at room temperature for 3 min as described previously 2 with the synthetic YFF peptide (Operon Biotechnologies). References 1. Yamashita Y, Kajigaya S, Yoshida K, et al. Sak serine/threonine kinase acts as an effector of Tec tyrosine kinase. J Biol Chem 21;276:3912-2. 2. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 27;448:561-6.
Choi et al. 3 Supplementary Figure Legends Figure 1. Tumor growth during the treatment with crizotinib. Contrast-enhanced computed tomography was performed on the patient before as well as 1 and 6 months after the onset of crizotinib treatment. The image obtained before the treatment (top panel) revealed large tumor nodules at the right hilum (indicated by the broken white circle) with multiple enlarged lymph nodes in the mediastinum, atelectasis in the right lung, and a massive effusion in the right pleural cavity. R, right; L, left. After 1 month of treatment with crizotinib, however, a marked reduction in the size both of the tumors and of the enlarged lymph nodes was apparent (middle panel). Crizotinib did not completely eradicate the pleural effusion during the treatment course. After 6 months of treatment, the hilar tumors had regrown with a concomitant expansion of the pleural effusion in both lungs as well as an increase in the size of the mediastinal lymph nodes (bottom two panels). The patient received an esophageal stent to relieve swallowing difficulty on 2 March 29. Figure 2. Positions of PCR primers. A schematic representation of the EML4-ALK protein is shown with the positions of two de novo mutations in the kinase domain indicated below. The positions of PCR primers for amplification of kinase-domain or fusion cdnas are indicated by the closed and open arrows, respectively. Figure 3. Deep sequencing of ALK kinase-domain cdnas. PCR products of ~1 bp from the EML4-ALK positive NSCLC cell line H2228 or from specimen IDs J-#1, J-#12, J-#113, J-#127, or LK-#33 (all positive for EML4-ALK) were sequenced with the GAII and multiplexed paired-end sequencing system. Characteristics of
Choi et al. 4 these specimens are described in Table 2 in Supplementary Appendix. Totals of 17,149, 18,724, 5,982, 76,27, 176,588, and 46,596 filter-passed reads were obtained for the H2228, J-#1, J-#12, J-#113, J-#127, and LK-#33 samples, respectively. The numbers for total reads (Total) and mismatched reads (Mismatch) are shown at each position of the kinase-domain cdnas with blue and red diamonds, respectively. Insets show magnified views for the 5 region of the cdnas for J-#1 and J-#113 (depicted by green rectangles). Figure 4. Genomic sequences surrounding the positions corresponding to G4374 and C4493 of ALK cdna. Genomic DNA isolated from cells in the pleural effusion of the patient was subjected to PCR with the primers 5 -GGTAAGAAGTGGCTCACTCTTGAG-3 and 5 -CACAACAACTGCAGCAAAGACTGG-3, and the products were ligated into the pt7blue-2 plasmid (Takara Bio). Inserts of the plasmids were then sequenced with the 313xl Genetic Analyzer, resulting in the identification of the G4374A change in 9 of the 32 PCR clones examined (left panel) and the C4493A change in 2 of the 32 clones (right panel). Substituted A nucleotides are shown in red. Figure 5. BA/F3 cells treated with crizotinib. BA/F3 cells expressing primary EML4-ALK, EML4-ALK(C1156Y), or EML4-ALK(L1196M) were incubated in the presence of the indicated concentrations of crizotinib for 48 h, after which cell morphology was examined by phase-contrast microscopy. Scale bar, 2 µm. Figure 6. BA/F3 cells treated with PDD. BA/F3 cells expressing primary EML4-ALK, EML4-ALK(C1156Y), or
Choi et al. 5 EML4-ALK(L1196M) were incubated in the presence of the indicated concentrations of PDD for 48 h, after which cell morphology was examined by phase-contrast microscopy. Scale bar, 2 µm. Figure 7. Alignment of ALK amino acid sequence with those of related tyrosine kinases. The amino acid sequence of human ALK (residues 115 to 12) is aligned with those of the corresponding regions of human INSR, human EGFR, human ABL1, human KIT, and human PDGFRA. Residue numbers are shown at the left. Regions corresponding to predicted α-helix (αc) and β-sheet (β4 and β5) structures are indicated. Amino acids corresponding to C1156 or L1196 of ALK are highlighted in pink or green, respectively.
Supplementary Table 1. Detection ratios of JAK3(V674A) cdna at various sequencing depths. Input ratio of Coverage mutant cdna 1 2 1 3 1 4 1-1 8.64 1-2 8.66 1-2 8.66 1-2 1-2 8.19 1-3 8.24 1-3 8.25 1-3 1-3 8.35 1-4 8.41 1-4 8.39 1-4 1-4 1.21 1-4 1.22 1-4 1.18 1-4 1-5 4.6 1-5 3.76 1-5 4.6 1-5 6.5 1-5 5.86 1-5 5.74 1-5 Human JAK3(V674A) mutant cdna was mixed with the wild-type JAK3 cdna at the ratios indicated in the left column. Each mixture was then subjected to deep sequencing with the GAII system. The mean value for the observed detection ratio of the mutant cdna reads among total reads at the mutation site was calculated with permutation tests (n = 1,) at each coverage level.
Supplementary Table 2. Characteristics of the patients with NSCLC from whom specimens were derived. Sample ID Age (years) Sex Specimen Pre- or posttreatment Pathologic subtype EML4-ALK variant* J-#1 28 M Sputum Pre Adenocarcinoma E13;A2 J-#12 56 F Primary tumor Pre Adenocarcinoma E13;A2 J-#113 28 M Pleural effusion Post Adenocarcinoma E13;A2 J-#127 49 F TBB Pre Adenocarcinoma E6;A2 LK-#33 62 M Primary tumor Pre Adenocarcinoma E13;A2 *EML4-ALK variants are denoted according to our novel nomenclature system (see http://atlasgeneticsoncology.org/tumors/inv2p21p23nscclungid5667.html). These two specimens were obtained from the subject of the present study. TBB, transbronchial tumor biopsy.
R L R L R L R L Before the crizotinib treatment (November 25, 28) After one month of the treatment (December 3, 28) After six months of the treatment (May 25, 29) Supplmentary Figure 1 Choi et al.
EML4-ALK EML4 EA-F-g-S ALK-TK-F ALK ALK-TK-R2 ALK-TK-R1 Kinase C1156Y L1196M Supplementary Figure 2 Choi et al.
Total Mismatch 8 4 T423C rs379585 5 1 15 3 15 G4374A C4493A 1 2 3 2 H2228 3 J-#1 12 J-#113 1 15 6 5 1 5 1 5 1 6 4 2 LK-#33 8 4 J-#12 2 1 J-#127 5 1 5 1 5 1 Supplementary Figure 3 Choi et al.
G4374A C4493A T G T A C T C C T G A T G G Supplmentary Figure 4 Choi et al.
Crizotinib (nm) 5 1 Primary C1156Y L1196M 2 µm Supplementary Figure 5 Choi et al.
PDD (nm) 5 1 Primary C1156Y L1196M 2 µm Supplementary Figure 6 Choi et al.
αc β4 β5 ALK INSR EGFR ABL1 KIT PDGFRA 115 KTLPEVCSEQDELDFLMEALIISKFN-HQNIVRCIGVSLQSLPRFILLELMA 157 KTVNESASLRERIEFLNEASVMKGFT-CHHVVRLLGVVSKGQPTLVVMELMA 745 KELREATSPKANKEILDEAYVMASVD-NPHVCRLLGICLTSTVQL-ITQLMP 271 KTLKEDTMEVEE--FLKEAAVMKEIK-HPNLVQLLGVCTREPPFYIITEFMT 623 KMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCC 627 KMLKPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIITEYCF Supplementary Figure 7 Choi et al.