Supplementary Table S1: Primers for DNA sequencing (A), quantification of relative mrna expression (B), and copy number analysis (C) A. DNA sequencing (1-4) Genes Primers Sequence EGFR exon 18 EGFR E18F 5 -TCCAAATGAGCTGGCAAGTG-3 EGFR E18R 5 -TCCCAAACACTCAGTGAAACAAA-3 EGFR exon 19 EGFR E19F 5 -CCCAGTGTCCCTCACCTTC-3 EGFR E19R 5 -GCAGGGTCTAGAGCAGAGCA-3 EGFR exon 20 EGFR E20F 5 -CATTCATGCGTCTTCACCTG-3 EGFR E20R 5 -CATATCCCCATGGCAAACTC-3 EGFR exon 21 EGFR E21F 5 -GCTCAGAGCCTGGCATGAA-3 EGFR E21R 5 -CATCCTCCCCTGCATGTGT-3 KRAS exon 1 KRAS E1F 5 -GGGGAGTATTTTAGTGTA-3 KRAS E1R 5 -GTCCTGCACGTAATATG-3 KRAS exon 2 KRAS E2F 5 -TCAGTCCTTGCCATTTT-3 KRAS E2R 5 -GCATGGCATAGCAAGAC-3 BRAF exon 11 BRAF E11F 5 -TCCCTCTCAGGCATAAGGTAA-3 BRAF E11R 5 -CGAACAGTGAATATTTCCTTTGAT-3 BRAF exon 15 BRAF E15F 5 -TCATAATGCTTGCTCTGATAGGA-3 BRAF E15R 5 -GGCCAAAAATTTAATCAGTGGA-3 NRAS exon 2 NRAS E2F 5 -ACCAAATGGAAGGTCACACTAGGGTTT-3 NRAS E2R 5 -ACAGGATCAGGTCAGCGGGC-3 NRAS exon 3 NRAS E3F 5 -TGAGGGACAAACCAGATAGGCAGA-3 NRAS E3R 5 -CCCTAGTGTGGTAACCTCATTTCCCCA-3 PIK3CA exon 9 PIK3CA E9F 5 -AATCATCTGTGAATCCAGAGG-3 PIK3CA E9R 5 -ATGCTGAGATCAGCCAAAT-3 PIK3CA exon 20 PIK3CA E20F 5 -CATTTGAGCAAAGACCTGAAGG-3 PIK3CA E20R 5 -TGAGCTTTCATTTTCTCAGTTATC-3 1
B. Relative mrna expression (2, 5-9) Genes Primers Sequence EGFR EGFR-F 5 -ATCATTTTCTCAGCCTCCA-3 EGFR-R 5 -GACATAACCAGCCACCTCC-3 HB-EGF HB-EGF-F 5 -GAAAGACTTCCATCTAGTCACAAAGA-3 HB-EGF-R 5 -GGGAGGCCCAATCCTAGA-3 EGF EGF-F 5 -AGCTAACCCATTATGGCA-3 EGF-R 5 -AGTTTTCACTGAGTCAGCTCCAT-3 Amphiregulin AR-F 5 -ATATCACATTGGAGTCACTGCCCA-3 AR-R 5 -GGGTCCATTGTCTTATGATCCAC-3 Epiregulin EPI-F 5 -TGCATGCAATTTAAAGTAACTTATTTGACTA-3 EPI-R 5 -ATCTTAAGGTACACAATTATCAAAGCTGA-3 β-actin β-actin-f 5 -CAATGAGCTGCGTGTGGCT-3 β-actin-r 5 -TAGCACAGCCTGGATAGCAA-3 FGFR1 FGFR1-F 5 -TAATGGACTCTGTGGTGCCCTC-3 FGFR1-R 5 -ATGTGTGGTTGATGCTGCCG-3 MAPK1 MAPK1-F 5 -AAGACACAACACCTCAGCAATG-3 MAPK1-R 5 -GTTGAGCAGCAGGTTGGAAG-3 AKT1 AKT1-F 5 -AACGAGGGGGAGTACATCAAGAC-3 AKT1-R 5 -CGCCACAGAGAAGTTGTTGA-3 AKT2 AKT2-F 5 -TAGCAGAATGCCAGCTGATG-3 AKT2-R 5 -ATCCACTCCTCCCTCTCGTC-3 AKT3 AKT3-F 5 -ATTATTGCAAAGGATGAAGTGGC-3 AKT3-R 5 -CGGTCTTTTGTCTGGAAGGA-3 2
C. Copy number analysis (3, 4, 8, 10) Genes Primers Sequence EGFR EGFR-F 5 -CAAGGCCATGGAATCTGTCA-3 EGFR-R 5 -CTGGAATGAGGTGGAGGAACA-3 MET MET-F 5 -ATCAACATGGCTCTAGTTGTC-3 MET-R 5 -GGGAGAATATGCAGTGAACC-3 HER2-F 5 -GCGGTGGGGACCTGACACTA-3 HER2 HER2-R 5 -CCTTCGGAGGGTGCCAGT-3 Probe 5 -Fam-CCCTCTGAAGAGGAGGCCCCCAGGTC-Tamra-3 gb2m-f 5 -TAAAACTTAATGTCTTCCTTTTTTTTCTC-3 gb2m gb2m-r 5 -AAACATTTTCTCAAGGTCAAAAACTTA-3 Probe 5 -Fam-CCTCCATGATGCTGCTTACATGTCTC-Tamra-3 PIK3CA PIK3CA-F 5 -ATCTTTTCTCAATGATGCTTGGCT-3 PIK3CA-R 5 -CTAGGGTCTTTCGAATGTATG-3 LINE-1 LINE1-F 5 -AAAGCCGCTCAACTACATGG-3 LINE1-R 5 -TGCTTTGAATGCGTCCCAGAG-3 COL8A1 COL8A1-F 5 -GGGCTAAGAAAGGCAAGAATGG-3 COL8A1-R 5 -GTGGGAAAGGTGCGGTTAGCT-3 MAPK1 MAPK1-F 5 -ACTCCTACCAGTTTACCCAATTTG-3 MAPK1-R 5 -AGTGGCAGGAGGCATTTCAC-3 References 1. Jang TW, Oak CH, Chang HK, Suo SJ, Jung MH. EGFR and KRAS mutations in patients with adenocarcinoma of the lung. The Korean journal of internal medicine. 2009;24:48-54. 2. Rexer BN, Ghosh R, Narasanna A, Estrada MV, Chakrabarty A, Song Y, et al. Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013;19:5390-401. 3. Shien K, Toyooka S, Yamamoto H, Soh J, Jida M, Thu KL, et al. Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells. Cancer Res. 2013;73:3051-61. 4. Yamamoto H, Shigematsu H, Nomura M, Lockwood WW, Sato M, Okumura 3
N, et al. PIK3CA mutations and copy number gains in human lung cancers. Cancer Res. 2008;68:6913-21. 5. Huppi K, Volfovsky N, Runfola T, Jones TL, Mackiewicz M, Martin SE, et al. The identification of micrornas in a genomically unstable region of human chromosome 8q24. Molecular cancer research : MCR. 2008;6:212-21. 6. Marek L, Ware KE, Fritzsche A, Hercule P, Helton WR, Smith JE, et al. Fibroblast growth factor (FGF) and FGF receptor-mediated autocrine signaling in non-small-cell lung cancer cells. Molecular pharmacology. 2009;75:196-207. 7. Ware KE, Marshall ME, Heasley LR, Marek L, Hinz TK, Hercule P, et al. Rapidly acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines through de-repression of FGFR2 and FGFR3 expression. PloS one. 2010;5:e14117. 8. Kostrzewska-Poczekaj M, Giefing M, Jarmuz M, Brauze D, Pelinska K, Grenman R, et al. Recurrent amplification in the 22q11 region in laryngeal squamous cell carcinoma results in overexpression of the CRKL but not the MAPK1 oncogene. Cancer biomarkers : section A of Disease markers. 2010;8:11-9. 9. Fornari F, Milazzo M, Chieco P, Negrini M, Marasco E, Capranico G, et al. In hepatocellular carcinoma mir-519d is up-regulated by p53 and DNA hypomethylation and targets CDKN1A/p21, PTEN, AKT3 and TIMP2. The Journal of pathology. 2012;227:275-85. 10. Bechmann T, Andersen RF, Pallisgaard N, Madsen JS, Maae E, Jakobsen EH, et al. Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer. Journal of cancer research and clinical oncology. 2013;139:995-1003. 4
Supplementary Table S2. Annotated genes excluding EGFR with high severity impact by GEMINI No. AZD9291 chr position gene variant reference alteration pfam domain cytobands aa change Impact 1 2 Pre Post Pre Post 7 128846346 SMO snp A T Frizzled chr7q32.1 K/* stopgain 17 7577548 TP53 indel GCCCATGCA G P53 chr17p13.1 None frameshift 3 178936042 PIK3CA snp G T PI3Ka chr3q26.32 E/* stopgain 4 55955083 KDR snp G T Pkinase chr4q12 S/* stopgain 4 1806179 FGFR3 indel G GC None chr4p16.3 None frameshift 4 1807834 FGFR3 snp A T Pkinase chr4p16.3 K/* stopgain None Pre 11 108201061 ATM snp G T FAT chr11q22.3 G/* stopgain 3 178921526 PIK3CA snp A T None chr3q26.32 K/* stopgain 3 Post 4 1806179 FGFR3 indel G GC None chr4p16.3 None frameshift 4 1807834 FGFR3 snp A T Pkinase chr4p16.3 K/* stopgain 4 153250851 FBXW7 snp A T WD40 chr4q31.3 L/* stopgain 18 48575204 SMAD4 snp C A MH1 chr18q21.2 Y/* stopgain Pre 4 1806179 FGFR3 indel G GC None chr4p16.3 None frameshift 2 212576821 ERBB4 snp A T Receptor chr2q34 C/* stopgain 4 55602666 KIT snp C T Pkinase chr4q12 R/* stopgain 4 Post 4 55972934 KDR snp C T None chr4q12 W/* stopgain 7 21659684 DNAH11 snp G T DHC_N2 chr7p15.3 E/* stopgain 10 89720797 PTEN indel GTACT G None chr10q23.3 None frameshift 11 108119794 ATM snp C T None chr11q22.3 Q/* stopgain 5
Supplementary Figure S1. Sanger sequencing of EGFR at exon 19 and position 790 before and after AZD9291 in four patients. 6
Supplementary Figure S2. A diagram describing the study scheme. 7
Supplementary Figure S3. Computed tomography of the chest showed response to radiotherapy (left), but progression during AZD9291 at a dose of 40mg once daily (middle) and after intra-patient dose escalation to 80mg once daily (right). 8
Supplementary Figure S4. Targeted exome sequencing results were visualized with Integrated Genome Viewers for the EGFR exon 19 in-frame deletion and EGFR T790M mutations in Patients 1 to 4 (A - D). 9
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Supplementary Figure S5. EGFR, MET, and HER2 per centromere evaluation probe ratio were summarized (A). Representative FISH results of EGFR and MET in Patient 1 showed high polysomy according to the Colorado scoring criteria and no MET amplification (B). Although EGFR amplification was observed before and after AZD9291, MET amplification was not observed in Patient 2 (C) 13
Supplementary Figure S6. Targeted exome sequencing showed PTEN deletion with Integrated Genome Viewers in post-azd9291 tumors of Patients 4. 14
Supplementary Figure S7. Copy number analysis and relative mrna expression of EGFR and alternative pathway genes. qpcr method was used to determine relative mrna expression levels as well as copy number of genes, according to the protocols provided by Applied Biosystems (Life Technologies). 15
Supplementary Figure S8. Cell proliferation assay of HCC4006 cells treated with 50 ng/ml FGF2 showed resistance to AZD9291 (A). AZD9291-resistant HCC4006AR1 and HCC4006AR2 cells showed higher sensitivities to selective FGFR1 inhibitors, PD173074 (B) and BGJ398 (C). 16
Supplementary Figure S9. Immunoblotting of oncogenic downstream signals for parental and AZD9291-resistant NSCLC cells after exposure to AZD9291. The expression levels of signaling molecules were shown after a 4 hour incubation at 0, 0.1 and 1 μm concentrations of AZD9291 (A). AZD9291-resistant NSCLC cells were resistant to AZD9291 plus selumetinib or BYL719 (B). 17
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Supplementary Figure S10. Cell proliferation assays were performed in parental and AZD9291-resistant NSCLC cells treated with AZD9291 for 72 hours (A and C). The copy number analysis and relative mrna expression of EGFR and alternative pathway genes in NSCLC cells were performed using a qpcr method (B and D). HCC4006AR1-2 cells with AXL up-regulation were resistant to an AXL inhibitor (E). 19
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Supplementary Figure S11. Images of the chest showed dramatic response to salvage radiotherapy (A), but refractory disease after one cycle of etoposide and carboplatin (B). Malignant pleural effusions were obtained for establishing SNU-2962A cells that expressed CD56, chromogranin A, and synaptophysin (C). SNU-2962A cells harbored no EGFR T790M mutation (D) and low RB1 mrna expression (E). SNU-2962A cells were resistant to AZD9291 (IC 50, 3774.6±462.7 nm), but sensitive to paclitaxel (IC 50, 0.998±0.046 nm) (F). 21
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