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NEJM -- Lynch TJ et al. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib. 350(21):2129-39. Supplementary Appendix Article: Lynch TJ et al. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non–Small-Cell Lung Cancer to Gefitinib. N Engl J Med 2004;350(21):2129-39.

Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Mutational Analysis

The polymerase chain reaction was used to amplify the 28 exons comprising the EGFR gene using DNA isolated from primary tumor tissue or tumor-derived cell-lines. Primer pairs used were: Exon 1, CAGATTTGGCTCGACCTGGACATAG (sense) and CAGCTGATCTCAAGGAAACAGG (antisense); Exon 2, GTATTATCAGTCACTAAAGCTCAC (sense) and CACACTTCAAGTGGAATTCTGC; Exon 3, CTCGTGTGCATTAGGGTTCAACTGG (sense) and CCTTCTCCGAGGTGGAATTGAGTGAC (antisense); Exon 4, GCTAATTGCGGGACTCTTGTTCGCAC (sense) and TACATGCTTTTCTAGTGGTCAG (antisense); Exon 5, GGTCTCAAGTGATTCTACAAACCAG (sense) and CCTTCACCTACTGGTTCACATCTG (antisense); Exon 6, CATGGTTTGACTTAGTTTGAATGTGG (sense) and GGATACTAAAGATACTTTGTCACCAGG (antisense); Exon 7, GAACACTAGGCTGCAAAGACAGTAAC (sense) and CCAAGCAAGGCAAACACATCCACC (antisense); Exon 8, GGAGGATGGAGCCTTTCCATCAC (sense) and GAAGAGGAAGATGTGTTCCTTTGG (antisense); Exons 9 and 10, GAATGAAGGATGATGTGGCAGTGG (sense) and CAAAACATCAGCCATTAACGG (antisense); Exon 11, CCACTTACTGTTCATATAATACAGAG (sense) and CATGTGAGATAGCATTTGGGAATGC (antisense); Exon 12, CATGACCTACCATCATTGGAAAGCAG (sense) and GTAATTTCACAGTTAGGAATC (sense); Exon 13, GTCACCCAAGGTCATGGAGCACAGG (sense) and CAGAATGCCTGTAAAGCTATAAC (antisense); Exon 14, GTCCTGGAGTCCCAACTCCTTGAC (sense) and GGAAGTGGCTCTGATGGCCGTCCTG (antisense); Exon 15, CCACTCACACACACTAAATATTTTAAG (sense) and GACCAAAACACCTTAAGTAACTGACTC (antisense); Exon 16, CCAATCCAACATCCAGACACATAG (sense) and CCAGAGCCATAGAAACTTGATCAG (antisense); Exon 17, GTATGGACTATGGCACTTCAATTGCATGG (sense) and CCAGAGAACATGGCAACCAGCACAGGAC (antisense); Exon 18, CAAATGAGCTGGCAAGTGCCGTGTC (sense) and GAGTTTCCCAAACACTCAGTGAAAC (antisense); Exon 19, GCAATATCAGCCTTAGGTGCGGCTC (sense) and CATAGAAAGTGAACATTTAGGATGTG (antisense); Exon 20, CCATGAGTACGTATTTTGAAACTC (sense) and CATATCCCCATGGCAAACTCTTGC (antisense); Exon 21, CTAACGTTCGCCAGCCATAAGTCC (sense) and GCTGCGAGCTCACCCAGAATGTCTGG (antisense); Exon 22, GAGCAGCCCTGAACTCCGTCAGACTG (sense) and CTCAGTACAATAGATAGACAGCAATG (antisense); Exon 23, CAGGACTACAGAAATGTAGGTTTC (sense) and GTGCCTGCCTTAAGTAATGTGATGAC (antisense); Exon 24, GACTGGAAGTGTCGCATCACCAATG (sense) and GGTTTAATAATGCGATCTGGGACAC (antisense); Exon 25, GCAGCTATAATTTAGAGAACCAAGG (sense) and GGTTAAAATTGACTTCATTTCCATG (antisense); Exon 26, CCTAGTTGCTCTAAAACTAACG (sense) and CTGTGAGGCGTGACAGCCGTGCAG (antisense); Exon 27, CAACCTACTAATCAGAACCAGCATC (sense) and CCTTCACTGTGTCTGCAAATCTGC (antisense); Exon 28, CCTGTCATAAGTCTCCTTGTTGAG (sense) and CAGTCTGTGGGTCTAAGAGCTAATG (antisense). Annealing temperatures were 58°C (exons 1,3, 4, 7-10, 12-25, 27, and 28), 56°C (exons 2, 5, 6, and 26), or 52°C (exon 11).

Nested PCR amplification of DNA extracted from archival tumor tissue was performed as follows. An initial PCR for exons 2, 5, 6, 7, 11, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 was generated using primers and conditions described above. Subsequently, 2 µl of this reaction was amplified in a secondary PCR using the following internal primer pairs: Exon 2, CAGGAATGGGTGAGTCTCTGTGTG (sense) and GTGGAATTCTGCCCAGGCCTTTC (antisense); Exon 5, GATTCTACAAACCAGCCAGCCAAAC (sense) and CCTACTGGTTCACATCTGACCCTG (antisense); Exon 6, GTTTGAATGTGGTTTCGTTGGAAG (sense) and CTTTGTCACCAGGCAGAGGGCAATATC (antisense); Exon 7, GACAGTAACTTGGGCTTTCTGAC (sense) and CATCCACCCAAAGACTCTCCAAG (antisense); Exon11, CTGTTCATATAATACAGAGTCCCTG (sense) and GAGAGATGCAGGAGCTCTGTGC (antisense); Exon12, GCAGTTTGTAGTCAATCAAAGGTGG (sense) and GTAATTTAAATGGGAATAGCCC (antisense); Exon14, CAACTCCTTGACCATTACCTCAAG (sense) and GATGGCCGTCCTGCCCACACAGG (antisense); Exon16, GAGTAGTTTAGCATATATTGC (sense) and GACAGTCAGAAATGCAGGAAAGC (antisense); Exon18, CAAGTGCCGTGTCCTGGCACCCAAGC (sense) and CCAAACACTCAGTGAAACAAAGAG (antisense); Exon 19, CCTTAGGTGCGGCTCCACAGC (sense) and CATTTAGGATGTGGAGATGAGC (antisense); Exon 20, GAAACTCAAGATCGCATTCATGC (sense) and GCAAACTCTTGCTATCCCAGGAG (antisense); Exon 21, CAGCCATAAGTCCTCGACGTGG (sense) and CATCCTCCCCTGCATGTGTTAAAC (antisense); Exon 22, GACGGGTCCTGGGGTGATCTGGCTC (sense) and GATTACATTATCATTAGTCATTATC (antisense); Exon 23, GTAGGTTTCTAAACATCAAGAAAC (sense) and GTGATGACATTTCTCCAGGGATGC (antisense); Exon 24, CATCACCAATGCCTTCTTTAAGC (sense) and GCTGGAGGGTTTAATAATGCGATC (antisense); Exon 25, GCAAACACACAGGCACCTGCTGGC (sense) and CATTTCCATGTGAGTTTCACTAGATGG (antisense); Exon 26, CACCTTCACAATATACCCTCCATG (sense) and GACAGCCGTGCAGGGAAAAACC (antisense); Exon 27, GAACCAGCATCTCAAGGAGATCTC (sense) and GAGCACCTGGCTTGGACACTGGAG (antisense).

Nested PCR amplifications for the remaining exons consisted of primary PCR using the following primers. Exon 1, GACCGGACGACAGGCCACCTCGTC (sense) and GAAGAACGAAACGTCCCGTTCCTCC (antisense); Exon 3, GTTGAGCACTCGTGTGCATTAGG (sense) and CTCAGTGCACGTGTACTGGGTA (antisense); Exon 4, GTTCACTGGGCTAATTGCGGGACTCTTGTTCGCAC (sense) and GGTAAATACATGCTTTTCTAGTGGTCAG (antisense); Exon 8, GGAGGATGGAGCCTTTCCATCAC (sense) and GAAGAGGAAGATGTGTTCCTTTGG (antisense); Exon 9, GAATGAAGGATGATGTGGCAGTGG (sense) and GTATGTGTGAAGGAGTCACTGAAAC (antisense); Exon 10, GGTGAGTCACAGGTTCAGTTGC (sense) and CAAAACATCAGCCATTAACGG (antisense); Exon 13, GTAGCCAGCATGTCTGTGTCAC (sense) and CAGAATGCCTGTAAAGCTATAAC (antisense); Exon 15, CATTTGGCTTTCCCCACTCACAC (sense) and GACCAAAACACCTTAAGTAACTGACTC (antisense); Exon 17, GAAGCTACATAGTGTCTCACTTTCC (sense) and CACAACTGCTAATGGCCCGTTCTCG (antisense); Exon 28a GCTCCTGCTCCCTGTCATAAGTC (sense) and GAAGTCCTGCTGGTAGTCAGGGTTG (antisense); Exon 28b, CTGCAGTGGGCAACCCCGAGTATC (sense) and CAGTCTGTGGGTCTAAGAGCTAATG (antisense). Secondary PCR amplification was carried out using primer pairs: Exon 1, GACAGGCCACCTCGTCGGCGTC (sense) and CAGCTGATCTCAAGGAAACAGG (antisense); Exon 3, CTCGTGTGCATTAGGGTTCAACTGG (sense) and CCTTCTCCGAGGTGGAATTGAGTGAC (antisense); Exon 4, GCTAATTGCGGGACTCTTGTTCGCAC (sense) and TACATGCTTTTCTAGTGGTCAG (antisense); Exon 8, CCTTTCCATCACCCCTCAAGAGG (sense) and GATGTGTTCCTTTGGAGGTGGCATG (antisense); Exon 9, GATGTGGCAGTGGCGGTTCCGGTG (sense) and GGAGTCACTGAAACAAACAACAGG (antisense); Exon 10, GGTTCAGTTGCTTGTATAAAG (sense) and CCATTAACGGTAAAATTTCAGAAG (antisense); Exon 13, CCAAGGTCATGGAGCACAGG (sense) and CTGTAAAGCTATAACAACAACCTGG (antisense); Exon 15, CCACTCACACACACTAAATATTTTAAG (sense) and GTAACTGACTCAAATACAAACCAC (antisense); Exon 17, GAAGCTACATAGTGTCTCACTTTCC (sense) and CACAACTGCTAATGGCCCGTTCTCG (antisense); Exon 28a, CCTGTCATAAGTCTCCTTGTTGAG (sense) and GGTAGTCAGGGTTGTCCAGG (antisense); Exon 28b, CGAGTATCTCAACACTGTCCAGC (sense) and CTAAGAGCTAATGCGGGCATGGCTG (antisense). Annealing temperatures for both primary and secondary amplifications were 58°C (exons 1,3, 4, 7-10, 12-17, 19-25, 27, and 28), 56°C (exons 2, 5, 6, and 26), or 52°C (exons 11 and 18).

PCR amplicons were purified using exonuclease I (United States Biochemical, Cleveland, OH), and shrimp alkaline phosphatase (United States Biochemical, Cleveland, OH) prior to sequencing. Purified DNA was diluted and cycle-sequenced using the ABI BigDye Terminator kit v1.1 (ABI, Foster City, CA) according to manufacturer's instructions. Sequencing reactions were electrophoresed on an ABI3100 genetic analyzer. Electropherograms were analyzed in both sense and antisense direction for the presence of mutations, using Sequence Navigator software in combination with Factura to mark heterozygous positions. All sequence variants were confirmed in multiple independent PCR amplifications and sequencing reactions.

Cancer-Derived Cell Lines

A panel of 14 lung cancer-derived cell lines was analyzed for EGFR mutations. These were derived from tumors of NSCLC (N = 5), small cell lung cancer (N = 6), adenosquamous (N = 1), bronchial carcinoid (N = 1), and unknown histology (N = 1). Specific cell lines were: NCI-H460, NCI-522, HOP-92, NCIH841, NCIH734, NCIH2228, NCIH596, NCIH727, NCIH446, NCIH1781, NCIH209, NCIH510, NCIH82, NCIH865. In addition, 94 cancer-derived cell lines were screened for mutations in exons 19 and 21. These represented the following histologies: breast cancer (BT549, BT483, UACC893, HS467T, HS578T, MCF7, MCF7-ADR, MDA-MB-15, MDA-MB-175, MDA-MB-231, MDA-MB-415, MDA-MB-436, MDA-MB-453, MDA-MB-468, T47D), ovarian cancer (ES-2, IGROV-1, MDAH2774, OV1063, OVCAR3, OVCAR4, OVCAR5, SKOV3, SW626), CNS cancers (SF-295, SNB-19, U-251, CCF-STTG1, SW-1088, SW-1783, T98G, M059K, A172, SK-N-DZ, SK-N-MC), leukemia (CCRF-CEM, K562, MOLT-4, RPMI8226, SR), prostate cancer (DU-145, PC-3), colon cancer (COLO-205, HCT-116, HCT-15, HT-29, SW-620), renal cancer (786-O, ACHN, CAKI-1, SN-12C, UO-31), melanoma (LOX-IMVI, M14, SKMEL2, UACC-62, MGH-MC-2, SKMEL21, MGH-PO-1, SKMEL119, MelJuso, K19, HS940, SKMEL63, HS939T, K4, WM115, WM164, A375, MGH-TH, MGH-MA, MGH-MC-1, WM1158, SKMEL39, MGH-BO-1, UACC903, MM455, SKMEL28, MGH-BA-1, SKMEL23, CHL1, HS597, LH, K1-MEL, K-11, MGH-LE)), osteosarcoma (SAOS-2), and head and neck cancers (O11, O13, 019, O28, O22, O29, O12). The head and neck cancer cell-lines were provided by Dr. James Rocco, Massachusetts General Hospital/Massachusetts Eye and Ear Infirmary. All other cell-lines are available through the American Type Culture Collection (Manassas, VA).


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