An Alternative Method for Screening EGFR Mutation Using RFLP in Non-small Cell Lung Cancer Patients Ichiro Kawada, MD, Kenzo Soejima, MD, PhD, Hideo.

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An Alternative Method for Screening EGFR Mutation Using RFLP in Non-small Cell Lung Cancer Patients Ichiro Kawada, MD, Kenzo Soejima, MD, PhD, Hideo Watanabe, MD, Ichiro Nakachi, MD, Hiroyuki Yasuda, MD, Katsuhiko Naoki, MD, PhD, Masafumi Kawamura, MD, PhD, Keisuke Eguchi, MD, PhD, Koichi Kobayashi, MD, PhD, Akitoshi Ishizaka, MD, PhD Journal of Thoracic Oncology Volume 3, Issue 10, Pages 1096-1103 (October 2008) DOI: 10.1097/JTO.0b013e318186fadd Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Scheme of digestion of PCR products and gel electrophoresis. PCR products were digested with corresponding enzymes (without digestion for exon 19), then were run on 2% agarose gel and the existence of mutations was assessed. Both ApaI and MscI digest wild type EGFR allele, while PvuII digests mutant EGFR allele. Journal of Thoracic Oncology 2008 3, 1096-1103DOI: (10.1097/JTO.0b013e318186fadd) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Demographic data of EGFR mutations in exon 18, 19, 21, and 20 by PCR-RFLP. A, The PCR products of exon18 treated with ApaI. B, The PCR products of exon19. C, The PCR products of the exon21 treated with MscI and PvuII, simultaneously. D, The PCR products of exon20 treated with NlaIII. WT, EGFR (wild type) vector; A4, EGFR (G719S) vector; H1650, lung cancer cell line with EGFR mutation (del E746-A750); A1, EGFR (L858R) vector; H1975, lung cancer cell line with EGFR mutation (L858R and T790M); SK-MES-1, lung cancer cell line without EGFR mutation. Journal of Thoracic Oncology 2008 3, 1096-1103DOI: (10.1097/JTO.0b013e318186fadd) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Sensitivity of PCR-RFLP analysis of EGFR mutations. A, In exon 19, H1650 was mixed with SK-MES-1 from 1- to 103-fold. A shorter band from the deleted allele was detected up to the level of 1 × 102-fold dilution. In exon 21, H1975 was mixed with SK-MES-1 from 1- to 103-fold. The mutant allele at the 154 bp band was detected up to the level of 1 × 102-fold dilution. SK-MES-1, lung cancer cell line without EGFR mutation; H1650, lung cancer cell line with EGFR mutation (del E746-A750); H1975, lung cancer cell line with EGFR mutation (L858R). B, PCR-RFLP was performed using either cDNA or genomic DNA (gDNA). The mutant allele in exon 19 was only detected by using cDNA in the case of malignant pleural effusion. In exon 21, the mutant allele (a 154 bp digested fragment) can be distinguished readily by using cDNA in the case of malignant pleural effusion of NSCLC. H1650; lung cancer cell line with EGFR mutation (del E746-A750), H1975; lung cancer cell line with EGFR mutation (L858R), SK-MES-1; lung cancer cell line without EGFR mutation, P; the case of malignant pleural effusion of NSCLC, M; marker. Journal of Thoracic Oncology 2008 3, 1096-1103DOI: (10.1097/JTO.0b013e318186fadd) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Result of PCR-RFLP analysis of EGFR mutations in exon 18, 19, and 21 in clinical samples. A, In exon 18, only patient 56 showed 2 fragments (237 bp and 195 bp, corresponding to mutant and wild type alleles, respectively). Direct sequencing analysis of PCR products is shown. Point mutation 2156G>C (G719D) (arrow) is detected in patient 56. B, In exon 19, patients 11, 28, and 46 showed 2 fragments corresponding to wild type and deletion mutant alleles. In-frame deletion was detected in patients 28. C, In exon 21, patients 3,12, and 41 showed 2 fragments (154 bp and 98 bp, corresponding to L858R mutant and wild type alleles, respectively). No L861Q mutation was observed in our specimens. Point mutation 2573T>G (L858R) was detected in patients 3, 12, and 41. Journal of Thoracic Oncology 2008 3, 1096-1103DOI: (10.1097/JTO.0b013e318186fadd) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions