Application of a Highly Sensitive Detection System for Epidermal Growth Factor Receptor Mutations in Plasma DNA Tomomi Nakamura, MD, Naoko Sueoka-Aragane,

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Application of a Highly Sensitive Detection System for Epidermal Growth Factor Receptor Mutations in Plasma DNA Tomomi Nakamura, MD, Naoko Sueoka-Aragane, MD, PhD, Kentaro Iwanaga, MD, PhD, Akemi Sato, PhD, Kazutoshi Komiya, MD, PhD, Naomi Kobayashi, MD, Shinichiro Hayashi, MD, PhD, Toshiya Hosomi, PhD, Mitsuharu Hirai, PhD, Eisaburo Sueoka, MD, PhD, Shinya Kimura, MD, PhD Journal of Thoracic Oncology Volume 7, Issue 9, Pages 1369-1381 (September 2012) DOI: 10.1097/JTO.0b013e31825f2821 Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 A, Principles of the WIP-QP method for detecting exon 19 deletions. It involves the following two steps: wild inhibiting PCR (WIP) and a quenching probe (QP) system. Wild Inhibitor nucleic acid (WI), complementary to wild type sequence corresponding to the deletion part, suppresses amplification of the wild type sequence by binding to wild-type template but not mutant, resulting in preferential amplification of the mutant sequence. Deletions in the amplified sequences were determined by the QP system, in which the fluorescence intensity of a TAMRA-conjugated guanine-specific quench fluorophore probe (QProbe) complementary to the wild type sequence containing the deletion part is monitored. Fluorescence intensity at different dissociation temperatures was measured to identify wild-type and mutant amplicons. B, Results with the WIP-QP method using control plasmids and genomic DNA isolated from cancer cell lines are shown. Mutant plasmids and wild type were mixed in the indicated ratios, and the assays were performed. As for the genomic DNA, DNA isolated from PC-9 or HCC4006 cells were mixed with A549, and the assays were performed. PC-9 and HCC4006 respectively contain the exon 19 deletion alleles Del E746-A750 and Del L747-A750. Both alleles were wild type of exon 19 in A549. Black and red arrows indicate wild type and mutant, respectively. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 A, Principles of the WIP-QP method for detecting exon 19 deletions. It involves the following two steps: wild inhibiting PCR (WIP) and a quenching probe (QP) system. Wild Inhibitor nucleic acid (WI), complementary to wild type sequence corresponding to the deletion part, suppresses amplification of the wild type sequence by binding to wild-type template but not mutant, resulting in preferential amplification of the mutant sequence. Deletions in the amplified sequences were determined by the QP system, in which the fluorescence intensity of a TAMRA-conjugated guanine-specific quench fluorophore probe (QProbe) complementary to the wild type sequence containing the deletion part is monitored. Fluorescence intensity at different dissociation temperatures was measured to identify wild-type and mutant amplicons. B, Results with the WIP-QP method using control plasmids and genomic DNA isolated from cancer cell lines are shown. Mutant plasmids and wild type were mixed in the indicated ratios, and the assays were performed. As for the genomic DNA, DNA isolated from PC-9 or HCC4006 cells were mixed with A549, and the assays were performed. PC-9 and HCC4006 respectively contain the exon 19 deletion alleles Del E746-A750 and Del L747-A750. Both alleles were wild type of exon 19 in A549. Black and red arrows indicate wild type and mutant, respectively. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 A, Principles of the WIP-QP method for detecting exon 19 deletions. It involves the following two steps: wild inhibiting PCR (WIP) and a quenching probe (QP) system. Wild Inhibitor nucleic acid (WI), complementary to wild type sequence corresponding to the deletion part, suppresses amplification of the wild type sequence by binding to wild-type template but not mutant, resulting in preferential amplification of the mutant sequence. Deletions in the amplified sequences were determined by the QP system, in which the fluorescence intensity of a TAMRA-conjugated guanine-specific quench fluorophore probe (QProbe) complementary to the wild type sequence containing the deletion part is monitored. Fluorescence intensity at different dissociation temperatures was measured to identify wild-type and mutant amplicons. B, Results with the WIP-QP method using control plasmids and genomic DNA isolated from cancer cell lines are shown. Mutant plasmids and wild type were mixed in the indicated ratios, and the assays were performed. As for the genomic DNA, DNA isolated from PC-9 or HCC4006 cells were mixed with A549, and the assays were performed. PC-9 and HCC4006 respectively contain the exon 19 deletion alleles Del E746-A750 and Del L747-A750. Both alleles were wild type of exon 19 in A549. Black and red arrows indicate wild type and mutant, respectively. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 A, Principles of the MBP-QP method for detecting L858R. The MBP-QP method consists of two steps: mutation-biased PCR (MBP) and a quenching probe (QP) system. In MBP, primer lengths differ for wild-type and mutant sequences, and five bases of random sequence are added to the reverse primers. QProbe is complementary to L858R, and the subsequent reaction procedure is the same as with the WIP-QP method. B, Results with the MBP-QP method using control plasmid and genomic DNA isolated from H1975 cells are shown. Mutant plasmids and wild type were mixed in the indicated ratios, and the assays were performed. DNA isolated from H1975 and A549 were mixed, and the assays were performed. H1975 cells harbor both a L858R and a wild type allele, whereas A549 contains only wild-type alleles. Black and red arrows indicate wild type and mutant, respectively. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 A, Principles of the MBP-QP method for detecting L858R. The MBP-QP method consists of two steps: mutation-biased PCR (MBP) and a quenching probe (QP) system. In MBP, primer lengths differ for wild-type and mutant sequences, and five bases of random sequence are added to the reverse primers. QProbe is complementary to L858R, and the subsequent reaction procedure is the same as with the WIP-QP method. B, Results with the MBP-QP method using control plasmid and genomic DNA isolated from H1975 cells are shown. Mutant plasmids and wild type were mixed in the indicated ratios, and the assays were performed. DNA isolated from H1975 and A549 were mixed, and the assays were performed. H1975 cells harbor both a L858R and a wild type allele, whereas A549 contains only wild-type alleles. Black and red arrows indicate wild type and mutant, respectively. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Results with the MBP-QP and WIP-QP methods for detecting L858R and exon 19 deletion, respectively, using lung cancer tissues. Black and red arrows indicate wild type and mutant, respectively. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Representative results from serial analysis using the MBP-QP method to detect the L858R EGFR mutation in plasma DNA from lung adenocarcinoma patients. A, Case 1 recurred as intrapulmonary metastasis after surgery, and L858R was detected in plasma DNA after appearance of metastatic lesions spread to the liver. B, L858R was detected before chemotherapy and disappeared after tumor regression in case 2. Black and red arrows indicate wild type and mutant, respectively. TKI, EGFR-TKI; chemo, chemotherapy; G, gefitinib; E, erlotinib; CT, computed tomography. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

Figure 5 A, Combination of EGFR activating/sensitive mutations with T790M in plasma DNA during treatment for lung adenocarcinoma. A serial analysis of the WIP-QP for exon 19 deletion and MBP-QP for T790M in plasma DNA form a lung adenocarcinoma patient is shown. Black and red arrows indicate wild type and mutant, respectively. TKI, EGFR-TKI; chemo, chemotherapy; G, gefitinib; E, erlotinib; CT, computed tomography. B, Disappearance of T790M from plasma DNA after chemotherapy. Serial analysis of WIP-QP for exon 19 deletion and MBP-QP for T790M in plasma DNA form a lung adenocarcinoma patient is shown. Black and red arrows indicate wild type and mutant, respectively. TKI, EGFR-TKI; chemo, chemotherapy; G, gefitinib; E, erlotinib; CT, computed tomography. Journal of Thoracic Oncology 2012 7, 1369-1381DOI: (10.1097/JTO.0b013e31825f2821) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions