A Sensitive Method for Detecting EGFR Mutations in Non-small Cell Lung Cancer Samples with Few Tumor Cells Miguel A. Molina-Vila, PhD, Jordi Bertran-Alamillo,

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A Sensitive Method for Detecting EGFR Mutations in Non-small Cell Lung Cancer Samples with Few Tumor Cells Miguel A. Molina-Vila, PhD, Jordi Bertran-Alamillo, MSc, Noemí Reguart, MD, PhD, Miquel Taron, PhD, Eva Castellà, MD, Mariona Llatjós, MD, Carlota Costa, PhD, Clara Mayo, PhD, Anna Pradas, MSc, Cristina Queralt, MSc, Mónica Botia, María Pérez-Cano, Esther Carrasco, MSc, Mireia Tomàs, MSc, Jose Luis Mate, MD, Teresa Moran, MD, Rafael Rosell, MD, PhD Journal of Thoracic Oncology Volume 3, Issue 11, Pages 1224-1235 (November 2008) DOI: 10.1097/JTO.0b013e318189f579 Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 A, Example of length analysis for a wild-type (top panel) and a mutated patient (bottom panel) for exon 19 of EGFR. The peaks corresponding to the samples are represented in blue, the molecular weight markers (75, 100, 139, 150, and 160 bp) in orange. The mutated patient harbors a 15-bp deletion. B, Example of Taqman assay for a subset of patients. The two green triangles correspond to the H1975 cell line and to a tumor harboring the L858R mutation. The red circles correspond to the PC-9 cell line and to several wild-type tumors. The gray squares are negative and extraction controls. Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Example of response to erlotinib (patient 13 in Table 3) harboring exon 19 delL746–A750, detected in pleural fluid. Top panel: CT scan prior to erlotinib treatment (A) and after 1 year of erlotinib treatment (B). Bottom panel: plain chest radiograph before erlotinib treatment (C) and after 1 year of erlotinib treatment (D). Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Sequencing chromatogram for EGFR exons 19 and 20 in a patient progressing to erlotinib (patient A in Table 4) showing heterogeneous distribution of the T790M mutation. Four areas of the tumor mass at the site of progression (PB1, PB2, PB3, and PB4) were analyzed. Top panel: length analysis for exon 19; the 104-kb peak showing the presence of a 15-bp deletion is indicated by an arrow. Bottom panel: sequencing results for exon 20; the presence of the T790M mutation as an additional T peak in the chromatogram is indicated with an asterisk. While the primary mutation (the 15-bp deletion delE746–A750) was present in all areas, the T790M mutation (C to T transition) was not detected in one area of the tumor (PB1). The result was confirmed by TaqMan assay. Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE S1 Sensitivity of the assay: Serially diluted genomic DNA from the cell lines PC-9 (harboring a deletion in exon 19) and H1975 (harboring both the T790M and the L858R mutations) was analyzed (A and C). Ten picogram of DNA were successfully amplified and the corresponding mutations detected. We also trypsinized tumor cells in culture, extended them on a slide and microdissected them in different quantities. Four tumor cells were sufficient to determine EGFR mutation status (B and D). Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE S1 Sensitivity of the assay: Serially diluted genomic DNA from the cell lines PC-9 (harboring a deletion in exon 19) and H1975 (harboring both the T790M and the L858R mutations) was analyzed (A and C). Ten picogram of DNA were successfully amplified and the corresponding mutations detected. We also trypsinized tumor cells in culture, extended them on a slide and microdissected them in different quantities. Four tumor cells were sufficient to determine EGFR mutation status (B and D). Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE S1 Sensitivity of the assay: Serially diluted genomic DNA from the cell lines PC-9 (harboring a deletion in exon 19) and H1975 (harboring both the T790M and the L858R mutations) was analyzed (A and C). Ten picogram of DNA were successfully amplified and the corresponding mutations detected. We also trypsinized tumor cells in culture, extended them on a slide and microdissected them in different quantities. Four tumor cells were sufficient to determine EGFR mutation status (B and D). Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE S1 Sensitivity of the assay: Serially diluted genomic DNA from the cell lines PC-9 (harboring a deletion in exon 19) and H1975 (harboring both the T790M and the L858R mutations) was analyzed (A and C). Ten picogram of DNA were successfully amplified and the corresponding mutations detected. We also trypsinized tumor cells in culture, extended them on a slide and microdissected them in different quantities. Four tumor cells were sufficient to determine EGFR mutation status (B and D). Journal of Thoracic Oncology 2008 3, 1224-1235DOI: (10.1097/JTO.0b013e318189f579) Copyright © 2008 International Association for the Study of Lung Cancer Terms and Conditions