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EGFR-GRB2 Protein Colocalization Is a Prognostic Factor Unrelated to Overall EGFR Expression or EGFR Mutation in Lung Adenocarcinoma  Maria I. Toki, MD,

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Presentation on theme: "EGFR-GRB2 Protein Colocalization Is a Prognostic Factor Unrelated to Overall EGFR Expression or EGFR Mutation in Lung Adenocarcinoma  Maria I. Toki, MD,"— Presentation transcript:

1 EGFR-GRB2 Protein Colocalization Is a Prognostic Factor Unrelated to Overall EGFR Expression or EGFR Mutation in Lung Adenocarcinoma  Maria I. Toki, MD, Daniel E. Carvajal-Hausdorf, MD, Mehmet Altan, MD, Joseph McLaughlin, MD, Brian Henick, MD, Kurt A. Schalper, MD, PhD, Konstantinos N. Syrigos, MD, PhD, David L. Rimm, MD, PhD  Journal of Thoracic Oncology  Volume 11, Issue 11, Pages (November 2016) DOI: /j.jtho Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

2 Figure 1 Validation of EGFR–growth factor receptor bound protein 2 (GRB2) proximity ligation assay (PLA) in NSCLC cell lines. (A) Automated Quantitative Analysis (AQUA) images of formalin-fixed paraffin-embedded cell lines with different EGFR expression levels and NSCLC formalin-fixed paraffin-embedded cell lines with different EGFR mutation status (EGFR-GRB2 PLA [red], cytokeratin [green], and 4’,6-diamidino-2-phenylindole [blue]). (B) Western blots of phosphorylated Tyr1068 EGFR, total EGFR, and GRB2. β-tubulin was used as a loading control. Blots are representative of two independent experiments. (C) Technical control of EGFR-GRB2 PLA. AQUA images of EGFR-mutant patient case with complete PLA reaction and without EGFR primary antibody (EGFR-GRB2 PLA [red], cytokeratin [green], and 4’,6-diamidino-2-phenylindole [blue]). Images in (A) and (C) are representative of two independent experiments. (D) Reproducibility and core-to-core variability of EGFR-GRB2 quantified by AQUA. QIF, quantitative immunofluorescence. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

3 Figure 2 EGFR–growth factor receptor bound protein 2 (GRB2) proximity ligation assay (PLA) and total EGFR correlation in an array with patients with NSCLC and different mutational status. (A) Quantitative immunofluorescence (QIF) score regression chart of EGFR-GRB2 PLA and EGFR in patients with NSCLC with known mutation status. (B) EGFR-GRB2 PLA and EGFR QIF score distribution (cell lines [blue], EGFR-mutant cases [green], KRAS-mutant cases [red], EGFR/KRAS wild- type cases [yellow]). (C) Images of formalin-fixed paraffin-embedded tumor sections of two patient cases with EGFR-GRB2 PLA and EGFR expression discordance. Yellow arrow points at an EGFR wild-type (WT) tumor with high EGFR-GRB2 PLA QIF scores but low EGFR expression. Red arrow points at a KRAS-mutant case with high EGFR expression, but low EGFR-GRB2 PLA signal (EGFR-GRB2 PLA [red], cytokeratin [green], and 4’,6-diamidino-2-phenylindole [blue]). Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

4 Figure 3 EGFR–growth factor receptor bound protein 2 (GRB2) proximity ligation assay (PLA) and EGFR quantitative immunofluorescence (QIF) scores across mutations and mutation sites in NSCLC tumors. (A) EGFR-GRB2 PLA QIF scores in NSCLC tumors with different mutation status. EGFR-mutant (p = 0.04) and EGFR/KRAS wild-type (WT) tumors (p = ) have significantly higher EGFR-GRB2 PLA QIF scores compared with KRAS-mutant tumors. (B) Total EGFR QIF scores in NSCLC tumors with different mutation status. No statistically significant difference across different mutations or mutation sites. Two-tailed Mann-Whitney U test: bars represent means with standard deviation. Asterisk denotes statistical significance at p < 0.05. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

5 Figure 4 EGFR–growth factor receptor bound protein 2 (GRB2) proximity ligation assay (PLA) as a prognostic marker. (A) Kaplan-Meier 5-year survival curves of patients with adenocarcinoma in Yale cohorts A and B, stratified according to EGFR-GRB2 PLA quantitative immunofluorescence (QIF) scores; p = (cohort A) and p = (cohort B), log-rank (Mantel-Cox) test. (B) Score distribution of EGFR-GRB2 QIF scores of patients with adenocarcinoma in Yale cohorts A and B. Journal of Thoracic Oncology  , DOI: ( /j.jtho ) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

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