Research ArticleCancer

Annotation of human cancers with EGFR signaling–associated protein complexes using proximity ligation assays

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Science Signaling  13 Jan 2015:
Vol. 8, Issue 359, pp. ra4
DOI: 10.1126/scisignal.2005906
  • Fig. 1 Characterization of EGFR:GRB2 PLA in cultured cell lines.

    (A) Images of PC9 NSCLC cells, which have activating mutations in EGFR, fluorescently labeled by EGFR:GRB2 PLA (red), with an antibody (Ab) targeting cytokeratin (green), and with DAPI (4′,6-diamidino-2-phenylindole) (blue). (B) Images of PC9 or H520 cells labeled as in (A). Immunoblot shows that H520 cells have little to no detectable EGFR. (C) Images of PC9 cells exposed to the EGFR inhibitor erlotinib and fluorescently labeled by EGFR:GRB2 PLA (red) and DAPI (blue). Images in (A) to (C) are representative of three independent experiments. Scale bars, 20 μm. (D and E) Western blots of PC9 cells exposed to the indicated concentrations of erlotinib. In (D), lysates were probed for activated EGFR with an antibody against Tyr1068 phosphorylated EGFR (pEGFR). β-Actin was used as a loading control. In (E), lysates were subjected to coimmunoprecipitation with an antibody to GRB2 and blotted for EGFR. Blots in (D) and (E) are representative of three independent experiments.

  • Fig. 2 EGFR:GRB2 PLA in NSCLC cell lines with known EGFR mutation status.

    (A) Images of confocal microscopy (representative z-stacks shown) of seven NSCLC cell lines labeled by EGFR:GRB2 PLA (red) and DAPI (blue). Images are representative of three independent experiments. Scale bars, 20 μm. (B) Immunoblots of phosphorylated Tyr1068 EGFR, total EGFR, and GRB2. β-Actin was used as a loading control. Blots are representative of three independent experiments. (C) Immunoblot of coimmunoprecipitation of endogenous EGFR using antibodies against GRB2 in lysates of the indicated NSCLC cell lines. Blot is representative of two independent experiments. IP, immunoprecipitation.

  • Fig. 3 EGFR:GRB2 PLA in NSCLC xenografts demonstrates erlotinib-mediated complex dissociation.

    (A and B) Images of sections of FFPE tumors from untreated or erlotinib-treated xenografts from NSCLC cell lines (A) or PDX tumors established from patients harboring an EGFR G179A mutation or a KRAS G12C mutation (B) that were labeled by EGFR:GRB2 PLA (red), an antibody against cytokeratin (green), and with DAPI (blue). Images are representative of three technical replicates. Scale bars, 100 μm (A) and 20 μm (B).

  • Fig. 4 EGFR:GRB2 PLA in PDX tumors in mice.

    (A and B) Graphs of the variability between replicate cores in a PDX tumor microarray labeled by (A) EGFR:GRB2 PLA or (B) EGFR AQUA. n = 289 tumors. For two PDX tumors, only a single core was available. PLA intensity was scored manually from 0 to 3+ based on the number of foci per cell by blinded observers. For (B), P < 0.0001, Pearson correlation. (C and D) Graphs of the relationship between EGFR:GRB2 PLA and EGFR AQUA for individual cores across all cancer types (C) or in NSCLC PDX tumors (D). Bars represent means ± 95% confidence interval (CI). For (C), n = 579 cores from 289 tumors. Three cores were missing either EGFR:GRB2 PLA or EGFR AQUA values. P < 0.0001, Kruskal-Wallis test. For (D), n = 119 cores from 60 tumors. Only a single core was available for one tumor. EGFR:GRB2 PLA scores were grouped as low (0 or 1) or high (2 or 3) . *P < 0.001, two-tailed Mann-Whitney U test. (E and F) Graphs of percentage of PDX tumors of the indicated types with EGFR:GRB2 PLA scores grouped as in (D). adeno, adenocarcinoma; squam, squamous cell carcinoma; NOS, not otherwise specified. n in parenthesis indicates the number of PDX tumors.

  • Fig. 5 Predictive capacity of EGFR:GRB2 PLA for cetuximab response in PDX tumors.

    (A) Contingency tables (2 × 2) demonstrating EGFR:GRB2 PLA analytical performance to predict robust response to cetuximab (defined as >70% tumor reduction in treated compared to untreated mice). All cancers: P = 0.006; NSCLC only: P = 0.18, two-tailed Fisher’s exact test. (B) Graphs of the percentage of tumor inhibition in mice treated with cetuximab (50 mg/kg; days 0, 7, and 14) compared to untreated mice and sacrificed on day 28. Bars are colored to reflect EGFR:GRB2 PLA score. Lines at 30% (representing 70% reduction in tumor size) demarcate the cutoff between responders (below) and nonresponders (above). Drug resistance–associated mutations and molecular events are also annotated. K, KRAS mutant (G12x); B, BRAF mutant; N, NRAS mutant; M, phosphorylated Met; A, phosphorylated AKT; H, phosphorylated HER3.

  • Fig. 6 EGFR:GRB2 PLA in NSCLC patient tumor specimens.

    (A) Distributions of EGFR:GRB2 PLA status in three NSCLC patient cohorts (MCC1, n = 103; MCC2, n = 149; MCC3, n = 98). (B) Relationship between EGFR:GRB2 PLA status and EGFR protein abundance determined by AQUA across individual cores in each cohort (MCC3 had two cores per patient). Bars represent means ± 95% CI. *P < 0.001, two-tailed Mann-Whitney U test. (C) Examples of EGFR:GRB2 PLA status across multiple histologies. Each column represents a single patient specimen. The frequency of high EGFR:GRB2 scores was higher in metastatic brain lesions (P < 0.0001, two-tailed Fisher’s exact test). (D) Relationship of EGFR:GRB2 PLA status with the indicated genotypes and with EGFR AQUA in the MCC2 cohort. * indicates P = 0.0112 (EGFR mutant), P < 0.0001 (KRAS mutant), P < 0.0001 (EGFR wild type), two-tailed Mann-Whitney U tests. (E) Survival curves for the MCC3 cohort stratified according to EGFR:GRB2 PLA or EGFR AQUA. P = 0.045 (EGFR:GRB2 PLA), P = 0.81 (EGFR AQUA), log-rank test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/359/ra4/DC1

    Fig. S1. PLA assay schematic.

    Fig. S2. Phosphorylated Tyr1068 EGFR in xenografts.

    Fig. S3. Distribution of PDX tumors.

    Fig. S4. Distribution of PDX tumors with cetuximab response data.

    Fig. S5. Interrater reliability and variability between duplicate cores.

    Fig. S6. Variability of EGFR:GRB2 PLA signal among EGFR mutant patients.

    Fig. S7. Overall survival analysis with extended follow-up.

  • Supplementary Materials for:

    Annotation of human cancers with EGFR signaling–associated protein complexes using proximity ligation assays

    Matthew A. Smith, Richard Hall, Kate Fisher, Scott M. Haake, Farah Khalil, Matthew B. Schabath, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Soner Altiok, Yian Ann Chen, Eric B. Haura*

    *Corresponding author. E-mail: eric.haura{at}moffitt.org

    This PDF file includes:

    • Fig. S1. PLA assay schematic.
    • Fig. S2. Phosphorylated Tyr1068 EGFR in xenografts.
    • Fig. S3. Distribution of PDX tumors.
    • Fig. S4. Distribution of PDX tumors with cetuximab response data.
    • Fig. S5. Interrater reliability and variability between duplicate cores.
    • Fig. S6. Variability of EGFR:GRB2 PLA signal among EGFR mutant patients.
    • Fig. S7. Overall survival analysis with extended follow-up.

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    Citation: M. A. Smith, R. Hall, K. Fisher, S. M. Haake, F. Khalil, M. B. Schabath, V. Vuaroqueaux, H.-H. Fiebig, S. Altiok, Y. A. Chen, E. B. Haura, Annotation of human cancers with EGFR signaling–associated protein complexes using proximity ligation assays. Sci. Signal. 8, ra4 (2015).

    © 2014 American Association for the Advancement of Science

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