Met activation in non-small cell lung cancer is associated with de novo resistance to EGFR inhibitors and the development of brain metastasis - PubMed (original) (raw)

. 2010 Jul;177(1):415-23.

doi: 10.2353/ajpath.2010.090863. Epub 2010 May 20.

Lynette M Sholl, Michael Peyton, John Reilly, Christopher Ware, Lenora Davis, Natalie Vena, Dyane Bailey, Beow Y Yeap, Michelangelo Fiorentino, Azra H Ligon, Bo-Sheng Pan, Victoria Richon, John D Minna, Adi F Gazdar, Giulio Draetta, Silvano Bosari, Lucian R Chirieac, Bart Lutterbach, Massimo Loda

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Met activation in non-small cell lung cancer is associated with de novo resistance to EGFR inhibitors and the development of brain metastasis

Elisa Benedettini et al. Am J Pathol. 2010 Jul.

Abstract

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.

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Figures

Figure 1

Figure 1

Met expression and phosphorylation in EGFR mutation containing NSCLC tumors are associated with poor response to EGFR TKI therapy. Kaplan–Meier curves of progression-free survival (PFS) for Met and Y1234/35 Met in 23 NSCLC patients before EGFR inhibitor therapy (A), and the subset of patients (n = 10) harboring EGFR mutations (B). C: Top panels (EGFR TKI nonresponsive) indicate elevated Met and Y1234/35 Met in the tumor specimen of a nonresponsive patient (Patient eight, Table one, PFS of three months); bottom panels (EGFR TKI responsive) indicate undetectable Met and Y1234/35 Met in the tumor from a responsive patient tumor (PFS = 40 months, patient five, Table one). FISH analysis revealed low Met copy gain in both patients (Table 1).

Figure 2

Figure 2

A: Gefitinib-induced growth inhibition of HCC827 is rescued by HGF. Gefitinib (1 μmol/L) addition was followed by addition of 50 ng/ml HGF. SU11274 (2 μmol/L) addition blocked HGF-mediated rescue. HGF rescue was maintained in 0.1% serum as indicated. Cell growth was measured after four days of treatment and is shown relative to untreated cells. Data represent mean ± SEM. ***P < 0.001. B: HGF restores signal transduction pathways in Gefitinib-treated cells. Gefitinib (1 μmol/L) was added to HCC827 cells, followed by addition of 50 ng/ml HGF. Seventy-two hours later, lysates were prepared and analyzed by SDS-PAGE and Western blotted for the indicated phospho proteins.

Figure 3

Figure 3

Colocalization of Met phosphorylation and EGFR mutation. A and B: Exon 19 EGFR deletion, detected with a delEGFR-specific antibody. C and D: Met Y1234/35 phosphorylation. IHC images on consecutive sections (patient 2, Table 1) reveal colocalization of delEGFR and Y1234/35 Met. Arrowheads indicate regions of positivity for both antibodies; arrows indicate regions harboring EGFR deletion but not Met activation. B and D are magnification (×400) of A and C (×100), respectively.

Figure 4

Figure 4

Met expression, phosphorylation, and gene copy gain are enriched in brain metastases relative to their paired NSCLC. A: Met and Y1234/35 Met were assessed by immunohistochemistry (H score) in primary NSCLCs and paired brain metastases. Both Met expression (P = 0.003) and Y1234/35 Met phosphorylation (P = 0.031) are significantly upregulated in metastases. Data represent mean ± SEM. B: Prevalence of MET copy gain (factoring MET copy number gain and % positive cells as in Materials and Methods) is higher in the metastatic lesions relative to the paired primary NSCLCs (P = 0.024). C: Met IHC (magnification ×200) and FISH analysis on a primary NSCLC and its paired brain metastasis (patient 10, Table 1). Met and Y1234/35 Met staining was heterogeneous/focal in the primary cancer but more widespread in the paired brain metastasis. MET FISH reveals low copy gain (MET copies, n = 3 to 5) in the primary lung tumor, and high copy gain (MET copies, n = 6 to 10) in the metastatic lesion.

Figure 5

Figure 5

MET is amplified and constitutively activated in the metastasis-derived H1993 cell line but not in the paired primary lung tumor–derived H2073 cell line. A: Quantitative PCR for MET copy number. MET is not amplified in H2073 cells but is amplified in H1993 cells. Data represent mean ± SEM. Immunoblot reveals elevated Met expression and Y1234/35 and Y1349 phosphorylation in H1993 cells but not in H2073 cells. B-actin is the loading control. B: Growth of H2073 cells is not inhibited by Met shRNA, whereas H1993 cell growth is inhibited. Data represent mean ± SEM. Immunoblot revealing efficient Met knockdown in H2073 and H1993 cells treated with Met shRNA M3 and less efficient knockdown with Met shRNA M2 and M1. L indicates Luciferase shRNA; 0, no shRNA. C: Migration assay was performed in the indicated cell lines as described in Methods. 0, initial time point; 72, 72 hours later; SU, addition of 2.5 μmol/L SU11274 Met inhibitor. Data represent mean ± SEM. *P < 0.5, ***P < 0.001. Results are representative of three independent experiments.

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