Improved diagnostics targeting c-MET in non-small cell lung cancer: expression, amplification and activation?

Abstract

Background: Several c-MET targeting inhibitory molecules have already shown promising results in the treatment of patients with Non-small Cell Lung Cancer (NSCLC). Combination of EGFR- and c-MET-specific molecules may overcome EGFR tyrosine kinase inhibitor (TKI) resistance. The aim of this study was to allow for the identification of patients who might benefit from TKI treatments targeting MET and to narrow in on the diagnostic assessment of MET.

Methods: 222 tumor tissues of patients with NSCLC were analyzed concerning c-MET expression and activation in terms of phosphorylation (Y1234/1235 and Y1349) using a microarray format employing immunohistochemistry (IHC). Furthermore, protein expression and MET activation was correlated with the amplification status by Fluorescence in Situ Hybridization (FISH).

Results: Correlation was observed between phosphorylation of c-MET at Y1234/1235 and Y1349 (spearman correlation coefficient rs = 0.41; p < 0.0001). No significant correlation was shown between MET expression and phosphorylation (p > 0.05). c-MET gene amplification was detected in eight of 214 patients (3.7%). No significant association was observed between c-MET amplification, c-MET protein expression and phosphorylation.

Conclusion: Our data indicate, that neither expression of c-MET nor the gene amplification status might be the best way to select patients for MET targeting therapies, since no correlation with the activation status of MET was observed. We propose to take into account analyzing the phosphorylation status of MET by IHC to select patients for MET targeting therapies. Signaling of the receptor and the activation of downstream molecules might be more crucial for the benefit of therapeutics targeting MET receptor tyrosine kinases than expression levels alone.

Fig. 1

Examples of NSCLC tissues stained for immunohistochemical quantification of MET differences between FFPE- and HOPE-fixed tumor samples, stained with MET specific antibody clone SP44 (original magnification x 400). a + b) same specimen. a FFPE: MET expression scored 0; b HOPE fixed sample; MET expression scored 1+. (c + d) one specimen. c FFPE: MET expression scored 1+; d HOPE fixed sample; MET expression scored 2+. (e + f) one specimen. e FFPE: MET expression scored 2+; f HOPE fixed sample; MET expression scored 3+

Fig. 2

a Distribution of positive and negative MET expression of NSCLC samples. b Intersection of MET positive Formalin- and HOPE- fixed NSCLC samples. c Mean of MET intensities of Formalin- versus HOPE- fixed NSCLC samples

Fig. 3

Examples for immunohistochemical Analysis of activated p-MET [Y1234/1235] and [Y1349] in NSCLC tumor tissues (original magnification x 400). a Phosphorylation of Y1234/1235. b Phosphorylation of Y1349

Fig. 4

MET gene copy numbers evaluated by FISH analysis

Fig. 5

Quantification of MET. Comparison of IHC, Western Blot Analysis and Real-Time PCR