MET Copy Number by Dual Color Brightfield In Situ Hybridization and Fluorescence In Situ Hybridization Correlates with Met Protein Expression in Lung Adenocarcinomas
Lynette Sholl, Heather Sun, Azra Ligon, Mohit Butaney, Pasi A Janne, Scott Rodig. Brigham and Women's Hospital, Boston, MA; Dana Farber Cancer Institute, Boston, MA
Background: Met pathway activation drives resistance to EGFR tyrosine kinase inhibitors in some patients with EGFR-mutated lung adenocarcinoma (ACA). Mechanisms of Met pathway activation include MET amplification, HGF-mediated signaling, and EGFR crosstalk. MET amplification and/or Met protein overexpression may predict response to combination EGFR and Met inhibitor therapy. This study correlates MET copy number (CN) by fluorescence and dual color in situ hybridization (FISH and ISH) with protein expression by immunohistochemistry (IHC) in a cohort of lung ACA enriched for EGFR mutations.
Design: Studies were performed on 4um formalin-fixed paraffin embedded tissue sections from 32 patients with advanced lung ACA and known EGFR mutation status (66% EGFR-mutated). MET CN was assessed by FISH using probes to the MET locus and CEN7 and by dual color ISH using probes to the MET locus and CEN7 with dual color open probe software (Ventana Medical Systems, Tucson, AZ). Met IHC was performed using Met antibody clone SP44 on the Benchmark XT with ultraView DAB detection per manufacturer's instructions (Ventana). MET CN was categorized by signals/cell as disomic (1-2), low (3-5) or high polysomy (6-9), or amplified (≥10 and/or MET signal clusters). IHC was scored from 0 as absent to 4 as strong membranous and cytoplasmic staining; populations of differing intensity were multiplied by % of cells staining and summed for an H score ranging from 0 to 400.
Results: FISH and ISH for MET CN were highly correlated (Spearman's ρ=0.72, p<0.0001). By ISH, 12 cases were disomic, 5 had low and 8 had high polysomy, and 7 were MET amplified with gene clusters. Gene amplification consistently occurred on a background of polysomy and was associated with significantly higher protein expression compared to tumors with disomy or low or high polysomy (mean H scores 354 vs. 165, 165, and 221, respectively; ANOVA p=0.0002). However, one EGFR WT tumor was MET disomic and had an IHC H score of 380.
Conclusions: FISH and dual-color brightfield ISH are comparable for assessing MET CN on FFPE tissue slides. MET CN gain is common in a cohort of lung ACA enriched for EGFR mutations, and MET amplification is highly correlated with strong cytoplasmic and membranous Met protein expression. Of note, strong Met expression can occur in the absence of MET amplification, an observation that suggests other mechanisms of upregulation, and may have implications for identification of patients for targeted Met therapy.
Monday, March 4, 2013 1:00 PM
Poster Session II # 286, Monday Afternoon