Abstract
16 Background: Overexpression of MET in gastroesophageal cancer (GEC) is associated with poor prognosis and potentially predictive of anti-MET therapeutic benefit. IHC has been the method chosen to quantify MET expression to date. However, IHC is semi-quantitative, suffers from cross-reactivity, and is low throughput. Moreover, MET IHC is hampered by antigenic instability in FFPE sections, limiting its utility to recently cut FFPE sections. Increasing recognition of the importance of other biomarkers in GEC suggests that ‘economic’ testing of scarce samples will be required. We sought to develop a MET quantitative assay within our ‘GEC-plex’ Liquid-Tissue-selected reaction monitoring (LT-SRM) MS test. Methods: We used trypsin digestion mapping of rMET to identify unique peptides for MS assay development. The assay was pre-clinically validated in 5 cell lines, where electrochemiluminescence (ECL) immunoassay measurement of MET was also performed. To assess the MET MS assay stability from archival FFPE sections, freshly cut FFPE tissue sections were immediately microdissected, processed and analyzed, while adjacent sections were processed and analyzed one year after cutting, to compare temporal quantification from the same FFPE samples (n=33). MET expression was assessed in GEC cases (n=121), and compared to IHC and FISH in select cases. Results: Tryptic digestion mapping of rMET showed that peptide TEFTTALQR was optimal for MET quantification. The LLOD for this peptide was 150 amol with CV<20%. Validation of the MET MS assay on 5 cell lines revealed concordance when compared to ECL (R2=0.99). The MET MS assay demonstrated temporal stability of serial sections cut from 33 samples analyzed one year apart: CVs<20%, R2=0.75. Analysis of 121 GEC FFPE tissues showed a broad range of MET expression levels (<150-4600 amol/ug), with 36/121 (29.7%) having detectable levels, similar to that observed using IHC. MS expression thresholds were determined that reliably identified MET gene amplification; sensitivity and specificity of these thresholds will be presented. Conclusions: ‘GEC-plex’ has a quantitative, sensitive, and specific MET MS assay that can be multiplexed along with other GEC biomarkers.
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