Her2 expression in gastroesophageal cancer (GEC) FFPE tissue using mass spectrometry (MS) and correlation with HER2 gene amplification.

82 Background: HER2+ GEC derived benefit from trastuzumab. Her2 IHC is semi-quantitative, subjective, and sensitive to antigen instability; HER2 FISH is laborious, expensive, and subjective. False positivity/negativity have been described. Also, these are low throughput assays; there is known molecular heterogeneity, with several putative biomarkers, and only scarce tissue to assess for each. We sought to evaluate the association of Her2 MS expression with HER2 FISH, along with other markers within the ‘GEC-plex’. Methods: We utilized a previously described unique Her2 peptide and quantification method (Hembrough et al J Clin Oncol 32,2014(suppl 3;abstr17)). The assay was run on 27 cell lines, in parallel with HER2:CEP17 FISH. Her2 expression thresholds were established for HER2 amplification using ROC curves. We adjusted for Her3, Egfr, and Met MS expression levels and sample HER2:CEP17 ratio heterogeneity in a multiple linear regression model. The model/cut-offs were then validated prospectively on GEC tissues (n=142). Results: Her2 MS on cell lines revealed concordance with FISH (HER2:CEP17) ratio (R 2 =0.75), which improved by adjusting for Her3 MS, Egfr MS, Met MS and HER2:CEP17 heterogeneity (R 2 >0.98), with significant interactions. IHC correlation with HER2:CEP17 ratio was poor (R 2 =0.12). A wide range of expression was noted within IHC3+ patients (<150-24671amol/ug). Her2 MS expression (>150 amol/ug) was seen in 72.9% (102/142) of tissues (range <150-24671amol/ug); 9.9% (14/142) had Her2 > 750 amol/ug - all were HER2 FISH amplified. 6/142 (4%) cases <500 amol/ug were clinically ‘HER2amplified’. IHC/FISH results for cases with 550-750 amol/ug demonstrated an ‘equivocal’ zone, not unlike ‘IHC 2+’, but less frequent (8/142, 5.6% vs 46/142 32.4%). Conclusions: The MS HER2 assay can be multiplexed with other GEC biomarkers. A robust model was developed that showed strong correlation of Her2 MS with HER2:CEP17 ratio, particularly when adjusting for covariates Egfr MS, Met MS, Her3 MS, and HER2 FISH heterogeneity. Future analysis will assess the established model as a tool for better prediction of therapeutic benefit from anti-Her2 therapy compared to current methods.