Mass spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: Comparison to IHC and FISH

BackgroundTrastuzumab showed survival benefit for Her2-positive gastroesophageal cancers (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, both assays are low throughput with various limitations.\n\nMethodsWe developed a selected reaction monitoring mass spectrometric (SRM-MS) assay and quantified levels (amol/ug) of Her2-SRM in cell lines (n=27) and GEC tissues (n=139). We compared Her2-SRM expression with IHC/FISH, seeking to determine optimal SRM expression cut-offs to identify HER2 amplification.\n\nResultsAfter demonstrating assay development, precision, and stability, Her2-SRM measurement was observed to be highly concordant with HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM-expression of Met, Egfr, Her3, and HER2-heterogeneity covariates, and their interactions (cell lines r2=0.9842; FFPE r2=0.7643). In GEC tissues, Her2-SRM was detected in 71.2% of cases, and 12.3% were identified as HER2+. ROC curves demonstrated HER2-SRM levels to have high specificity (100%) at an upper-level cut-off of >750 amol/g and sensitivity (75%) at lower-level cut-off of <450 amol/ug. We observed an equivocal-zone between 450-750 amol/ug, analogous to IHC2+, but less frequent (9-16% of cases versus 36-41%).\n\nSignificanceCompared to IHC, SRM-MS provided more objective and quantitative Her2 expression with excellent HER2/CEP17 FISH correlation and fewer equivocal cases. Along with the multiplex capability for other relevant oncoproteins, these results demonstrated a refined HER2 expression assay for clinical application.