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

Gastric Cancer. 2016 Oct;19(4):1066-1079. doi: 10.1007/s10120-015-0566-0. Epub 2015 Nov 18.


Background: Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results.

Methods: We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/μg) of Her2-SRM protein in cell lines (n = 27) and GEC tissues (n = 139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cutoffs in order to identify HER2 gene amplification.

Results: After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with the HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM expression of the covariates Met, Egfr, Her3, and HER2 heterogeneity, as well as their interactions (cell lines r (2) = 0.9842; FFPE r (2) = 0.7643). In GEC tissues, Her2-SRM protein was detected at any level in 71.2 % of cases. ROC curves demonstrated that Her2-SRM protein levels have a high specificity (100 %) at an upper-level cutoff of >750 amol/µg and sensitivity of 75 % at a lower-level cutoff of <450 amol/μg for identifying HER2 FISH-amplified tumors. An "equivocal zone" of 450-750 amol/µg of Her2-SRM protein was analogous to IHC2+ but represented fewer cases (9-16 % of cases versus 36-41 %).

Conclusions: Compared to IHC, targeted SRM-Her2 proteomics provided more objective and quantitative Her2 expression with excellent HER2/CEP17 FISH correlation and fewer equivocal cases. Along with its multiplex capability for other relevant oncoproteins, these results demonstrate a refined HER2 protein expression assay for clinical application.

Keywords: Clinical biomarker assay; Companion diagnostic; Esophageal; Gastric; Gastroesophageal adenocarcinoma; HER2 (ERBB2) amplification; Her2 expression; Multiplex protein expression analysis in FFPE tissue; SRM-MS; Selected reaction monitoring mass spectrometry; Stomach cancer.

Publication types

  • Comparative Study

MeSH terms

  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / metabolism
  • Gene Amplification
  • Humans
  • Immunoenzyme Techniques
  • In Situ Hybridization, Fluorescence / methods*
  • Proteomics / methods*
  • Receptor, ErbB-2 / genetics*
  • Receptor, ErbB-2 / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Stomach Neoplasms / genetics*
  • Stomach Neoplasms / metabolism*
  • Stomach Neoplasms / pathology


  • Biomarkers, Tumor
  • ERBB2 protein, human
  • Receptor, ErbB-2