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

By Daniel V.T. Catenacci, Wei-Li Liao, Lei Zhao, Emma Whitcomb, Les Henderson, Emily O’Day, Peng Xu, Sheeno Thyparambil, David Krisman, Kathleen Bengali, Jamar Uzzell, Marlene Darflur, Fabiola Cecchi, Adele Blackler, Yung-Jue Bang, John Hart, Shu-Yuan Xiao, Sang Mee Lee, Jon Burrows, Todd Hembrough

Posted 30 Apr 2015
bioRxiv DOI: 10.1101/018747 (published DOI: 10.1007/s10120-015-0566-0)

Abstract Background: Trastuzumab 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. Methods: We 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. Results: After 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/µg 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%). Significance: Compared 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.

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