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bioRxiv · 10.1101/018747

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

Daniel Catenacci·Wei-Li Liao·Lei Zhao·Emma Whitcomb·Les Henderson·peng Xu·Emily O'Day·Sheeno Thyparambil·Kathleen Bengali·Jamar Uzzell·Marlene Darflur·Fabiola Cecchi·Adele Blackler·David Krisman·Yung-Jue Bang·John Hart·Shu-Yuan Xiao·Sang Mee Lee·Jon Burrows·Todd Hembrough
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Abstract

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.

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Daniel Catenacci, Wei-Li Liao, Lei Zhao, Emma Whitcomb, Les Henderson, peng Xu, Emily O'Day, Sheeno Thyparambil, Kathleen Bengali, Jamar Uzzell, Marlene Darflur, Fabiola Cecchi, Adele Blackler, David Krisman, Yung-Jue Bang, John Hart, Shu-Yuan Xiao, Sang Mee Lee, Jon Burrows, Todd Hembrough. 2015-04-30. Mass spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: Comparison to IHC and FISH. https://doi.org/10.1101/018747

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