PT  - JOURNAL ARTICLE
AU  - Daniel V.T. Catenacci
AU  - Wei-Li Liao
AU  - Lei Zhao
AU  - Emma Whitcomb
AU  - Les Henderson
AU  - Emily O’Day
AU  - Peng Xu
AU  - Sheeno Thyparambil
AU  - David Krisman
AU  - Kathleen Bengali
AU  - Jamar Uzzell
AU  - Marlene Darflur
AU  - Fabiola Cecchi
AU  - Adele Blackler
AU  - Yung-Jue Bang
AU  - John Hart
AU  - Shu-Yuan Xiao
AU  - Sang Mee Lee
AU  - Jon Burrows
AU  - Todd Hembrough
TI  - Mass spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: Comparison to IHC and FISH
AID  - 10.1101/018747
DP  - 2015 Jan 01
TA  - bioRxiv
PG  - 018747
4099  - http://biorxiv.org/content/early/2015/04/30/018747.short
4100  - http://biorxiv.org/content/early/2015/04/30/018747.full
AB  - 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.