Journal
GASTRIC CANCER
Volume 19, Issue 4, Pages 1066-1079Publisher
SPRINGER
DOI: 10.1007/s10120-015-0566-0
Keywords
Her2 expression; HER2 (ERBB2) amplification; Gastric; Esophageal; Gastroesophageal adenocarcinoma; Stomach cancer; SRM-MS; Selected reaction monitoring mass spectrometry; Companion diagnostic; Clinical biomarker assay; Multiplex protein expression analysis in FFPE tissue
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Funding
- NIH [CA139160-01A, CA178203-01A1]
- UCCCC (University of Chicago Comprehensive Cancer Center) Award in Precision Oncology-CCSG (Cancer Center Support Grant) [P30 CA014599]
- Cancer Research Foundation Young Investigator Award
- ALLIANCE for Clinical Trials in Oncology Foundation Young Investigator Award
- Oncoplex Dx Collaborative Research Agreement
- LLK (Live Like Katie) Foundation Award
- Sal Ferrara II Fund for PANGEA
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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. We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/mu g) 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. 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/A mu g and sensitivity of 75 % at a lower-level cutoff of < 450 amol/mu g for identifying HER2 FISH-amplified tumors. An equivocal zone of 450-750 amol/A mu g of Her2-SRM protein was analogous to IHC2+ but represented fewer cases (9-16 % of cases versus 36-41 %). 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.
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