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.
Footnotes
Grant Support: This work was supported by NIH K12 award (CA139160-01A), NIH K23 award (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, and the Sal Ferrara II Fund for PANGEA (to D.V.T.C).
Nomenclature: HER2 - reference to the pathway in general; HER2 - reference to the gene; Her2 - reference to the protein.