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

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, these low-throughput assays often produce discordant or equivocal results. Methods We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/ug) 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 cut-offs to identify HER2 gene amplification. Results After demonstrating assay development, precision, and stability, Her2-SRM protein 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 protein was detected in 71.2% of cases. ROC curves demonstrated Her2-SRM protein 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 to identify HER2 FISH amplified tumors. An ‘equivocal-zone’ of 450-750 amol/ug of Her2-SRM protein was analogous to ’IHC2+#x2019;, but represented fewer cases (9-16% of cases versus 36-41%). Conclusions 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 the multiplex capability for other relevant oncoproteins, these results demonstrated a refined HER2 protein expression assay for clinical application.

MET tyrosine kinase receptor expression and amplification as prognostic biomarkers of survival in gastroesophageal adenocarcinoma

“…Protein concentrations in the remaining 51 samples (18.1%) ranged from 200-3245.5 amol/μg. 11,27 Using previous established expression levels in an independent cohort, 11 and minimum p-value and hazard modeling, 400 amol/μg was tested and established as the cut-off for ‘Met-positive’ expression, as this value was determined to have the most significant effect on OS (Figure 4A). As such, 22 tumors (7.8%) were classified as Met-positive in this study.…”

“…11 This is also quite analogous to HER2 amplification, as recently described. 27 Therefore, the ease of categorizing to binary subsets (≥ or < 2 ratio) should be weighed against the weaker observed correlation with expression when combining higher and lower FISH ratios within one category. This may have implications regarding the strength of FISH as a prognostic and predictive biomarker.…”

“…SRM is a novel technology demonstrating clinical utility with Met and Her2, 11,27,39 additionally having the advantage of multiplex peptide analysis, 22 analogous to NGS for genomic aberrations. The results of this study suggest that amplification by FISH and/or overexpression by SRM should be incorporated into multivariate models when assessing the prognostic significance of other novel covariates.…”

Novel Targeted Therapies for Esophagogastric Cancer

“…Higher throughput assays, including mass spectrometry and next-generation sequencing (NGS), have emerged with the potential to refine diagnostic accuracy as well as possessing multiplexing capability to assess for other relevant aberrations. 5,20,21 Assessment of ERBB2 amplification by cell free DNA (cfDNA) is also emerging as a potential non-invasive strategy, potentially for serial assessment of ERBB2 status over time in a non-invasive manner, 22 given reports supporting the concept of tumor evolution including the potential loss of ERBB2 expression over time. 21,23–25 …”

“…Recently, the degree of amplification has been shown to correlate closely with absolute protein expression level, again closely associated with clinical benefit. 20,29 The variations in absolute amplification/expression across various trials, as well as lack of antibody-dependent cell-mediated cytotoxicity (ADCC) with lapatinib as compared to trastuzumab, serve as two of many potential explanations when contrasting outcomes of ToGA and LOGiC. In the second line, the phase III Asian TyTAN trial enrolled patients regardless of HER2 expression (FISH ≥2 were eligible), where 31% of patients enrolled were FISH+ and IHC 0/1+.…”