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Updated: May 17, 2026
Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers
Published on: December 5, 2017
Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping
Julia D Wulfkuhle1, Daniela Berg, Claudia Wolff
1Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia 20110, USA.
Purpose:
Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.
Experimental Design:
Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC).
Results:
RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC(+) population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC(-) tumors and, identical to that seen with FISH/IHC(+) tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation.
Conclusions:
Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC(-)/FISH(-)/pHER2(+) tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.
Insights
This study shows that some breast cancers with low HER2 protein levels still exhibit active HER2 signaling. This activation can be detected using reverse-phase protein microarrays (RPMA) and is linked to downstream signaling.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Targeting HER2 (Human Epidermal growth factor Receptor 2) protein is crucial in breast cancer treatment.
- Current methods often measure total HER2, not its signaling network activity, potentially missing key activation pathways.
Purpose of the Study:
- To investigate the correlation between phosphorylated and total HER2 levels.
- To understand HER2 signaling network activation in breast cancer using reverse-phase protein microarrays (RPMA).
- To assess HER2 pathway activation in tumors with varying HER2 expression levels.
Main Methods:
- Analyzed 415 patient samples (frozen and FFPE) using RPMA.
- Measured total and phosphorylated levels of HER receptor family proteins and downstream signaling molecules.
- Compared RPMA results with fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC).
Main Results:
- RPMA measurements of total HER2 highly correlated with FISH/IHC data (>90%).
- HER2 phosphorylation was observed in FISH/IHC-negative tumors.
- HER2 activation in these tumors correlated with EGFR and HER3 phosphorylation and downstream signaling.
Conclusions:
- Molecular profiling using RPMA identified HER2 pathway activation in tumors negative for HER2 by IHC/FISH but positive for phosphorylated HER2 (pHER2).
- This activation is independent of total HER2 levels.
- Functional signaling through HER3 and EGFR was observed in these pHER2(+) tumors.
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