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. 2012 Dec 1;18(23):6426-35.
doi: 10.1158/1078-0432.CCR-12-0452. Epub 2012 Oct 8.

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

Julia D Wulfkuhle  1 , Daniela Berg, Claudia Wolff, Rupert Langer, Kai Tran, Julie Illi, Virginia Espina, Mariaelena Pierobon, Jianghong Deng, Angela DeMichele, Axel Walch, Holger Bronger, Ingrid Becker, Christine Waldhör, Heinz Höfler, Laura Esserman; I-SPY 1 TRIAL Investigators; Lance A Liotta, Karl-Friedrich Becker, Emanuel F Petricoin 3rd
Affiliations

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

Julia D Wulfkuhle et al. Clin Cancer Res. .

Abstract

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.

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Conflict of interest statement

Conflicts of Interest:

Stock ownership in Theranostics Health, LLC: (JDW, VE, MP,LAL, EFP);

Uncompensated consultancy Theranostics Health, LLC: (JDW, VE, LAL, EFP);

Advisory role in Theranostics Health, LLC: (LAL, EFP)

Figures

Figure 1
Figure 1. Correlation of RPMA measurements of HER2 and pHER2 protein levels with IHC and FISH results for frozen breast tumor tissue specimens and identification of a HER2-/pHER2+ subpopulation of tumors
A. RPMA measurements of HER2 protein expression in frozen, microdissected breast tumor epithelium were plotted against reported FISH status (left panel) or IHC results (right panel). A threshold intensity value that generated no false-positive values in either data set is shown as a dashed line. B. pHER2 relative intensity values measured by RPMA are plotted against FISH status (left) or IHC status (right) for frozen, microdissected tissue samples. A tentative threshold pHER2 intensity value of 0.15 is indicated by a dashed line. C. Immunoblot validation of pHER2 RPMA relative expression levels in frozen, microdissected tissues. Samples of microdissected tumor cells from 4 HER2- tumors (–4) and one HER2+ tumor (5) were probed with antibody against pHER2(Y1248) to verify relative protein expression level differences measured in the same cases by RPMA. SKBR3 and MD-MBA-231 cell lysates were run as high and low level expression controls, respectively, and serve as an internal control for antibody specificity. Immunoblotting of beta-actin was used for normalization of protein loading. Central FISH and IHC status for HER2 as well as HER2 status by RPMA measurements for each tumor are shown. Immunoblot images are cropped for clarity.
Figure 2
Figure 2. Correlation of RPMA measurements of HER2 and pHER2 protein levels with IHC and FISH results for FFPE breast tumor tissue specimens and identification of a HER2-/pHER2+ subpopulation of tumors
A, B. HER2 concentration thresholds optimally distinguishing HER2 IHC+ (HER2 IHC score 3+; 2+/FISH+) from HER2 IHC- (HER2 IHC score 0; 1+; 2+/FISH-) tumor samples were established within training sets for FFPE surgical specimens (A) (left panel; HER2 concentration threshold 12pg HER2/ng protein), as well as core biopsies (B) (left panel; HER2 concentration threshold 7pg HER2/ng protein). Threshold values were validated using independent tissue samples for each study set (right panels). Thresholds for positive and negative samples are marked by horizontal dashed lines. C. pHER2 relative intensity values measured by RPMA are plotted against HER2 IHC status for the FFPE surgical validation study set. A tentative threshold intensity value of 1000RU is indicated by a dashed line. RU=relative units.
Figure 3
Figure 3. Co-activation of various HER family receptors with activation of HER2
Unsupervised hierarchical clustering of various activated HER family relative expression levels in FFPE surgical validation specimens (A) or frozen, microdissected breast tumor tissues (B). Green case labels indicate IHC+/FISH+, black indicate IHC-/FISH-, red indicate IHC-/FISH borderline tumors (horizontal axis). Endpoints examined are clustered on the vertical axis. Black arrows indicate IHC-/FISH-/pHER2+ tumors. Within the heatmap, red color represents higher levels of relative activity/expression; black represents intermediate levels and green represents lower levels of relative activity/expression.
Figure 4
Figure 4. The IHC-/pHER2+ subpopulation of frozen, microdissected tumors demonstrate full HER-signaling pathway activation
(A) Pathway diagram of selected HER signaling pathway downstream proteins measured in frozen-LCM tissues. (B) Comparison of HER pathway activation levels of the proteins displayed in (A) between the IHC-/pHER2- cohort (N=87), IHC-/pHER2+ cohort (N=9) and IHC+/pHER2+ cohort (N=28) P-value results for ANOVA multiple mean comparisons are shown, with statistically different cohorts marked by an asterisk (*).
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