Measurement of Domain-Specific HER2 (ERBB2) Expression May Classify Benefit From Trastuzumab in Breast Cancer

Abstract

Background: Studies have shown that antibodies targeting the intracellular (ICD) or extracellular domains (ECD) of human epidermal growth factor receptor 2 (HER2) are equivalent when traditional methods are used. We describe a new method to quantify ICD and ECD expression separately and assess the prognostic value of domain-specific HER2 results in patients who received adjuvant trastuzumab therapy.

Methods: We measured HER2 protein expression with quantitative immunofluorescence (QIF) in tissue microarrays (TMA) using two different antibodies targeting the ICD (CB11 and A0485) and ECD (SP3 and D8F12). We assessed the prognostic value of ICD and ECD expression in 180 patients from a clinical trial of adjuvant chemotherapy followed by trastuzumab (HeCOG 10/05). We performed an exploratory univariate domain-specific, disease-free survival (DFS) analysis and compared DFS functions with Kaplan-Meier estimates. All statistical tests were two-sided.

Results: HER2 ICD expression by QIF showed slightly higher sensitivity to predict ERBB2 (HER2) gene amplification than ECD expression, which was more specific and had higher positive predictive value. In the HeCOG 10/05 trial specimens, 15% of cases showed discordant results for ICD and ECD expression. High ECD was statistically associated with longer DFS (log-rank P = .049, HR = 0.31, 95% CI = 0.144 to 0.997), while ICD status was not. Among patients with low ECD, there was no difference in DFS by ICD status. However, when ICD was high, high ECD was statistically associated with longer DFS (log-rank P = .027, HR = 0.23, 95% CI = 0.037 to 0.82) compared with low ECD.

Conclusion: Quantitative measurements of HER2 ICD and ECD expression in breast cancer suggest a subclassification of HER2-positive tumors. Trastuzumab-treated patients with high ECD showed better DFS than patients with low ECD. This suggests differential benefit from trastuzumab therapy based on HER2 ECD expression.

Figure 1.

Standardization of human epidermal growth factor receptor 2 (HER2) protein measurement using automated, quantitative immunofluorescence. HER2 antibody assays directed against the intracellular (ICD) and extracellular (ECD) domains were tested in a HER2 standardization tissue microarray. A-D) Distribution of HER2 A0485 (ICD) scores at different dilutions. Two populations and clustering of amplified cases (black) can be observed when the optimized titer is reached (C). E-F) Distribution of HER2 CB11 (ICD) and SP3 (ECD) scores at their optimized titer. The red dotted line shows the Joinpoint cutpoint for each assay. AU = arbitrary units of fluorescence; QIF = quantitative immunofluorescence.

Figure 2.

Domain-specific human epidermal growth factor receptor 2 (HER2) measurement reveals differential expression of the protein’s intracellular (ICD) and extracellular domain (ECD) in breast cancer cases. A-B) Representative pictures of a breast cancer case from the HER2 standardization tissue microarray show intense ICD signal (CB11) in the tumor in (A), while having lower ECD signal (B) (SP3) in the same area. C-D) depict the difference in chromogen deposition using the ICD and ECD assays in the same tumor. More intense signal is observed for HER2 ICD than for ECD. Scale bars = 100 μM.

Figure 3.

Domain-specific human epidermal growth factor receptor 2 (HER2) subgrouping in breast cancer cases. Scatterplots show the HER2 intracellular (ICD [CB11]) and extracellular domain (ECD [SP3]) scores from the HER2 standardization tissue microarray (TMA) (A) and HeCOG 10/05 trial (B), along with cutpoints for each assay. Discordant ICD-high/ECD-low cases are allocated in the bottom right quadrant (10.5% and 15.1% in the HER2 standardization TMA and HeCOG 10/05, respectively).

Figure 4.

Characterization of differential human epidermal growth factor receptor 2 (HER2) intracellular (ICD) and extracellular domain (ECD) expression in HER2-positive, trastuzumab-treated patients from HeCOG 10/05. A-D) Association of differential HER2 domain expression and survival in trastuzumab-treated patients. Kaplan-Meier graphical analysis of disease-free survival according to ICD/ECD status. Low and high groups were determined using population-based cutpoints (see Methods). The number of patients at risk in each group and the respective log rank P values are indicated in the chart. All tests were two-sided. CI = confidence interval; DFS = disease-free survival; HR = hazard ratio.