Abstract P3-07-06: Objective measurement of HER2 (ERBB2) intracellular and extracellular domain spatial co-localization stratifies benefit from adjuvant trastuzumab
Authors: Carvajal-Hausdorf DE , Toki M , Schalper KA , Pusztai L , Psyrri A , Kalogeras KT , Kotoula V , Fountzilas G , Rimm DL 
Published: May 2016

Abstract
Background: The ASCO/CAP guidelines consider chromogen-based immunohistochemistry (IHC) as the primary assay to determine HER2 status in breast cancer. U. S. Food and Drugs Administration (FDA) approved HER2 antibody assays target the protein's intracellular domain (ICD). Studies suggest that quantitative, domain-specific measurement of HER2 might predict benefit from trastuzumab therapy, further classifying traditional HER2-positive breast cancer. Here we define a method of simultaneous, objective measurement of HER2 ICD and extracellular (ECD) domains, and determine its effect on trastuzumab benefit in the adjuvant setting.
Methods: We measured co-expression of HER2 ICD and ECD using a proximity ligation assay (PLA) and quantitative immunofluorescence (QIF) in a HER2 standardization tissue microarray (TMA) with CLIA-lab defined HER2 status. Previously validated, standardized HER2 antibodies were used to detect ICD and ECD (CB11 and SP3, respectively). We determined the relationship between HER2 PLA scores, HER2 clinical status and domain-specific scores. Finally, we measured HER2 ICD/ECD PLA in 180 patients from a clinical trial of adjuvant chemotherapy followed by trastuzumab (HeCOG 10/05). Median cut-point was used to stratify patients according to HER2 PLA scores. Cut-points for HER2 ICD and ECD were obtained using Joinpoint software. All statistical tests were two-sided.
Results: In the standardization TMA, HER2 PLA levels were associated to HER2 CLIA status (P&lt;0.0001). There was a good correlation between HER2 PLA scores and HER2 ICD and ECD (R2=0.57 and R2=0.54, respectively). In trastuzumab-treated patients from HeCOG 10/05, a similarly good correlation was observed between HER2 PLA scores and HER2 ICD and ECD (R2=0.41 and R2=0.3, respectively). In univariate analysis, HER2 PLA-low status was associated with ER-positive status (P=0.005). There was no association with age, histological grade, tumor size, lymph node status and TNM stage. Although all tumors were HER2-positive, HER2 PLA-high status was significantly associated with longer 5-year disease-free survival (DFS) (log-rank P=0.036, HR=0.32, 95% CI: 0.132-0.935). HER2 PLA status was superior to ICD status (log-rank P=0.67) and numerically comparable to ECD status (log-rank P=0.049, HR=0.31, 95% CI: 0.144-0.997) to predict benefit from adjuvant trastuzumab, as previously published by our group. HER2 PLA-high status was independent predictor of better outcome in a Cox proportional hazards model including age, histological grade, ER status, tumor size, lymph node status and TNM stage.
Discussion: Using an objective, quantitative HER2 assay for synchronous, domain-specific measurement, we stratified benefit from adjuvant trastuzumab treatment in patients from a prospective cohort. Our results further support the concept that benefit from HER2 ECD-targeted therapies might be modulated by the presence of truncated HER2 protein variants and that tyrosine kinase inhibitors (ICD-directed) may be advantageous for a subset of HER2-positive patients. Furthermore, this technique that uses two antibodies has the potential to increase both sensitivity and specificity of the IHC assay to predict response to HER2 pathway inhibitors.
Citation Format: Carvajal-Hausdorf DE, Toki M, Schalper KA, Pusztai L, Psyrri A, Kalogeras KT, Kotoula V, Fountzilas G, Rimm DL. Objective measurement of HER2 (ERBB2) intracellular and extracellular domain spatial co-localization stratifies benefit from adjuvant trastuzumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-06.