Abstract P6-07-14: New method to measure functional HER2-driven signaling activity in primary tumor cells identifies HER2-negative breast cancers with abnormal HER2 signaling activity: New group of patients may benefit from anti-HER2 therapy
Authors: Laing L , Burns D , MacNeil I , Rich B , Huang Y , Myhre S , Soltani S , Sullivan B 
Published: May 2019

Abstract
Background: Clinical trials have indicated a weak correlation between HER2 expression levels and HER2 targeted therapy benefit. Other biological factors, such as HER2 signaling activity, may be important to measure, in addition to expression and amplification of HER2, when identifying patients eligible for HER2 therapies. To measure the HER2-driven signaling activity of a patient's tumor cells, a new assay using an impedance biosensor, the CELx HER2 Signaling Profile (CELx HSP) Test, was developed. This study set out to provide an initial assessment of the CELx HSP Test, specifically to: 1) quantify HER2-driven signaling activity (HER2S) in cell lines and primary epithelial cells; 2) assess the correlation between HER2 expression levels and HER2 signaling activity; 3) define a preliminary cut-point between normal and abnormal HER2 signaling; and 4) estimate the proportion of HER2- primary breast cancer tumors with abnormal HER2 signaling.
Methods: A training set of de-identified fresh breast tissue specimens was obtained from 50 patients, 34 with HER2- breast cancer (IHC 0 or 1+) and 16 healthy patients. Cell samples were comprised of epithelial cells extracted and cultured from each specimen. Reference human breast cancer cell lines (9 HER2+, 10 HER2-) were also tested, including two cell lines used as controls in IHC HER2 tests. Real time live cell response to specific HER2 agonists (NRG1b or EGF) and with or without an antagonist (pertuzumab, an FDA-approved HER2 dimerization inhibitor) was measured and quantified using an xCELLigence RTCA impedance biosensor (ACEA Biosciences, San Diego, CA). From these responses, the net amount of HER2 participation in HER2 signaling initiated by the HER2 agonists (“HER2S”) was determined. Fluorescence cytometry was used to measure HER2 expression levels of each cell sample.
Results: Of the HER2– breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI=10%-37%) had net HER2 signaling activity that was greater than the median HER2S of the HER2+ cell lines. There was no categorical correlation between HER2 IHC status (+ or -) and HER2 signaling activity (abnormal or normal) (Pearson's Chi-Square = 3.68; Phi Max = -0.78, Contingency Coefficient 0.28). The median HER2S, or net HER2 signaling activity, was comparable for the HER2- tumor, HER2- cell line, and the healthy patient samples (Md = 100, 117, 77, respectively). The median HER2S in HER2+ cell lines (Md = 248) is approximately 2.5-3.0 fold greater than the median of the other groups. A HER2S above 250 was considered abnormal or test positive, and was defined as the cut-point. The HER2S for the two IHC HER2 test control cell lines, SKBR3 for HER2+ and MDA-MB231 for HER2-, was 544 and 0.
Conclusions: These findings provide strong evidence that measurement of HER2 signaling activity may provide clinically relevant information, particularly for HER2- breast cancer patients. These results suggest a new group of HER2- breast cancer patients with abnormal HER2-driven signaling may benefit from anti-HER2 therapy. Additional studies are underway to confirm these findings and to analytically validate the CELx HSP test.
Citation Format: Laing L, Burns D, MacNeil I, Rich B, Huang Y, Myhre S, Soltani S, Sullivan B. New method to measure functional HER2-driven signaling activity in primary tumor cells identifies HER2-negative breast cancers with abnormal HER2 signaling activity: New group of patients may benefit from anti-HER2 therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-14.