A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity

Abstract

The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2−, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2− breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%–37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

New HER2-negative breast cancer subtype responsive to anti-HER2 therapy identified

“…Previous analysis of 34 HER2-negative breast tumor cell samples using the CELx HSF test indicated that 20.5% (95% CI 10.0-37.1) of these samples were HER2-/ HSFs+ (Huang et al 2016(Huang et al , 2017. Further work screening multiple HER2+ and HER2− cell lines and patient samples demonstrated that there was no significant correlation between HER2 receptor expression level and the HER2-initiated signaling activity quantified by the CELx HSF Test (Huang et al 2017).…”

“…Methods for sample collection, storage, transport, tissue extraction, and primary cell culture have been previously described (Huang et al 2016(Huang et al , 2017. Briefly, patient breast tumor tissue was delivered from the participating clinic to the laboratory in less than 30 h at 0-8 °C, accessioned, and then minced with scalpels to < 2-mm pieces and either used fresh or cryopreserved until further analysis (Unisol, Cell and Tissue Systems, Charleston, SC).…”

“…Flow cytometry was performed using a BD FACSCalibur (BD Biosciences, San Jose, CA) with cells harvested at the time of CELx HSF Test using methods previously described by others (Huang et al 2016;Lim et al 2009). Data were analyzed with FlowJo 2 software (version 10.4) (FlowJo, Ashland, OR).…”

“…The impedance value, defined as the cellular adhesion signal, or CAS, is dependent on the type and density of adhesion proteins on the cell surface and is regulated by cell response to specific perturbations such as the addition of receptor ligands. We have demonstrated that this assay precisely measures impedance response to functional real-time HER2-driven signaling activity (Huang et al 2016).…”

“…The total contribution of HER2-specific involvement in pathway signaling is determined by measuring the change in the growth factor-induced CAS in the presence of HER2specific dimerization inhibitor. A high ΔCAS is indicative of abnormally high HER2-involved signal (Huang et al 2016(Huang et al , 2017.…”