Background: 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. The CELx HER2 Signaling Function (CELx HSF) Test measures HER2 signaling activity in live tumor cells using a label-free impedance biosensor to identify HER2-negative breast cancer patients likely to be responsive to treatment with anti-HER2 therapies. Previous studies quantified HER2-driven signaling activity in a training set (N=34) of primary tissue samples from HER2-negative breast cancer patients and found 21% of the samples had abnormal HER2 signaling. Other studies confirmed that anti-HER2 therapies, such as trastuzumab, pertuzumab, afatinib, and neratinib, are as effective in inhibiting HER2-driven signaling activity in HER2- tumor cells as they are in HER2+ tumor cells. This study set out to confirm the prevalence of abnormal HER2 signaling amongst HER2-negative breast cancer patients in a larger sample (N=114) and to characterize the sensitivity and specificity of the CELx HSF Test.

Methods: A validation set of de-identified fresh breast tumor specimens were obtained from 114 HER2- breast cancer patients. Real time live cell response to specific HER2 agonists (NRG1b or EGF) with or without an antagonist (HER2 dimerization inhibitor) was measured using an impedance biosensor. From these responses, the net amount of HER2 participation in HER2 signaling initiated by the HER2 agonists was quantified. Samples with HER2 signaling activity levels above a previously determined cut-off value were identified as abnormal.

Results: Of the HER2- breast tumor cell samples tested, 27 of 114 patients (23.7%; 95% CI=17%-32%) had abnormal HER2 signaling activity. Little or no correlation was found between a patient's HER2 signaling activity and their estrogen receptor status or tumor grade. To compare the results obtained from the training set of 34 patients and the current set of 114 patients, the Kolmogorov-Smirnov two-sample test was applied (D=0.17, P-value 0.45) and found no significant difference between the training and validation sets. A normal mixture model was fitted to the new 114 patient data set and found that HER2- breast cancer patients fall into three distinct groups (abnormal, normal, low). Patients falling into the abnormal group had mean HER2 signaling scores 4.5 standard deviations above the mean score of the normal group. A ROC curve constructed with this data projects that both the sensitivity and specificity of the CELx HSF Test would be greater than 90%.

Conclusions: These results confirm that a clinically relevant proportion of HER2- breast cancer patients, approximately 20%, have tumors with abnormal HER2-signaling activity and may benefit from HER2 therapy. With high specificity and sensitivity, the CELx HSF test may be suitable as a companion diagnostic to identify new patients eligible to receive HER2 therapies. An interventional trial to evaluate the efficacy of trastuzumab and pertuzumab in HER2- patients selected with the CELx HSF test is underway.

Citation Format: Laing LG, Burns DJ, MacNeil IA, Rich BE, Myhre S, Soltani S, Sullivan BF. Use of a functional signal profiling test with high sensitivity and specificity to determine the prevalence of abnormal HER2-driven signaling activity in the HER2-negative breast cancer patient population: New patient group may benefit from HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-22.