Development of a test that measures real-time HER2 signaling function in live breast cancer cell lines and primary cells

Abstract

Background: Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies.

Methods: A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor.

Results: The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line.

Conclusions: Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.

Keywords: Breast cancer; Breast tumor; CELx HSF Test; Cancer diagnostic; HER2-negative; HER2-positive; Oncology; Primary epithelial cells; Signaling pathway; Targeted therapeutics.

Figures

Fig. 1

Representative CI versus time-course curves for basic cell attachment. Human breast cancer BT474 cells (a) or R56 patient-derived primary breast tumor cells (b) were seeded in a sensor plate and allowed to adhere, spread, and proliferate. Impedance was recorded as Cell Index (CI) versus time for 100 h after seeding. Cell attachment, stabilization, proliferation, and confluent phases are shown as indicated. c Representative images captured by an inverted phase contrast microscope (magnification: X40) showing cell morphology of BT474 and breast cancer R56 primary cells. Scale bar, 100 μm

Fig. 2

Dose–response curves of EGF and NRG1b stimulation of HER2 signaling in SKBr3 cells. SKBr3 cells were seeded in the sensor plates and stimulated with serial titrations of a EGF (0 pM to 1200 pM) or b NRG1b (0 pM to 1350 pM). Instrument data for CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. Log plots of dose-response curves with error bars of EGF and NRG1b stimulation are shown in the insets for a and b, respectively

Fig. 3

Dose–response curves showing the effects of HER2 inhibitors on EGF- and NRG1b-directed HER2 signaling. a Neither pertuzumab nor lapatinib has significant effect on baseline cell signal determined before agonist addition. SKBr3 cells were seeded in sensor plates and treated with pertuzumab (10 μg/mL), lapatinib (200nM), or vehicle (control) 18 h prior to stimulation with NRG1 or EGF. CELx curves are displayed using Delta CI values to easily compare the relative change in signals from the time point of drug addition. The time points for drug addition and growth factor (GF) addition are indicated by black arrows. b and c SKBr3 cells were seeded in sensor plates and treated with serial titrations of lapatinib (0 nM to 3200 nM) or pertuzumab (0 μg/mL to 40 μg/mL) two hours prior to stimulation with EGF or NRG1b. Dose–response curves of drug inhibition on NRG1b and EGF-driven cell index signals are displayed

Fig. 4

The PI3K/AKT pathway significantly contributes to the ligand-driven HER2 signaling activities detected by CELx HSF tests. a and b SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the PI3K/AKT pathway inhibitor GSK1059615 (0 nM to 810 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (a) or EGF (600 pM) (b). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. Dose–response curves of GSK1059615 inhibition on NRG1b and EGF-driven HER2 signals are shown in the insets

Fig. 5

Comparison of EGF–HER2, NRG1b–HER2, and IGF-1–IGF-1R signaling systems in CELx assays. Human breast cancer T47D cells pre-seeded in sensor plates were treated with (a) pertuzumab (10 μg/mL), (b) lapatinib (200 nM), (c) linsitinib (200 nM), or (d) GSK1059615 (300 nM) two hours prior to stimulation with NRG1b (800 pM), EGF (600 pM), or IGF-1 (8 nM). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (NRG1b, EGF, or IGF-1) was added. Blue curves, unstimulated cells (baseline); Green curves, cells stimulated with ligand (NRG1b, EGF, or IGF-1); Red curves, cells stimulated with ligand in the presence of drug (pertuzumab, lapatinib, linsitinib, or GSK1059615)

Fig. 6

CELx HSF Test signals in HER2+ and HER2- breast cancer cell lines. a HER2+ cell lines (n = 9) and HER2- cell lines (n = 10) were evaluated with the CELx HSF test as described in the Methods. The sum of NRG1b- and EGF-driven HER2 signals that can be inhibited by the HER2-specific mAb pertuzumab was approximated as response units for all cell lines and plotted. b Comparison of NRG1b-driven CELx signals in AU565, BT483, SKBr3 (HER2+ reference cell line), and MDA-MB231 (HER2- reference cell line) and sensitivities to HER2-targeted drugs (pertuzumab, lapatinib, and afatinib). c HER2 expression levels in HER2+ (n = 9) and HER2- cell lines (n = 10) were determined by fluorescence flow cytometry (mean fluorescence channel units, MFC) and plotted against the corresponding HER2 signal determined by CELx HSF test (response units) for each cell line. No correlation between the two parameters was observed (P = 0.204, R2 = 0.0929). Empty circles, HER2- cell lines; Filled circles, HER2+ cell lines. The locations of BT483, AU565, SKBr3 (HER2+ reference cell line) and MDA-MB231 (HER2- reference cell line) are indicated

Fig. 7

Subtypes of CELx HSF curves. Representative CELx time-course curves representing HER2+/HSF+ (HER2+ cells having high HER2 signaling activities) (a), HER2+/HSF- (HER2+ cells having low HER2 signaling activities) (b), HER2-/HSF+ (HER2- cells having high HER2 signaling activities) (c), and HER2-/HSF- (HER2- cells having low HER2 signaling activities) (d) are shown. For display purposes, NRG1b and EGF-driven HER2 CELx signals are shown in separated panels. CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. Red curves, unstimulated cells (control); Green curves, cells stimulated with ligand (NRG1b or EGF); Blue curves, cells stimulated with ligand in the presence of drug (pertuzumab)

Fig. 8

Validation of CELx HSF test in patient tissue specimen-derived primary cells ex vivo. Primary epithelial cells derived from two HER2- (R39 and R49) patients with breast cancer and one healthy control subject (R62) were subjected to CELx HSF tests. Responses of NRG1b-driven HER2 CELx signals with and without pertuzumab for these primary cells are plotted along with those for the HER2+ reference cell line (SKBr3) and the HER2- reference cell line (MDA-MB231) as bar charts. Black bars, cells stimulated with NRG1b; Grey bars, cells stimulated with NRG1b in the presence of pertuzumab. HER2- Patient R39 has approximately 80% of the NRG1 CELx signal of HER2+ cell line SKBr3

Fig. 9

The PI3K/AKT pathway significantly contributes to the ligand-driven HER2 signaling activities detected by CELx HSF tests in patient-derived breast tumor primary cells. Patient R54 breast tumor-derived primary cells (15,000 cells per well) pre-seeded in sensor plates were treated with a serial titration of GSK1059615 (0 nM to 2700 nM) two hours prior to stimulation with NRG1b (800 pM) (a) or EGF (600 pM) (b). The dose-dependent inhibitory effect of GSK1059615 on ligand-driven HER2 signals is shown