Abstract 3144: Detection of HER2-HER3 heterodimers in patient circulating tumor cells using proximity ligation assay
Authors: Soliman Hatem , Khambati Fatema 
Published: July 2016

Abstract
BACKGROUND: The HER2 protein is a key oncogene in approximately 20% of breast cancers, and preferentially forms heterodimers with other members of the HER family (such as HER3) to activate its signaling cascade. Dual HER2 signaling blockade by using both trastuzumab and pertuzumab (a HER2 dimerization inhibiting monoclonal antibody) yields superior clinical benefit in HER2+ patients compared to trastuzumab monotherapy. A phase 1/2 clinical trial is ongoing to assess the safety and efficacy of the combination of gemcitabine with trastuzumab and pertuzumab in metastatic HER2+ pretreated patients. (NCT02139358) One of the correlative experiments is looking at the feasibility of detecting HER2-HER3 heterodimers using a proximity ligation assay on circulating tumor cells isolated from patients on treatment as a possible pharmcodynamic marker.
METHODS: Whole blood was collected in two 10cc Cellsave tubes (Veridex, Cat#: 7900005) at baseline, week 4, 7, 10. Samples were drawn before treatment was administered, to avoid contamination of the samples with cytotoxic agents. The blood was subjected to red cell lysis with 1XRBC lysis buffer (Santa Cruz). The lysed cells were fixed with 4% paraformaldehyde followed by negative selection utilizing CD45+ magnetic beads (EasySep CD45 depletion kit, cat#18259). The CD45 depleted cell suspension was re-fixed with 4% PFA on coated glass slides following cytospin. These cells were then stained for PLA to test for HER2-HER3 using Sigma Aldrich DuoLink PLA kit + DAPI fluorescence mounting medium (DUO92013) with anti-HER2 (PA5, Thermo Scientific, diluted 1:50) and anti-HER3 (MA5-12675, Thermo Scientific diluted 1:50) as primary antibodies plus cytokeratin (CK) and CD45 antibodies. A set of HER2+ and HER2- PLA controls using SKBR3 were included in the analysis. Control samples using SKBR3 cells were used to optimize the method.
RESULTS: Using the spiked SKBR3 HER2+ blood samples it was possible to isolate CK+ CD45- DAPI+ CTCs demonstrating HER2-HER3 heterodimers following optimization of the PLA method. Using this method in the first four patients we successfully collected CTCs in 7/11 samples processed. In those 7 CTC+ samples, 4 contained CTCs with positive HER2-HER3 PLA signals.
CONCLUSIONS: We have demonstrated a feasible methodology for the detection of HER2-HER3 heterodimers on the surface of circulating tumor cells from HER2+ metastatic breast cancer patients. This can provide investigators with a non-invasive method to monitor the pharmacodynamics of pertuzumab therapy on tumor cells. The collection of these samples is ongoing and correlation with benefit from therapy will be analyzed in the phase 2 portion of the study.
Citation Format: Hatem Soliman, Fatema Khambati. Detection of HER2-HER3 heterodimers in patient circulating tumor cells using proximity ligation assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3144.