Abstract 1970: Quantitative analysis of HER2, HER3, and Ɣ-H2AX expression on circulating tumor cells for clinical trials

Background: Circulating Tumor Cells (CTCs) are an attractive target for pharmacodynamic measurements in clinical trials, providing quantitative biomarker expression of the tumor, independent of tissue biopsy. The increasing challenge to obtain tissue samples continues to focus attention on non-invasive liquid biopsy approaches. The RareCyte platform offers a highly sensitive method for CTC enumeration and protein expression on CTCs. In this study, we describe the development of assays to quantify HER2, HER3, and Ɣ-H2AX protein expression on CTCs. Each of the single biomarker assays allow for the inclusion of an additional biomarker of interest to be measured on each CTC. Methods: Cell lines with known positive or negative expression for each biomarker were spiked into healthy donor blood to generate model CTCs. Nucleated cells were collected from these samples using the AccuCyte® sample preparation kit and spread onto microscope slides. Slides were fixed and stained with antibodies targeting cytokeratin, EpCAM, and CD45 to detect CTCs and exclude white blood cells (WBCs), as well as primary antibodies to HER2, HER3 or Ɣ-H2AX to evaluate protein expression on identified CTCs. Slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope, to quantitatively measure Mean Fluorescence Intensity (MFI) of biomarkers to assess protein expression on individual CTCs. Results: Assays for the detection of HER2, HER3, and Ɣ-H2AX on CTCs were designed and optimized to maximize the signal-to-background ratio. The final assays were run on replicate slides of positive and negative cell lines over 3 independent days to evaluate the analytic performance of each assay. Thresholds for biomarker positivity were established based on MFI measurements from positive and negative control slides. The accuracy of all three biomarker assays was >90%. Both repeatability (intra-run precision) and inter-run precision, assessed by the Coefficient of Variations (CV), were <20% for all three assays. The HER2 biomarker assay was tested on 16 cell lines with varying levels of HER2 expression, demonstrating its ability to confirm HER2 protein levels and distinguish between cell lines with known HER2 expression (0, 1+, 2+, 3+). Conclusions: We developed biomarker assays for quantifying protein expression on individual CTCs, that demonstrate high accuracy and reproducibility for assessing biomarker expression of tumors through liquid biopsy and are currently being utilized in clinical studies. Recent findings suggest that patients may respond to targeted therapies even when their tumors have low or ultra-low HER2 expression, if HER2 expression is detected on CTCs. Quantifying target protein expression on CTCs offers a promising approach for patient selection and longitudinal, non-invasive monitoring of cancer patients, generating valuable data for clinical trial sponsors. Citation Format: Erin G. Bayer, Brock Bartels, Rachel Ponting, Casey Helmicki, Arturo B. Ramirez. Quantitative analysis of HER2, HER3, and Ɣ-H2AX expression on circulating tumor cells for clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1970.