Abstract 670: Optimizing fixed flow cytometry for breast cancer biomarker expression in representative samples and FFPE curls
Authors: Samantha M. Hill, Hannah L. Veloz, Brian Hanley, Tracy Davis, Lisa L. Gallegos, Harold Sasano, Samra Turajlic, Nelson R. Alexander
Journal: Cancer Research (2025)

Abstract Introduction: Flow cytometry is a well-established method to analyze cell populations using antibody-based fluorescent detection of protein biomarkers. In this study, we demonstrate the ability to generate intact single cells and perform flow cytometry analysis with two types of formalin fixed tissue: FFPE curls and Representative Samples (RS). RS are homogenized, well-mixed tissue samples from formalin fixed tumors dissected from leftover surgical material. We demonstrate biomarker expression results which correlate with IHC scores. This new method for biomarker quantification may be considered alongside other methods (e.g. digital pathology). Methods: Intact single cells were dissociated from RS and FFPE curls using a non-enzymatic, mechanical dissociation method. Cells were stained in suspension for Cytokeratin 8&amp;18 (CK8&amp;18), Ki67, Her2, and DNA content was assessed via DAPI staining and analyzed by flow cytometry. Samples were analyzed on a BD FACSMelody or BD LSR II flow cytometer, and analysis was performed using FCS Express 7 software. Immunohistochemistry (IHC) was performed on the Benchmark ULTRA to compare Ki67 expression (n=78) and Her2 expression (n=16) to the flow analysis. Ki67 expression by IHC was assessed using the international Ki67 working group scoring methods to generate a positive percentage. Her2 expression by IHC was assessed by a pathologist and assigned a score ranging 0 to 3+. Results: Formalin fixed tissue such as RS and FFPE curls can be mechanically dissociated into single cells with intact surface biomarkers. These single cells can be stained in suspension, analyzed via flow cytometry and generate correlating data with both weighted IHC-based scores and clinical IHC scores. The flow analysis of Her2 positive percentage correlates with the IHC scores showing an increasing trend and significant difference between scores for both FFPE and RS. Ki67 expression varied by tumor region using IHC analysis, however by flow cytometry showed a strong correlation with a weighted average across multiregional quantification of Ki67 expression. Conclusion: We demonstrate that millions of intact single cells can be generated from RS and FFPE curls for breast tissue, using non-enzymatic mechanical dissociation methods. Staining in suspension and flow cytometry analysis can be performed in a day for rapid biomarker quantification. Flow cytometry has the potential to analyze FFPE samples using workflows and technologies that have existed in the hematopathology space for decades. Citation Format: Samantha M. Hill, Hannah L. Veloz, Brian Hanley, Tracy Davis, Lisa L. Gallegos, Harold Sasano, Samra Turajlic, Nelson R. Alexander. Optimizing fixed flow cytometry for breast cancer biomarker expression in representative samples and FFPE curls [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 670.