Abstract 3708: Characterization of EpCAM-positive cells for specific detection of disseminated cancer cells in bone marrow of early breast cancer patients
Authors: Roth Jonas , Raya Elia , Hücker Sarah , Koerkel-Qu Huiqin , Seitz Stephan , Klein Christoph 
Published: March 2024

Abstract
Obviously, breast cancer cells that disseminated from the primary tumor before surgical resection are responsible for metastasis. However, detection of disseminated cancer cells (DCC) in breast cancer patients during minimal residual disease is still difficult, impeding the study of early systemic cancer and the extraction of clinically relevant diagnostic or therapeutic information. Others and we have shown previously that EpCAM is a suitable DCC detection marker, however not entirely specific in bone marrow. To improve DCC detection, we here compared different methods to enrich DCC exploiting information from our ongoing single cell RNA-seq study. Based on our ongoing scRNA-seq study (see poster of Elia Raya), we generated a qPCR assay to classify candidate DCC based on an expression signature of four genes. This assay categorizes plasma cell-like EpCAM+ cells from healthy donors and candidate DCC from breast cancer patients with high fidelity. With this assay at hand, we compared two enrichment strategies: depletion of CD11b, CD319 and Glycophorin A positive cells from density gradient enriched mononuclear cells (MNC) vs. positive selection of EpCAM+ cells and subsequent addition of markers that separate out DCC among EpCAM+ cells. For this we applied 10x genomics profiling of EpCAM+ cells from bone marrow. We depleted and screened the bone marrow of 221 patients suffering from non-metastatic early breast cancer for EpCAM-positive cells. From 87 patient samples we could isolate EpCAM-positive cells. We performed single cell RNA-sequencing on 64 EpCAM-positive cells from 54 patients of which 84% were classified as cancer cells and 16% of bone marrow origin. Applying the selected gene signature, 76% of isolated cells were classified as DCC, 16% were undefined and 8% as non-cancer bone marrow cell (NCC). Upon positive selection of EpCAM+ cells implementing additional markers, we isolated 53 cells from 14 patients. Of these, 68% showed a DCC or DCC-like-signature, 15 % a NCC and 17% an undefined profile. To get a better understanding of these EpCAM-positive confounder populations we currently perform a scRNA-seq profiling using 10X on the enriched EpCAM+ cells. Our data shows that EpCAM is a promising marker to identify DCCs in the bone marrow of early cancer patients, but it also indicates that we find several groups of confounder cells. To differentiate between these cell types during staining and in our qPCR assay we are currently testing additional markers.
Citation Format: Jonas Roth, Elia Raya, Sarah Hücker, Huiqin Koerkel-Qu, Stephan Seitz, Christoph Klein. Characterization of EpCAM-positive cells for specific detection of disseminated cancer cells in bone marrow of early breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3708.