Multi-Parameter Analysis of Disseminated Tumor Cells (DTCs) in Early Breast Cancer Patients with Hormone-Receptor-Positive Tumors

SIMPLE SUMMARY: Despite successful treatment of the primary tumor, recurrence occurs in about 30% of breast cancer patients. This could be due to disseminated tumor cells (DTCs), which split off during the early stages, metastasize to the bone marrow and cause relapse years after initial diagnosis, preferentially in hormone-receptor-positive patients. Currently, standardized DTC analysis is based on the detection of epithelial cells in the bone marrow. In this study, we established a sequential multi-parameter staining procedure to investigate phenotypical and therapy-related features of these rare cells. We found distinct receptor profiles that enormously differed from the primary tumor tissue and might have clinical implications. Especially, patients with luminal A tumors revealed DTCs with potential therapeutic targets. We analyzed particular non-epithelial DTC subgroups and clusters that were never described before. Our findings indicate that characterization rather than quantification of DTCs might be relevant for prognosis and treatment decisions. ABSTRACT: Background: Patients with hormone-receptor-positive (HR+) breast cancer are at increased risk for late recurrence. One reason might be disseminated tumor cells (DTCs), which split off in the early stages of the disease and metastasize into the bone marrow (BM). Methods: We developed a novel multi-parameter immunofluorescence staining protocol using releasable and bleachable antibody–fluorochrome-conjugates. This sequential procedure enabled us to analyze six distinct phenotypical and therapy-related markers on the same DTC. We characterized BM aspirates from 29 patients with a HR+ tumor and a known positive DTC status—based on the standardized detection of epithelial cells in BM. Results: Using the immunofluorescence staining, a total of 153 DTCs were detected. Luminal A patients revealed a higher DTC count compared with luminal B. The majority of the detected DTCs were CK-positive (128/153). However, in 16 of 17 luminal A patients we found HER2-positive DTCs. We detected CK-negative DTCs (25/153) in 12 of 29 patients. Of those cells, 76% were Ki67-positive and 68% were HER2-positive. Moreover, we detected DTC clusters consisting of mixed characteristics in 6 of 29 patients. Conclusions: Using sequential multi-parameter imaging made it possible to identify distinct DTC profiles not solely based on epithelial features. Our findings indicate that characterization rather than quantification of DTCs might be relevant for treatment decisions.