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. 2023 Jan 17;15(3):568.
doi: 10.3390/cancers15030568.

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

Theresa König  1 , Senol Dogan  1 , Anne Kathrin Höhn  2 , Laura Weydandt  1 , Bahriye Aktas  1 , Ivonne Nel  1
Affiliations

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

Theresa König et al. Cancers (Basel). .

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.

Keywords: HER2; bone marrow; breast cancer; disseminated tumor cells; dormancy; hormone receptor; proliferation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic of sequential immunofluorescence staining followed by image acquisition and releasing/bleaching of fluorochromes.
Figure 2
Figure 2
Immunofluorescent staining of ZR75-1 cells (white arrows) and T98G cells (yellow arrows) mixed with bone marrow cells at 20-fold magnification. (A) Before and after treatment with the releasing enzyme. Hence, secondary fluorescence of Pan-CK was successfully quenched and another set of antibodies could be applied. (B) Before and after bleaching with increased light exposure in channels Y3 and Y5. Shown is a loss of the secondary fluorescent activity of markers Ki67 and HER2 to prepare the sample for another set of antibodies.
Figure 3
Figure 3
Sequential staining of positive control cell lines ZR75-1 (white arrows) and T98G (yellow arrows) at 20-fold magnification. Shown are three different tumor cells for each staining round. For comparison, hematopoietic bone marrow cells, which are distinctly smaller and only stain positive for DAPI, are marked with green arrowheads.
Figure 4
Figure 4
Staining of ZR75-1 cells (white arrows), which stain positive for ER (red) while remaining negative for N-cadherin, next to T98G cells (yellow arrows), which stain positive for N-cadherin (yellow) and negative for ER at 20-fold magnification.
Figure 5
Figure 5
Sequential immunofluorescent staining of a disseminated tumor cell in the bone marrow of a patient exhibiting the profile CK+CD133+HER2+Ki67+Ncad+ at 20-fold magnification. The DTC expressed all stained markers (DAPI, CD133, CK, Ki67, HER2, N-cadherin) except ER, although the sample derived from a patient with an ER-positive primary tumor.
Figure 6
Figure 6
Quantification of DTCs per patient and division into subgroups defined by the number of DTCs detected in a bone marrow sample using sequential immunofluorescent staining.
Figure 7
Figure 7
Quantification of DTC subpopulations in the (A) CK-positive and (B) CK-negative DTC subpopulations.
Figure 8
Figure 8
Distribution of DTC phenotypes among the HR+ cohort of 29 patients.
Figure 9
Figure 9
DTC cluster consisting of two cells staining positive for CK at 20-fold magnification.
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