Characterization of Disseminated Tumor Cells (DTCs) in Patients with Triple-Negative Breast Cancer (TNBC)
Abstract
1. Introduction
2. Materials and Methods
2.1. Study Population and Informed Consent
2.2. Bone Marrow Aspirates
2.3. Cell Culture for Reference Slides
2.4. Sequential Immunofluorescence Staining and DTC Imaging
2.5. Release of Fluorochromes from APC- and FITC- Conjugated Antibodies for Further Staining
2.6. Immunohistochemical Staining of Matching Tumor Tissue
2.7. Statistical Analysis
3. Results
3.1. Staining of Reference Slides Using Sequential Immunofluorescence
3.1.1. Control Cells for Marker Panels
3.1.2. Calculation of the Release Rate
3.1.3. Characterization of Control Cells
3.2. Identification and Quantification of DTC-Subtypes in Bone Marrow Samples
3.3. DTC Subtypes in Primary and Recurrent TNBC Patients
3.4. DTC Status and Subtypes After NACT in Patients with Primary TNBC Reaching pCR vs. Non-pCR
3.5. DTC Subtype Detection vs. Standard Detection of CK+ Cells
3.6. Correlation of DTC Analysis to Clinical Parameters
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
TNBC | Triple Negative Breast Cancer |
DTCs | Disseminated Tumor Cells |
NACT | Neoadjuvant Chemotherapy |
pCR | Pathologic Complete Remission |
CK | Cytokeratin |
Vim | Vimentin |
HER2 | Human Epidermal Growth Factor Receptor 2 |
PD-L1 | Programmed Cell Death 1 Ligand 1 |
BM | Bone Marrow |
EMT | Epithelial–Mesenchymal Transition |
ER | Estrogen Receptor |
PR | Progesterone Receptor |
IF | Immunofluorescence |
IHC | Immunohistochemical |
FISH | Fluorescence In Situ Hybridization |
CISH | Chromogenic In Situ Hybridization |
CNB | Core Needle Biopsy |
DMEM | Dulbecco’s Modified Eagle Medium |
RPMI | Roswell Park Memorial Institute |
PBS | Phosphate-Buffered Saline |
DAPI | 4′,6′-Diamidin-2-phenylindol |
APC | Allophycocyanin |
FITC | Fluoresceinisothiocyanat |
PE | Phycoerythrin |
TPS | Tumor Proportion Score |
CPS | Combined Positive Score |
CTCs | Circulating Tumor Cells |
Appendix A
Pat.-ID | Total DTCs | Total CK+ DTCs | Total CK- DTCs | Total Vim+ DTCs | Total Ki67+ DTCs | Total PD-L1+ DTCs | Total HER2+ DTCs |
---|---|---|---|---|---|---|---|
731 | 3 | 3 | 0 | 3 | 2 | 0 | 0 |
744 | 7 | 3 | 4 | 5 | 5 | 5 | 0 |
762 | 14 | 6 | 8 | 7 | 13 | 13 | 0 |
801 | 3 | 3 | 0 | 2 | 1 | 2 | 0 |
844 | 11 | 7 | 4 | 7 | 6 | 9 | 2 |
1085 | 10 | 1 | 9 | 2 | 7 | 9 | 6 |
1123 | 10 | 5 | 5 | 3 | 7 | 8 | 0 |
1139 | 15 | 11 | 4 | 15 | 14 | 15 | 4 |
1230 | 3 | 1 | 2 | 1 | 1 | 3 | 3 |
1320 | 2 | 2 | 0 | 1 | 1 | 1 | 0 |
1892 | 1 | 1 | 0 | 1 | 1 | 1 | 0 |
1893 | 3 | 3 | 0 | 3 | 2 | 3 | 0 |
2017 | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
2037 | 4 | 3 | 1 | 3 | 4 | 3 | 0 |
2045 | 8 | 5 | 3 | 7 | 7 | 2 | 0 |
2108 | 6 | 5 | 1 | 5 | 6 | 0 | 0 |
2284 | 12 | 9 | 3 | 11 | 11 | 2 | 0 |
2492 | 10 | 10 | 0 | 6 | 6 | 1 | 0 |
2539 | 1 | 1 | 0 | 1 | 0 | 0 | 0 |
2607 | 7 | 7 | 0 | 7 | 5 | 1 | 0 |
2735 | 4 | 4 | 0 | 0 | 0 | 0 | 0 |
2769 | 3 | 2 | 1 | 3 | 3 | 1 | 0 |
2803 | 5 | 3 | 2 | 5 | 5 | 0 | 0 |
2833 | 7 | 6 | 1 | 5 | 6 | 0 | 0 |
2909 | 2 | 2 | 0 | 1 | 1 | 0 | 0 |
2917 | 6 | 5 | 1 | 3 | 3 | 0 | 0 |
2927 | 8 | 6 | 2 | 7 | 6 | 0 | 1 |
2953 | 16 | 12 | 4 | 11 | 10 | 4 | 0 |
2973 | 1 | 1 | 0 | 1 | 1 | 0 | 0 |
3122 | 1 | 1 | 0 | 1 | 1 | 0 | 0 |
3217 | 3 | 3 | 0 | 3 | 3 | 3 | 1 |
3306 | 3 | 3 | 0 | 3 | 3 | 2 | 2 |
3313 | 2 | 2 | 0 | 0 | 0 | 0 | 0 |
3339 | 8 | 5 | 3 | 5 | 6 | 2 | 0 |
3353 | 2 | 1 | 1 | 1 | 1 | 1 | 1 |
3427 | 7 | 6 | 1 | 7 | 7 | 6 | 0 |
3457 | 1 | 0 | 1 | 1 | 1 | 1 | 0 |
3558 | 11 | 10 | 1 | 11 | 9 | 0 | 0 |
3569 | 12 | 9 | 3 | 11 | 12 | 0 | 0 |
3573 | 1 | 1 | 0 | 1 | 1 | 0 | 0 |
3605 | 5 | 4 | 1 | 4 | 3 | 2 | 0 |
3625 | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
3648 | 7 | 4 | 3 | 6 | 6 | 2 | 0 |
3652 | 3 | 3 | 0 | 1 | 2 | 0 | 0 |
3684 | 3 | 3 | 0 | 3 | 1 | 1 | 1 |
sum | 253 | 184 | 69 | 184 | 190 | 103 | 21 |
mean | 5.62 | 4.09 | 1.53 | 4.09 | 4.22 | 2.29 | 0.467 |
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n = 80 | % | |
---|---|---|
Age | ||
Median (years) | 50 | |
Range | 26–86 | |
Menopausal status | ||
pre- | 34 | 42.5 |
peri- | 7 | 8.8 |
post- | 39 | 48.8 |
Tumor stage | ||
Primary | 67 | 83.8 |
Recurrent | 13 | 16.3 |
Ki67 (CNB) | ||
average (%) | 52 | |
range (%) | 2–90 | |
Nodal status N0 N1 | 59 21 | 73.8 26.2 |
HER2 score (CNB) | ||
0 | 64 | 80.0 |
1+ | 12 | 15.0 |
2+/CISH neg | 4 | 5.0 |
Grade (CNB) | ||
G1 | 3 | 3.8 |
G2 | 28 | 35.0 |
G3 | 47 | 58.8 |
n.a. | 2 | 2.5 |
NACT | ||
yes | 64 | 80.0 |
combined with pembrolizumab | 9 | 14.1 |
no | 16 | 20.0 |
pCR | 39 | 60.9 |
Cells/Marker | ZR75-1 | T98G | CaSki | MCF7 | Hematopoietic Cells |
---|---|---|---|---|---|
Cytokeratin | + | - | + | + | - |
Vimentin | - | + | + | - | + |
CD45 | - | - | - | - | + |
PD-L1 | - | - | + | - | both * |
Ki67 | + | + | + | + | - |
HER2 | + | - | - | - | - |
DAPI | + | + | + | + | + |
Marker | Patients with DTCs, Positive for Marker (n) | Tumor Tissue * Positive (n) | Tumor Tissue * Negative (n) | Missing Data (n) |
---|---|---|---|---|
HER2 | 9 | 4 | 3 | 2 |
PD-L1 | 27 | 10 | 10 | 7 |
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Eckardt, A.; Nel, I.; Weydandt, L.; Brochwitz, E.; Höhn, A.K.; Winter, K.; Aktas, B. Characterization of Disseminated Tumor Cells (DTCs) in Patients with Triple-Negative Breast Cancer (TNBC). Cells 2025, 14, 857. https://doi.org/10.3390/cells14120857
Eckardt A, Nel I, Weydandt L, Brochwitz E, Höhn AK, Winter K, Aktas B. Characterization of Disseminated Tumor Cells (DTCs) in Patients with Triple-Negative Breast Cancer (TNBC). Cells. 2025; 14(12):857. https://doi.org/10.3390/cells14120857
Chicago/Turabian StyleEckardt, Anne, Ivonne Nel, Laura Weydandt, Elisa Brochwitz, Anne Kathrin Höhn, Karsten Winter, and Bahriye Aktas. 2025. "Characterization of Disseminated Tumor Cells (DTCs) in Patients with Triple-Negative Breast Cancer (TNBC)" Cells 14, no. 12: 857. https://doi.org/10.3390/cells14120857
APA StyleEckardt, A., Nel, I., Weydandt, L., Brochwitz, E., Höhn, A. K., Winter, K., & Aktas, B. (2025). Characterization of Disseminated Tumor Cells (DTCs) in Patients with Triple-Negative Breast Cancer (TNBC). Cells, 14(12), 857. https://doi.org/10.3390/cells14120857