Skip to main content

Advertisement

Log in

Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen

  • Research Paper
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

Purpose The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galβ1-3GalNAcα-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment. Methods BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy. Results 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1–10). By enrichment, a median of 23.3 mio cells (0.8–218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1–105, P < .0001). Blood from 1/11 pts before and 5/11 (45%) after enrichment showed CTCs (med. 2, 1–20), at a med. of 12.4 mio (2.6–38.5) cells analysed. Comparing BM and blood of the same patients after enrichment, 5 were positive in both compartments, 4 showed DTC-BM without presence of CTCs. Conclusion The positive immunomagnetic enrichment technique with TF-antibodies enables to analyse larger sample volumes and increase tumor cell detection rate. This could allow monitoring and characterisation of CTCs as targets for therapies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

APAAP:

Alkaline phosphatase anti-alkaline phosphatase

BM:

Bone marrow

CK:

Cytokeratin

CTC:

Circulating tumor cells

DTC:

Disseminated tumor cells

EpCam:

Epithelial cell adhesion molecule

FCS:

Fetal calf serum

HER2:

Human epidermal growth factor receptor 2

MACS:

Magnetic activated cell sorting

MNC:

Mononuclear cells

PB:

Peripheral blood

PBS:

Phosphate buffered saline

PCR:

Polymerase chain reaction

TF:

Thomsen-Friedenreich

References

  1. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC et al (2005) A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 353(8):793–802

    Article  PubMed  CAS  Google Scholar 

  2. Janni W, Gastroph S, Hepp F, Kentenich C, Rjosk D, Schindlbeck C et al (2000) Prognostic significance of an increased number of micrometastatic tumor cells in the bone marrow of patients with first recurrence of breast carcinoma. Cancer 88(10):2252–2259

    Article  PubMed  CAS  Google Scholar 

  3. Braun S, Kentenich C, Janni W, Hepp F, de Waal J, Willgeroth F et al (2000) Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 18(1):80–86

    PubMed  CAS  Google Scholar 

  4. Martin VM, Siewert C, Scharl A, Harms T, Heinze R, Ohl S et al (1998) Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS. Exp Hematol 26(3):252–264

    PubMed  CAS  Google Scholar 

  5. Weihrauch MR, Skibowski E, Draube A, Geller A, Tesch H, Diehl V et al (2002) Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies. Clin Exp Metastasis 19(7):617–621

    Article  PubMed  CAS  Google Scholar 

  6. Kasimir-Bauer S, Otterbach F, Oberhoff C, Schmid KW, Kimmig R, Seeber S (2003) Rare expression of target antigens for immunotherapy on disseminated tumor cells in breast cancer patients without overt metastases. Int J Mol Med 12(6):969–975

    PubMed  CAS  Google Scholar 

  7. Woelfle U, Breit E, Pantel K (2005) Influence of immunomagnetic enrichment on gene expression of tumor cells. J Transl Med 3(1):12

    Article  PubMed  CAS  Google Scholar 

  8. Rao CG, Chianese D, Doyle GV, Miller MC, Russell T, Sanders RA Jr et al (2005) Expression of epithelial cell adhesion molecule in carcinoma cells present in blood and primary and metastatic tumors. Int J Oncol 27(1):49–57

    PubMed  CAS  Google Scholar 

  9. Thurm H, Ebel S, Kentenich C, Hemsen A, Riethdorf S, Coith C et al (2003) Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy. Clin Cancer Res 9(7):2598–2604

    PubMed  CAS  Google Scholar 

  10. Taubert H, Blumke K, Bilkenroth U, Meye A, Kutz A, Bartel F et al (2004) Detection of disseminated tumor cells in peripheral blood of patients with breast cancer: correlation to nodal status and occurrence of metastases. Gynecol Oncol 92(1):256–261

    Article  PubMed  Google Scholar 

  11. Naume B, Borgen E, Nesland JM, Beiske K, Gilen E, Renolen A et al (1998) Increased sensitivity for detection of micrometastases in bone-marrow/peripheral-blood stem-cell products from breast-cancer patients by negative immunomagnetic separation. Int J Cancer 78(5):556–560

    Article  PubMed  CAS  Google Scholar 

  12. Springer GF, Desai PR, Scanlon EF (1976) Blood group MN precursors as human breast carcinoma-associated antigens and “naturally” occurring human cytotoxins against them. Cancer 37:169–176

    Article  PubMed  CAS  Google Scholar 

  13. Glinsky VV, Glinsky GV, Glinskii OV, Huxley VH, Turk JR, Mossine VV et al (2003) Intravascular metastatic cancer cell homotypic aggregation at the sites of primary attachment to the endothelium. Cancer Res 63(13):3805–3811

    PubMed  CAS  Google Scholar 

  14. Cao Y, Karsten UR, Liebrich W, Haensch W, Springer GF, Schlag PM (1995) Expression of Thomsen-Friedenreich-related antigens in primary and metastatic colorectal carcinomas. A reevaluation. Cancer 76(10):1700–1708

    Article  PubMed  CAS  Google Scholar 

  15. Wolf MF, Ludwig A, Fritz P, Schumacher K (1988) Increased expression of Thomsen-Friedenreich antigens during tumor progression in breast cancer patients. Tumour Biol 9(4):190–194

    PubMed  CAS  Google Scholar 

  16. Schindlbeck C, Jeschke U, Schulze S, Karsten U, Janni W, Rack B et al (2005) Characterisation of disseminated tumor cells in the bone marrow of breast cancer patients by the Thomsen-Friedenreich tumor antigen. Histochem Cell Biol 123(6):631–637

    Article  PubMed  CAS  Google Scholar 

  17. Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR et al (2000) Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 342(8):525–533

    Article  PubMed  CAS  Google Scholar 

  18. Waseem A, Karsten U, Leigh IM, Purkis P, Waseem NH, Lane EB (2004) Conformational changes in the rod domain of human keratin 8 following heterotypic association with keratin 18 and its implication for filament stability. Biochemistry 43(5):1283–1295

    Article  PubMed  CAS  Google Scholar 

  19. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC et al (2005) A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 353(8):793–802

    Article  PubMed  CAS  Google Scholar 

  20. Fehm T, Braun S, Muller V, Janni W, Gebauer G, Marth C et al (2006) A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer 107(5):885–892

    Article  PubMed  Google Scholar 

  21. Slade MJ, Coombes RC (2007) The clinical significance of disseminated tumor cells in breast cancer. Nat Clin Pract Oncol 4(1):30–41

    Article  PubMed  Google Scholar 

  22. Muller V, Stahmann N, Riethdorf S, Rau T, Zabel T, Goetz A et al (2005) Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity. Clin Cancer Res 11(10):3678–3685

    Article  PubMed  Google Scholar 

  23. Kahn HJ, Presta A, Yang LY, Blondal J, Trudeau M, Lickley L et al (2004) Enumeration of circulating tumor cells in the blood of breast cancer patients after filtration enrichment: correlation with disease stage. Breast Cancer Res Treat 86(3):237–247

    Article  PubMed  Google Scholar 

  24. Baldus SE, Zirbes TK, Hanisch FG, Kunze D, Shafizadeh ST, Nolden S et al (2000) Thomsen-Friedenreich antigen presents as a prognostic factor in colorectal carcinoma: A clinicopathologic study of 264 patients. Cancer 88(7):1536–1543

    Article  PubMed  CAS  Google Scholar 

  25. Takanami I (1999) Expression of Thomsen-Friedenreich antigen as a marker of poor prognosis in pulmonary adenocarcinoma. Oncol Rep 6(2):341–344

    PubMed  CAS  Google Scholar 

  26. Hirao T, Sakamoto Y, Kamada M, Hamada S, Aono T (1993) Tn antigen, a marker of potential for metastasis of uterine cervix cancer cells. Cancer 72(1):154–159

    Article  PubMed  CAS  Google Scholar 

  27. Schindlbeck C, Jeschke U, Schulze S, Karsten U, Janni W, Rack B et al (2007) Prognostic impact of Thomsen-Friedenreich tumor antigen and disseminated tumor cells in the bone marrow of breast cancer patients. Breast Cancer Res Treat 101(1):17–25

    Article  PubMed  CAS  Google Scholar 

  28. Kruger W, Datta C, Badbaran A, Togel F, Gutensohn K, Carrero I et al (2000) Immunomagnetic tumor cell selection–implications for the detection of disseminated cancer cells. Transfusion 40(12):1489–1493

    Article  PubMed  CAS  Google Scholar 

  29. Naume B, Borgen E, Beiske K, Herstad TK, Ravnas G, Renolen A et al (1997) Immunomagnetic techniques for the enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood. J Hematother 6(2):103–114

    PubMed  CAS  Google Scholar 

  30. Choesmel V, Anract P, Hoifodt H, Thiery JP, Blin N (2004) A relevant immunomagnetic assay to detect and characterize epithelial cell adhesion molecule-positive cells in bone marrow from patients with breast carcinoma: immunomagnetic purification of micrometastases. Cancer 101(4):693–703

    Article  PubMed  Google Scholar 

  31. Woelfle U, Breit E, Zafrakas K, Otte M, Schubert F, Muller V et al (2005) Bi-specific immunomagnetic enrichment of micrometastatic tumour cell clusters from bone marrow of cancer patients. J Immunol Methods 300(1–2):136–145

    Article  PubMed  CAS  Google Scholar 

  32. Janni W, Wiedswang G, Fehm T, Jueckstock J, Borgen E, Rack B et al (2006) Persistence of disseminated tumor cells (DTC) in bone marrow (BM) during follow-up predicts increased risk for relapse up-date of the pooled European data. San Antonio Breast Cancer Symposium 2006; Abstract # 18

  33. Wiedswang G, Borgen E, Schirmer C, Karesen R, Kvalheim G, Nesland JM et al (2006) Comparison of the clinical significance of occult tumor cells in blood and bone marrow in breast cancer. Int J Cancer 118(8):2013–2019

    Article  PubMed  CAS  Google Scholar 

  34. Cristofanilli M, Broglio KR, Guarneri V, Jackson S, Fritsche HA, Islam R et al (2007) Circulating tumor cells in metastatic breast cancer: biologic staging beyond tumor burden. Clin Breast Cancer 7(6):471–479

    Article  PubMed  Google Scholar 

  35. Gaforio JJ, Serrano MJ, Sanchez-Rovira P, Sirvent A, Delgado-Rodriguez M, Campos M et al (2003) Detection of breast cancer cells in the peripheral blood is positively correlated with estrogen-receptor status and predicts for poor prognosis. Int J Cancer 107(6):984–990

    Article  PubMed  CAS  Google Scholar 

  36. Xenidis N, Perraki M, Kafousi M, Apostolaki S, Bolonaki I, Stathopoulou A et al (2006) Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients. J Clin Oncol 24(23):3756–3762

    Article  PubMed  CAS  Google Scholar 

  37. Pierga JY, Bonneton C, Vincent-Salomon A, de Cremoux P, Nos C, Blin N et al (2004) Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clin Cancer Res 10(4):1392–1400

    Article  PubMed  CAS  Google Scholar 

  38. Meng S, Tripathy D, Frenkel EP, Shete S, Naftalis EZ, Huth JF et al (2004) Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res 10(24):8152–8162

    Article  PubMed  Google Scholar 

  39. Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC et al (2006) Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res 2(14 Pt 1):4218–4224

    Article  Google Scholar 

  40. Shigeoka H, Karsten U, Okuno K, Yasutomi M (1999) Inhibition of liver metastases from neuraminidase-treated colon 26 cells by an anti-Thomsen-Friedenreich-specific monoclonal antibody. Tumour Biol 20(3):139–146

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Mrs S. Schulze and Ms S. Hofmann for excellent technical assistance. This study contains material analysed by Ms J. Stellwagen in preparation of her thesis to achieve the degree of MD at Ludwig-Maximilians University, Medical Faculty, in Munich, Germany.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Christian Schindlbeck.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schindlbeck, C., Stellwagen, J., Jeschke, U. et al. Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen. Clin Exp Metastasis 25, 233–240 (2008). https://doi.org/10.1007/s10585-007-9137-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10585-007-9137-z

Keywords

Navigation