Annals of Surgical Oncology

, Volume 12, Issue 9, pp 753–760 | Cite as

The Detection of Isolated Tumor Cells in Bone Marrow Comparing Bright-Field Immunocytochemistry and Multicolor Immunofluorescence

  • David N. Krag
  • Roberto Kusminsky
  • Edward Manna
  • Abiy Ambaye
  • Donald L. Weaver
  • Seth P. Harlow
  • Michael Covelli
  • Mary A. Stanley
  • Laurence McCahill
  • Frank Ittleman
  • Bruce Leavitt
  • Martin Krag
Article

Abstract

Background

The detection of isolated tumor cells in bone marrow by immunocytochemistry (ICC) has been reported to predict progression of early-stage breast cancer. The most common staining procedure uses bright-field ICC with cytokeratin (CK) antibodies to label isolated tumor cells. However, this method can result in false-positive staining events. We used multicolor immunofluorescence (IF) to develop a more specific assay for detecting isolated tumor cells in marrow samples from breast cancer patients.

Methods

We compared ICC and IF side by side for detection of cancer cells and false-positive staining events on bone marrow aspirates from breast cancer patients, bone marrow from healthy donors, and healthy donor blood spiked with cancer cells. The primary target for isolated tumor cell detection was CK for both methods. IF used an additional set of antibodies to label hematopoietic cells (HCs).

Results

The detection rate of CK+ events in breast cancer patient bone marrow aspirates was 18 (58%) of 31 for ICC and 21 (68%) of 31 for IF. However, with IF, 17 of 21 CK+ cases were stained with HC markers and thus were identified as false-positive events. A surprisingly high CK+ event rate was observed in healthy donor blood and marrow. In all healthy donor samples, CK+ events were readily identified as HCs by IF. Detection sensitivity of spiked cancer cells in donor blood was similar for both methods.

Conclusions

There is a high frequency of CK+ events in blood and marrow, and it is important to note that this is observed both in patients with and those without cancer. IF with multiple HC markers allows straightforward discrimination between CK+ cells of hematopoietic and nonhematopoietic origin.

Keywords

Immunocytochemistry Bone marrow Cytokeratin Immunofluorescence 

Notes

Acknowledgments

The authors thank Elaine Cahoon, Patricia Lutton, Jennifer Spano, and Jenne Wax at the University of Vermont, Vermont Cancer Center, and Augusta Kosowicz of the Charleston Area Medical Center for Cancer Research for their assistance in protocol development, patient recruitment, and sample delivery. We also thank Julie Malloy for administrative assistance and Joseph Tessitore for obtaining material from the primary breast tumors at Fletcher Allen Health Care. Supported by The University of Vermont General Clinical Research Center (GCRC MO1 RR00109), the National Institute of Health (PHS CA74137-06S1), the Charleston Area Medical Center Foundation and Charleston Area Medical Center Institute, Charleston, WV.

References

  1. 1.
    American Joint Committee on Cancer. AJCC Cancer Staging Handbook. 6th ed. New York: Springer, 2002Google Scholar
  2. 2.
    Mansi JL, Easton D, Berger U, et al. Bone marrow micrometastases in primary breast cancer: prognostic significance after 6 years’ follow-up. Eur J Cancer 1991;27:1552–5PubMedGoogle Scholar
  3. 3.
    Pantel K, Muller V, Auer M, Nusser N, Harbeck N, Braun S. Detection and clinical implications of early systemic tumor cell dissemination in breast cancer. Clin Cancer Res 2003;9:6326–34Google Scholar
  4. 4.
    Diel IJ, Kaufmann M, Costa SD, et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996;88:1652–8Google Scholar
  5. 5.
    Solomayer EF, Diel IJ, Salanti G, et al. Time independence of the prognostic impact of tumor cell detection in the bone marrow of primary breast cancer patients. Clin Cancer Res 2001;7:4102–8Google Scholar
  6. 6.
    Braun S, Cevatli BS, Assemi C, et al. Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy. J Clin Oncol 2001;19:1468–75PubMedGoogle Scholar
  7. 7.
    Wiedswang G, Borgen E, Karesen R, et al. Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003;21:3469–78CrossRefPubMedGoogle Scholar
  8. 8.
    Naume B, Wiedswang G, Borgen E, et al. The prognostic value of isolated tumor cells in bone marrow in breast cancer patients: evaluation of morphological categories and the number of clinically significant cells. Clin Cancer Res 2004;10:3091–7Google Scholar
  9. 9.
    Wiedswang G, Borgen E, Karesen R, et al. Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin Cancer Res 2004;10:5342–8Google Scholar
  10. 10.
    Gerber B, Krause A, Muller H, et al. Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001;19:960–71PubMedGoogle Scholar
  11. 11.
    Gebauer G, Fehm T, Merkle E, Beck EP, Lang N, Jager W. Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001;19:3669–74PubMedGoogle Scholar
  12. 12.
    Janni W, Hepp F, Rjosk D, et al. The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence. Cancer 2001;92:46–53CrossRefPubMedGoogle Scholar
  13. 13.
    Janni W, Gastroph S, Hepp F, et al. Prognostic significance of an increased number of micrometastatic tumor cells in the bone marrow of patients with first recurrence of breast carcinoma. Cancer 2000;88:2252–9CrossRefPubMedGoogle Scholar
  14. 14.
    Leinung S, Wurl P, Schonfelder A, Weiss CL, Roder I, Schonfelder M. Rating of isolated disseminated tumor cells in bone marrow in comparison with other factors of prognosis in breast carcinoma. Int J Surg Investig 2000;2:193–202PubMedGoogle Scholar
  15. 15.
    Gebauer G, Fehm T, Merkle E, Jaeger W, Mitze M. Micrometastases in axillary lymph nodes and bone marrow of lymph node-negative breast cancer patients—prognostic relevance after 10 years. Anticancer Res 2003;23:4319–24PubMedGoogle Scholar
  16. 16.
    Lagrange M, Ferrero JM, Lagrange JL, et al. Non-specifically labelled cells that simulate bone marrow metastases in patients with non-metastatic breast cancer. J Clin Pathol 1997;50:206–11PubMedGoogle Scholar
  17. 17.
    Ellis G, Ferguson M, Yamanaka E, Livingston RB, Gown AM. Monoclonal antibodies for detection of occult carcinoma cells in bone marrow of breast cancer patients. Cancer 1989;63:2509–14PubMedGoogle Scholar
  18. 18.
    Molino A, Pelosi G, Turazza M, et al. Bone marrow micrometastases in 109 breast cancer patients: correlations with clinical and pathological features and prognosis. Breast Cancer Res Treat 1997;42:23–30CrossRefPubMedGoogle Scholar
  19. 19.
    Mathieu MC, Friedman S, Bosq J, et al. Immunohistochemical staining of bone marrow biopsies for detection of occult metastasis in breast cancer. Breast Cancer Res Treat 1990;15:21-6CrossRefPubMedGoogle Scholar
  20. 20.
    American Joint Committee on Cancer. AJCC Cancer Staging Manual. 6th ed. New York: Springer-Verlag, 2002Google Scholar
  21. 21.
    Borgen E, Naume B, Nesland JM, et al. Standardization of the immunocytochemical detection of cancer cells in BM and blood: 1. Establishment of objective criteria for the evaluation of immunostained cells. Cytotherapy 1999;1:377–88CrossRefGoogle Scholar
  22. 22.
    Brugger W, Buhring HJ, Grunebach F, et al. Expression of MUC-1 epitopes on normal bone marrow: implications for the detection of micrometastatic tumor cells. J Clin Oncol 1999;17:1535–44PubMedGoogle Scholar
  23. 23.
    Borgen E, Beiske K, Trachsel S, et al. Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998;185:427–34CrossRefPubMedGoogle Scholar
  24. 24.
    Braun S, Pantel K, Muller P, et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525–33CrossRefPubMedGoogle Scholar
  25. 25.
    Ahr A, Scharl A, Muller M, et al. Cross-reactive staining of normal bone-marrow cells by monoclonal antibody 2E11. Int J Cancer 1999;84:502–5CrossRefPubMedGoogle Scholar
  26. 26.
    Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781–91CrossRefPubMedGoogle Scholar

Copyright information

© The Society of Surgical Oncology, Inc. 2005

Authors and Affiliations

  • David N. Krag
    • 1
  • Roberto Kusminsky
    • 2
  • Edward Manna
    • 1
  • Abiy Ambaye
    • 3
  • Donald L. Weaver
    • 3
  • Seth P. Harlow
    • 1
  • Michael Covelli
    • 2
  • Mary A. Stanley
    • 1
  • Laurence McCahill
    • 1
  • Frank Ittleman
    • 1
  • Bruce Leavitt
    • 1
  • Martin Krag
    • 4
  1. 1.Department of SurgeryUniversity of Vermont College of MedicineBurlington
  2. 2.Charleston Area Medical Center InstituteCharleston
  3. 3.Department of Surgical PathologyFletcher Allen Health CareBurlington
  4. 4.Department of Orthopaedics and RehabilitationUniversity of Vermont College of MedicineBurlington

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