Urological Research

, Volume 22, Issue 1, pp 3–8 | Cite as

Immunocytochemical detection and phenotypic characterization of micrometastatic tumour cells in bone marrow of patients with prostate cancer

  • R. Oberneder
  • R. Riesenberg
  • M. Kriegmair
  • U. Bitzer
  • R. Klammert
  • P. Schneede
  • A. Hofstetter
  • G. Riethmüller
  • K. Pantel
Original Paper


Monoclonal antibodies (mAbs) specific for cytokeratins are potent probes for the identification of disseminated individual epithelial tumour cells in mesenchymal organs such as bone marrow. We have used a monoclonal antibody (mAB) against cytokeratin 18 (CK18) for the detection of individual metastatic tumour cells in bone marrow aspirates from 84 patients with carcinoma of the prostate. CK18+cells were detected in a sensitivity of 1 per 8×105 marrow cells using the alkaline phosphatase anti-alkaline phosphatase (APAAP) system for staining. We were able to detect CK18+tumour cells in the marrow of 33% of patients with stage N0M0 prostate cancers. The incidence of CK18+cells showed a significant correlation with established risk factors, such as local tumour extent, distant metastases and tumour differentiation. For further characterization of such cells in patients with prostate cancer, we developed an immunocytochemical procedure for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate-specific antigen (PSA). In a first step, cells were incubated with a murine mAb against PSA, followed by goldconjugated goat anti-mouse antibodies. In a second step, a biotinylated mAb to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. CK18+cells co-expressing PSA were found in bone marrow aspirates from 5 out of 14 patients with carcinomas of the prostate. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hyperplasia (BPH). Thus the prostatic origin of CK+cells in bone marrow of patients with prostate cancer has been directly demonstrated for the first time in this work. In conclusion, the approaches presented appear to be reliable methods of identifying and phenotyping individual prostatic carcinoma cells and may help to identify those patients with prostate cancer who are at high risk of relapse.

Key words

Prostate cancer Micrometastasis Bone marrow Double immunocytochemistry Cytokeratin Prostate-specific antigen 


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  1. 1.
    Bitzer U, Riesenberg R, Oberneder R, Kriegmair M, Pantel K, Hofstetter A (1992) Doppelmarkierung von CK18 and PSA an Gewebe, Zellkultur und Mikrometastasen. Urologe A 31 [Suppl]Google Scholar
  2. 2.
    Blatt WF, Robinson SM (1968) Membrane ultrafiltration: the diafiltration technique and its application to microsolute exchange and binding phenomena. Anal Biochem 26:151Google Scholar
  3. 3.
    Bonnard C, Papermaster DS, Kraehenbuhl J-P (1984) The streptavidin-biotin bridge technique: application in light and electron microscope immunocytochemistry. In: Polak JM, Varndell IM (eds) Immunolabelling for electron microscopy. Elsevier Science, Amsterdam, p 95Google Scholar
  4. 4.
    Bosch FX, Leube RE, Achtstätter T, Moll R, Franke WW (1988) Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ. J Cell Biol 106:1635Google Scholar
  5. 5.
    Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:219Google Scholar
  6. 6.
    Cote RJ, Rosen PP, Lesser ML, Old LJ, Osborne MP (1991) Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 9:1749Google Scholar
  7. 7.
    Danscher G (1981) Localization of gold in biological tissue. A photochemical method for light and electron microscopy. Histochemistry 71:81Google Scholar
  8. 8.
    Debus E, Weber K, Osborn M (1982) Monoclonal cytokeratin antibodies that distinguish simple from stratified squamous epithelia: Characterization on human tissues. EMBO J 1:1641Google Scholar
  9. 9.
    Debus E, Moll R, Franke WW, Weber K, Osborn M (1984) Immunohistochemical distinction of human carcinomas by cytokeratin typing with monoclonal antibodies. Am J Pathol 114:121Google Scholar
  10. 10.
    De Valck V, Renmans W, Segers E, Leunissen J, De Waele M (1991) Light microscopical detection of leucocyte cell surface antigens with a one-nanometer gold probe. Histochemistry 95:483Google Scholar
  11. 11.
    Donn F, Bruns T, Meyerinck L von, Schulz M, Becker W-M, Becker H, Klosterhalfen H (1989) Monoclonal antibody that defines the prostate specific antigen. Prostate 14:237Google Scholar
  12. 12.
    Franke WW, Moll R (1987) Cytoskeletal components of lymphoid organs. Synthesis of cytokeratin 8 and 18 and desmin in subpopulation of extrafollicular reticulum cells of human lymph nodes, tonsil and spleen. Differentiation 36:145Google Scholar
  13. 13.
    Gallee MPW, Vroonhoven CCJ van, Korput HAGM van der, Kwast TH van der, Kate FJW ten, Romijn JC, Trapman J (1986) Characterization of monoclonal antibodies raised against the prostatic cell line PC-82. Prostate 9:33Google Scholar
  14. 14.
    Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13Google Scholar
  15. 15.
    Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP (1983) LNCaP model of human prostatic carcinoma. Cancer Res 43:1809Google Scholar
  16. 16.
    Huang WM, Gibson SJ, Facer P, Gu J, Polak JM (1983) Improved section adhesion for immunocytochemistry using high molecular weight polymers of l-lysine as a slide coating. Histochemistry 77:275Google Scholar
  17. 17.
    Klammert R, Oberneder R, Kriegmair M, Riesenberg R, Hofmann T, Hader C, Hofstetter A (1992) Nachweis und klinische Relevanz einer frühen Tumorzellaussaat bei Nieren-und Blasentumoren. Urologe A 31 [Suppl]Google Scholar
  18. 18.
    Kriegmair M, Oberneder R, Klammert R, Hader C, Riesenberg R, Pantel K (1992) Cytokeratin marking of disseminated tumour cells—a new prognostic parameter for urological tumors? In: Klapdor R (ed) Tumor-associated antigens, oncogens, receptors, cytokines in tumor diagnosis and therapy at the beginning of the nineties. Zuckschwerdt, Munich, p 490Google Scholar
  19. 19.
    Lin CT, Liu JW, Song GX, Wu JY, Lam KW, Yam LT, Li CY (1986) Immunoultrastructural demonstration of prostatic acid phosphatase isoenzyme 2 in prostate carcinoma. J Urol 136:173Google Scholar
  20. 20.
    Lindemann F, Schlimok G, Dirschedl P, Witte J, Riethmüller G (1992) Prognostic significance of micrometastatic tumour cells in bone marrow of colorectral cancer patients Lancet 340:685Google Scholar
  21. 21.
    Loos JA, Roos D (1974) Ficoll-isopaque grandients for the determination of density distributions of human blood lymphocytes and other reticulo-endothelial cells. Exp Cell Res 86:333Google Scholar
  22. 22.
    Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11Google Scholar
  23. 23.
    Moll R, Achtstätter T, Becht E, Balcarova-Ständer J, Ittensohn M, Franke WW (1988) Cytokeratins in normal and malignant transitional epithelium. Am J Pathol 132:123Google Scholar
  24. 24.
    Nadji M, Tabei SZ, Castro A, Chu TM, Murphy GP, Wang MC, Morales AR (1981) Prostatic-specific antigen: an immunohistologic marker for prostatic neoplasms. Cancer 48:1229Google Scholar
  25. 25.
    Oberneder R, Kriegmair M, Pantel K, Bitzer U (1991) Methodik des immunzytochemischen Nachweises disseminierter Tumorzellen urologischer Tumoren. Urologe A 30[Suppl]:96Google Scholar
  26. 26.
    Oberneder R, Kriegmair M, Pantel K, Bitzer U, Hofstetter A, Riethmüller G (1991) Immunocytological detection of micrometastatic tumor cells in bone marrow of patients with stage M0 genitourinary tract malignancies. J Urol 145 [Suppl] 314aGoogle Scholar
  27. 27.
    Oberneder R, Kriegmair M, Klammert R, Riesenberg R, Hader C, Pantel K, Riethmüller G, Hofstetter A (1992) Nachweis und phänotypische Charakterisierung von mikrometastatischen Tumorzellen beim Nierenzellkarzinom. In: Hofstetter A, Kriegmair M (eds) Aktuelle Kontroversen in der Therapie des Nierenzellkarzinoms Klinische und experimentelle Urologie, vol 24, Zuckschwerdt, Munich, pp 86Google Scholar
  28. 28.
    Pantel K, Schlimok G, Kutter D, Schaller G, Genz T, Wiebecke B, Backmann R, Funke I, Riethmüller G (1991) Frequent down-regulation of major histocompatibility class I antigen expression on individual micrometastatic carcinoma cells. Cancer Res 51:4712Google Scholar
  29. 29.
    Riesenberg R, Oberneder R, Kriegmair M, Epp M, Bitzer U, Hofstetter A, Braun S, Riethmüller G, Pantel K (1993) Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumor cells. Histochemistry 99:61Google Scholar
  30. 30.
    Roth J (1982) Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antiens in paraffin sections. J Histochem Cytochem 30:691Google Scholar
  31. 31.
    Schlimok G, Funke I, Holzmann B, Göttlinger G, Schmidt G, Häuser H, Swierkot S, Warnecke HH, Schneider B, Koprowski H, Riethmüller G (1987) Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. Proc Natl Acad Sci USA 84:8672Google Scholar
  32. 32.
    Schlimok G, Funke I, Bock B, Schweiberer B, Witte J, Riethmüller G (1990) Epithelial tumor cells in bone marrow of patients with colorectal cancer: immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 8:831Google Scholar
  33. 33.
    Schlimok G, Funke I, Pantel K, Strobel F, Lindemann F, Witte J, Riethmüller G (1991) Micrometastatic tumour cells in bone marrow of patients with gastric cancer: methodological aspects of detection and prognostic significance. Eur J Cancer 27:1461Google Scholar
  34. 34.
    Song GX, Lin CT, Wu JY, Lam KW, Li CY, Lam LT (1985) Immunoelectron microscopic demonstration of prostatic acid and phosphatase in human hyperplastic prostate. Prostate 7:63Google Scholar
  35. 35.
    Stein BS, Vangore S, Petersen RO (1984) Immunoperoxidase localization of prostatic antigens. Comparison of primary and metastatic sites. Urology 24:146Google Scholar
  36. 36.
    Trainer DL, Kline T, McCabe FL, Faucette LF, Feild J, Chaikin M, Anzano M, Rieman D, Hoffstein S, Li D-J, Gennaro D, Buscarino C, Lynch M, Poste G, Greig R (1988) Biological characterization and oncogenes expression in human colorectal carcinoma cell lines. Int J Cancer 41:287Google Scholar
  37. 37.
    Warhol MJ, Longtine JA (1985) The ultrastructural localization of prostatic specific antigen and prostatic acid phosphatase in hyperplastic and neoplastic human prostates. J Urol 134:607Google Scholar
  38. 38.
    Wernert N, Seitz G, Achtstätter T (1987) Immunohistochemical investigation of different cytokeratins and vimentin in the prostate from the fetal period up to adulthood and in prostate carcinoma. Pathol Res Pract 182:617Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • R. Oberneder
    • 1
  • R. Riesenberg
    • 1
  • M. Kriegmair
    • 1
  • U. Bitzer
    • 1
  • R. Klammert
    • 1
  • P. Schneede
    • 1
  • A. Hofstetter
    • 1
  • G. Riethmüller
    • 2
  • K. Pantel
    • 2
  1. 1.Klinikum GroßhadernUrologische UniversitätsklinikMünchen 70Germany
  2. 2.Institut für ImmunologieLudwig-Maximilians-UniversitätMünchenGermany

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