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

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 x 10(5) 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 gold-conjugated 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).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells.

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18+/PSA- HT29 colon carcinoma cells. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. 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 hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)