IMMUNOCYTOCHEMICAL DETECTION AND PHENOTYPIC CHARACTERIZATION OF MICROMETASTATIC TUMOR-CELLS IN BONE-MARROW OF PATIENTS WITH PROSTATE-CANCER

Monoclonal antibodies (mAbs) specific for cytokeratins are potent prob es for the identification of disseminated individual epithelial tumour cells in mesenchymal organs such as bone marrow. We have used a monoc lonal antibody (mAB) against cytokeratin 18 (CK18) for the detection o f 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 phosp hatase anti-alkaline phosphatase (APAAP) system for staining. We were able to detect CK18+ tumour cells in the marrow of 33% of patients wit h stage N0M0 prostate cancers. The incidence of CK18+ cells showed a s ignificant correlation with established risk factors, such as local tu mour extent, distant metastases and tumour differentiation. For furthe r characterization of such cells in patients with prostate cancer, we developed an immunocytochemical procedure for simultaneous labelling o f cytokeratin component no. 18 (CK18) and prostate-specific antigen (P SA). In a first step, cells were incubated with a murine mAb against P SA, followed by gold-conjugated goat anti-mouse antibodies. In a secon d step, a biotinylated mAb to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alka line phosphatase, which were developed with Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhanceme nt. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 14 patients with carcinomas of the prostate. The specifi city 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 i n bone marrow of patients with prostate cancer has been directly demon strated for the first time in this work. In conclusion, the approaches presented appear to be reliable methods of identifying and phenotypin g individual prostatic carcinoma cells and may help to identify those patients with prostate cancer who are at high risk of relapse.