bromochloroacetic-acid and Bone-Marrow-Diseases

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)

Topics: Bone Marrow Diseases; Bone Neoplasms; Carcinoma; Cells, Cultured; Humans; Immunohistochemistry; Keratins; Male; Phenotype; Prostate-Specific Antigen; Prostatic Neoplasms; Staining and Labeling

Present diagnostic techniques do not allow the detection of early metastatic spread of tumor cells, although this spread largely determines the clinical course of patients with small primary cancers. By use of monoclonal antibody CK2 to the epithelial cytokeratin component number 18 (CK18), individual disseminated carcinoma cells present in bone marrow of cancer patients can now be identified (G. Schlimok, I. Funke, B. Holzmann, G. Göttlinger, G. Schmidt, H. Häuser, S. Swierkot, H. H. Warnecke, B. Schneider, H. Koprowski, and G. Riethmüller, Proc. Natl. Acad. Sci. USA, 84: 8672-8676, 1987; F. Lindemann, G. Schlimok, P. Dirschedl, J. Witte, and G. Riethmüller, Lancet, 340: 685-689, 1992). In the present study, we applied this approach to patients with operable non-small cell lung cancer. CK18 was expressed on 84 of 88 (95.5%) primary adenocarcinomas and squamous cell carcinomas. Irrespective of primary tumor histology, single aspirates of iliac bone marrow from 18 of 82 (21.9%) lung cancer patients exhibited between 1 and 531 CK18+ cells/4 x 10(5) nucleated marrow cells. The specificity of our assay is underlined by the small rate of "false-positive" cells being observed in only 2 of 117 (1.7%) marrow samples from control patients with no evidence for an epithelial malignancy at the time of aspiration. Comparison with established risk factors demonstrated positive correlations (P < 0.05) between the size and histological grade of the primary carcinoma and cytokeratin positivity in iliac bone marrow. In contrast, the association with the metastatic involvement of regional lymph nodes was only weak (P = 0.09). Following a median observation period of 13 months, patients who displayed cytokeratin-positive cells in iliac bone marrow at the time of primary surgery relapsed more frequently as compared to patients with a negative marrow finding (66.7 versus 36.6%; P < 0.05). This difference was even more pronounced by comparing the rates of manifest skeleton metastasis observed in both groups (26.7 versus 2.4%; P < 0.005). Finally, colabeling of CK18+ cells in marrow with monoclonal antibodies to proliferation-associated markers, such as the nucleolar antigen p120 or the tyrosine kinase receptor erbB2, exemplified the oncogenic capacity of CK18+ micrometastatic cells. In conclusion, CK18+ cells present in the bone marrow of patients with apparently operable non-small cell lung cancer exhibit the potential to form solid metastases. Therefore, the approach presente

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Bone Marrow Diseases; Carcinoma, Non-Small-Cell Lung; Female; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Male; Middle Aged; Proto-Oncogene Proteins; Receptor, ErbB-2; Risk Factors

Using conventional examination (CE) of H&E stained slides from bone marrow aspirates, metastases can be detected in approximately 25% of patients with small cell lung cancer. We investigated a panel of monoclonal antibodies using immunohistochemistry in the diagnosis of bone marrow infiltration from SCLC and compared the results with CE. Seven monoclonal antibodies raised against epithelial antigens (CAM 5.2, MOV 15, NCCST 433, PE 35, LCA1/L38, HMFG 1 AND HMFG 2) were applied on bone marrow sections from three groups of patients (pts): (1) 19 pts in whom SCLC-metastases were detected by CE, (2) 44 pts with SCLC in whom metastases could not be detected by CE, and (3) 20 pts with non-malignant bone marrow diseases. All the antibodies except LCA1/L38 were positive in 60-90% of the slides with infiltrating tumour cells in group 1. No positive tumour cells were detected in group 2. A few plasma cells and megakaryocytes were slightly positive for MOV 15 and NCCST 433, but no other positive cells were detected in group 3. In conclusion, the monoclonal antibodies used in this study may be useful for diagnostic purposes when a suspicious looking infiltration is detected by CE. However, these antibodies could not detect metastatic tumour cells in the bone marrow sections from patients in whom CE did not reveal any tumour cells.

Topics: Antibodies, Monoclonal; Bone Marrow Diseases; Carcinoma, Small Cell; Humans; Keratins; Lung Neoplasms; Membrane Glycoproteins; Mucin-1; Neoplasm Metastasis; Survival Analysis

Twenty percent (n = 6) of Stage III or IV breast cancer patients (n = 30) had bone marrow metastases detected in bilateral bone marrow biopsy/aspiration preparations using standard histologic preparations. Each metastasis was also detected by four separate monoclonal antibodies (MAbs) which recognize breast carcinoma associated antigens (DF3, anti-EMA, HMFG-2, and CAM5.2). These MAbs were then utilized to stain other bone marrow preparations (n = 81) to determine their utility for the detection of micrometastatic breast carcinoma. MAbs HMFG-2, anti-EMA, and DF3 were each strongly reactive with bone marrows containing histologically-evident metastatic breast carcinoma (18/18). These anti-epithelial membrane antigen MAbs, however, were also reactive with rare plasma cells and immature cells (as well as cell clusters) in some of the control bone marrow samples tested, including those from normal patients and patients with hematologic disorders. They also reacted with some of the preparations from patients with leukemia and lymphoma, and with uninvolved marrows from patients with non-epithelial malignancies. The anti-keratin MAb CAM5.2, in contrast, reacted with 83% (15/18) breast cancer metastases and failed to stain any cells in the various categories of control marrow preparations. These data suggested that MAb CAM5.2 might be utilized to immunohistochemically differentiate micrometastatic breast carcinoma from immature myeloid or erythroid elements. Each MAb was then reacted with histologically uninvolved marrow preparations from the remaining 24 of 30 breast cancer patients in an attempt to identify occult breast carcinoma metastases. While MAbs HMFG-2, DF3, and anti-EMA demonstrated reactive cells in some of these marrows, this reactivity was similar to that seen with control preparations. MAb CAM5.2, in contrast, was negative with all specimens. These data suggest that MAb CAM5.2 may be a useful immunologic probe for the detection and confirmation of metastatic breast carcinoma in bone marrow, while more caution must be employed in the interpretation of results obtained using MAbs anti-EMA, DF3, and HMFG-2.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Bone Marrow Diseases; Bone Marrow Examination; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Child; Child, Preschool; Female; Humans; Keratins; Middle Aged; Neoplasm Metastasis; Neoplasm Staging

The present study describes 11 cases (10 carcinomas, one rhabdomyosarcoma) in which immuno-alkaline phosphatase labelling with monoclonal antibodies was used to demonstrate metastatic cells in routine smears of aspirated bone marrow. Carcinoma cells were detected using antibodies against epithelial cytokeratins, milk fat globule membrane antigen and carcinoembryonic antigen, and rhabdomyosarcoma cells with monoclonal anti-desmin. In four of the carcinoma cases it had not been possible to identify malignant cells in routinely stained marrow smears, whilst the case of disseminated rhabdomyosarcoma had initially been diagnosed (and treated) as a case of acute lymphoblastic leukaemia. The anti-cytokeratin antibody was found to be the most valuable of the anti-epithelial reagents used, since it labelled malignant cells in all of the 10 cases of carcinoma and gave the strongest reactions. These results suggest that immunocytochemical labelling should be used in cases of suspected carcinoma whenever conventional examination of marrow smears yields negative results, and furthermore (as illustrated by the case of rhabdomyosarcoma) that the technique is of value for identifying the true nature of poorly differentiated neoplasms in bone marrow.

Topics: Aged; Antibodies, Monoclonal; Antigens, Neoplasm; Bone Marrow Diseases; Carcinoembryonic Antigen; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Membrane Proteins; Middle Aged; Mucin-1; Neoplasm Metastasis; Rhabdomyosarcoma