Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase

Abstract

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I–II breast cancer patients were stained with anti‐cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)‐based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti‐CK MAbs AE1/AE3 or A45‐B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n=257), anti‐CK‐positive cells were detected in 26·6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control‐positive cells were detected in 5·4 per cent of patients. Some of the false‐positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly‐reactive cells as CD45‐negative and human Ig kappa/lambda‐positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens. © 1998 John Wiley & Sons, Ltd.

Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer

“…MMG is a protein associated with most breast cancers, and RT-PCR determination of MMG expression in BM has been found to be specific and sensitive. 24 Its expression may inversely correlate with tumor burden and prognosis. 30 We elected to study BM rather than peripheral blood cells for several reasons: firstly, bone and BM are usual sites of metastasis by breast cancer; secondly, most studies to date have focused on BM micrometastases, and have found a correlation among presence of tumor cells and survival; [15][16][17][18] thirdly, guidelines for analysis and evaluation of isolated tumor cells in the BM have been issued by a scientific society (ISHAGE, now ISCT); 31 and finally, BM samples were already available because of chimerism analysis, without the need to perform fastidious ad hoc sampling.…”

“…23 Immunocytochemistry is relatively easy to apply, not requiring sophisticated devices, and standardization criteria have been reported. 24 We have recently found that a nested RT-PCR assay for the expression of maspin and mammaglobin (MMG) genes is a sensitive and specific assay for detection of breast cancer cells in the BM. 25 In comparison with immunocytochemical assay, RT-PCR has the advantage of being operator-independent, and allows a permanent record of the results.…”

The presence of bone marrow cytokeratin-immunoreactive cells does not predict outcome in gastric cancer patients

“…Also the different immunocytochemical procedures used in the various studies might be a source of bias. Different patterns of specificity and sensitivity among the various MoAbs used are known to influence the tumour-cell detection (Borgen et al, 1998;Page et al, 1999). Due to these problems, also in gastric cancer there was a wide range of positivity (from 25% to 53%).…”

“…The magnitude of 10 6 mononuclear cells has been shown to provide a higher probability of detecting even one or two positive cells (Borgen et al, 1998;Funke and Schraut, 1998). Another aspect that might be a source of pitfalls is that during the cytospin procedure there is a loss of mononuclear cells and the number of cells rescued on the slides is lower than that counted in the suspension before starting the procedure.…”

“…As already pointed out, false positive immunocytochemical reactions have been demonstrated between haematopoietic and tumour cells (Borgen et al, 1998). Bone marrow tumour cell contamination is still a grey area and it is risky not to take cell morphology into account; experienced observers are therefore necessary for the evaluation of bone marrow cytospins.…”