Effects of Different Immunolabeling Techniques on the Detection of Small-cell Lung Cancer Cells in Bone Marrow

Abstract

SUMMARY Recent reports have suggested that the immunodetection of tumor cells in bone marrow of small-cell lung cancer (SCLC) patients is by far more effective than traditional cytohistological methods and that this may be clinically relevant. This study aimed to evaluate whether the level of detection of tumor cells in bone marrow is affected by different immunostaining methods. Using two anti-NCAM monoclonal antibodies (MAbs), we compared four different "sandwich" methods on cytospin preparations of the N592 human SCLC cell line and of bone marrow aspirates from 37 SCLC patients. Our data indicate that the combination of the alkaline phosphatase-anti-alkaline phosphatase and streptavidinbiotin-alkaline phosphatase complex methods provides the best results in terms of sensitivity and specificity, and of intensity of immunoreaction and absence of staining background. Moreover, bone marrow micrometastases detected by this method were prognostically relevant and identified, among patients with apparently limited disease according to conventional staging procedures, a subgroup with shorter survival. We suggest that the choice of a sensitive immunostaining technique may significantly increase the detection rate of SCLC cells in bone marrow, mirroring the biological aggressiveness of the disease.

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

“…The detection of the presence of epithelial cells in bone marrow has been studied as a prognostic factor in many epithelial malignancies such as breast (Braun et al, 2000), small- (Pasini et al, 1998;Pelosi et al, 1999a) and non small-cell lung cancer (Passlick et al, 1999) and oesophageal cancer (Thorban et al, 1996). Bone marrow isolated tumour cells are also evident in many patients with gastric cancer, but its prognostic significance is still uncertain (Funke and Schraut, 1998;Hermanek et al, 1999).…”

“…The preparation of the samples and the immunocytochemical procedures were performed as previously described with some modifications (Pelosi et al, 1999a). Briefly, bone marrow aspirates (4 to 6 ml each) were collected in heparinized syringes, diluted with an equivalent volume of RPMI 1640 and sedimented onto a Ficoll-Hypaque density-gradient.…”

Immunocytochemical detection of bone marrow micrometastases in colorectal carcinoma patients, using a monoclonal antibody to villin

“…A first step in working with tissue‐specific markers is to select a protein and its antibody. Some that have already been tested include monoclonal antibodies against NCAM for detection of micrometastases in bone marrow aspirates of small‐cell lung cancer patients (31).…”

Immunocytochemical assessment of bone marrow aspirates for monitoring response to chemotherapy in small-cell lung cancer patients

“…Moreover, BMA from healthy volunteers and patients with unrelated, non-malignant diseases provided the control group for non-carcinoma patients. In a set of experiments we used BMA from healthy volunteers and from patients with unrelated, non-malignant diseases artificially contaminated with a known number of tumour cells at different dilutions in order to assess the sensitivity of our immunocytochemical method in the detection of NCAM-reactive tumour cells as described elsewhere (Pelosi et al, 1999). Results of these artificial contamination tests showed that NCAM-positive tumour cells were detected in expected quantities in all mixtures up to the final dilution of 1/10 5 bone marrow cells (data not shown).…”

“…Results of these artificial contamination tests showed that NCAM-positive tumour cells were detected in expected quantities in all mixtures up to the final dilution of 1/10 5 bone marrow cells (data not shown). Double immunostains for NCAM and low molecular weight cytokeratins were carried out in both BMA and artificial mixtures to differentiate, in doubtful cases, true tumour epithelial cells from other NCAM-positive non-tumour cells, including natural killer (NK) cells, as described elsewhere (Pelosi et al, 1999).…”