ISAC XX Abstracts on Disc
Presented by Purdue University Cytometry Laboratories

Proliferative Potential of Neuroblastoma Cells Disseminated in the Hematopoietic System
 
Gabor Mehes1, Claudia M. Hattinger2, Thomas L÷rch3, Peter F. Ambros1
  1. International Society for Analytical Cytology
    Department of Pathology H-7643 Pecs P.O. Box 99
    Pecs, Austria
  2. Childrens Cancer Research Institute
    St. Anna Kinderspital
    Kinderspitalgasse 6
    Vienna, Austria, A-1090
  3. MetaSystems GmbH
    Altlussheim, FRG
    Germany

Abstract Number: 7220     Clinical Cytometry  –  Solid Tumors
 
Introduction: Early tumor cell dissemination and minimal residual disease are of increasing clinical interest, however, little is known about the biological impact of rare tumour cells.
Aims: In this study, automatic microscopical analyses were performed to elucidate the proliferative status of minimally disseminated tumour cells in the bone marrow of neuroblastoma patients.
Methods: Automated fluorescence microscopical analyses (MRDetect, MetaSystems) of bone marrow samples from multiple punction sites of 18 neuroblastoma patients were performed. A total of 76 cytospin preparations was automatically scanned and analysed for GD2/TRITC and Ki-67/FITC immunolabelling to demonstrate the Ki-67+ fraction of GD2+ tumour cells present in the bone marrow. Demonstration of tumor cell specific cytogenetic aberrations and exclusion of false GD2 immunostaining could be performed by sequential FISH analysis of the selected cells after automatic relocation.
Results: Through the automatic scanning approach, as few as 1 GD2 positive tumour cell out of 106 bone marrow mononuclear cells (MNCs) could be identified and quantified. The overall Ki-67 labelling index in these cells was found to be between 0.00 and 0.78. However, marked differences were found between samples obatined before and after CT. Tumor cell populations in the initial samples showed a high Ki-67 labelling index (mean 0.35œSD 0.15), independent of the tumor cell load in the bone marrow. In samples taken after CT, a significant decrease of the Ki-67 labelling index (0.18œ0.16) was observed. BM samples with less than 100 GD2+ tumor cells/106 MNCs showed a significantly lower GD2+/Ki-67+ fraction, than samples with a tumour load over 100 tumour cells/106 MNCs (0.12œ0.13 vs.0.37œ0.09, respectively). Despite of a general decrease, in 8 out of 31 samples with rare tumor cells, a retained Ki-67 labelling index (>0.2) was observed.
Conclusions: Rare tumour cells in the bone marrow may represent cell clones with high proliferative capacity. The determination of the Ki-67 labelling index, with special emphasis on residual tumour cells after CT, might be of therapeutical importance in the follow up of stage 4 tumours.
 
Keywords: micrometastasis; cell cycle; immunocytochemistry; automated microscopy