Abstract 4391

Introduction

The Recently described NZB miRNA15a/16-1 mutation is syntenic to 13q14 deletion in CLL. The mir15a/16-1 complex is thought to regulate bcl-2 expression. Therefore the level of bcl-2 expression was examined more closely as it might be used to differentiate mono-allelic deletion from the diploid CLL clone.

Patients and Methods

Multicolor flow-cytometry using a panel of anti-bcl-2 FITC (clone 124, DAKO Cytomation, Glostrup, Denmark) and CD19, CD20, CD5, CD23, CD38, kappa, lambda, and CD45 was used. Bcl-2 expression was evaluated using the conventional bcl-2 index (Mean fluorescence intensity (MFI) B cell/ MFI T cells) in 25 untreated CLL patients, and 10 normal donor controls. A modified ratio based on normal remaining (NR) polyclonal B cells was also used (MFI clone/MFI NR) with a new gating strategy for bcl-2 expression analysis.

Results

The mean of the conventional bcl-2 index is significantly higher in CLL patients [1.52 ± 0.53(SD)] compared to the control [0.5 ± 0.19(SD)], p<0.001 Student‘s t test. When comparing the mean of conventional bcl-2 index (clone/T cell) versus the mean of the modified bcl-2 index (clone/NR) [1.78 ±0.61 (SD)] in only CLL patients there was no significant difference. On the other hand, the rank order analysis of the conventional and modified bcl-2 index showed an r=0.7, p< 0.001 indicating that both methods give similar results. In addition to the high correlation between the conventional and the modified ratio, the modified ratio was multimodal in distribution.

Conclusions

Bcl-2 expression varies largely among the lymphocyte sub-sets. The choice of the polyclonal B cells allows better segregation of multimodal bcl-2 expression. Since bcl-2 mRNA is a validated mir15a/16 target, increased bcl-2 expression maybe a direct result of and a surrogate marker for mir15a/16 levels and/or the 13q14 status. There are three possibilities: no deletion (diploid), heterozygous deletion, or homozygous deletion. This needs to be verified by fluorescence in situ hybridization (FISH) analysis of sorted CLL B cells based on bcl-2 expression.

Disclosures:

No relevant conflicts of interest to declare.

Topics:
bcl2 gene, cd19 antigens, cd20 antigens, cd23 antigen, cd45 antigens, flow cytometry, fluorescein-5-isothiocyanate, fluorescent in situ hybridization, infectious mononucleosis, rna, messenger

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2009 by The American Society of Hematology
2009
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