The G(-248)A polymorphism in the promoter region of the Bax gene does not correlate with prognostic markers or overall survival in chronic lymphocytic leukemia.

The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.

High expression of lipoprotein lipase in poor risk B-cell chronic lymphocytic leukemia.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavy-chain variable region genes (IGV(H)) compared to those with mutated IGV(H) genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N=51) or unmutated (N=53) IGV(H) (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV(H) mutational status (R=0.614; P<0.0001). High LPL expression predicted unmutated IGV(H) status with an odds ratio of 25.90 (P<0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P=0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

High expression of activation-induced cytidine deaminase (AID) mRNA is associated with unmutated IGVH gene status and unfavourable cytogenetic aberrations in patients with chronic lymphocytic leukaemia.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation of B-cells. We investigated the expression of AID mRNA by real-time polymerase chain reaction (PCR) in peripheral blood mononuclear cells of 80 patients with B-CLL. AID expression was detected in 45 of 80 patients (56%) at various levels, but was undetectable in 35 patients (44%). AID PCR positivity was associated with unmutated IGV(H) gene status (22 of 25 patients; P=0.002) and unfavourable cytogenetics (18 of 23 patients with deletion in 11q or loss of p53; P=0.040). Using a threshold level of 0.01-fold expression compared to Ramos control cells, even more significant associations were observed (P=0.001 for IGVH; P=0.002 for cytogenetics). A correlation was observed between individual AID levels and the percentage of V(H) homology (R=0.41; P=0.001). AID positivity predicted unmutated IGV(H) status with an odds ratio of 8.31 (P=0.003) and poor risk cytogenetics with an odds ratio of 3.46 (P=0.032). Significance was retained after adjustment for Binet or Rai stages. AID mRNA levels were stable over time. These data suggest a potential role of AID as a prognostic marker in B-CLL.

Topology of double minutes (dmins) and homogeneously staining regions (HSRs) in nuclei of human neuroblastoma cell lines.

Amplification of the MYCN gene is a characteristic feature of many neuroblastomas and is correlated with aggressive tumor growth. Amplicons containing this gene form either double minutes (dmins) or homogeneously staining regions (HSRs). To study the nuclear topology of these tumor-specific and transcriptionally active chromatin structures in comparison to chromosome territories, we performed fluorescence in situ hybridization with a MYCN probe and various chromosome paint probes, confocal laser scanning microscopy, and quantitative three-dimensional image analysis. The dmins formed dot-like structures in interphase nuclei and were typically located at the periphery of complexly folded chromosome territories; dmins noted in the chromosome territory interior were often detected within an invagination of the territory surface. Interphase HSRs typically formed extremely expanded structures, which we have never observed for chromosome territories of normal and tumor cell nuclei. Stretches of HSR-chromatin often extended throughout a large part of the cell nucleus, but appeared well separated from neighboring chromosome territories. We hypothesize that dmins are located within the interchromosomal domain (ICD) space and that stretches of HSR-chromatin align along this space. Such a topology could facilitate access of amplified genes to transcription and splicing complexes that are assumed to localize in the ICD space.

Separate and variably shaped chromosome arm domains are disclosed by chromosome arm painting in human cell nuclei.

Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.