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 heavychain 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 ¼ 51) or unmutated (N ¼ 53) IGV H (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV H mutational status (R ¼ 0.614; Po0.0001). High LPL expression predicted unmutated IGV H status with an odds ratio of 25.90 (Po0.0001) 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 ¼ 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.

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 ¼ 0.002) and unfavourable cytogenetics (18 of 23 patients with deletion in 11q or loss of p53; P ¼ 0.040). Using a threshold level of 0.01-fold expression compared to Ramos control cells, even more significant associations were observed (P ¼ 0.001 for IGV H ; P ¼ 0.002 for cytogenetics). A correlation was observed between individual AID levels and the percentage of V H homology (R ¼ 0.41; P ¼ 0.001). AID positivity predicted unmutated IGV H status with an odds ratio of 8.31 (P ¼ 0.003) and poor risk cytogenetics with an odds ratio of 3.46 (P ¼ 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.

Stimulation of primary human monocytes from several donors by cross-linking of Fc gamma receptor type I (Fc gamma RI) and Fc gamma RII gave rise to calcium mobilization and protein tyrosine phosphorylation. These early events were not observed without cross-linking. CD45, a transmembrane tyrosine phosphatase, when co-cross-linked with either Fc gamma RI or Fc gamma RII, could prevent Fc gamma RI and Fc gamma RII-mediated calcium mobilization and protein tyrosine phosphorylation. When interleukin (IL)-6 production was measured, we noted a strong IL-6 production after activation of primary human monocytes by cross-linking of Fc gamma RI or Fc gamma RII. In contrast to calcium mobilization and tyrosine phosphorylation of proteins, IL-6 production was not affected by co-cross-linking of CD45 with either Fc gamma RI or Fc gamma RII. Interestingly, cross-linking of the CD45 itself was sufficient to induce IL-6 production. Our results show that the CD45 molecule is important in modulating early events following stimulation of primary human monocytes by cross-linking of Fc gamma RI or Fc gamma RII. However, triggering of CD45 alone can also induce IL-6 production, indicating that CD45 ligation itself can give signals and may have an important role in cytokine induction pathways.

Introduction: To establish surrogate markers for IgVH mutation status in B-CLL, we performed microarray analysis with mutated and unmutated patient samples. Among the most differentially expressed genes was PEG10, a maternally imprinted gene located on chromosome 7q21. PEG10 is known to be expressed during placental development and in hepatocellular carcinoma. Here we have investigated the association of PEG10 with B-CLL subtypes and risk factors as well as its transcriptional regulation. In addition, we studied its expression in other hematologic malignancies. Methods and Results: In our microarray experiments, PEG10 was highly expressed (2.1-fold) in unmutated samples. We confirmed these results by real time PCR in 42 B-CLL patients and found a significant correlation between PEG10 expression and unmutated IgVH status (P=0.007). High PEG10 expression was associated with chromosomal del(11q) (P=0.008), a shorter time to treatment (P=0.009) as well as a positive Coombs test (P=0.04). High expression was found in B-CLL and sorted CD19+ B-CLL cells, plasmoblastic DLBCL as well as in the Burkitt’s lymphoma cell line Ramos. In normal tissues, relatively high levels were seen in brain, liver, lung and kidney, but these expression levels were lower than in CD19+ B-CLL cells. Expression in sorted B cells from healthy donors was lower than two orders of magnitude. Intracellular expression of the PEG10 protein in B-CLL cells was confirmed by immunofluorescence studies using antibodies that recognize specifically PEG10-RF1. To address the question whether PEG10 overexpression results from loss of imprinting, we performed allele-specific as well as methylation-specific PCR. Imprinting was maintained in 6 patients with B-CLL. Analysis of a mother and son, both having B-CLL, confirmed that at least in this familial case the maternal PEG10 allele was silenced as expected. Interestingly, similar expression patterns were observed for the neighbouring gene sarcoglycan epsilon indicating a common mechanism of deregulation of the 7q21 locus in high risk B-CLL. Conclusions: We show that PEG10 is differentially expressed in B-CLL and is associated with del(11q), a shorter time to treatment and unmutated IgVH mutation status, suggesting its role as a marker for high risk B-CLL. PEG10 overexpression is unlikely to result from loss of imprinting. Further studies of the gene locus at 7q21 are in progress.

The prognosis of patients with B-CLL is largely determined by the karyotype of the malignant clone. Microarray technology has facilitated linkage between chromosomal aberrations and gene expression signatures. We have investigated the gene expression profile associated with trisomy 12 (+12). Expression data were obtained by microarray analysis of mRNA from unselected PBMNC of 4 patients with +12 and compared with 16 B-CLL controls. 146 genes were at least 2-fold over- or underexpressed in samples with +12. Five of the 16 genes showing the strongest correlation with +12 were selected for further analysis (HIP1R FC=3,43; MYF6 FC=3,92; P2RY14 FC=−9,59; RASGRP3 FC=−3,85; SLC2A6 FC=2,13) and validation by real time PCR: HIP1R located on chromosome 12q24, with a fold change (FC) of 3,43, MYF6 (chromosome 12q21, FC=3,92), P2RY14 (chromosome 3q21–q25, FC-9,59) RASGRP3 (chromosome 2p25.1–p24.1, FC=−3,85). SLC2A6 (chromosome 9q34, FC=2,13). Quantitative PCR was performed with mRNA from 61 patients (29 with +12, 32 B-CLL controls) and 2 healthy donors. Only 3 genes were significantly associated with +12 compared to the B-CLL-controls in this evaluation: HIP1R (3,486; p<0,0001), MYF6 (1,498; p=0,005), P2RY14 (1,216; p=0,013). (Table1). Two of these genes (HIP1R, MYF6) are located on chromosome 12 indicating a “gene dosage effect”, while P2RY14 is localized on a different chromosome suggesting trans-acting processes. We have used expression of HIP1R as a surrogate marker for trisomy 12. The predicted sensitivity was 79,3% and the predicted specifity was 90,6. Analysis of CD19+ selected B-CLL and normal B-cells revealed that MYF6 is exclusively expressed in normal or malignant B-cells in peripheral blood. We confirmed that MYF6 is highly specific for skeletal muscle, however strong expression was found in normal tonsils, DLBCL, and other B-cell malignancies. Our data link a specific gene expression signature with trisomy 12. 3 novel marker genes were identified, which could be used as diagnostic tools. The linkage with P2RY14 suggests that +12 influences the expression of genes from other chromosomes. Table 1 Mean + 12, N=29 Mean B-CLL controls, N=32 p-value Locus Fold change microarray HIP1R 0,7819 0,2243 0,000 12q24 3,43 MYF6 37,55 25,06 0,005 12q21 3,92 P2RY14 −0,2873 0,3494 0,013 3q21–q25 −9,59 RASGRP3 0,8404 1,0774 0,055 2p25.1–p24.1 −3,85 SLC2A6 29,78 22,37 0,080 9q34 2,13