p53 protein expression in chronic lymphocytic leukemia.

Alterations in the function of the p53 pathway are frequently described in chronic lymphocytic leukemia (CLL), mostly associated with deletion of 17p13 and/or mutations of the TP53 gene. In the present study, we investigated 103 CLLs for the impact of protein expression of full-length p53 and its isoforms ß and γ. A strong correlation between deletions of 17p13 and an accumulation of full-length p53 protein was found and was associated with a worse outcome compared to CLL with normal p53 (treatment-free survival p < 0.001, overall survival p = 0.04). Interestingly, the relative expression levels between full-length p53 protein and its isoforms ß and γ were significantly altered in CLL even without deletions of 17p13, compared to normal B-cells (p = 0.005). Furthermore, CLLs with higher p53 protein ratios showed worse clinical courses compared to CLLs with lower p53 protein ratios. Taken together, the differential expression of p53 isoforms could disrupt the p53 response and contribute to CLL pathogenesis.

Telomeres and prognosis in patients with chronic lymphocytic leukaemia.

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length (ΔTL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p = 0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r = 0.509, p < 0.001). With respect to prognosis, a threshold ΔTL of -4.2 kb was the best predictor for progression-free and overall survival. ΔTL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the ΔTL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the ΔTL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.

Molecular analysis of IgD-positive human germinal centres.

It is controversially discussed whether human IgM(+)IgD(+)CD27(+) B cells, which carry somatically mutated Ig variable region (IgV) genes, are derived from germinal centres (GC) B cells or originate from another developmental pathway. GC composed of IgM(+)IgD(+) B cells, which co-express the CD70 surface marker, have been described in approximately 10% of tonsils. As IgM(+)IgD(+)CD27(+) B cells might be generated in such GC, we characterized IgD(+) tonsillar GC cells. GC dominated by IgD(+) B cells were present in 10 of 67 tonsils analyzed. Three GC were additionally positive for CD70. Detailed analysis of one such GC by microdissection and single-cell DNA PCR revealed IgD(+) GC B cells undergoing somatic hypermutation during clonal expansion. However, further analysis of this GC as well as five additional microdissected GC by reverse transcription (RT)-PCR for clonally related Igmu and Igdelta transcripts indicated that the B-cell clones in five of these six IgD(+) GC belong to the IgD-only B cell subset, which has deleted the Cmu gene, and that only one GC harboured a large IgM(+)IgD(+) B-cell clone. Hence, most IgD(+) GC consist of IgD-only B cells and fully developed IgM(+)IgD(+)(CD70(+)) GC are very rare. This indicates that the rare IgM(+)IgD(+) GC B-cell clones from IgD(+) GC contribute little to the large population of IgM(+)IgD(+)CD27(+) B cells. Finally, an RT-PCR analysis with clone-specific primers for two IgD(+) GC B-cell clones showed an absence of IgG or IgA class-switched clone members, indicating strict regulation of class switching and a selective production of IgD(+) B cells from such clones.

Preferential expression of truncated isoforms of FOXP1 in primary central nervous system lymphoma.

Forkhead box P1 (FOXP1) protein is a transcription factor involved in cell signaling and regulation of gene expression. The overexpression of FOXP1 in a subgroup of systemic diffuse large B-cell lymphomas has been associated with an exceptionally poor clinical outcome. Data on FOXP1 expression in primary central nervous system lymphomas (PCNSL), that is, diffuse large B-cell lymphomas confined to the central nervous system, are not yet available. We analyzed 43 PCNSL from immunocompetent patients. Immunohistochemistry showed expression of FOXP1 protein in 21 (88%) of 24 cases. All 19 PCNSL analyzed by quantitative gene expression analysis showed overexpression of truncated FOXP1 Isoforms 3 and 9 and downregulation of normal-size FOXP1 compared with nonmalignant germinal center B cells, the normal counterpart of PCNSL tumor cells. Thus, truncated FOXP1 isoforms are preferentially overexpressed in PCNSL as they are in diffuse large B-cell lymphomas. Although the mechanisms are presently unclear, this overexpression may contribute to a poor prognosis in PCNSL.

Recurrent inactivation of the PRDM1 gene in primary central nervous system lymphoma.

Primary lymphomas of the CNS (PCNSLs) show molecular features of the late germinal center exit B-cell phenotype and are impaired in their terminal differentiation as indicated by a lack of immunoglobulin class switching. Because the positive regulatory domain I protein with ZNF domain (PRDM1/BLIMP1) is a master regulator of terminal B-cell differentiation into plasma cells, we investigated a series of 21 PCNSLs for the presence of mutations in the PRDM1 gene and alterations in the expression pattern of the PRDM1 protein. Direct sequencing of all coding exons of the PRDM1 gene identified deleterious mutations associated with abrogation of PRDM1 protein expression in 4 of 21 (19%) PCNSLs. Thus, similar to systemic diffuse large B-cell lymphomas, PRDM1 may be a tumor suppressor in some PCNSL and contribute to lymphomagenesis by impairing terminal differentiation.

EBV transformation overrides gene expression patterns of B cell differentiation stages.

EBV-associated Hodgkin lymphoma (HL) and some post-transplant lymphoproliferative disease (PTLD) cases originate from pro-apoptotic germinal center (GC) B cells that have acquired destructive somatic Ig V gene mutations and were presumably rescued from apoptosis by EBV. To find out whether B cell receptor-crippled GC B cells acquire features of HL and/or PTLD cells upon EBV-infection and to reveal the impact of EBV on expression of B cell differentiation markers, we compared lymphoblastoid cell lines (LCLs) from GC B cells (including BCR-crippled GC-LCLs) to monoclonal LCLs from naïve B cells (N-LCLs). In addition, we analyzed the controversially discussed effect of EBV-infection on the GC B-cell-specific process of somatic hypermutation in vitro. Irrespective of their cellular origin, LCLs expressed CD20, CD30, CD38, AID, Pu.1, and with one exception Syk, but lacked expression of the GC B cell marker BCL-6. Interestingly, the T cell transcription factor GATA-3 that is aberrantly expressed in HL was induced in most GC-LCLs and the memory B cell marker CD27 was activated in N-LCLs. Remarkably, only 4 of 24 GC-LCLs showed significant somatic hypermutation activity, demonstrating that EBV usually silences hypermutation upon infection of GC B cells. Notably, one of three N-LCL showed a low level of intraclonal diversification. Thus, EBV-infection deregulates multiple differentiation factors and processes in B cells, leading to a largely homogenous phenotype of EBV-infected B cells in latency III.

Trisomy 19 is associated with trisomy 12 and mutated IGHV genes in B-chronic lymphocytic leukaemia.

The occurrence of trisomy 19 was investigated in 705 cases of B-chronic lymphocytic leukaemia (CLL) by metaphase cytogenetic and/or fluorescence in situ hybridisation analyses. Trisomy 19 was detected in 11 cases (1.6%), all of which also carried a trisomy 12; nine of 10 had mutated IGHV genes. In contrast, B-CLL cases with trisomy 12 lacking trisomy 19 mostly had unmutated IGHV genes. Karyotypes of the present study and the literature identified a strong correlation to trisomy 18 in addition to trisomy 12. Trisomy 19 seems to be a secondary event in B-CLL with trisomy 12, mostly originating from mutated B cells.

Transcriptional profiling of the nuclear factor-kappaB pathway identifies a subgroup of primary lymphoma of the central nervous system with low BCL10 expression.

Recent studies point to a role of nuclear factor (NF)-kappaB signaling in a subset of diffuse large B cell lymphomas. We have analyzed the expression of 21 genes encoding NF-kappaB family members, upstream modulators, and targets in 32 primary central nervous system lymphomas (PCNSLs) by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Compared with nonmalignant germinal center centroblasts, expression of BCL10, REL, IAP1, and TRAF1 was significantly lower in PCNSLs, whereas that of BAX, BCLXL, BCL2, MALT1, CARD9, CARD10, CARD11, CARD14, CCND2, cFLIP, RELA, RELB, NFKB1, NFKB2, and IRF4 was higher. Hierarchical clustering of gene expression data revealed two distinct subgroups of PCNSLs, which were characterized by significantly different transcriptional levels, predominantly of BCL10, but also of REL and IAP1. Thus, these quantitative RT-PCR data with expression of genes of the NF-kappaB family as well as NF-kappaB-regulated genes together with immunohistochemical detection of nuclear RELA and REL indicate activation of the NF-kappaB pathway in PCNSLs, which may contribute to their high proliferative activity and the low level of apoptosis.

The GNAS1 T393C polymorphism is associated with disease progression and survival in chronic lymphocytic leukemia.

PURPOSE: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal mature B cells. The G protein Galphas subunit has been linked to proapoptotic processes in cancer cell lines. The TT genotype of the GNAS1 T393C polymorphism is associated with increased Galphas transcript levels and a more favorable clinical course in different solid cancers. EXPERIMENTAL DESIGN: We retrospectively genotyped 144 patients with B-CLL to examine a potential association between T393C genotypes with progression-free survival (time from diagnosis to initiation of chemotherapy) and overall survival. RESULTS: The C-allele frequency in the patient group was 0.57 and not significantly different from that of healthy blood donors. Median progression-free survival was significantly different between genotypes (TT 130 months; TC 100 months; CC 31 months; P = 0.0066). Multivariable analysis showed that besides of ZAP-70 (P = 0.005) and Binet stage (P < 0.001), the T393C polymorphism was an independent prognostic factor for progression-free survival [hazard ratio (HR) CC versus TT 2.7; P = 0.010]. In Binet A stages, ZAP-70-positive patients with CC genotypes had a HR of 4.4 to receive first therapy compared with ZAP-70-negative patients with T-alleles (P = 0.0001). Regarding overall survival, CC genotypes (median overall survival, 197 months) were at highest risk for death compared with T-alleles (median overall survival, 310 months) in both univariate (HR, 4.8; P < 0.0001) and multivariable analysis (HR, 5.6; P = 0.002). CONCLUSIONS: Here, we show that the GNAS1 T393C status is a novel independent prognostic marker in patients with B-CLL. These results could help to define patients who could benefit from an early individualized therapy.