BCL2 Mutations Research Articles

Bcl10/Malt1 Signaling Is Essential for TCR-Induced NF-κB Activation in Thymocytes but Dispensable for Positive or Negative Selection

During T cell development in the thymus, high-affinity/avidity TCR engagement induces negative selection by apoptosis, while lower affinity/avidity TCR interactions lead to positive selection and survival of thymocytes. Yet, the mechanisms that discriminate between positive and negative selection are not fully understood. One major regulator of survival and apoptosis in lymphoid cells is the transcription factor NF-kappaB. Several reports have indicated key roles for NF-kappaB in positive and negative selection. In peripheral T cells, TCR ligation activates NF-kappaB through a selective pathway that involves protein kinase Ctheta, Bcl10, and Malt1. While protein kinase Ctheta is dispensable for thymic TCR signaling, the molecular roles of Bcl10 and Malt1 in thymocytes have not been investigated. In the present study, we show that both Bcl10 and Malt1 are essential for TCR signaling in thymocytes as a genetic disruption of either molecule blocks TCR-induced NF-kappaB activation in these cells. To investigate the function of this pathway in thymic selection, we introduced the Bcl10 or Malt1 mutations into three well-established TCR transgenic mouse models. Surprisingly, using several in vivo or in vitro assays, we were unable to demonstrate a role for TCR-induced NF-kappaB activation in either positive or negative selection. Thus, while TCR signaling to NF-kappaB controls the activation of mature T cells, we suggest that this pathway is not involved in the positive or negative selection of thymocytes.

Somatic hypermutation and mRNA expression levels of the BCL‐6 gene in patients with hepatitis C virus‐associated lymphoproliferative diseases*

Chronic hepatitis C virus (HCV) infection leads to mixed cryoglobulinaemia (MC) and B-cell non-Hodgkin lymphoma (B-NHL). Aberrant somatic hypermutation and deregulation of the oncogene BCL-6 is associated with lymphomagenesis. Recently, HCV was shown to induce BCL-6 mutations in vitro. The BCL-6 gene (area B) was cloned and sequenced from peripheral blood mononuclear cells (PBMC) of 21 chronically HCV-infected patients with or without MC and B-NHL, and six healthy controls. Mutational frequencies, genetic complexity and diversity were calculated. BCL-6 mRNA from PBMC was quantified by real-time polymerase chain reaction, and additional sustained virologic responders to antiviral therapy and HBV patients served as controls. The overall/recurrent mutational frequencies tended to be lower in MC and B-NHL patients when compared with controls (P = 0.15 and 0.06, respectively). Genetic complexity was significantly lower in MC and B-NHL patients (P = 0.025). BCL-6 mRNA concentration was decreased in all HCV patients when compared with healthy controls, sustained virologic responder and HBV patients (P = 0.005). Although HCV can induce BCL-6 mutations in vitro, lower mutational frequencies and decreased BCL-6 mRNA expression in vivo suggest no major role of aberrant somatic hypermutation in HCV-associated MC and B-NHL.

Bortezomib induces different apoptotic rates in B-CLL cells according to IgVH and BCL-6 mutations

B-cell chronic lymphocytic leukemia (B-CLL) is a remarkably heterogeneous disorder. Some patients have an indolent disease whereas others undergo a more agressive presentation needing treatment. New therapeutics approaches are necessary for the treatment of B-CLL. Bortezomib (Btz), is a proteasome inhibitor, currently undergoing clinical trials whose function, at least in part, by stabilizing the IkappaBalpha protein and inhibiting NFkappaB activation. The objective of this work was to study the effects of Btz on isolated human B-CLL cells, in vitro, and to correlate the differential rates of apoptosis induction with biological variables. 31 B-CLL samples, from patients in stage A of Binet were used for this study, and the apoptotic effect of Btz on these cells was measured. Our data show that Btz treatment of B-CLL cells induces apoptosis in a time and dose-dependent manner. The apoptosis induction is mediated in part by inhibition of NFkappaB and is dependent on caspases activation. Interesting, in IgVH mutated cells, Btz have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status. Btz is a promising pharmacologic agent for the treatment of B-CLL, but its efficacy seems to be related to IgVH and BCL-6 mutational status, therefore, it could be interesting to further investigate the mechanisms involved in the different behavior of the cells in response to apoptosis induction by this drug.

Molecular, cytogenetic, and immunophenotypic characterization of follicular lymphoma grade 3B; a separate entity or part of the spectrum of diffuse large B-cell lymphoma or follicular lymphoma?

We studied a histological homogeneous group of 29 cases with the diagnosis of follicular lymphoma (FL) grade 3B (FL3Bs). In a previous study, we subdivided this group in 3 subgroups based on (1) aberrations of the 3q27 region, (2) lack of 3q27 and t(14;18), and (3) the presence of a t(14;18). In this study, we further characterized the FL3B lymphomas that are currently part of the spectrum of FL in the WHO classification, taking into account other cytogenetical aberrations, immunohistochemistry for P53, bcl2, bcl6, and CD10, rearrangement of the proto-oncogene myc, and mutation of the tumor suppressor gene TP53. With respect to P53, bcl2, bcl6 expression, myc rearrangement, and TP53 mutation, FL3B represents a homogeneous group. CD10 expression and gain of chromosome 7, considered to be typical FL markers, were more common in the FL3B t(14;18)-positive subgroup. The lack of CD10 expression and gain of chromosome 7 in most cases in the other 2 subgroups suggest that those cases have a closer relation to diffuse large B-cell lymphomas.

Molecular Cytogenetic Evidence of t(14;18)(IGH;BCL2) in a Substantial Proportion of Primary Cutaneous Follicle Center Lymphomas

In contrast to nodal follicular lymphoma, limited data exist on genetic changes in primary cutaneous follicular lymphoma (primary cutaneous follicle center lymphoma according to WHO-EORTC). The detection rate of the BCL2 rearrangement, representing the characteristic t(14;18)(q32;q21) underlying follicular lymphoma, by polymerase chain reaction (PCR) has been reported to vary over a wide range (0%-41%), and only a few cases have been studied by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). In this study, 27 primary cutaneous follicle center lymphomas were analyzed by FISH and the results compared with those obtained by PCR. FISH demonstrated translocations affecting the immunoglobulin heavy chain locus (IGH) in 14 of 27 cases (52%): a t(14;18)(q32;q21) involving BCL2 was found in 11 cases (41%), a t(3;14)(q27;q32) affecting BCL6 in 2 cases (7%), and in 1 case the partner gene of IGH could not be identified. Interestingly, PCR did not detect BCL2 rearrangement in any case. These data suggest that the t(14;18)(q32;q21) frequently occurs in primary cutaneous follicular lymphoma. The reason(s) why BCL2 rearrangements escape the detection by PCR is (are) not clear but could be due to BCL2 mutations, breakpoints outside the amplified DNA, or a high load of somatic mutations.

A spindle cell variant of diffuse large B-cell lymphoma possesses genotypic and phenotypic markers characteristic of a germinal center B-cell origin

Lymphoma with prominent spindle cell features, the so-called spindle cell lymphoma, is an unusual morphological variant of diffuse large B-cell lymphoma. Five new cases of spindle cell lymphoma have been analyzed by a multiparameter approach in order to clarify its clinical and biological features. All patients presented advanced stage disease with extranodal involvement. Vagina was the most common extranodal site. All patients received chemotherapy and are alive in complete remission. Morphologically, all five cases exhibited proliferation of spindle cells with a vaguely storiform pattern highly suggestive of spindle cell neoplasms of nonlymphoid origin. In contrast, the results of immunohistochemical analysis indicated that all five cases were hematolymphoid neoplasms of the B-cell lineage. These lymphomas consisted of a B-cell clonal population which exhibited somatic immunoglobulin and BCL-6 mutations as well as BCL-6 protein expression. The neoplastic spindle cells therefore closely resemble B cells residing in the germinal center. The absence of MUM1 expression in neoplastic spindle cells suggested that neoplastic spindle cells may be related to the early phases of intragerminal center maturation of B cells. The germinal center phenotype, with restricted expression of BCL-6, was associated with the presence of a primary extranodal origin, normal lactate dehydrogenase levels, and good response to treatment.

Distinctive Patterns of BCL6 Molecular Alterations in Different Subsets of Diffuse Large B-Cell Lymphoma and Their Functional Consequences.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B cell-like (ABC), and primary mediastinal (PM) DLBCL. The BCL6 gene on chromosome 3q27 is a transcriptional repressor and is required for GC formation and function. Two molecular alterations involving the BCL6 gene are commonly observed in DLBCL: chromosomal translocations and mutations in the 5′ non-coding region.The functional consequences of BCL6 translocation and mutation have not been studied in the context of DLBCL subgroups. Therefore, we examined the frequency of translocations and the spectrum of BCL6 mutations in exon 1 and intron 1 in different DLBCL subgroups. We correlated these findings with BCL6 mRNA and protein expression, as well as the expression of BCL6 target genes. Fluorescence in situ hybridization (FISH) using a break-apart probe detected BCL6 translocations in 24 of 112 (21%) DLBCL cases. Surprisingly, the frequency of BCL6 translocation was higher in the ABC subgroup than the GCB subgroup (10 of 40; 25% vs 5 of 44; 11%, respectively) and the PM subgroup had the highest incidence (4 of 8; 50%). Expression of BCL6 protein was detected by immunohistochemistry in 75 of 138 cases (54%), including 15 of 44 (34%) in the ABC subgroup, 46 of 57 (80%) in the GCB subgroup and 4 of 10 (40%) in the PM subgroup. A good correlation of protein and mRNA expression, as assessed by cDNA microarrays (NEJM 346:1937–47; 2002) was observed in the GCB subgroup but not in the other subgroups. BCL6 mutations were detected in 71 of 128 cases (55%) of DLBCL with a higher frequency in the GCB subgroup (34 of 50; 68%) and PM subgroup (10 of 12; 90%) than the ABC subgroup (14 of 43; 32%). Interestingly, there was a distinctive pattern of distribution of BCL6 mutations in the DLBCL subgroups. For example, 11 of 100 (11%) of the mutations targeted exon 1 and 8 of these 11 mutants were observed in the GCB subgroup. They preferentially affected the BCL6, STAT1 or predicted CEBP binding sites. High BCL6 mRNA and protein expression was generally associated with mutants affecting the 3′ BCL6 binding sites. We also examined the expression of 25 known BCL6 target genes in the different subgroups of DLBCL and observed the repression of this set of genes mainly in the GCB but not in the ABC subgroup, and the presence of a translocation did not correlate with target gene suppression.In conclusion, the frequency of BCL6 translocation and mutation is distinctly different in the DLBCL subgroups and BCL6 expression has different regulatory influences on target genes in different subgroups of DLBCL. Exon 1 mutations preferentially occur in the GCB subgroup and affect transcription factor binding sites. However, BCL6 translocations did not show good correlation with BCL6 expression or functional repression of target genes. The influence of the reciprocal partner genes in the translocations on the pathogenesis of DLBCL warrants further investigation.

Significant Differences in the IGVH and BCL6 Mutation Status in Aggressive B-Cell Lymphomas with and without MYC Breakpoints.

Somatically mutated IGVH regions are a hallmark of germinal center (GC) B-cells. Moreover, aberrant somatic hypermutation (SHM) of oncogenes, like changes in the 5′ non-coding region of the BCL6 gene occurring in 75% of DLBCL, are a mechanism of oncogene activation independently of chromosomal translocations.Large scale mutational screening using DHPLC and direct sequencing was applied to determine the somatic hypermutation status of clonal IGVH as well as several oncogenes like BCL6 and MYC in a series of more than 250 aggressive B-cell lymphomas included in the Deutsche Krebshilfe funded network “Molecular Mechanisms in Malignant Lymphoma”. Mutation patterns were correlated with the results of molecular cytogenetic analyses. MYC breakpoints detected by FISH clearly differentiated two groups of aggressive B-NHL with significantly different VH mutation status. MYC-positive lymphomas by FISH carried VH genes with a mutation frequency significantly lower compared to aggressive lymphomas lacking these features (median 4.8% vs.11.,0%, p<0.0001). Similarly, the mutation frequency of BCL6 was significantly lower in MYC-positive than in MYC-negative lymphomas (median 0.13% vs 0.25% p=0,020). We observed a bias in VH gene usage in both groups with an overrepresentation of VH4 (40% in both groups) and VH3 gene (24% in MYC-positive, 40% in MYC-negative). The group of MYC-positive lymphomas was heterogeneous with regard to the pattern of chromosomal aberrations. (IGH, MYC, BCL2, BCL6, MALT1 and REL loci). Lymphomas with IG-MYC fusion lacking breakpoints in BCL2, BCL6, MALT1 or REL loci as well as non-MYC associated IGH translocations displayed a median IGVH mutation frequency of 4,8% vs. 12,2% in those cases with additional chromosomal translocations (p=0.0060).Molecular classification of aggressive B-cell lymphomas according to MYC breakpoints distinguishes subgroups with significant differences in the VH and BCL6 mutation frequencies.

Characterization of the Proteomic and Genomic Profiles of Chronic Lymphocytic Leukemia Patients with Distinct Clinical Prognosis According to the Mutational Status of the IgVH and BCL6 and Expression Level of CD38 and ZAP70.

Introduction: In recent years several molecular prognostic factors have been identified in Chronic Lymphocytic Leukemia B (B-CLL). These include mutations in the variable region of the immunoglobulin genes (IgVH), somatic mutations in the BCL6 gene (Leukemia ( 2004) 18, 743–746) and the expression level of CD38 and ZAP70. However its biological significance is not clear. In order to identify novel molecular markers with prognostic and therapeutic value we have analyzed the proteomic and genomic profile of 40 B-CLL patients (Binet stage A).Material and methods: 100 μg of total PBMC proteins were used for IEF followed by 2D electrophoresis. Image analysis of scanned gels was used to identify statistically significant differentially expressed proteins. Image acquisition and analysis were performed using the Image Master Platinum software. Selected spots were subjected to automatic digestion and the proteins were identified by MALDI-TOF (Voyager DE-Pro, Applied Biosystems) peptide mass fingerprint using the Protein Prospector software. To confirm the initial identification amino acid sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with a nESI-QTRAP mass spectrometer from Applied Biosystems. 100 ng of total RNA was used to analyze the expression profile by cDNA microarrays (Whole Human Genome U133 Plus 2.0 Array from Affymetrix). Differential gene expression has been analyzed using the corresponding module (pomelo) from the GEPAS web tools (http://www.gepas.org ).Results and Discussion: We found 126 proteins and 25 genes exhibiting differential expression (p<0.05, Student t-test, FDR-adjusted for multiple testing contrasts). We found 34 proteins and 18 genes with different expression according to the mutational status of IgVH. We also classified patients according to mutations in the BCL6 gene, which we previously showed clinical relevance (Leukemia ( 2004) 18, 743–746), and found 36 proteins and 2 genes with differential expression. Next, we compared samples attending to the mutational status of both genes, results are summarized in the following table:Compared groups attending to the mutational status of IgVH and BCL6 genestargetsIgVH wt/BCL6wt IgVH mut/BCL6 mut16 proteins / 10 genesIgVH mut/BCL6 wt28 proteins / 9 genesOf particular interest is the comparision between BCL6 status in IgVH mutated patients where we found 27 proteins with differential expression but no difference in the gene expression profile. This result suggest that the bad prognostic associated to BCL6 mutation may be due solely to postransductional modification of proteins.CD38 and ZAP70 expression level have also been associated with bad clinical prognosis, however, we found no significant difference in the gene expression profile according to CD38 or ZAP70 expression level.We are currently assessing a wider number of patients in order to obtain stronger conclusions. The study of these proteins and genes may lead to a better understanding of the different clinical behaviour of these B-CLL forms.

Loss of BCL-2 in the progression of oral cancer is not attributable to mutations

Background: BCL-2 and BAX are important in the regulation of apoptosis. There have been reports of loss of BCL-2 in basal cells of oral epithelial dysplasia (OED) and in oral...

Genetic predictors for drug resistance in soft tissue sarcoma: a review of publications in 2004

Sarcomas are generally managed through a multidisciplinary approach including surgery, radiotherapy, and chemotherapy. Metastatic tumor cells frequently develop resistance to cytotoxic agents. Several molecular mechanisms of drug resistance have been uncovered recently. Among genes governing the apoptosis pathway, overexpression of the bcl2 family or mutations of p53 have recently been reported to be involved in drug resistance in sarcoma. The multidrug resistance genes are involved in the efflux of chemotherapeutic agents. Identification of members of this family of genes may predict resistance for developing new strategies. Several histologic subtypes of sarcomas have specific mechanisms of resistance. Methotrexate is one of the four active drugs for osteosarcoma and penetrates in tumor cell through specific carrier (reduced folate carrier) whose levels of expression or changes in amino acid sequence correlate to resistance to methotrexate. Specific molecular alteration defining nosological entities among sarcoma also correlates to drug resistance. Thirty percent of human sarcomas are characterized by specific translocations that may predict, for specific subtypes, survival and response to treatment. Finally, gastrointestinal tumors harbor specific activating mutations in KIT or PDGFRA genes, which are responsive to imatinib. Specific mutations of the KIT and PDGFRA genes have now repeatedly been found correlated to response or resistance to imatinib. The molecular alterations present in tumor cells predict influence sensitivity or resistance to chemotherapy. Ultimately, this may delineate specific therapeutic strategies for both cytotoxic and targeted therapies in sarcomas.

Targeted somatic mutation of the BCL6 proto-oncogene and its impact on lymphomagenesis

Cloning translocation breakpoints which cluster suspiciously to specific chromosomal loci has proved fruitful, leading to the identification of genes implicated in the onset of hematological malignancy. One of the most notable is BCL6, located on chromosome 3q27. The BCL6 is now known to encode a nuclear transcriptional repressor, with pivotal roles in germinal center (GC) formation and regulation of lymphocyte function, differentiation and survival. Unusually, the BCL6 gene locus is also actively targeted by the somatic mutation (SM) mechanism, at a rate indicative of specific, regulated events in both normal and malignant B-cells. These mutations occur in approximately 30% of normal centrocytes and centroblasts, but not in naive or pre-GC B-cells. They are also observed in approximately 70% of diffuse large B-cells lymphomas, approximately 30% of follicular lymphomas (FL) and at various frequencies in many lymphoma subtypes. Mutations are generated in the 5' proximity of the BCL6 promoter, including the first intron and are mainly single nucleotide substitutions, but with insertions and deletions also observed. Mutations in BCL6 occur independently of translocations, although mutational levels can be dramatically influenced by aberrantly translocated chromosomal elements, which map in the vicinity of the gene. Indeed, SMs are directly implicated in the generation of chromosomal translocations, as suggested by the overlap of the breakpoint cluster region and the mutational cluster domain. The prognostic value of the overall level of BCL6 mutations in specific lymphoma populations is, in the main, not as yet fully resolved. The accumulation of mutations in BCL6 during high grade transformation of FL, a mutational clustering and specific recurrent mutations suggest that some mutations may be selected for by their effect on the survival of the tumoral clone. In fact, it is now clear that SM can target and disrupt regulatory motifs in BCL6 to result in upregulated gene expression. Exogenous factors can also perturbate SM in BCL6. Viral infection elevates BCL6 mutational activity, suggesting a potential link with onset of virus-associated lymphoma. These findings to date reveal several mechanisms which can influence specific mutations targeting BCL6, and which may contribute to lymphomagenesis by dysregulating control of BCL6 expression.

Avaliação morfológica e imuno-histoquímica de linfomas gástricos primários

INTRODUÇÃO: Os linfomas gástricos primários representam cerca de 50% dos linfomas não-Hodgkin extranodais e de 2% a 8% das neoplasias malignas do estômago. A maioria é formada pela proliferação de linfócitos B, que ocorre a partir do tecido linfóide associado à mucosa. Esse tecido, inexistente no estômago em condições normais, surge como conseqüência de gastrite crônica, na maioria das vezes associada à bactéria Helicobacter pylori. A inflamação crônica provocada pela infecção bacteriana parece sensibilizar linfócitos T, levando a estímulo constante de linfócitos B, que passam a proliferar inicialmente em padrão reacional. Posteriormente surgem alterações genômicas nessas células, que provocam ganho proliferativo, seleção clonal e transformação neoplásica, dando origem a linfoma de baixo grau. Alterações genéticas adicionais, como mutações em p53, podem induzir transformação para alto grau. MATERIAL E MÉTODOS: Foram estudados 32 casos de linfomas gástricos: 15 de baixo e 17 de alto grau. Foram investigados sexo e idade dos pacientes, características morfológicas das lesões,índice de proliferação celular e colonização pelo Helicobacter pylori. Verificou-se a expressão de CD20, Ki-67, p53, BCL-2 e BCL-6 por imuno-histoquímica. RESULTADOS: Os linfomas gástricos ocorreram mais freqüentemente em homens, e a faixa etária mais elevada foi observada no grupo de baixo grau. Lesões linfoepiteliais foram observadas em 93% dos linfomas desse grupo. O índice de proliferação celular e a expressão do p53 foram mais elevados no grupo de alto grau. A expressão do BCL-2 foi mais elevada nos linfomas de baixo grau. Não houve diferença significante quanto à expressão do BCL-6 nos dois grupos. CONCLUSÃO: Os resultados sugerem a participação dos genes p53 e BCL-2 na patogênese e na evolução dos linfomas gástricos.

Proteomic Profile Study of Chronic Lymphocytic Leukemia-B Patients with IGVH and BCL6 Mutated or Unmutated Genes.

Introduction: Chronic lymphocytic leukemia B (CLL-B) is the most frequent chronic lymphoproliferative disorder in western countries. The majority of patients are diagnosed in incipient Binet stage A. Some prognostic factors have been identified, as mutations in the genes coding for immunoglobulin variable regions (IgVH). Complementary somatic mutations in the BCL6 gene have been observed in 25% of CLL-B patients, although its clinical relevance remains unclear.Objective: To identify molecular markers of different patient groups of lymphoproliferative disorder through analysis of their proteomic profiles.Material and Methods: 15 samples of peripheral blood lymphocytes B from Binet stage A CLL-B patients were analyzed. Among them, 5 carried mutations in IgVH and BCL6 (M) genes, 5 had no mutations (IgVH, BCL6 (U)), and 5 with mutations in IgVH(M) but without mutations in BCL6(U). 100 μg of total proteins were isoelectrofocussed using DryStrips (13 cm, pI 4–7), followed by 15% SDS-PAGE, and Coomasie Blue staining. Image acquisition and analysis were performed with the Image Master Platinum software (Amersham Biosciences). Spots showing differential expression were subjected to automatic tryptic digestion. Proteins were identified by MALDI-TOF mass fingerprinting using the Protein Prospector software. Selected peptide ions were sequenced by collision-induced dissociation (CID) using an ESI-QTRAP instrument.Results: The mean number of spots per gel was 214, which showed a mean percentage matching among the three patient groups of 77%.Statistics of the 2D-IEF/SDS-PAGE separations of the total lymphocyte B protein from patients with different mutational status of their IgVH and BCL6 genes.IgVH, BCL6 (M) vs. IgVH(M), BCL6(U)IgVH (M), BCL6 (U) vs. IgVH, BCL6(U)IgVH, BCL6 (M) vs. IgVH, BCL6(U)Spot media per gel228/180180/235228/235Matching percentage80.2%79.3%70.5%Number of spots with p<0.05(*)622(*) Test t of student.MALDI-TOF mass fingerprint and/or CID MS/MS identification of spots with differential expresion among the 3 patient groups, and their level of expression in each mutational situation (Center value of volume % in each of the gels).ProteinIgVH, BCL6(M)IgVH(M), BCL6(U)IgVH, BCL6(U)14-3-3 Protein Z (*)0.820.30nsHeterochromatin-specific nonhistone protein (*)0.290.02nsNADH-Ubiquinone reductase (*)0.120.02nsTubulin α (*)0.541.28nsVimentin (*)0.270.98nsSimilar Beta Tubulin (*)0.401.85nsCytoskeleton associated protein 1 (**)ns0.030.26Endoplasmatic-reticulum-lumenal protein 28 (**)ns0.550.21Chaperonin TCP-10.0050.050.18Nucleophosmin0.171.511.22ns: no statistical significanceConclusions: Proteomic profiling of lymphocyte proteins of the three patient groups revealed 10 proteins with differential expression, which can be divided into the following categories:The expression of six proteins (*), changed with the mutational status of BCL6 gene.The expression of two proteins (**), correlated with the mutational status of the IgVH gene.Nucleophosmin exhibited increased expression in patients with unmutated BCL6 gene.Chaperonin TCP-1 expression was higher in patients with unmutated IgVH gene.Work supported by Redes Tematicas de Grupo Grant G03/179 of the Instituto de Salud Carlos III, Madrid, Spain

Treatment of B-CLL Cells with Bortezomib and Rituximab Reduces Cell Viability In Vitro..

Introduction: In B-CLL, somatic mutation of IgVH genes defines a group of patients with favorable prognosis, whereas the absence of IgVH mutations is correlated with a worse outcome. Our previous data suggested that BCL-6 mutations identify a subgroup of patients with high risk of progression despite the presence of mutated IgVH gene, but the clinical significance of this molecular alteration remains uncertain. New approaches are now being tested for the treatment of B-CLL. Proteasome inhibitor, Bortezomib (Btz), and monoclonal antibodies specific for surface antigens, Rituximab (Rtx), represent potential therapeutic strategy.Objetives: To study the effects of Btz and Rtx on viability of CLL cells in culture, and to correlate the responses to the mutational status of the samples.Material and Methods: PBMC from 29 B-CLL patients (Binet stage A) were isolated by Ficoll-Hypaque gradient centrifugation. Samples were culture in RPMI supplemented with 20% FCS. Complement rabbit serum was added to the cultures in Rtx treated cells. Cells were exposed to various concentrations of Btz (0.1, 1 and 10 uM), Rtx (10 ug/ml) or both together during 24 hours. Then, cells were stained with Propidium Iodide and subjected to flow cytometry analysis.Results: Btz and Rtx reduce viability of B-CLL cells in a dose and time dependent manner. The kinetic of both drugs were different, whereas Rtx reaches its maximun effect within 3 hours after treatment, Btz do the same after 48 hours. Combined treatment (Btz + Rtx) increases the effect of each one separately. All differences between treatments with increased doses of Btz + Rtx vs Btz alone were statistically significant. In the group of IgVH mutated patients, there were a significant difference in the response to Btz and Rtx in BCL-6 mutated (mut) and unmutated (unmut) groups (n= 10 and 8 respectively), whereas no significant differences were observed in the number of lymphocytes, lymphocyte doubling time, CD38 expression and cytogenetic alterations.Effects of Btz and Rtx on IgVH mutated samples: correlation with BCL-6 mutationsBCL-6Mean viability (%)SDp *Controlunmut82.138.700.42mut86.366.01Btz 0.1 uMunmut49.0510.390.006mut64.2812.16Btz 1 uMunmut39.867.190.040mut53.2713.35Btz 10 uMunmut30.217.410.041mut43.9914.73Rtx 10 ug/mlunmut48.3114.510.050mut34.6212.51Btz 0.1 uM + Rtxunmut32.9614.150.153mut24.5010.01Btz 1 uM + Rtxunmut25.9910.880.315mut19.897.53Btz 10 uM + Rtxunmut19.279.040.491mut16.317.52* U. Mann Whitney testConclusion: Our data show that Btz and Rtx have in vitro activity on B-CLL cells, being used alone or in combination. Interesting, in IgVH mutated cells, Btz and Rtx have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status, but the biological significance of this differential responses remains unclear

Histogenesis and Pathogenesis of Primary Breast Lymphoma.

Primary lymphoma of the breast accounts for <2% of extranodal non-Hodgkin's lymphoma and generally displays morphology consistent with diffuse large B-cell lymphoma (DLBCL). The pathogenesis and histogenesis of primary breast lymphoma are currently unknown and represented the aim of this study. Thirteen cases of primary breast lymphoma (all DLBCL) were analyzed for physiological somatic hypermutation (SHM) of IgV and BCL-6 genes, as well as for aberrant SHM of PAX-5, PIM-1, RhoH/TTF and c-MYC genes. A functional IgVH rearrangement was identified in 8/13 (61.5%) primary breast lymphomas. All IgVH genes displayed SHM, with a mutation frequency ranging from 4.0% to 25.9%, suggesing that primary breast lymphomas derive from GC-related B-cells. The IgVH gene families utilized included VH4 (4/8 cases), VH3 (3/8 cases) and VH2 (1/8 case). Three cases utilized the same VH 4.30.1/4-31 gene. Mutations of BCL-6 were detected in 7/13 (53.8%) cases, further confirming the finding that primary lymphomas of the breast derive from GC-experienced B cells. Analysis of aberrant SHM of proto-oncogenes was performed on selected regions known to contain >90% of mutations found in lymphoma. Overall, mutations in at least one of the four proto-oncogenes targeted by aberrant SHM were found in 9/13 (69%) primary breast lymphomas, whereas mutations in more than one gene were found in 4/9 (44%) cases. Each of the four proto-oncogenes was altered in a significant fraction of primary breast lymphomas (PAX-5 in 4/9 cases; RhoH/TTF in 5/9 cases; PIM-1 in 5/9 cases and c-MYC in 2/9 cases). The overwhelming majority of mutations was represented by single base-pair substitutions (n=36), whereas in only one instance a deletion of a short DNA stretch was observed. Among the 36 single base-pair substitutions detected in primary breast lymphoma, the transition/transversion ratio was 1.76 (expected 0.5; p<0.01; Chi square test), reflecting the mutational profile seen in nodal DLBCL of immunocompetent hosts. In PIM-1, a fraction of mutations led to amino acid substitution, with potential functional consequences. In particular, three primary breast lymphomas displayed four missense mutations localized within the serine-threonine kinase domain of PIM-1. The association of primary breast lymphoma with aberrant SHM of proto-oncogenes expands the types of aggressive lymphomas marked by this molecular abnormality and provides clues for understanding breast lymphoma pathogenesis. In particular, missense mutations in the PIM-1 coding region can deregulate its function, whereas mutations of the 5′ regulatory regions of PAX-5, RhoH/TTF and c-MYC are expected to influence the expression and regulation of these genes in a fashion similar to that reported for the BCL-6 gene in B-cell lymphoma. Consistent with the role of PAX-5 in B-cell differentiation, of RhoH/TTF in signal transduction, and of c-MYC in B-cell growth and fate, deregulation of these genes by aberrant hypermutation may contribute to breast lymphoma pathogenesis by multiple pathways.

BCL-6 Overexpression Predicts Poor Response to Reduced Immune Suppression Therapy in Children with Post-Transplantation Lymphoproliferative Disease (PTLD).

Background: PTLD is a well known complication of organ transplantation. While the understanding of this disease is increasing, the diagnosis and treatment remain challenging. The clinical, morphological, and molecular findings in PTLD vary widely. Reduction of immune suppression is the treatment of choice, but is effective in only 20–50% of patients. Mutations in the BCL6 gene have been demonstrated in some lymphomas and PTLD and may have prognostic implications. A single study, in which most subjects were adults, has suggested that BCL6 mutation in PTLD predicts a lack of response to reduced immunosuppression (IS).Objectives: To determine if BCL-6 overexpression predicts poor response to reduced IS or decreased survival in children with PTLD.Methods: Clinical data of patients identified with PTLD at The Children's Hospital (TCH) from 1992–2002 were obtained, including age, type of organ transplanted, subtype of PTLD, treatment received, response to treatment, relapse free survival (RFS), and overall survival (OS). Archived specimens of lesions obtained at the time of diagnosis of PTLD were evaluated for the presence of BCL-6 expression by immunohistochemistry.Results: Between 1992 and 2002, 486 organ transplants (211 heart, 107 hematopoietic stem cell (HSC), 86 liver, 82 kidney) were performed at TCH. During that same period, 20 children were diagnosed with 21 cases of PTLD. Eleven patients had liver transplants (12.7% of transplants), 5 had heart transplants (2.4%), 3 had HSC transplants (2.8%), and 1 had a kidney transplant (1.2%). Histologic classifications of PTLD included 1 follicular hyperplasia, 9 polymorphic PTLD, and 11 lymphomas. Only 5 of 20 patients responded to reduced IS. Overall mortality was 45% (9/20). BCL-6 was overexpressed in 7 of 18 evaluable specimens from 20 patients. None of the patients with BCL-6 overexpression responded to reduced IS (p=0.05). BCL-6 overexpression was associated with monomorphic histology (p=0.02), but did not predict RFS or OS.Conclusions: BCL-6 expression is associated with monomorphic histology and non-response to reduced IS in children with PTLD. Larger, prospective studies of BCL-6 mutation and expression are needed to verify the clinical significance of these findings.

BCL-6 mutations in pulmonary lymphoproliferative disorders: demonstration of an aberrant immunological reaction in HIV-related lymphoid interstitial pneumonia.

We used a PCR and sequence procedure to analyze the Ig V(H) gene and the mutations in the 5' regulatory regions of BCL-6 genes in pulmonary lymphoproliferative disorders (mucosa-associated lymphoid tissue (MALT) lymphoma, HIV-related, EBV-related, and virus-negative lymphocytic interstitial pneumonia (LIP)). Eight of 20 (40%) pulmonary MALT lymphoma and 10 of 20 LIP (5 of 5 (100%) HIV-related, 2 of 5 (40%) EBV-related, and 3 of 10 (30%) virus-negative LIP) cases showed BCL-6 gene mutations. Intraclonal heterogeneity of the BCL-6 mutations was observed only in pulmonary MALT lymphoma cases whose Ig V(H) genes also showed intraclonal heterogeneity. Ongoing BCL-6 mutations might reflect re-entry into a germinal center pathway to further mutations. BCL-6 mutations in pulmonary MALT lymphoma and HIV-negative LIP showed some features (high transition to transversion ratio, standard polarity, and RGYW/WRCY bias) of Ig V(H) gene hypermutation, leading to the view that pulmonary MALT lymphomas and HIV-negative LIP are under the influence of germinal center hypermutation mechanisms. Because BCL-6 mutations in HIV-related LIP cases did not demonstrate features of Ig V(H) gene hypermutation, immunological reactions in HIV-related LIP are the result of a process different from that found in HIV-negative pulmonary lymphoproliferative disorders.

Neurotrophin-3 and a CREB-mediated signaling pathway regulate Bcl-2 expression in oligodendrocyte progenitor cells.

Our previous results suggested that the transcription factor CREB mediates the actions of neuroligands and growth factor signals that coupled to different signaling pathways may play different roles along oligodendrocyte (OLG) development. We showed before that CREB phosphorylation in OLG progenitors is up-regulated by neurotrophin-3 (NT-3); and moreover CREB is required for NT-3 to stimulate the proliferation of these cells. We now show that treatment of OLG progenitors with NT-3 is also accompanied by an increase in the levels of the anti-apoptotic protein Bcl-2. Interestingly, the presence of a putative CREB binding site (CRE) in the Bcl-2 gene raised the possibility that CREB could also be involved in regulating Bcl-2 expression in the OLGs. Supporting this hypothesis, the NT-3 dependent increase in Bcl-2 levels is abolished by inhibition of CREB expression. In addition, transient transfection experiments using various regions of the Bcl-2 promoter and mutation of the CRE site indicate a direct role of CREB in regulating Bcl-2 gene activity in response to NT-3. Furthermore, protein-DNA binding assays show that the CREB protein from freshly isolated OLGs indeed binds to the Bcl-2 promoter CRE. Together with our previous results, these observations suggest that CREB may play an important role in linking proliferation and survival pathways in the OLG progenitors.

Bcl-6 mutation status provides clinically valuable information in early-stage B-cell chronic lymphocytic leukemia.

In B-cell chronic lymphocytic leukemia (B-CLL), somatic mutation of IgVH genes defines a subgroup with favorable prognosis, whereas the absence of IgVH mutations is correlated with a worse outcome. Mutations of the BCL-6 gene are also observed in a subset of B-CLL, but the clinical significance of this molecular alteration remains uncertain. We examined the distribution of IgVH and BCL-6 gene mutations in 95 well-characterized patients with Binet stage A B-CLL, and correlated them with clinical, laboratory, cytogenetic findings and disease progression. Mutations of the BCL-6 gene were observed only in cases harboring mutated IgVH. Unexpectedly, coexistence of IgVH and BCL-6 mutations was correlated with shorter treatment-free interval (TFI) compared to cases harboring only IgVH mutation (median, 55 months vs not reached; P=0.01), resembling the clinical course of unmutated IgVH cases (median TFI, 44 months). As expected, deletions of 17p13 (P53 locus) and 11q22 (ATM locus) were observed in cases with unmutated IgVH, except one patient who showed mutations of both IgVH and BCL-6. No other statistically significant differences were observed among the genetic subgroups. Our data indicate that BCL-6 mutations identify a subgroup of Binet stage A B-CLL patients with a high risk of progression despite the presence of mutated IgVH gene.