B-cell Chronic Lymphocytic Leukemia Subgroups Research Articles

T-cell independent, B-cell receptor-mediated induction of telomerase activity differs among IGHV mutation-based subgroups of chronic lymphocytic leukemia patients

AbstractAlthough B-cell chronic lymphocytic leukemia (B-CLL) clones with unmutated IGHV genes (U-CLL) exhibit greater telomerase activity than those with mutated IGHV genes (M-CLL), the extent to which B-cell receptor (BCR) triggering contributes to telomerase up-regulation is not known. Therefore, we studied the effect of BCR stimulation on modulating telomerase activity. The multivalent BCR ligand, dextran conjugated anti-μ mAb HB57 (HB57-dex), increased telomerase activity and promoted cell survival and proliferation preferentially in U-CLL cases, whereas the PI3K/Akt inhibitor LY294002 blocked HB57-dex induced telomerase activation. Although both U-CLL and M-CLL clones exhibited similar membrane proximal signaling responses to HB57-dex, telomerase activity and cell proliferation, when inducible in M-CLL, differed. B-CLL cells stimulated using bivalent F(ab′)2 -goat anti-μ antibody (goat anti-μ) exhibited higher membrane proximal response in U-CLL than M-CLL cells, whereas telomerase activity, cell survival, and proliferation were induced to lower levels than those induced by HB57-dex. In normal B lymphocytes, HB57-dex induced less protein phosphorylation but more cell proliferation and survival than goat anti-μ. Although both anti-BCR stimuli induced comparable telomerase activity, normal CD5+ B cells preferentially exhibited higher hTERT positivity than their CD5− counterparts. These findings provide an understanding of how BCR-mediated signals impact telomerase modulation in IGHV mutation-based subgroups of B-CLL and normal B cells.

Comprehensive Analysis of BAFF Binding Receptor Profiles and Receptor Occupancy in B Cell Chronic Lymphocytic Leukemia: Identification of Discrete Phenotypic Subgroups.

B cell-activating factor (BAFF) is known to regulate normal B cell development and homeostasis primarily by signaling through the high affinity receptor, BAFF-R, one of three BAFF binding receptors (BBRs). BAFF also binds two other receptors, BCMA and TACI with lesser affinity. We have recently shown that normal peripheral blood (PB) B cells express high levels of prebound soluble BAFF, which is lost upon B cell activation. Because of BAFF's activity on normal B cells, we have been interested in the roles of BAFF and BBRs in B cell chronic lymphocytic leukemia (B-CLL). We and others have demonstrated that BAFF promotes primary CLL B cell survival and that serum BAFF levels are elevated in some patients. Although CLL B cells are known to express BBRs, a comprehensive and quantitative analysis of BBR levels and CLL B cell capacity to bind BAFF has not yet been done. We began this study by characterizing the level of soluble BAFF bound to freshly isolated CLL B cells, measured by both western blot analysis and flow cytometry. To assess receptor occupancy, cells were incubated with or without exogenous BAFF before assessing anti-BAFF reactivity and changes in median fluorescence intensity (ΔMFI; defined by dividing the MFI of the anti-BAFF antibody by the MFI of the isotype matched control antibody) were calculated. Normal B cells have higher detectable levels of bound BAFF with a ΔMFI ranging from 16 to 35 (mean=22.2). Upon addition of exogenous BAFF, the ΔMFI range increased to 27–96.6 (mean=49.1; n=8). Thus, despite evidence of prebound BAFF, clearly not all BBRs were occupied on normal PB B cells. By contrast, the levels of prebound BAFF on CLL B cells were significantly lower with a ΔMFI ranging from 1 to 13.1 (mean=2.7; n=36). Of note, 10/36 patients did not exhibit increased anti-BAFF reactivity upon incubation with exogenous BAFF (mean fold induction=0.8) whereas 26/36 patients displayed a mean fold induction of anti-BAFF reactivity of 3.5. These observations prompted us to next quantitate CLL B cell BBR expression. All patient CLL B cells expressed BAFF-R but at significantly lower levels than observed in normal B cells (p=0.0009). When CLL patients were categorized into IGHV mutated (M; n=22) and unmutated (UM; n=24), UM patients were observed to express higher levels of BAFF-R (ΔMFI =8.9) than M patients (ΔMFI =5.24). Regarding TACI, we previously demonstrated that normal memory B cells uniformly express TACI (ΔMFI =12.7; n=10) and there is a small population of activated naïve B cells that express TACI at lower levels (ΔMFI =8.3; n=10). In our CLL cohort, 14/22 M patients were TACI+ (ΔMFI =7.0) and 19/24 UM patients were TACI+ (ΔMFI =4.7). Finally, whereas normal PB B cells completely lack BCMA expression, 7/22 M and 4/22 UM patients expressed BCMA. Thus, using the BBR profile and analysis of expression levels relative to normal PB B cells, the following subgroups of B-CLL can be defined:1.BAFF-R+;2.BAFF-R/TACI+;3.BAFF-R/BCMA+;4.BAFF-R/TACI/BCMA+.It remains to be determined if these BBR profiles correlate with aspects of clinical disease. In addition, given the putative importance of BAFF in this disease, it is interesting to note that in general, CLL B cells display overall lower levels of prebound BAFF. Current studies are focused on determining whether this reflects CLL B cell activation status, increased competition for BAFF, and/or reduced levels of BBR expression.

GLI1 Transcription Factor: A Potential Therapeutic Target in B-CLL.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. Therefore, identification of molecules responsible for the increased resistance to apoptosis is warranted. The identified molecules could be further targeted to develop effective therapy. Emerging evidence on the mechanism of resistant to apoptosis in several cancers suggests that GLI1 transcription factor, target of hedgehog signaling, may have a potential role in the increased resistance to apoptosis in B-CLL. However such studies have not been reported for B-CLL. Therefore, we investigated the role of sonic hedgehog (Shh)-GLI signaling in the pathogenesis B-CLL using 38 different patient samples and different parameters. The results were analyzed based on good and poor clinical outcome B-CLL subgroups identified on the basis of cytogenetic abnormality and/or CD38 expression levels. Microarray and RT-PCR analyses demonstrated active Shh-GLI signaling molecules in B-CLL cells including GLI1. Our results demonstrate that GLI1 transcripts were significantly (p = 0.003) over-expressed in B-CLL cells of poor clinical outcome patient subgroups (17pdel, 11qdel, trisomy12; N = 20) compared to good clinical outcome (13qdel, normal karyotype; N = 18) as observed in microarray analysis and confirmation by real time PCR. In addition, similar observation was also made with poor prognosis (high CD38 expression) versus good prognosis (low CD38 expression) B-CLL subgroups. Furthermore, higher expression of GLI1 was associated with significantly (p = 0.02) shorter time to treatment compared to lower GLI1 expressing B-CLL patients as determined by Kaplan-Meier survival analysis using log rank test, suggesting the increased expression of GLI1 and its association with the disease progression in poor clinical outcome B-CLL patient sub-groups. Addition of exogenous Shh on the significantly (p<0.01) increased cell survival and up-regulation of GLI1 transcripts, and decreased cell survival and downregulation of GLI1 transcripts in the presence of cyclopamine, known inhibitor of signaling, as determined by MTT assay and RT-PCR in vitro, suggesting the role of GLI1 in the survival of B-CLL. Downregulation of GLI1 expression in B-CLL cells using antisense oligonucleotides resulted in the significantly (p<0.01) increased susceptibility of the B-CLL cells to fludarabine mediated cytotoxicity compare to fludarabine alone or antisense alone or B-CLL cells treated with irrelevant oligonucleotides as determined by MTT assay and Annexin V method for identifying apoptotic cells. There was a significant decrease in BCL2 expression in GLI1-downregulated B-CLL cells suggesting that GLI1 may regulates BCL2 and thereby regulate cell survival/apoptosis in B-CLL. Together, these results demonstrate a role for GLI1 transcription factor for the increased resistance to apoptosis and thereby disease progression in B-CLL, and hence may be a potential therapeutic target to further improve treatment for B-CLL.

Bulky lymphadenopathy with poor clinical outcome is associated with ATM downregulation in B-cell chronic lymphocytic leukemia patients irrespective of 11q23 deletion

B-cell chronic lymphocytic leukemia (B-CLL) is the most common B-cell leukemia among older populations in Western countries. The clinical course of B-CLL is heterogeneous: in some patients the disease course is indolent, in others it is aggressive. The B-CLL subgroups with chromosome 11q23 deletion have been associated with aggressive disease course involving ATM deletion, extensive bulky lymphadenopathy (BLA), and inferior clinical outcome. Using real-time reverse transcriptase–polymerase chain reaction, we found that ATM was consistently underexpressed in B-CLL patients with BLA, irrespective of 11q23 deletion status. In addition, B-CLL patients who presented with BLA had a significantly shorter time to treatment (2 months) than did patients without BLA (74 months). Moreover, gene expression analysis in B-CLL patients with and without BLA revealed differences in expression for genes involved in apoptosis, cell cycle, and cell adhesion. These results indicate an association between BLA and reduced expression of ATM, suggesting a role for ATM in disease progression in B-CLL.

Analysis of B-CLL Transcriptomic and Proteomic Profiles: Differences between Molecular Subgroups.

INTRODUCTION: B cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder with a variable clinical course. Patients (pts) with unmutated (unmut) IgVH gene show a shorter progression free survival and overall survival than the patients with IgVH mutated (mut). To understand the differences between molecular subgroups of B-CLL we have studied transcriptomic and proteomic profiles on samples from 40 B-CLL pts (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 differentially expressed proteins. Image acquisition and analysis were performed using the Ludesi software (http://www.ludesi.com). 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 results, sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with a nESI-QTRAP mass spectrometer from Applied Biosystems. Eight proteins were validated by Western Blot.Total RNA from B cells was used to analyze the expression profile by hybridization with Whole Human Genome U133 Plus 2.0 Array from Affymetrix. Normalization, differential gene expression and functional annotations were analyzed using the GEPAS suite (http://www.gepas.org). qPCR using TaqMan primers/probes was used for validation of the microarray dataRESULTS: When we compared IgVH mut vs unmut transcriptomic and proteomic profiles, we found more than 600 differentially expressed genes and 12 proteins ( fdr <0.05 adjusted for multiple test contrast and p<0.05, Mann-Whitney's test, for gene and proteins, respectively). In tables 1 and 2 we show some of the most differentially expressed gene/proteins in each group of pts. Validation of results from microarrays and proteomic data using qPCR and Western blot are in progress. We obtained positive correlation between transcriptomic and proteomic profiles, (corr=0.21, p=0.04, Pearson's correation test) suggesting that common features are found using both approximations.CONCLUSION: We found a number of interesting gene/proteins that could be able to differentiate molecular subgroups of B-CLL pts. The study of these proteins and genes may lead to better understand the different clinical behaviour of IgVH mut and unmut B-CLL forms, but validation with a larger group of pts is still necessary.Table 1: Genes differentially expressedIgVH mutIgVH unmutBCL11AMGC9913DUSP22RGS4PDLIM5CRY1RDH13GGT2PHF15DMDSVHTUBB6ADAM29LPLITPKBITGA9RBKSBCL7ANFATC1MYEOVRIN3PPP1R9AGenes annotated using their gene symbolTable 2:Proteins differentially expressedIgVH mutIgVH unmutVIMERP29COTL1CCT2S100A9PSMB10HSPD1Proteins were annotated according to their gene symbol

Expression of CD49c and CD49d Chains of β1 Integrins Identifies B-CLL Subsets with Different Prognosis and Distinct Immunophenotypic Profiles.

Introduction: Although expression of β1 integrins was analyzed in B-cell chronic lymphocytic leukemia (B-CLL), little is known about the clinical behaviour and the phenotyic profile of B-CLLs with different patterns of β1 integrin expression.Methods: Expression of β1 integrins (α1-α6) was investigated by flow cytometry along with other 30 surface molecules (B-cell markers, cell-adhesion-molecules, activation inducers, complement activity regulators) in 155 B-CLLs, 106 characterized for clinical stage (Rai's stages), 121 for IgVH mutations, 79 for ZAP-70 expression and 109 with survival data.Results: In agreement with previous studies, also in our B-CLL series CD49c, CD49d and, to a lesser extent, CD49e resulted the most expressed β1 integrins, with mean expression values, evaluated as % of positive cells, of 59%, 36% and 26% (range 1-100%), respectively. Since expression of CD49c and CD49d have been recently identified by us as part of the immunophenotypic signatures of B-CLLs with different prognosis (J Cell Physiol 204, 113, 2005), the present study was designed to define: 1) the real prognostic impact and the optimal cutoffs for CD49c and CD49d splitting patients into two groups with different survivals; 2) the immunophenotypic and molecular features of B-CLL subsets expressing different levels of CD49c and CD49d. 1) By applying the maximally selected log-rank statistics, we identified cutoff values of 40% and 30% of positive cells for CD49c and CD49d, respectively; therefore, B-CLL cases were divided in CD49chigh or CD49clow and CD49dhigh or CD49dlow if the expression of the molecules was above or below their respective cutoffs. The CD49chigh B-CLL subset (n=78) displayed longer survival as compared to CD49clow cases (n=31; p=2.0x10-2, log-rank test); conversely, CD49dhigh B-CLLs (n=46) had shorter survivals, as compared to the CD49dlow subset (n=62; p=1.2x10-3). 2) Supervised analyses were performed by comparing the expression of a wide panel of surface antigens in CD49chigh vs. CD49clow and CD49dhigh vs. CD49dlow B-CLLs. In addition, B-CLL subgroups, as identified by CD49c and CD49d expression, were compared for IgVH mutations (2% cutoff), ZAP-70 expression (30% cutoff) and Rai's stages distribution (stages 0-I vs. II-IV). According to these analyses, CD62L, CD22, CD55, CD29, CD11c, CD54 and CD40 were the molecules significantly over-expressed in good prognosis CD49chigh B-CLLs, as compared to CD49clow cases (p<0.05; t-test); consistently, CD49chigh vs. CD49clow B-CLLs had mutated:unmutated (M:UM) ratios of 2.2 vs. 0.6 (p=9.2x10-3; Chi-square Test). Bad prognosis CD49dhigh B-CLLs, as compared to CD49dlow cases, over-expressed CD38, CD29, CD49e, CD79b and displayed a lower expression of CD11c (p<0.05). In the same subsets, M:UM ratios were 0.6 (CD49dhigh) or 3.4 (CD49dlow; p=4.2x10-5; Chi-square Test); finally, the frequency of both ZAP-70+ cases and cases with advanced Rai's stages (II-IV) was significantly higher (p=5.6x10-4 and 2.9x10-2, respectively) in CD49dhigh B-CLLs.Conclusion: CD49c and CD49d identify B-CLL subsets with peculiar clinico-biological features, allegedly originating from different normal B cell counterparts; under a clinical standpoint, CD49c and CD49d may be employed as additional prognosticators for B-CLLs.

Surface-antigen expression profiling (SEP) in B-cell chronic lymphocytic leukemia (B-CLL): Identification of markers with prognostic relevance

Studies of gene expression profiling (GEP) have been successfully used for the identification of molecules to be employed as potential prognosticators. With the aim of identifying the immunophenotypic profile of B-CLL subsets with different prognoses, we investigated by flow cytometry the expression of 36 surface antigens in 117 cases, 113 with survival data. In analogy with GEP, results were analyzed by applying unsupervised hierarchical algorithms (surface-antigen expression profiling, SEP). Distinct immunophenotypic groups (A, B1, B2 and C) were identified, group C (57/117) with longer survivals, as compared to groups A (23/117), B1 (16/117) and B2 (21/117). The immunophenotypic signatures of these groups were characterized by the coordinated and differential over-expression of: i) CD62L, CD54 and CD49c (group C); ii) CD38 and CD49d (group A); iii) none of the above markers (group B1 and B2). Other molecules were either not expressed, widely expressed by all samples, or were variably expressed within the observed B-CLL subgroups, although without a clearly distinguishable pattern. By employing an identical approach for investigating the reactivity of B-cell panel monoclonal antibodies (B-mAbs) in B-CLLs (29 cases) and in 19 B and non-B leukemia/lymphoma cell lines, we found mAbs (B012, B001, B006, B018, B019, B020, B017) mainly unreactive in all the samples, mAbs (B002, B010, B013, B014, B015) strongly reactive in B-CLLs and B-cell lines but not in non-B-cell lines, and mAbs recognizing antigens variably expressed in cell lines and B-CLLs. A hierarchical clustering focused on B-CLLs alone, combining reactivity values for B-mAbs with the expression of CD62L and CD38, these latter antigens identified as leader markers of B-CLL subsets with different prognosis, demonstrated a correlation between CD62L expression and the reactivity of B007, B003, B011 and B005 mAbs. These mAbs may represent potentially novel markers with prognostic relevance in B-CLLs.

Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.

Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes. One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes. Aiming at microarray-based subclassification, class predictors were constructed using sets of differentially expressed genes, which yielded in zero or low misclassification rates. Furthermore, a significant number of the differentially expressed genes clustered in chromosomal regions affected by the respective genomic losses/gains. Deletions affecting chromosome bands 11q22-q23 and 17p13 led to a reduced expression of the corresponding genes, such as ATM and p53, while trisomy 12 resulted in the upregulation of genes mapping to chromosome arm 12q. Using an unsupervised analysis algorithm, expression profiling allowed partitioning into predominantly VH-mutated versus unmutated patient groups; however, association of the expression profile with the VH mutational status could only be detected in male patients. The finding that the most significantly differentially expressed genes are located in the corresponding aberrant chromosomal regions indicates that a gene dosage effect may exert a pathogenic role in B-CLL. The significant difference in the partitioning of male and female B-CLL samples suggests that the genomic signature for the VH mutational status might be sex-related.

Recent advances in the molecular biology and immunobiology of chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) has long been viewed as a relatively homogeneous disease caused by the accumulation of monoclonal immature, immunoincompetent B cells with faulty apoptotic capacities. However, recent evidence, reviewed here, demonstrates that at least two different B-CLL subgroups exist with different clinical courses and outcomes. The malignant cells from both B-CLL subgroups are antigen-experienced cells that have a normal apoptotic apparatus and turnover continually. The leukemic cells of the two B-CLL subgroups have engaged antigen before transformation, although primarily the cells of patients in the poor outcome subgroup can respond to antigens following transformation. The difference in the ability to respond to antigen as a full-fledged B-CLL probably accounts for the different biological features and clinical outcomes of the patients in these subgroups.

Biological and Clinical Heterogeneity of B-cell Chronic Lymphocytic Leukemia

B-cell chronic lymphoproliferative disorders have been recognized as a heterogeneous group of neoplastic diseases affecting the lymphoid system. They are frequently characterized by leukemic manifestations with peripheral blood and/or bone marrow involvement. B-cell chronic lymphocytic leukemia (B-CLL), a disease which is now accepted as derived from immunologically competent antigen-activated B-cells, is the most common in the western countries. Although for several decades it was considered as a unique entity, a body of evidences is now emerging indicating the biological heterogeneity of B-CLL. Molecular and immunophenotypic data induce to consider two main subgroups of B-CLL exist at the cellular level: in fact, B-CLL cases can be divided into two categories according to IgV gene mutation status. One group develops from "memory" B-cells after antigenic stimulation in the germinal center of secondary follicles, displays mutated gene IgV and lacks CD38 expression; the second one derives from the so-called "naïve" antigen inexperienced B-cells, does not present IgV gene mutations and displays CD38 surface molecule. The two groups seem to be characterised by different clinical outcomes with the "mutated" group expected to have a more benign course than that arising from a "naïve" B-cell. Thus, the definition of the biological features of these different groups could be of help for clinicians in order to recognize patients needing more intensive therapeutic approaches.

Lack of somatic hypermutation of IG V(H) genes in lymphoid malignancies with t(2;14)(p13;q32) translocation involving the BCL11A gene.

The t(2;14)(p13;q32.3) involving the BCL11A and IGH genes is a rare but recurrent chromosomal aberration in B-cell malignancies. Hitherto, juxtaposition of BCL11A and IGH has only been described in B-cell chronic lymphocytic leukemia (B-CLL) and immunocytoma. As subgroups of B-CLL can be distinguished by the pattern of somatic mutation of immunoglobulin variable (V) genes we investigated four lymphomas with IGH/BCL11A involvement for IGH hypermutation. Clonal V(H) gene rearrangements were amplified; in all four cases, sequencing of the amplificates revealed the rearranged V(H) genes to lack somatic mutations. These results suggest that t(2;14)(p13;q32.3) is associated with a subset of B-CLL/immunocytoma characterized by non-mutated IG genes deriving from pre-germinal center B cells. As the translocations in both informative cases are targeted to the switch regions of the IGG2 gene, which is mainly used in T cell-independent immune responses, these translocations presumably occurred in activated B cells in the course of T cell-independent immune responses outside the germinal center.

Expression of the T1 (CD5, p67) surface antigen in B‐CLL and B‐NHL and its correlation with other B‐cell differentiation markers

The T1 surface antigen (CD5,p67) expression on blood lymphocytes (PBL) and lymphoid cells from lymph node biopsies (LN) from 31 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 79 with B non-Hodgkin lymphoma (B-NHL), was detected in 25 B-CLL (80 per cent) and in 11 B-NHL (13 per cent) belonging to the following histologic subtypes: lymphocytic of CLL type (DLWD) one case, lymphoplasmacytoid (DLWD) four cases, centrocytic (DLPD) five cases, immunoblastic (DH) one case. All B-CLL and the T1 + B-NHL were also tested with monoclonal antibodies against the Common Acute Lymphoblastic Leukemia Antigen, B cells (FMC7, FMC8, BA1, Y29-55), T cells (OKT11a), HLA-DR and HLA-DQ monomorphic determinants. All the B-CLL and the T1+ B-NHL were CALLA-, BA1+, Y29.55+. FMC7+ cells were detected in large numbers six B-CLL (three T1+ and three T1-) and in four centrocytic lymphomas. FMC8 reacted with 70 per cent of leukemias (where it stained 30 per cent of neoplastic cells) and with 8/9 T+ B-NHL. HLA-DR and HLA-DQ molecules were detected in 100 per cent and 90 per cent of cases respectively. In vitro treatment of HLA-DQ- or T1- B-CLL with phorbol ester TPA led to the expression of these antigens as well as of the receptors for Interleukin 2 and MLR3 activation antigen. Surface membrane Ig (SIg) was detected in 79 per cent of cases, its density measured by FACS analysis varied, even markedly, from case to case. Among the B-CLL, cells with high SIg content were either T1+ or T1- and more likely FMC7+. The SIg- cases were seven B-CLL (five T1+ and two T1-) and two B-NHL, in which, however, cytoplasmic IgM was detected. This study reveals the existence of four major B-CLL subgroups: T1- SIg-, T1+ SIg+, T1+ SIg+, T1- SIg+. It also indicates that the T1 antigen may be transitionally present during B-cell differentiation and that its expression may precede that of SIg as supported by the in vitro studies. In addition, the finding that some B-NHL are T1+ suggests that they derive similarly to the B-CLL from a common progenitor.