Sensitivity to Wnt Pathway Inhibition in CLL Is Associated with Specific Gene Expression Signatures

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 801-801
Author(s):  
Lili Wang ◽  
Alex K Shalek ◽  
Jellert Gaublomme ◽  
Nir Yosef ◽  
Jennifer R Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Wnt Pathway ◽  
Differentially Expressed ◽  
Differential Sensitivity ◽  
Specific Gene ◽  
Disease Heterogeneity

Abstract Abstract 801 We have recently found that the Wnt/b-catenin signaling pathway plays a key role in chronic lymphocytic leukemia (CLL). We were, however, intrigued by the question of whether this aberrant pathway may function differently in independent leukemias, and contribute to disease heterogeneity. To assess differential activity of the Wnt pathway across patients, we tested the effects of blocking Wnt activation on CLL cell survival. We knocked down a key downstream gene, LEF1, which is the most differentially expressed gene in CLL compared to normal B cells (based on gene expression microarrays). Addressing this question requires genetic manipulation of primary normal and malignant human B cells, and yet these cells are notoriously difficult to transfect. We therefore focused on developing a method for introducing siRNAs into normal and malignant B cells. We adapted a novel delivery system consisting of vertical silicon nanowires (SiNWs, Shalek et al PNAS 2010) that penetrate the plasma membrane in a minimally invasive fashion and deliver biomolecular cargo directly into the cytoplasm. We achieved consistent and reliable delivery of fluorescently labeled siRNAs (at 50–200 pmol) into normal and CLL B cells. siRNA was delivered to >90% of cells with >85% cell viability remaining after 48 hours. We used this platform to knockdown LEF1 in 20 CLL-B and 5 normal CD19+ B cell samples, and examined cell survival 48 hours after siRNA delivery using an ATP-based CellTiter-Glo assay. Indeed, our studies revealed a heterogeneous response among CLL-B cells to LEF1 inhibition. As a group, CLL-B cells were significantly more sensitive to LEF1 knockdown with a survival rate of 77% (12% s.e.m) compared to 97% (13% s.e.m) in normal B cells. CLL B cells from different patients showed differential sensitivity to LEF1 knockdown, with 8 non-responders, 8 intermediate responders and 4 strong responders (i.e. significant death). Sensitivity to LEF1 inhibition did not correlate with known CLL cytogenetic prognostic factors. To determine if the differential response to LEF1 knockdown was associated with specific gene signatures, we examined gene expression data generated from CLL-B cells from 12 (4 strong, 3 intermediate, and 5 non-responders) of the 20 CLLs tested (using the Affymetrix U133 Plus 2 Array). To increase statistical power, we used each CLL's expression profile (using only genes that showed variability across samples) to create clusters of ∼19 CLLs that showed similar expression profiles (using microarray data from our compendium of 177 additional CLLs). We further reduced the number of genes to ∼4000 genes by retaining only those whose expression levels were significantly different in at least one associated cluster relative to normal CD19+ B cell controls (T-test, FDR<10−4; p-values converted using the Benjamini-Hochberg method). These analyses led to the identification of several hundred genes whose expression correlated significantly with LEF1 knockdown's effect on cell viability. Analysis of these differentially expressed genes identified several potentially important pathways. Ongoing analyses include the identification and validation of a molecular signature for this effect. This signature could enable rapid identification of patients who would be most responsive to therapy with LEF1 inhibitors, which are under development along with other Wnt pathway inhibitors. Disclosures: No relevant conflicts of interest to declare.

B-Cell Scaffold Protein with Ankyrin Repeats (BANK1) Is Differentially Expressed in ZAP-70 Negative Compared to ZAP-70 Positive Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2938-2938
Author(s):  
Frank Dicker ◽  
Susanne Schnittger ◽  
Claudia Schoch ◽  
Alexander Kohlmann ◽  
Wei-Min Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Differentially Expressed Genes ◽  
Expression Profiling ◽  
Lymphocytic Leukemia ◽  
Differentially Expressed ◽  
Cell Scaffold

Abstract The lack of somatic mutations of the immunoglobulin variable heavy chain (IgVH) gene has been established as poor prognostic marker for chronic lymphocytic leukemia (CLL) patients at early stage disease. Expression of the non receptor tyrosine kinase zeta chain associated protein (ZAP-70) was proposed as a surrogate marker for an unmutated IgVH, however, up to 30% discordant samples have been reported depending on the respective study. B cell receptor (BCR) mediated signaling is enhanced by ZAP-70 expression in CLL cells in vitro and ZAP-70 expression also tends to decrease the time from diagnosis to treatment irrespective of the IgVH status. Therefore, we wanted to identify differentially expressed genes between the ZAP-70 positive and negative CLLs by gene expression profiling of peripheral blood mononuclear cells (PBMCs) using Affymetrix microarrays (HG-U133 Plus 2.0). ZAP-70 expression was analyzed by quantitative real time PCR of CD19 purified (purity &gt; 99%) PBMCs (n=62) using a LightCycler instrument. Expression of ZAP-70 mRNA was normalized against the housekeeping gene ABL and a relative quantitation against Jurkat T cells as a calibrator was performed. Results are expressed as normalized ratio and a cut-off of 0.5 normalized ratio gave the best correlation to the IgVH status with 77% concordant samples between ZAP-70 expression and the IgVH status. The discordant samples consisted of 5 unmutated IgVHs in the ZAP-70 negative group and 9 mutated in the ZAP-70 positive group. In a second step PBMCs of the same samples were analyzed by gene expression profiling and differentially expressed genes were identified by t-test. Among the two best genes that could be used in a classification algorithm (SVM) to distinguish between the 2 subsets with 92% accuracy were ZAP-70 and B cell scaffold protein with ankyrin repeats (BANK1). The expression of BANK1 was increased 3–4-fold in the ZAP-70 negative compared to the ZAP-70 positive CLL subset (P = 0,001). In the literature, BANK1 has been identified in human BCR expressing B cells and seems to be B cell restricted. In B cells the scaffolding protein BANK1 enhances BCR-mediated Ca2+-signaling, a signaling pathway that is also enhanced by ZAP-70 expression in CLL B cells. Based on these data we show that increased BANK1 expression correlates with a ZAP-70 negative status in CLL B cells. The functional consequences of BANK1 expression in the ZAP-70 negative subset of CLL B cells, which are usually associated with a more favorable prognosis, still need to be established further.

Gene Expression Signature of B-Cell Chronic Lymphocytic Leukemia with Trisomy 12.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2079-2079
Author(s):  
Edit Porpaczy ◽  
Martin Bilban ◽  
Elisabeth Kroemer ◽  
Georg Heinze ◽  
Michaela Gruber ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Signature ◽  
Fold Change ◽  
Diagnostic Tools ◽  
Marker Genes ◽  
Specific Gene ◽  
Expression Signature ◽  
Trisomy 12

Abstract 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&lt;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

scholarly journals Constitutive NF-κB and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4540-4548 ◽  
Author(s):  
Lingchen Fu ◽  
Yen-Chiu Lin-Lee ◽  
Lan V. Pham ◽  
Archito Tamayo ◽  
Linda Yoshimura ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
B Lymphocyte ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Non Hodgkin Lymphoma ◽  
Survival Pathway ◽  
Differentiation And Proliferation

AbstractB-lymphocyte stimulator (BLyS), a relatively recently recognized member of the tumor necrosis factor ligand family (TNF), is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors, TACI, BCMA, BAFF-R, to regulate B-cell survival, differentiation, and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study, we examined BLYS gene expression, function, and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells, including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL), playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors, NF-κB and NFAT, are involved in regulating BLyS expression through at least one NF-κB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-κB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B.

Gene Expression Profiling of Highly Purified Malignant and Non-Malignant Cells: Characterization of the Tumor-Microenvironment Cellular Synapse in De Novo Follicular Lymphoma (FL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 355-355 ◽  
Author(s):  
Karin Tarte ◽  
Céline Pangault ◽  
John de Vos ◽  
Philippe Ruminy ◽  
Fabienne Sauvee ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
De Novo ◽  
Differentially Expressed ◽  
Malignant Cells ◽  
Activated T Cell

Abstract Genetic and functional studies have demonstrated that FL cells retain the major features of normal germinal center (GC)-derived B cells, in particular the dependency on an active crosstalk with their specialized microenvironment. In agreement, microarray analyses have recently revealed that FL patient outcome is primarily predicted by molecular characteristics of tumor-infiltrating immune cells instead of tumor cells. However, our knowledge of the crucial interactions between malignant and non-malignant cells in FL remains limited by the use of whole biopsy specimen to perform gene expression profiling (GEP). We thus conducted GEP on both CD19pos B cells and CD19negCD22neg non-B cells purified from lymph nodes of 17 patients with de novo FL & 4 normal donors (CD20pos &gt;94.5%, median=98.2%) and 9 de novo FL patients & 5 normal donors (CD20pos&lt;6.7%, median=0.5%), respectively. Biotinylated cRNA were amplified according to the small sample labelling protocol and hybridized onto HGU133 Plus 2.0 arrays (Affymetrix). Raw data were normalized using GC-RMA methodology (ArrayAssist, Stratagene) and finally, based on a CV&gt;80, 10870 probesets were selected for further analyses. Unsupervised hierarchical clustering (Eisen’s software) allowed the correct classification of the 35 samples into the 4 groups: FL B-cell, Normal B-cells, FL non-B cells, and Normal non-B cells. Supervised analyzes were done using asymptotic non-parametric Mann-Whitney U-test (fold change ≥2, P&lt;0.01) and confirmed by permutation analysis (500 permutations, false discovery rate &lt;5%) using SAM software. We first established the list of the 841 probesets that were differentially expressed between FL and normal B-cells containing, 355 probesets overexpressed in malignant B cells including genes involved in GC B-cell biology (BCL6, MTA3, ID2, CD80, SDC4) and oncogenes as well (BCL2, AURK2) and conversely, 486 probesets downregulated in malignant B cells involving several interferon-stimulated genes for example. We then looked for the FL-specific microenvironment signature and pointed out the 1206 probesets that were differentially expressed between FL and normal non-B cells. Interestingly, all these genes were upregulated in the lymphoma context. Among them, we identified a striking follicular helper T-cell (TFH) signature (CXCR5, ICOS, CXCL13, CD200, PDCD1, SH2D1A) and an activated T-cell signature (IFNG, FASLG, GZMA, ZAP70, CD247). Notably, the TFH and activated T-cell signatures were not merely a surrogate for the number of T cells since many standard T-cell genes (i.e. CD2, CD4, CD7, LEF1, CD8A) were not induced in the FL microenvironment. Finally, in order to draw an overview of the FL-specific synapse between B and non-B cell compartments, we isolated a group of 2323 probesets that were differentially expressed between both compartments in FL and not in normal context. Using Ingenuity Pathway Analysis software we then identified among them FL-specific functional networks, including an IL-4- & an IL-15-centered pathway. Altogether, these data shed new light on our understanding of FL biology and could be a source of new therapeutics targeting the interplay between B cells and their microenvironment.

B-Cell Chronic Lymphocytic Leukemia Cells Exposed to the Non-Genotoxic p53 Activator Nutlin-3 Are Characterized by a Specific Gene Expression Signature.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4374-4374
Author(s):  
Michele Dal-Bo ◽  
Paola Secchiero ◽  
Massimo Degan ◽  
Riccardo Bomben ◽  
Dania Benedetti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Differentially Expressed Genes ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Differentially Expressed ◽  
Specific Gene ◽  
P53 Mutations ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4374 Introduction p53 plays a key role in determining the clinical features of B cell chronic lymphocytic leukemia (CLL). Disruption of p53 by point mutations, deletion at 17p13, or both, occurs in a fraction of cases at diagnosis and predicts poor survival and chemorefractoriness. In cells with functional p53, p53 activity is inhibited through interaction with MDM2. In fact, p53 can be activated upon exposure of cells to inhibitors of p53/MDM2 interaction, like Nutlins. Exposure of CLL cells to Nutlin-3 is effective in raising the levels of p53 protein with subsequent induction of cell cycle arrest and/or apoptosis, independently of the most relevant prognostic markers. The aim of the present study was to analyze the gene expression profile (GEP) induced by Nutlin-3 exposure in primary CLL cells from p53wt and p53del/mut cases. Patients and methods purified cells from 24 PB CLL samples, all characterized for IGHV mutational status, CD38 and ZAP-70 and p53 mutations (16 p53wt CLL, 8 p53del/mut CLL of which 6 with del17p13 and p53 mutations, 1 with del17p13 alone, and 1 with p53 mutations alone), were exposed to 10 mM Nutlin-3 for 24 hours. GEP was performed using a dual labelling strategy; the differential expression of the below reported genes were validated by quantitative real-time PCR. Results i) signature of Nutlin-3 exposure in p53wt CLL: 144 differentially expressed genes (143 up-regulated, 1 down-regulated) were correlated with response to Nutlin-3. Among the over-expressed genes, several genes were related to apoptosis (e.g. BAX, BBC3, E124, IKIP, FAS, LRDD, FLJ11259, TRIAP1, GADD45, TP53INP1, ISG20L1, ZMAT3, TNFRS10C, TNFRSF10B/TRAIL-R2), while other genes (e.g. MDM2, CDKN1A, PCNA) were up-regulated by Nutlin-3 as a part of a negative feed-back mechanism. Of note, this signature was not shared by 3/16 p53wt cases (identified as “non-responder” p53wt CLL) and 7/8 p53del/mut cases (identified as “non-responder” p53del/mut CLL); consistently, cells from these cases were also significantly resistant to the in-vitro cytotoxic effects of Nutlin-3; ii) signature of Nutlin-3 “non-responder” p53wt CLL: by comparing the constitutive GEP of 13 “responder” versus 3 “non-responder” p53wt CLL, we obtained 278 differentially expressed genes, 149 up-regulated and 129 down-regulated in “non-responder” p53wt CLL. Among up-regulated genes, we focused on MDM4/MDMX, a gene whose product was known to have an inhibitor activity of p53-dependent transcription and to form Nutlin-3 resistant complexes with p53. Among down-regulated genes, validations were made for BIRC4BP, whose product is known to act as an antagonist of the anti-apoptotic protein XIAP; iii) signature of Nutlin-3 “non-responder” p53del/mut CLL: by comparing the constitutive GEP of 13 “responder” versus 7 “non-responder” p53del/mut cases, we obtained 72 differentially expressed genes, 26 up-regulated and 46 down-regulated (31/46 located at the 17p segment) in “non-responder” p53del/mut CLL. Validations were made for several genes whose products display pro-apoptotic activities (e.g. PSMB6, RPL26 and ZBTB4, located at 17p segment, and GNAZ located at chromosome 22) among down-regulated genes, and ARHGDIA, whose gene product displays anti-apoptotic activities and mediates cellular resistance to chemotherapeutic agents, among up-regulated genes. Notably, CLL cells (n=43) displayed constitutively higher levels of MDM4/MDMX (p<0.0001) and ARHGDIA (p=0.0002) transcripts than purified normal B cells (n=15), irrespectively to the major biologic prognosticators. Conclusions specific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53wt or p53del/mut CLL. These findings may help to identify novel molecular targets for CLL therapy. Disclosures: No relevant conflicts of interest to declare.

Loss of the B-lineage–specific gene expression program in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1505-1512 ◽  
Author(s):  
Ines Schwering ◽  
Andreas Bräuninger ◽  
Ulf Klein ◽  
Berit Jungnickel ◽  
Marianne Tinguely ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Expression Profiles ◽  
Specific Gene ◽  
Mrna Levels ◽  
Study Gene Expression ◽  
B Lineage

Hodgkin and Reed-Sternberg (HRS) cells represent the malignant cells in classical Hodgkin lymphoma (HL). Because their immunophenotype cannot be attributed to any normal cell of the hematopoietic lineage, the origin of HRS cells has been controversially discussed, but molecular studies established their derivation from germinal center B cells. In this study, gene expression profiles generated by serial analysis of gene expression (SAGE) and DNA chip microarrays from HL cell lines were compared with those of normal B-cell subsets, focusing here on the expression of B-lineage markers. This analysis revealed decreased mRNA levels for nearly all established B-lineage–specific genes. For 9 of these genes, lack of protein expression was histochemically confirmed. Down-regulation of genes affected multiple components of signaling pathways active in B cells, including B-cell receptor (BCR) signaling. Because several genes down-regulated in HRS cells are positively regulated by the transcriptional activator Pax-5, which is expressed in most HRS cells, we studied HL cell lines for mutations in the Pax-5gene. However, no mutations were found. We propose that the lost B-lineage identity in HRS cells may explain their survival without BCR expression and reflect a fundamental defect in maintaining the B-cell differentiation state in HRS cells, which is likely caused by a novel, yet unknown, pathogenic mechanism.

scholarly journals c-Myb expression is critical to maintain proliferation and glucose metabolism of large pre-B cells

2020 ◽  
Author(s):  
Andrea R. Daamen ◽  
Rowena B. Crittenden ◽  
Timothy P. Bender
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
B Cells ◽  
Glucose Metabolism ◽  
B Cell ◽  
Cell Survival ◽  
Myb Transcription Factor ◽  
Cell Cycle Exit ◽  
Cell Stage ◽  
B Cell Survival

AbstractThe c-Myb transcription factor is required for the differentiation of CD19+ B-lineage cells and plays significant roles from the specification of the B cell lineage to the survival of pro-B cells. c-Myb coordinates the survival of pro-B cells with the expression of genes required for transition to the large pre-B cell stage of differentiation. However, it is not known if c-Myb is important for the proliferative expansion or subsequent differentiation into small pre-B cells. Here we demonstrate that c-Myb expression is important for large pre-B cell survival, proliferation, and differentiation into small pre-B cells. Utilizing genome-wide analysis, we found that c-Myb was important for maintaining glucose uptake and utilization and exogenous expression of Glut1 and Hk1 rescued large pre-B cell recovery and survival. Furthermore, we found that c-Myb is important for repression of Ikaros and Aiolos and our c-Myb-dependent gene signature was enriched in an Ikaros footprint of genes that drive cell cycle exit and the large to small pre-B cell transition. However, upon loss of c-Myb expression, inhibition of Ikaros activity was able to restore certain Ikaros-mediated gene expression changes but was insufficient to rescue recovery of large pre-B cell numbers. We found that c-Myb regulates glucose utilization and glucose-dependent survival through Hk1 in an Ikaros-independent manner. Thus, c-Myb regulation of glucose metabolism is critical to maintain large pre-B cell survival while repression of the Ikaros-mediated gene expression program is critical to prevent premature cell cycle exit and premature differentiation into small pre-B cells.

B-Cell Prolymphocytic Leukemia (B-PLL) and Chronic Lymphocytic Leukemia (CLL) Express Distinct Genomic Profiles.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4372-4372
Author(s):  
Ilaria Del Giudice ◽  
Nnenna Osuji ◽  
Tim Dexter ◽  
Estella Matutes ◽  
Vasantha Brito-Babapulle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Clinical Features ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Differentially Expressed ◽  
Specific Gene ◽  
Prolymphocytic Leukemia

Abstract B-cell prolymphocytic leukemia (B-PLL) is a rare disease, originally described in the early seventies and now recognized as a specific entity by the WHO classification. Diagnosis is based on clinical features and lymphocyte morphology (>55% circulating prolymphocytes), as no specific immunophenotypic or cytogenetic marker is available. According to WHO, cases of chronic lymphocytic leukemia (CLL) with increased prolymphocytes (CLL/PL) (>10% and <55%) should not be considered as B-PLL, because they have different genetic features. However, the existence of B-PLL as a separate entity from CLL has been questioned. We investigated the gene expression profiles of B-PLL and CLL to identify key genetic differences potentially useful for the diagnosis or involved in their different natural history. We retrospectively selected cryopreserved samples from 10 de-novo B-PLL and 10 untreated CLL. Diagnosis was well-defined by clinical features, lymphocyte morphology and immunophenotype. Matutes immunophenotypic score was 4–5 in all CLL; 3 cases showed CLL/PL morphology. B-PLL scored 0–3, with 3 CD5+ cases. Diagnosis of B-PLL was corroborated by excluding a leukemic form of MCL; t(11;14) assessed by fluorescent in situ hybridization (FISH) was absent in all but one case which, originally diagnosed as B-PLL, was reclassified as leukemic MCL. Five B-PLL and 2 CLL showed del(p53) by FISH. Total RNA was extracted from frozen blood mononuclear cells containing ≥95% purity of malignant cells, determined by flow cytometry. cDNA synthesis followed by biotin-labelled cRNA synthesis was carried out as per Affymetrix protocols. Microarray experiments were performed by MRC geneservice (UK HGMP Resource Centre), using the Affymetrix Human U133PLUS2 GeneChip array (54K probes). Hierarchical clustering was performed on samples using a filtered set of 9878 genes with >4 different algorithms. Prediction analysis for microarray (PAM) and significance analysis of microarray data (SAM) were used to evaluate class performance, and to partition genes using a priori defined labels of morphology, immunophenotype and cytogenetics. Unsupervised analysis reproducibly partitioned samples into two homogeneous distinct groups, corresponding to the diagnoses of B-PLL and CLL. SAM analysis identified 3957 differentially expressed transcripts (false discovery rates <1%), >77% of which showed an over 2-fold difference in expression between the groups. PAM analysis refined a sub-group of 46 genes which most efficiently differentiated the two diseases. Differentially expressed genes included those encoding surface antigens, oncogenes, transcription factors, adhesion molecules or involved in cell cycle/cell proliferation, lipidic metabolism and catalytic protein activity. Comparison of CD5 positive (13) versus CD5 negative (7) cases and cases with (7) or without (13) del(p53) showed no reliable class prediction. Our study formally demonstrates that B-PLL and CLL are two distinct diseases, each showing a specific gene expression. B-PLL has a homogeneous genomic profile irrespective of its heterogeneity in laboratory features. Validation of a model based on the expression of few genes predictive of diagnosis is on going. Further analysis of these data may also identify specific genes involved in B-PLL pathogenesis and drug resistance.

The Biology of the Germinal Center

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 210-215 ◽  
Author(s):  
Yasodha Natkunam
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Raw Material ◽  
Specific Gene ◽  
Molecular Machinery ◽  
Selection Gene ◽  
Gene Expression Studies

Abstract The immune system requires the production of high affinity antibodies of different subclasses to accomplish its many effector functions. Specific steps in B-cell ontogeny that occur within germinal centers of secondary lymphoid organs create much of the diversity in the immune system. This process also provides the raw material for the genesis of B-cell lymphomas as misdirection of the molecular machinery that regulate these steps can cause chromosomal translocations, prevent apoptosis and promote proliferation of abnormal clones. Many recent avenues of investigation have elucidated that the germinal center is a dynamic microenvironment where B-cells undergo repeated rounds of mutation and selection. Gene expression studies have further shown that malignancies derived from germinal center B-cells elaborate specific gene expression signatures that derive from neoplastic cells as well as elements of the host response such as T-cells and macrophages. This review will examine the current understanding of B-cell development in the germinal center and the key molecules involved in this process. Interactions between lymphoma cells and their cellular partners and models in the growth and development of follicular lymphoma will be presented.

The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 846-855 ◽  
Author(s):  
Pablo G. Longo ◽  
Luca Laurenti ◽  
Stefania Gobessi ◽  
Simona Sica ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Leukemic Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Antiapoptotic Proteins

Sustained engagement of the B-cell receptor (BCR) increases apoptosis resistance in chronic lymphocytic leukemia (CLL) B cells, whereas transient stimulation usually has an opposite effect. The antiapoptotic BCR signal has been associated with prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the relative contribution of the Akt and ERK kinases in regulating CLL B-cell survival, we introduced constitutively active mutants of Akt and MEK in primary CLL B cells and evaluated changes in the expression of relevant pro- and antiapoptotic proteins. Sustained activation of Akt resulted in increased leukemic cell viability and increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and X-linked inhibitor of apoptosis protein (XIAP), thus largely recapitulating the effects of sustained BCR stimulation. Constitutively active MEK2 also up-regulated XIAP, but did not show a significant impact on leukemic cell survival. Down-regulation of Mcl-1 by siRNA treatment induced rapid and potent apoptosis in CLL B cells and blocked the antiapoptotic effect of sustained BCR stimulation, whereas down-regulation of Bcl-xL and XIAP did not affect leukemic cell viability. These data demonstrate that Akt and Mcl-1 are major components of a survival pathway that can be activated in CLL B cells by antigen stimulation.

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