Normal B-cell Subsets Research Articles

A Multiple Myeloma Classification System That Associates Normal Bone Marrow B-Cell Subset Phenotypes with Disease Stage and Prognosis

IntroductionToday’s diagnostic tests for multiple myeloma reflect the criteria of the updated WHO classification based on biological, morphological and clinical heterogeneity. It has been the concept behind the present study to extend our current biological classification and provide a new tool to generate insight into the stage of clonal differentiation and oncogenesis.The goal of the present study was to generate B-cell subset associated gene signatures (BAGS) from the BM hierarchy used to assign individual phenotypic cell of origin (pCOO) subtypes in patients and validate its pathogenetic impact by prognostic evaluation.MethodsBone marrows (BM, n=7) were harvested from sternum during cardiac surgery. B-cell subsets were phenotyped by Euroflow standard for multiparametric flow cytometry and FACS-sorted for microarray analysis on the Human Exon 1.0 ST Arrays platform. This combination allowed us to generate five BAGS for PreBI, PreBII, immature (Im), naïve (N), memory (M) B-cells, and plasma cells (PC) of normal BM. The BAGS classifier was based on all median-centred probe sets from the data set by regularized multinomial regression with 6 discrete outcomes corresponding to each B-cell subset and the elastic net penalty using the algorithm implemented in the R-package glmnet24. Each clinical data set was probe-set-wise adjusted to have a zero median and the same variance as in the BM data set. The associated cell of origin subset for each patient in each data set was predicted by the BAGS classifier by assigning the class with the highest predicted probability score above 0.45 and otherwise UC. All statistical analyses were done with R version 3.0.2. Survival analysis was performed by the Kaplan-Meier method and log-rank tests. Clinical data sets were from University of Arkansas for Medical Sciences UAMS (n=559), the Dana Farber Cancer Institute DFCI (n=170) both available on the GEO website.ResultsFirst, we verified the quality of the sampled B-cell subsets based on the expression patterns of differentiation marker genes. Next, we constructed the BAGS-classifier provided by 38-68 gene probe sets (n), capable of assigning samples to each of the six subtypes of PreBI (n=38), PreBII (n=82), Im (n=71), N (n=68), M (n=52) and PC (n=43).Second, we assigned individual myeloma cases in the 2 patient cohorts including a total of 729 myeloma patients. The resultant assignments generated patient BAGS subtypes with diagnostic frequencies of 0,5-0,6 % preBI, 7-8 % preBII, 27-30 % Im, 8-9 % N, 36-37 % M, 1-5 % PC and 15-16 % UC subtypes. The frequencies were not different between the cohorts.Third, the BAGS subtypes was associated significantly with overall survival (P = 8.6 x 10-5) for high dose melphalan and autologous stem cell transplanted UAMS patients1, as illustrated in Figure 1. The most significant impact was observed within the PreB-II (light blue) and M (acid green) subtypes conferred with significant inferior prognosis compared to the Im (amethyst), N (apple green) and PC (blue) subtypes. The PreB-II and M subtypes in the UAMS cohort were correlated to the ISS stage I-III with 33%, 49% and 69% of the cases, respectively.Fourth, we compared the BAGS subtypes and the TC classification2 with no observed correlations (results not shown).ConclusionsOur data support a new COO defined BAGS classification based on the normal BM B-cell subset phenotypes with impact on staging and prognosis in multiple myeloma and a new diagnostic platform, which may result in more effective disease management.References 1) Zhan F, Huang Y, Colla S et al. The molecular classification of multiple myeloma. Blood. 2006 Sep 15;108(6):2020-8. 2) Bergsagel PL, Kuehl WM, Zhan F, Sawyer J, Barlogie B, Shaughnessy J Jr. Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood. 2005 Jul 1;106(1):296-303. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inter and Intra-Clonal Heterogeneity in Multiple Myeloma and Waldenstrom Macroglobulinemia

Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies.Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software.Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented.Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

MicroRNAome Expression in Chronic Lymphocytic Leukemia: Comparison with Normal B-cell Subsets and Correlations with Prognostic and Clinical Parameters

Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial. We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood. Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone-like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival. This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL.

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

Hodgkin-Reed-Sternberg Cells in Classical Hodgkin Lymphoma Show Alterations of Genes Encoding the NADPH Oxidase Complex and Impaired Reactive Oxygen Species Synthesis Capacity

The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.

Impact Of The Number Of Prior Treatment Lines Received On The Distribution Of Peripheral Blood Leukocyte Subsets In Advanced-Stage B-Cell Chronic Lymphocytic Leukemia (CLL)

AbstractIntroduction It is currently well-known that B-cell chronic lymphocytic leukemia (CLL) patients have an impaired immune function -particularly in advanced disease-, which significantly contributes to a higher risk of infections. The introduction of new effective therapeutic agents, such as the purine analogues (or alkylating agents with concomitant properties of purine analogues) plusanti-CD20 monoclonal antibodies, have significantly increased the rate of complete responses in CLL, but so far their impact on the overall immune function and the spectrum of infections occurring in CLL patients after therapy, remains to be fully understood. AimTo evaluate the effect of the number of treatment lines received on the different normal circulating leucocyte cell populations, including normal B-cell subsets, in advanced-stage treated CLL. Material and MethodsThe distribution of peripheral blood (PB) leukocytes was analyzed in 85 untreated CLL patients, and compared to that of 63 patients who had previously been treated with 1 line of treatment (n=39) or >1 line of treatment (n=24), and who failed to respond. Analysis was performed by 8-color flow cytometry with monoclonal antibodies against CD3, CD4, CD5, CD8, TCRgd, CD19, CD20, CD27, CD38, CD45, CD56, sIgM, sIgA, sIgG, sIgLambda and sIgKappa. ResultsThe absolute count of circulating malignant B cells was not significantly different (p>0.05) in the untreated vs. previously treated patients who received 1 or >1 line of treatment (77,627±84,211 vs 67,994±72,087 vs 59,282±74,206 cells/uL; respectively). In contrast, as compared to untreated patients, PB normal B cells were found to be reduced in patients who had received either 1 line or >1 line of treatment (89±142 vs 36±57 and 23±32 cells/uL, p=0.004 and p<0.001, respectively), but at similar levels between the two groups of previously treated patients (p>0.05). When dissecting the normal B-cell subsets, therapy-related decreased B-cell numbers were mostly due to a reduced number of circulating memory B cells (67±98 vs 21±45 and 15±26 cells/uL; p=0.001 and p<0.001, respectively), including all memory isotypes: IgM (24±31 vs 5±13 and 5±6 cells/uL, p<0.001), IgG (25±59 vs 10±32 and 6±13 cells/uL; p=0.008) and IgA (18±30 vs 5±10 and 6±12 cells/uL; p=0.001 and p=0.003). No significant differences were found as regards the absolute count of immature (5±11 vs 8±26 and 3±11 cells/uL; p>0.05) and naïve (16±55 vs 6±18 and 4±8 cells/uL; p>0.05) B cells, nor for circulating plasma cells (3±18 vs 5±21 and 2±6 cells/uL; p>0.05), regardless of the therapy status.As compared to untreated patients, the absolute count of CD4+ T cells and CD4/CD8 double negative TCRαβ cells were significantly lower in patients with >1 line of treatment (1,836±1,340 vs 1,256±1,027 and 131±165 vs 56±97 cells/uL, p=0.03 and p=0.007 respectively) but not in those who had received only 1 line (1,477±1,349 and 201±747 cells/uL; p>0.05). In contrast, therapy did not show a significant impact on the absolute count of PB T CD8+ and TCRgd cells. No statistically significant differences were observed in the number of PB innate immune subpopulations including, neutrophils, eosinophils, basophils, monocytes, NK cells and dendritic cells. ConclusionsWhile there are no differences regarding the number of leukemic cells, previously treated patients have significantly reduced counts of total and memory (all isotypes) normal B-cell subsets when compared to untreated patients. Together with this, CD4+ helper T cells could also be compromised after more than 1 line of treatment. Monitoring of these therapy-related immune defects could contribute to a better management of infectious complications in advanced-stage CLL patients. Disclosures:No relevant conflicts of interest to declare.

Long Non-Coding RNAs Are Commonly Deregulated In Hodgkin Lymphoma

Hodgkin's lymphoma (HL) is a B-cell neoplasm characterized by a minority of neoplastic cells within an extensive inflammatory background. Long non-coding RNAs (lncRNAs) have diverse roles in transcriptional, posttranscriptional and epigenetic mechanisms in normal cell physiology. Deregulation of lncRNA expression has been implicated in a number of cancers. Up to date it is unclear to what extent lncRNAs are involved in the pathogenesis of Hodgkin lymphoma (HL).We designed a custom made microarray based on a published lncRNA catalog of 10,509 lncRNA loci (Cabili et al., 2011). The array also contains probes for all protein coding genes allowing simultaneous detection of the lncRNA and mRNA expression pattern. We analyzed 6 HL cell lines and three samples of purified tonsillar naive, germinal center (GC) and memory B cells. To get more insight into the potential function of individual differentially expressed lncRNAs we determined lncRNA enrichment in cytoplasmic and nuclear fractions of two HL cell lines. Furthermore, we studied to what extent lncRNAs are enriched in the Argonaute 2 (Ago2) containing complexes as an indication for interaction with miRNAs.In a comparison between the three different normal B-cell subsets, we observed 565 differentially expressed lncRNA probes. Overall, naive and memory B cell subsets showed a highly similar expression pattern while GC B cells showed a distinct lncRNA expression pattern. A significant differential expression between HL and normal GC B-cells was observed for 717 lncRNA probes. Interestingly, 109 of those overlap with the 565 lncRNAs differentially expressed between the normal B cell subsets. Differential expression for 4 in HL up and 2 downregulated lncRNAs was confirmed using qRT-PCR. To get a first indication about their potential function, we evaluated the subcellular location of the lncRNAs by comparing the abundance in nuclear and cytoplasmic fractions relative to the total fraction. Overall, 26% of the expressed lncRNAs were consistently enriched in the nuclear fraction and 10% were predominantly enriched in the cytoplasmic fraction. Of the 717 differentially expressed lncRNAs 82 were consistently enriched in the nucleus and 16 in the cytoplasm. Our Ago2 pull-down experiments revealed that 9% of the mRNAs and 3% of the expressed lncRNAs are consistently enriched in Ago2 complexes. In total, we observed evidence for miRNA binding for 17 of the in HL differentially expressed lncRNAs.In conclusion, we identified a specific lncRNA expression pattern in GC B cells and observed for more than 700 lncRNAs a differentially expression pattern between HL and normal GC B-cells. For 98 lncRNAs we found a specific subcellular enrichment and for 17 we observed enrichment in the Ago2 fraction, indicative of miRNA-lncRNA interactions. Follow-up studies will determine to what extent these lncRNAs contribute to the pathogenesis of Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.

Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy

Recent findings have suggested biological classification of B-cell malignancies as exemplified by the “activated B-cell-like” (ABC), the “germinal-center B-cell-like” (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and “recurrent translocation and cyclin D” (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy.

Common variable immunodeficiency and idiopathic primary hypogammaglobulinemia: two different conditions within the same disease spectrum

Patients with hypogammaglobulinemia who do not fulfill all the classical diagnostic criteria for common variable immunodeficiency (reduction of two immunoglobulin isotypes and a reduced response to vaccination) constitute a diagnostic and therapeutic dilemma, because information concerning the clinical and immunological characteristics of these patients with idiopathic primary hypogammaglobulinemia is not available. In 44 common variable immunodeficiency and 21 idiopathic primary hypogammaglobulinemia patients we determined the clinical phenotypes and performed flow cytometric immunophenotyping to assess the pathophysiological B-cell patterns and memory B-cell subset counts. Age-matched B-cell subset reference values of 130 healthy donors were generated. Severe pneumonia and bronchiectasis occurred at similar frequencies in idiopathic primary hypogammaglobulinemia and common variable immunodeficiency. Although IgG levels were only moderately reduced compared to common variable immunodeficiency, 12 of 21 idiopathic primary hypogammaglobulinemia patients required immunoglobulin replacement. Non-infectious disease-related clinical phenotypes (autoimmune cytopenia, polyclonal lymphocytic proliferation and persistent unexplained enteropathy) were exclusively observed in common variable immunodeficiency and were associated with early peripheral B-cell maturation defects or B-cell survival defects. T-cell dependent memory B-cell formation was more severely affected in common variable immunodeficiency. Furthermore, 14 of 21 idiopathic primary hypogammaglobulinemia patients showed normal peripheral B-cell subset counts, suggestive for a plasma cell defect. In conclusion, idiopathic primary hypogammaglobulinemia patients who do not fulfill all diagnostic criteria of common variable immunodeficiency have moderately decreased immunoglobulin levels and often a normal peripheral B-cell subset distribution, but still suffer from serious infectious complications.

IL10 Receptor a Is a Novel Therapeutic Target That Is Epigenetically Disregulated in Low Grade Lymphomas with Plasmacytic Differentiation.

Abstract 2383Waldenstrom's Macroglobulinemia (WM) is the clinical manifestation of lymphoproliferative disorders characterized by a clonal lymphoplasmacytic proliferation, excessive IgM secretion and elevated serum viscosity.Most common B-cell non-Hodgkin lymphomas associated with WM are Lymphoplasmacytic Lymphomas (LPL) and Marginal Zone Lymphomas (MZL), especially those displaying plasmacytic differentiation (MZL-P). In order to elucidate common pathogenetic mechanisms that result in WM across lymphoma types, we profiled the methylome of 6 LPLs, 10 MZL-Ps, as well as normal B-cell subsets purified from human tonsillar tissue –germinal center B-cell controls (GCB, n=3) and plasma cell (PC, n=3) controls, using HELP assay and high-density oligonucleotide microarray from RocheNImblegen that queries DNA methylation level of 50,000 cytosine residues distributed among 14,000 gene promoters.Unsupervised hierarchical clustering approach using Ward’s method and Eucledian distances separated normal GCB and PC from the lymphoma cases and identified bi-directional changes in gene methylation: with aberrant gain or loss of methylation at specific genomic locations. We further utilized a t-test and identified 208 probesets that were differentially methylated between controls and lymphomas at p<0.01 and mean log-ratio difference between 2 groups >1.5 corresponding to 30% methylation difference. Ingenuity pathway analysis revealed CDKN1A and TGFb networks as the most aberrantly methylated in lymphomas.Remarkably, we observed that Interleukin 10 Receptor a (IL10RA), was aberrantly hypomethylated in both lymphoma subtypes, resulting in its aberrant overexpression. IL10RA is a subunit of the IL10 cytokine receptor that is known to be a key factor in terminating the inflammatory responses via signaling through the JAK/STAT pathway resulting in STAT3 activation, which also plays an integral part in GCB differentiation and commitment to the plasma cell lineage. We thus hypothesized that aberrant epigenetic upregulation of IL10RA might promote survival and expansion of lymphoma cells. We predicted that stimulating IL10RA with IL10 ligand may lead to increased cell growth, while the blockade of IL10RA would inhibit cell growth. We selected 2 anti-IL10RA antibodies, which have previously been reported to have receptor blocking properties in vivo in mice. We tested 1ug/ml, 5ug/ml and 10ug/ml of each antibody and 10 ng/ml of the stimulatory IL10 ligand and observed that ligand provided stimulatory effect on the growth rate of a panel of B-cell lymphoma, including LPL cell line, while both anti-IL10RA antibodies had marked growth inhibitory effects. We further determined that growth inhibition resulted from dramatic induction of apoptosis in cells treated with the blocking anti-IL10RA antibodies. Further studies revealed that induction of apoptosis followed specific inhibition of signaling through JAK1/2 and phosphorylation of STAT3Y705 immediately after treatment and inhibition of signaling through MAPK and phosphorylation of STAT3S727 at later treatment time points.In conclusion, we determined that IL10RA is aberrantly methylated and overexpressed in subtypes of low grade lymphomas exhibiting plasmacytic differentiation and manifesting as WM. Hence, strategies targeting this pathway should be explored as potential therapy for WM (irrespective of the underlying type of lymphoma). Disclosures:No relevant conflicts of interest to declare.

Analyzing primary Hodgkin and Reed-Sternberg cells to capture the molecular and cellular pathogenesis of classical Hodgkin lymphoma

The pathogenesis of classical Hodgkin lymphoma (cHL), the most common lymphoma in the young, is still enigmatic, largely because its Hodgkin and Reed-Sternberg (HRS) tumor cells are rare in the involved lymph node and therefore difficult to analyze. Here, by overcoming this technical challenge and performing, for the first time, a genome-wide transcriptional analysis of microdissected HRS cells compared with other B-cell lymphomas, cHL lines, and normal B-cell subsets, we show that they differ extensively from the usually studied cHL cell lines, that the lost B-cell identity of cHLs is not linked to the acquisition of a plasma cell-like gene expression program, and that Epstein-Barr virus infection of HRS cells has a minor transcriptional influence on the established cHL clone. Moreover, although cHL appears a distinct lymphoma entity overall, HRS cells of its histologic subtypes diverged in their similarity to other related lymphomas. Unexpectedly, we identified 2 molecular subgroups of cHL associated with differential strengths of the transcription factor activity of the NOTCH1, MYC, and IRF4 proto-oncogenes. Finally, HRS cells display deregulated expression of several genes potentially highly relevant to lymphoma pathogenesis, including silencing of the apoptosis-inducer BIK and of INPP5D, an inhibitor of the PI3K-driven oncogenic pathway.

Monoclonal B‐cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B‐cell subsets

Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.Little is known about the distribution of normal lymphoid cells and their subsets in the peripheral blood (PB) of subjects with monoclonal B-cell lymphocytosis (MBL). In our study, we compared the absolute number of PB lymphoid cells and their subpopulations in 95 MBL cases with normal lymphocyte counts vs. 617 age-/sex-matched non-MBL healthy subjects (controls), using highly sensitive flow cytometry. MBL cases showed significantly reduced numbers of normal circulating B-cells, at the expense of immature and naive B-cells; in addition, CD4+CD8+ double-positive T-cells and CD8+ T-cells were significantly lower and higher vs. controls, respectively. Moreover, most normal B-cell subsets were significantly decreased in PB at >1% MBL-counts, vs. "low-count" MBL cases, and lower amounts of immature/naive B-cells were detected in biclonal (particularly in cases with coexisting CLL-like- and non-CLL-like B-cell clones) vs. monoclonal MBL subjects. In summary, our results show imbalanced (reduced) absolute numbers of recently produced normal circulating B-cells (e.g., immature and naıve B-cells) in MBL, which becomes more pronounced as the MBL cell count increases.

Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis

AbstractmiRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1–positive MCL (n = 30) and cyclin D1–negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1–negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.

The B lineage transcription factor E2A regulates apoptosis in chronic lymphocytic leukemia (CLL) cells

Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by the accumulation of B lymphocytes with an antigen-experienced activated CD19(+)CD5(+) clonal phenotype. Clinically, ∼50% of cases will behave more aggressively. Here, we investigate the role of the major B-cell transcription factor E2A, a known regulator of B-cell survival and proliferation, to CLL persistence. We show that E2A is elevated at the mRNA and protein levels relative to normal B-cell subsets. E2A silencing in primary CLL cells leads to a significant increase in spontaneous apoptosis in both CD38(+) (aggressive) and CD38(-) (indolent) cases. Moreover, E2A knockdown synergizes with the immunomodulatory drug lenalidomide to reduce CLL viability. E2A is known to restrain the proliferation of primary B and T lymphocytes at multiple stages of maturation and we report that targeted E2A disruption increases the frequency of Ki-67(+) CLL cells in the absence of effects on de novo proliferation. At the molecular level, E2A siRNA-treated CLL cells display reduced expression of key genes associated with survival and cell cycling including p27, p21 and mcl-1, of which the former two are known E2A target genes. Thus, E2A, a key transcription factor associated with the B-cell activation profile, regulates apoptosis in CLL and may contribute to disease pathology.

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

AbstractWe previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients.

LEF-1 is a prosurvival factor in chronic lymphocytic leukemia and is expressed in the preleukemic state of monoclonal B-cell lymphocytosis

AbstractThe canonical Wnt signaling pathway is pathogenic in a variety of cancers. We previously identified aberrant expression of the Wnt pathway transcription factor and target gene lymphoid enhancer binding factor-1 (LEF1) in chronic lymphocytic leukemia (CLL). This suggested that the Wnt signaling pathway has a role in the biology of CLL. In this study, we performed a Wnt pathway analysis using gene expression profiling and identified aberrant regulation of Wnt pathway target genes, ligands, and signaling members in CLL cells. Furthermore, we identified aberrant protein expression of LEF-1 specifically in CLL but not in normal mature B-cell subsets or after B-cell activation. Using the T cell–specific transcription factor/LEF (TCF/LEF) dual luciferase reporter assay, we demonstrated constitutive Wnt pathway activation in CLL, although the pathway was inactive in normal peripheral B cells. Importantly, LEF-1 knockdown decreased CLL B-cell survival. We also identified LEF-1 expression in CD19+/CD5+ cells obtained from patients with monoclonal B-cell lymphocytosis, suggesting a role for LEF-1 early in CLL leukemogenesis. This study has identified the constitutive activation and prosurvival function of LEF-1 and the Wnt pathway in CLL and uncovered a possible role for these factors in the preleukemic state of monoclonal B-cell lymphocytosis.

BCL6 modulates tonic BCR signaling in diffuse large B-cell lymphomas by repressing the SYK phosphatase, PTPROt

AbstractTonic B-cell receptor (BCR) signaling is a key survival pathway during normal B-cell ontogenesis and in a subset of diffuse large B-cell lymphomas (DLBCLs). We previously demonstrated that BCR-dependent DLBCL cell lines and primary tumors underwent apoptosis after treatment with an ATP-competitive inhibitor of the BCR-associated spleen tyrosine kinase (SYK). These “BCR-type” tumors also have more abundant expression of the transcriptional repressor, BCL6, and increased sensitivity to BCL6 inhibition. Herein, we evaluated potential connections between BCL6-mediated transcriptional repression and SYK-dependent BCR signaling. In transcriptionally profiled normal B-cell subsets (naive, germinal center, and memory B cells) and in primary DLBCLs, there were reciprocal patterns of expression of BCL6 and the SYK tyrosine phosphatase PTPROt. BCL6 repressed PTPROt transcription via a direct interaction with functional BCL6 binding sites in the PTPROt promoter. Enforced expression of BCL6 in normal naive B cells and RNAi-mediated depletion of BCL6 in germinal center B cells directly modulated PTPROt expression. In “BCR-type” DLBCLs, BCL6 depletion increased PTPROt expression and decreased phosphorylation of SYK and the downstream adaptor protein BLNK. Because BCL6 augments BCR signaling and BCL6 and SYK are both promising therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.

Mirna Profiling Reveals Specific Patterns for Normal B Cell Subsets and B Cell Lymphomas with a Unique Burkitt Lymphoma Profile

Abstract: MicroRNAs (miRNAs) are small (22–23nt) noncoding RNAs, which negatively regulate gene expression by inhibiting protein translation. MiRNAs play an important role in various cellular processes such as hematopoiesis. Deregulated expression is associated with development of B-cell lymphomas. The aim of this study was to define miRNA expression profiles to identify differentially expressed miRNAs in normal B cell subsets and malignancies derived from these subsets. Using Agilent Human miRNA microarrays the expression levels of 556 miRNAs were determined in 7 Mantle cell lymphoma (MCL), 7 Follicular lymphoma (FL), 7 paediatric Burkitt lymphoma (BL) and 13 Chronic lymphocytic lymphoma (CLL; 8 ZAP70 pos and 5 ZAP70 neg) cases, as well as in naïve, germinal center (GC), memory B cells and plasma cells obtained from 3 paediatric tonsils. Median normalisation was performed on all array data using GeneSpring GX 9.0.5. Differentially expressed miRNAs were defined by at least a four fold difference using ANOVA (p<0.05). Quantitive (q)RT-PCR was used for validation of the arrays. In the normal B-cell subsets 23 differentially expressed miRNAs were found with a four fold difference. Hierarchical clustering revealed a distinct pattern of these subsets. Specifically in GC B cells miR-15b, miR-17-5p and its seed family member miR- 106a were upregulated in comparison to the naïve, memory B cells and plasma cells. MiR-150, miR-221 and miR-222 were downregulated. Only miR-146a was significantly differentially expressed between naïve and memory B cell subsets. In the lymphoma cases a total of 59 miRNAs were differentially expressed. Hierarchical clustering of these miRNAs grouped all four lymphoma entities separately. ZAP70 positive and negative CLL cases did not cluster separately and none of the miRNAs were differentially expressed. Interestingly, 35 miRNAs were specifically deregulated in the Burkitt lymphomas as compared to MCL, FL and CLL, including miR-18a (up), miR-29a, miR-150, miR-155 and miR-222 (down). Almost all of these miRNAs were found not to be differentially expressed within the normal B cell subsets suggesting that these miRNAs might play a specific role in the biologic behaviour of BL. Comparison of BL to normal GC B cells revealed 15 miRNAs upregulated, including miR-126 and miR-145 (fold change >40), and 79 miRNAs downregulated, including miR-150, miR-155, miR-15, miR-16, miR- 17-5p, miR-21 and miR-222. In comparison with GC B cells FL showed 11 upregulated miRNAs, e.g. miR-143 and miR-145 (fold change >40) and 49 downregulated miRNAs. In comparison with memory B cells 21 miRNAs in the CLL group were upregulated, including miR-143 and miR-145 (fold change > 40) and 83 miRNAs were downregulated. Finally MCL was compared to the naïve B cells and showed 23 upregulated miRNAs with miR-126 showing the highest fold change (>40) and 70 downregulated miRNAs. In conclusion, we have identified specific miRNA profiles for MCL, BL, FL and CLL and also for normal B cell subsets. The differentially expressed miRNA profiles contain several miRNAs that have been shown to be directly or indirectly involved in B cell malignancies but also contain new potentially interesting miRNAs. We also have found several miRNAs that may play a tumor specific role in BL and it can be speculated that miRNAs contained in this profile may target genes related to the more aggressive clinical phenotype of BL.

Formin-Like Gene Expression in Chronic Lymphocytic Leukemia Is Associated with Unfavorable Prognostic Factors and Clinical Outcome

Formin proteins are large proteins evolutionarily conserved that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. The human leukocyte formin, named as FMNL1, corresponds to an extended cDNA of the 5′-truncated KW-13 which was reported as a tumor-associated antigen in CLL, using a serologic identification by recombinant expression cloning (SEREX). In addition, FMNL1 co-immunoprecipitates with P-Akt in CLL cells, suggests that this protein may contribute to regulate cell survival. The aim of this study was to analyze the expression of FMNL1 in normal B-cell subsets and in a series of 73 patients (median age, 59 years; male/female 40/33; Binet A: 90.2%) with CLL. FMNL1 expression was analyzed by Western Blot, Immunohistochemistry and by Real-Time RT-PCR using expression in Jurkat as baseline. In normal lymphocytes subsets, FMNL1 was only expressed in memory B-cells, and in T-cells, whereas germinal center lymphocytes were negative. Among lymphoid B-cell malignancies, FMNL1 was expressed in mantle-cell lymphomas, Burkitt's lymphomas and in DLBCL of GCB-type. In CLL cases mean of FMNL1 expression by QRT-PCR was 2.18 AU (SD, 1.01 AU). Using an arbitrary cut-off of 3.2 AU, cases with increased expression of FMNL1 were associated with a younger age at diagnosis (< 50 yrs), elevated lymphocyte count, high serum β2-microglobulin (β2-m) levels, increased ZAP-70 and CD38 expression, shorter time to progression, and shorter survival as compared to cases with low FMNL1 expression. No relationship was observed with genetic abnormalities. In summary, increased expression of FMNL1 gene couples with adverse clinical and biological parameters in patients with CLL. Finally, the interaction between FMNL1 and AKT protein in lymhoproliferative disorders is under investigation.

Gene Expression Analysis of B-Cell Post Transplant Lymphoproliferative Disorders Provides Insights into Disease Biology.

Background: B-cell post transplant lymphoproliferative disorders (PTLDs) encompass a diverse group of entities, the majority representing EBV-driven lymphoproliferations, which arise in the setting of iatrogenic immunosuppression. Their pathogenesis, especially the genetic relationship of PTLD with other types of B-cell non-Hodgkin lymphomas (B-NHLs), is not well understood and the association of polymorphic PTLD (P-PTLD) with monomorphic PTLD (M-PTLD) is presently unclear. Thus, in order to address these issues we analyzed gene expression profiles of PTLD and compared them with those derived from other types of B-NHL, normal B-cell subsets, and B-cell derived cell lines.Material and methods: Paraffin embedded and H&E stained sections were used for morphologic analysis and PTLD were classified according to the WHO classification. Phenotypic characterization was performed using a comprehensive panel of antibodies and in situ hybridization for EBER. Frozen tissue from PTLD, different B-NHL, purified normal B-cell subsets, EBV-transformed lymphoblastoid cell lines (LCLs) and multiple myeloma cell lines, was used to generate complementary RNA (cRNA) that was hybridized to HGU95 arrays (Affymetrix). Gene expression data were analyzed using unsupervised and supervised clustering algorithms and classifiers were generated to determine the relationship between PTLD and cells corresponding to different stages of mature B-cell development.Results: The 12 PTLD analyzed represented an infectious mononucleosis-like (IM-like) lesion, 5 P-PTLD, and 6 M-PTLD. All P-PTLD and 5 M-PTLD had late-germinal center (GC)/post-GC phenotypes and 8 PTLD, including the IM-like lesion, were EBV+, while 4 PTLD were EBV−. The expression profiles of late GC/post GC PTLD appeared homogeneous and distinct from other B-NHL, however no segregation of P-PTLD and M-PTLD or EBV+ and EBV− PTLD was noted. Surprisingly, on supervised analysis, only 4 genes were expressed at higher levels in PTLD compared to B-NHL and normal B-cell subsets (BLIMP-1, XBP-1, IFITM3, PCMT1) and only 2 genes were differentially upregulated in EBV+ PTLD (CD27 ligand, MTDH). The expression profiles of PTLD appeared most similar to LCLs.Conclusions: B-cell PTLDs of late-GC/post-GC phenotype represent a distinct type of B-NHL, which are possibly derived from activated B-cells. Despite morphologic distinctions, P-PTLD and M-PTLD have overlapping genetic profiles suggesting disease heterogeneity within different categories.