Demylinating Peripheral Neuropathy and Lymphoplasmacytic Lymphoma

Abstract Abstract 5090 Lymphoplasmacytic lymphoma (LPL) is a neoplasm of small B lymphocytes, plasmacytoid lymphocytes, and plasma cells, which does not fulfill criteria for any of the other small B-cell lymphoid neoplasms (WHO 2008). Neuropathy has been described in association with Waldenstrom's macroglobulinemia, but less so with LPL. We present 7 cases of neuropathy and LPL to highlight the variable presentations. 1) A 56 year old man developed sensory ataxia with an IgM kappa non-MAG paraprotein. CSF protein was 97. Stabilized on MMF, he slowly worsened. Plasma exchange (PE) was given with improvement. Bone marrow (BM) 4 years into his course revealed LPL. Rituximab was given, and his PE reduced. 2) A 49 year old woman developed progressive weakness with rapid decline in January 2010. NCS showed demyelinating polyneuropathy, and CSF protein was 179. An IgM kappa non-MAG paraprotein was found. BM was normal. PE was given with improvement but later prednisone, IVIg and rituximab twice did not help. Repeat BM revealed 2% clonal CD20+ CD5negCD10neg B cells by flow cytometry. PE was given with modest improvement. Cyclophosphamide 1 gm/m2 monthly was given with improvement. 3) A 53 year old man noted imbalance and distal weakness. NCS showed absent SAPs and prolonged distal and F wave latencies. An anti-MAG positive, IgM kappa paraprotein was found. In 2002 BM was normal. He received rituximab weekly × 4 doses. 7 years later, he developed anemia and worsening neuropathy. Repeat BM showed 0.5% CD20+CD5negCD10neg kappa-restricted B cells by flow cytometry and weekly rituximab was reinitiated. 4) A 62 year old man developed weakness and areflexia. NCS showed asymmetric demyelinating polyneuropathy. Biclonal gammopathy of IgM kappa and IgG kappa was found. BM showed LPL by histopathology. Prednisone was given with improvement. Later two courses of weekly rituximab were given. 5) A 55 year old woman developed asymmetric weakness. NCS showed asymmetric demyelinating polyneuropathy. MRI showed enlargement, abnormal signal intensity, and abnormal enhancement of bilateral radial, median, and ulnar nerves. She was found to have an IgG kappa paraprotein and LPL on BM biopsy. She was treated with rituximab, cyclophosphamide, fludarabine, and PE. In each case, the primary feature driving the need for therapy was the neuropathy and not the underlying hematologic process. Further, worsening neuropathy in 3 cases led to repeat bone marrow biopsies revealing a clonal B cell process and a diagnosis of lymphoplasmacytic lymphoma. Thus, in the presence of an IgM monoclonal gammopathy and peripheral neuropathy, we suggest bone marrow examination for LPL and then consideration of therapy directed toward the abnormal B cell clone. Disclosures: No relevant conflicts of interest to declare.

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Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽

10.1182/blood.v122.21.4943.4943 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4943-4943

Author(s):

Charles Repetti ◽

Hsueh-Hua Chen ◽

Yongbao Wang ◽

Vanessa A Jones ◽

Albert K Ho ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Somatic Mutation ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Sequence Variant ◽

Low Grade ◽

Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15). Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

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ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Blood ◽

10.1182/blood.v118.21.2844.2844 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2844-2844

Author(s):

Noelia Purroy ◽

Eva Calpe ◽

Pau Abrisqueta ◽

Cecilia Carpio ◽

Carles Palacio ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

Tumor Growth ◽

B Cell ◽

Cell Lines ◽

Ectopic Expression ◽

Xenograft Model ◽

Lymphoid Organs ◽

Transfected Cells

Abstract Abstract 2844 Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×106 cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process. Disclosures: No relevant conflicts of interest to declare.

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IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽

10.1182/blood.v120.21.1554.1554 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1554-1554

Author(s):

Lucy S. Hodge ◽

Steve Ziesmer ◽

Frank J Secreto ◽

Zhi-Zhang Yang ◽

Anne Novak ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Bone Marrow Microenvironment ◽

Waldenström’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Multiparameter Flow Cytometry (MFC) for Identification of the Waldenström's Clone in IgM MGUS and Waldenstrom's Macroglobulinemia (WM): New Criteria for Differential Diagnosis and Risk Stratification

Blood ◽

10.1182/blood.v120.21.936.936 ◽

2012 ◽

Vol 120 (21) ◽

pp. 936-936

Author(s):

Bruno Paiva ◽

Maria-Carmen Montes ◽

Ramón García-Sanz ◽

Jennifer Alonso ◽

Natalia de las Heras ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

B Cells ◽

B Cell ◽

Light Chain ◽

Conflicts Of Interest ◽

Phenotypic Characterization ◽

International Prognostic Scoring System ◽

Multiparameter Flow Cytometry ◽

Risk Of Progression

Abstract Abstract 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P<.001), as well of light chain restricted B cells (75%, 96% and 99%; P<.001). In contrast, no differences were found for the percentage of PC (median of 0.3%), but light chain restricted PC progressively increased from IgM MGUS to smoldering and symptomatic WM (70%, 85% and 97%; P<.001). Accordingly, only 1% of IgM MGUS patients showed >10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P<.001). Likewise, only 1% of IgM MGUS patients showed 100% light chain restricted B cells, in contrast to 19% and 40% of smoldering and symptomatic WM (P<.001); similar results being also found using a cutoff of 100% light chain restricted PC. Subsequently, we explored whether the percentages of BM and light chain restricted B cells and PC could predict time to progression (TTP) from smoldering into symptomatic WM, as well as overall survival (OS) in symptomatic WM. In smoldering WM, B cells (>10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs <100%: 26m vs 145m; P<.001) but not PC, predicted risk of progression. On the multivariate analysis that included serum M-spike (±3g/dL), BM infiltration (±50% lymphoplasmacytic cells), BM B-cells and light chain restricted B cells (by MFC), only the later retained independent prognostic value (HR: 19.8, P=.001). Upon analyzing factors influencing survival in symptomatic WM patients, cases with >10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs <100% light chain restricted B cells (median OS 44m vs 78m; P=.001). The later marker was independent (HR: 2.6; P=.004) of the International Prognostic Scoring System (HR: 2.2; P=.006). Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P<.001), CD25+ (61%, 88% and 90%; P<.001) and sIgM+ (88%, 95% and 97%; P=.002) B cells from IgM MGUS to smoldering and symptomatic WM. This underlies that the accumulating light chain restricted clonal B cells show a characteristic Waldenstrom's phenotype (CD22dim/CD25+/IgM+). Of note, a bimodal (from - to +) expression for the B cell memory marker CD27 was found in >50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

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The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

Blood ◽

10.1182/blood-2019-131641 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 79-79

Author(s):

Zev J. Greenberg ◽

Darlene A. Monlish ◽

Rachel L. Bartnett ◽

Jeffrey J. Bednarski ◽

Laura G. Schuettpelz

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Development ◽

B Cell Development ◽

Cellular Migration ◽

Physical Interaction ◽

Hematopoietic Stem ◽

Human Phenotype

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

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Demethylase Activity of Aid during Germinal Center B Cell Maturation Could Contribute to Lymphomagenesis

Blood ◽

10.1182/blood.v124.21.59.59 ◽

2014 ◽

Vol 124 (21) ◽

pp. 59-59 ◽

Cited By ~ 1

Author(s):

Maria Del Pilar Dominguez ◽

Matt Teater ◽

Nyasha Chambwe ◽

David Redmond ◽

Bao Vuong ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Cytosine Methylation ◽

Conflicts Of Interest ◽

Retroviral Vectors ◽

Dna Hypomethylation ◽

Demethylase Activity

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors that arise from germinal center B cells (GCBs) and post-GCBs and are noted for their heterogeneity and variable clinical outcomes. Epigenetic modifications like DNA methylation of cytosine nucleotides have emerged as important mechanisms of gene regulation and have been implicated in carcinogenesis. Our previous genome-wide studies in primary samples revealed profound alterations in the cytosine methylation patterning of DLBCLs. We also found that expression of activation-induced deaminase (AID) was significantly associated with the loss of methylation in DLBCL patients and was predominantly identified within computationally predicted AID-binding RGYW motifs. AID is a cytidine deaminase required for class switch recombination and somatic hypermutation (SHM) of immunoglobulin genes in GCBs. The enzymatic machinery that mediates these processes is error-prone and may introduce point-mutations and changes in DNA methylation, resulting in genomic and epigenomic instability. Since AID can also function as a demethylase during embryonic development, we asked whether AID has demethylase activity during transit of B cells through the GCs and if its overexpression can contribute to lymphomagenesis through disrupting DNA methylation. To address this question, we studied the epigenetic function of AID in GCBs and GC-derived lymphomas. We characterized the methylome of naïve B cells (NBs) and GCBs isolated from human tonsils and spleens of immunized mice by enhanced Reduced Representation Bisulfite Sequencing (eRRBS). We observed that the transition from NBs to GCBs was characterized by DNA hypomethylation, with 60,000 and 8,000 differentially methylated CpGs (DMCs) that were hypomethylated in GCBs compared to NBs, in human and mouse respectively. We also found that hypomethylated regions were enriched for the putative AID binding site RGYW (Wilcoxon P <.001). Furthermore, AID knockdown in lymphoma cells (RAMOS) resulted in preferential hypermethylation at AID-binding sites (Chi square P ~ 0). We then isolated DNA from splenic NBs and GCBs from Aicda-/- (AID-deficient) and Aicda+/+ (wild type) mice and performed eRRBS analysis, obtaining single nucleotide resolution for 2.5-3 million represented CpGs. We observed that most of the 8,000 hypoDMCs identified between GCBs and NBs in Aicda+/+ mice were absent in Aicda-/- mice (800 hypoDMCs between GCBs and NBs Aicda-/- cells), implying that AID is a regulator of DNA methylation in GCBs. In addition, those AID-dependent hypoDMCs were predominantly localized in introns (35%), and also in promoters (10%) and exons (10%). We then defined differentially methylated regions (DMRs) based on the following criteria: ≥ 5 DMCs and methylation difference ≥10%, with >250bp between DMRs. We identified DMRs that get hypomethylated in GCBs in the Aicda+/+ mice, but are not hypomethylated in Aicda-/- GCBs, corresponding to >200 genes that represent AID epigenetic targets. These genes include factors involved in B cell function and differentiation like PAX5, BCL2L1, IRF8 and others. Not unexpectedly, many of epigenetic targets are also known targets for SHM, but some are novel targets that only demonstrate evidence of epigenetic deregulation. We also analyzed the transcriptome of NBs and GCBs from Aicda-/- and Aicda+/+ mice by RNA-seq and detected an increase in DNMT1 expression in Aicda-/- cells compared to Aicda+/+ cells. There were no significant changes in expression of other factors involved in modification of cytosine methylation, such as DNMT3a/3b, TET1/2/3, UNG or MSH2/6. Finally, we performed bone marrow transplantation experiments using VavP-Bcl2 mice, which are known to develop GC-derived lymphomas. We transplanted VavP-Bcl2 bone marrow cells infected with AID-expressing retroviral vectors into C57BL/6 mice and monitored the progression of the resulting BCL2-driven lymphomas. Our preliminary results indicate that high AID expression is correlated with a more aggressive phenotype of the disease. We are currently analyzing the epigenetic targets of AID in both normal GCBs and tumors, in order to find genes that could be epigenetically deregulated and contribute to the formation of lymphomas. Our results demonstrate for the first time that AID functions as a demethylase in GCBs in vivo and suggest that the epigenetic role of AID could contribute to lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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In Vitro Study Of The Mechanisms Involved In The Bone Marrow Mesenchymal Stromal Cell Modulatory Effect On B Cell Function

Blood ◽

10.1182/blood.v122.21.1053.1053 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1053-1053

Author(s):

Eliana Amati ◽

Giulio Bassi ◽

Mariano Di Trapani ◽

Francesco Liotta ◽

Francesco Annunziato ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Conflicts Of Interest ◽

Cpg Oligodeoxynucleotide ◽

Ifn Γ ◽

Cell Inhibition

Abstract Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators of T cell activation and proliferation, mainly through the production of partially defined soluble factors, including the IFN-g-induced tryptophan-degrading enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is still controversial, due to both biological peculiarities of B cells, which do not produce IFN-γ, a key MSC-triggering cytokine, and to different and poorly comparable experimental approaches. Human purified B cells, either resting or activated for 4 days with a stimulation cocktail (CD40 ligand + enhancer polyhistidine mAb MAB050 + rhIL-2 + mouse F(ab’)2 anti-human IgM/IgA/IgG + CpG oligodeoxynucleotide 2006) were co-cultured with MSC, either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-γ + TNF-α for 48 hours), or with MSC supernatants. CD27-positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and intracellular activation status (through signaling network analysis by Phosphoflow) were assessed. Our results showed that MSC are normally supportive cells, not intrinsically capable of suppressing B cell proliferation, and require inflammatory priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair significantly activated B cell growth in a cell contact-independent manner, as supernatant is sufficient to provide maximal inhibition of B cell proliferation. B cell inhibition by MSC is not related to either induction of B cell apoptosis or early signaling events necessary for B cell activation. In addition, IDO pathway triggered in IFN-γ-primed MSC seems to have a role also in B cell inhibition by interfering with the tryptophan metabolism. Overall, B cell behavior following the interaction with MSC depends on the functional state of both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation. Disclosures: No relevant conflicts of interest to declare.

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Oncogenesis of CRLF2 Overexpression and Effect of JAK2 Inhibitor in CRLF2 Overexpressed B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-125259 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2757-2757

Author(s):

Yan Gu ◽

Chunhua Song ◽

Sinisa Dovat ◽

Qinglong Guo ◽

Qinyu Ge ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Proliferation ◽

B Cell ◽

Conflicts Of Interest ◽

Cox Model ◽

Lymphoblastic Leukemia ◽

Normal Bone Marrow ◽

Jak2 Inhibitor ◽

Normal Bone

Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P<0.0001). Patients with CRLF2 overexpression had a significantly higher WBC count (53*10^9/L vs. 29.5*10^9/L, P=0.041). Survival analysis showed that the patients with CRLF2 overexpression had a worse EFS and OS, the differences were statistically significant (11 months vs. 26 months, P=0.043 and 15 months vs. 32 months, P=0.015). Also, the CRLF2 expression is determined with flow cytometry after staining with FITC-CRLF2 antibody in 28 samples. The correlation analysis was performed on the CRLF2 expression detected by qPCR and flow cytometry, respectively. A significant positive correlation of the two methods was observed(r=0.957, P<0.0001). These data not only indicate that CRLF2 overexpression is a marker of poor outcome, but also reveal the qPCR might be a simple and quick method for screening CRLF2 overexpression in the clinic compared to flow cytometry which is commonly used. We further found that expression of CRLF2 or CRLF2 F232C mutant into Nalm6 and 697 B-ALL cells dramatically increase the CRLF2 mRNA level, which is 69 times than vector-only control. Moreover, CRLF2 or CRLF2 F232C significantly promotes the cell proliferation of Nalm6 and 697 cells compared to vector only (P<0.001). In addition, JAK2 inhibitor (BBT594) treatment showed the significant dose-dependent cell proliferation arrest and clonogenic inhibition in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells compared to vector-only control. Furthermore, in vivo we observed the 5-fold higher signal intensity of leukemia engraftment in the mice injected with Nalm6-CRLF2-luc or Nalm6-F232C-luc compared to that of Nalm6-vector-luc control 1-3 weeks after the injection(P<0.001). The Nalm6-CRLF2-luc and Nalm6-F232C-luc infiltrations were observed in bone marrow, central nervous system, liver and spleen of the mice. Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.

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The Complex Role of KDM6A in B-Cell Development and Function

Blood ◽

10.1182/blood-2018-99-119538 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 513-513

Author(s):

Ling Tian ◽

Monique Chavez ◽

Lukas D Wartman

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Bone Marrow Cells ◽

Cell Development ◽

Young Female ◽

B Cell Development ◽

And Function

Abstract Loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell lymphoid malignancies, including B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma. Germline inactivating mutations in KDM6A cause a neurodevelopmental disorder called Kabuki syndrome that is associated with recurrent infections and hypogammaglobulinemia.1 The role of KDM6A in normal B-cell development and function, as well as how the somatic loss of KDM6A contributes to B-cell malignancies, has not been completely defined. To address this issue, we generated a conditional knockout mouse of the KDM6A gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate KDM6A in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) male and female KDM6A conditional KO mice. We found a significant shift from lymphoid to myeloid differentiation in the bone marrow and peripheral blood of these mice. Young, female KDM6A-null mice had mild splenomegaly. Their spleens had an increased number of neutrophils (Gr-1+CD11b+ cells) and erythrocyte progenitors (CD71+Ter119+ cells) and a decreased number of B-cells (B220+ cells). These changes became more pronounced with age and were specific to the female, homozygous KDM6A knockout mice. Furthermore, analysis of B-cell maturation showed that the loss of KDM6A was associated with decreased immature (B220+IgM+ cells) and mature, resting B-cells (B220+IgD+ cells) in the spleen. Similar changes were present in the bone marrow (decreased B220+IgM+ cells and B220+CD19+ cells) and peripheral blood (decreased B220+IgM+, B220+IgD+ and B220+CD19+ cells). Early B-cell development is also altered in KDM6A-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female KDM6A-null mice bone marrow. Next, we performed flow cytometry to catergorize the Hardy fractions of early B-cell development on bone marrow isolated from young, female KDM6A-null mice. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D, which was likely indicative of an incomplete block in B-cell differentiation after the Fraction A stage. When bulk bone marrow cells isolated from young, female KDM6A-null mice were plated in methylcellulose supplemented with interleukin-7, we observed a significantly decreased colony formation compared with bone marrow cells isolated from wildtype littermates. This pre-B lymphoid progenitor cell plating phenotype was expected given the flow cytometry results of decreased B-cell progenitors outlined above. We examined the effect of the loss of KDM6A expression on germinal center (GC) formation in the spleen following immunization with NP-CGG (4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Ratio 16). Two weeks after NP-CGG immunization, we observed a significant decrease in follicular B-cells (FO) and a significant increase in GC B-cells as compared to wildtype littermates (Figure 1). The result is significant as GC B-cells are thought to be the cell-of-origin of follicular and DLBCL. To determine if inactivation of KDM6A affected antibody production, we measured IgM, IgG, IgE and IgA levels by ELISA from serum isolated from young, female KDM6A-null mice. Results revealed higher levels of IgM and lower levels of IgG in serum from KDM6A-null mice, which is suggestive of a class switch recombination (CSR) defect. Concordant with this result, we observed that the loss of KDM6A impaired CSR to IgG1 in splenic B cells after in vitro stimulation for three days with lipopolysaccharide (LPS), an anti-CD180 antibody and interleukin-4. Moreover, we observed a striking defect in the production of plasma cells from KDM6A-null B-cells after LPS stimulation. Taken together, our data shows that KDM6A plays an important, but complex, role in B-cell development and that loss of KDM6A impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies.Stagi S, et al. Epigenetic control of the immune system: a lesson from Kabuki syndrome. Immunol Res. 2016; 64(2):345-359. Disclosures No relevant conflicts of interest to declare.

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The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽

10.1182/blood.v114.22.1465.1465 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1465-1465

Author(s):

Jason Mullenix ◽

Kimi Y Kong ◽

Kristin Severns Owens ◽

Jason Rogers ◽

Shannon FitzPatrick ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Mature Mirnas ◽

Cell Development ◽

B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

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