Onset of Regulatory B Cells Occurs at Initial Stage of B Cell Dysfunction in Multiple Myeloma

Introduction :Multiple myeloma(MM) is caused by aggregation of clonal plasma cells, clinically presenting as evolvement in order from monoclonal gamma globulin disease (MGUS), smoldering MM (SMM), symptomatic MM to plasma cell leukemia. Regulatory B cells (Bregs), only a small immunosuppressive subgroup of B cells, have been recently identified in the setting of autoimmune diseases, immune thrombocytopenia (ITP) and gastric cancer . The role of Bregs in MM remains poorly defined. Here, the study was carried out on how Bregs correlate with evolution of MM, as well as how Bregs would be influenced by bortezomib, which is currently the first-line anti-MM agent. Methods :All patients met the International Myeloma Working Group (IMWG) Criteria for the Diagnosis of MM. Mononuclear cells (MNCs) were isolated from bone marrow(BM)but not peripheral blood(PB)at specified time points. Cell numbers were quantified by hemocytometer. The ratios of Bregs and B cells were delected by flow cytometry (FCM). Propidium iodide was widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic. The results were expressed as the mean ± SD. Comparisons between 2 groups were performed with Student's t-test. Multiple groups (≥3) were analyzed by one-way ANOVA, and paired groups were analyzed by two-way ANOVA or Student t test. Data were graphed and analyzed using GraphPad Prism 6.0. P < 0.05 was considered statistically significant. Results: The study firstly found that Bregs' ratio increased at very beginning stage of MM and headed for extinction during progression of MM. It showed that Bregs' ratios were 11.7 ± 6.3%, 11 ± 8.6%, 15.8 ± 6.8%, and 4.9 ± 2.1% at stage of MGUS (n=5), SMM (n=4), newly diagnosed MM (NDMM) (n=9), and relapsed or refractory MM (RRMM) (n=6), respectively (p<0.05). We then obtained mononuclear cells from NDMM samples. It revealed a positive correlation on both ratios and absolute numbers between Breg subgroup and B cell groups when B cells' ratio was higher than 5% of BMMNCs, whereas Bregs' ratio was rarely detected when B cells' ratio was lower than 5%. Subsequently, a retest was made to observe how Breg subgroup would be influenced by using bortezomib to target B-cell reservoir in MM. We added bortezomib of different concentrations to MNCs from NDMM samples within culture medium RPMI1640 (Ruikos Biotechnologies) for 24 hours (n=6). Bregs and B cells were totally killed when treated with high-dose bortezomib (n=3), while partially killed with low-dose bortezomib (n=3). To further explore how bortezomib could influence Bregs, another 6 samples were enrolled to demonstrate that the apoptotic absolute number of Bregs significantly increased after treatment of high-dose bortezomib (135615 ± 92085 v.s. 81132 ± 52908, P ≤ 0.05). Conclusions : Bregs were strongly entwined with the whole group of preserved B cell in MM. Bregs began to increase at very beginning stage of MM when B cells were preserved, accompanying with transition from MGUS to diagnostic MM. Bregs would substantially decreased while B-cell reservoir diminished during MM progression or by B cell targeted bortezomib. Disclosures Zou: the National Natural Science Foundation of China: Research Funding. Guo:the National Natural Science Foundation of China: Research Funding. Cui:the National Natural Science Foundation of China: Research Funding. Zhang:the National Natural Science Foundation of China: Research Funding. Pan:the National Natural Science Foundation of China: Research Funding.

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Myeloma Heterogeneity within Its Complex Immune Ecosystem

Blood ◽

10.1182/blood-2019-132193 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4354-4354

Author(s):

Jana Jakubikova ◽

Danka Cholujova ◽

Gabor Beke ◽

Zachary R Hunter ◽

Teru Hideshima ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Cell Distribution ◽

Advisory Committees ◽

Cell Clusters

Multiple myeloma (MM), the second most common hematologic malignancy worldwide, is a B cell malignancy characterized by high frequency of intra-clonal diversity within malignant plasma cells (PC) in the bone marrow (BM). To better understand the myeloma heterogeneity within its complex pathophysiology, we performed large-scale data-driven mass cytometry (CyTOF) analysis in cohort of 188 bone marrow (BM) samples from multiple myeloma (MM) patients compared to 10 age-matched healthy donors (HD). Our design focused on profiling of PC intra and inter-neoplastic heterogeneity based on molecular perturbations of transcriptional factors and signaling regulators and stemness-controlling markers ensuring development of B cell lymphopoiesis within myelomagenesis encompassing the different clinical spectra of pre-malignant/asymptomatic (16 MGUS and 25 SMM) and active symptomatic stages (43 NDMM and 104 relapsed or relapsed/refractory MM patients) of MM pathogenesis. Moreover, interaction of PC disease status with the immune ecosystem of myeloma microenvironment was evaluated as well. To distinguish tumor-driven specific immune changes from myeloma immune ecosystem, we observed that cell frequency of cytotoxic naïve and effector cells, g/dT, and early monocytes, myelocytes and erythroblasts immune subsets was significantly reduced in both premalignant and active MM stages. In contrast, mostly innate immune clusters including non-canonical monocytes, myeloblasts, and mature neutrophils, erythroblasts and platelets were present at a higher frequency across all MM stages versus HD. To evaluate cell distribution of B lymphopoiesis in MM disease stages, switched memory B cells and plasmablasts clusters were upregulated in premalignant stage MGUS compared to HD. Similar observations were detected in SMM and NDMM versus HD, with the highest abundance of PC clusters in NDMM. The downregulation of cell distribution in B cell progenies, immature and transitional B cells, and un-switched memory B cell clusters was observed in NDMM and relapsed/refractory MM patients. Furthermore, MM patients treated with Revlimid-Velcade-Dexamethasone therapy had decrease frequency of specific PC clusters and un-switched and transitional B cell clusters. In addition, our data revealed immunophenotyping aberrancies present not only in PC clusters but also across all myeloma B lymphomagenesis in BM samples from MM patients. In-depth characterization of malignant plasma cells, significant variations were detected in PC clusters of MM cohort based on different expression of IRF4, c-Myc, CD28, CD117, and FGFR-3, however with homogenous expression of sXBP1, and MMSET which differ in all 4 MM stages compared to HD. Significant upregulation of CD47 was showed in all PC clusters of MM cohort. Moreover, PC clusters differ in intra-clonal expression of self-renewing/stemness markers CD184, Notch-1, Oct3/4, KLF-4, Sox-2, and Nanog, supporting the idea of sub-clonal variations insight of MM tumor. This study might provide the rational for prediction of MM patient status and design of targeted therapy in MM on personalized bases. This work was supported by REA grant agreement No. 609427-SASPRO 0064/01/02, TRS-2015-00000170, APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Jamroziak:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Richardson:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Kastritis:Prothena: Honoraria; Genesis: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

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B-Cell Markers Predict Response to Venetoclax in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2108.2108 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2108-2108 ◽

Cited By ~ 2

Author(s):

Vikas A. Gupta ◽

Scott Newman ◽

Nizar J. Bahlis ◽

Jonathan J Keats ◽

Shannon Matulis ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Gene Signature ◽

Myeloma Cell ◽

Bcl2 Family

Abstract BCL2 family members such as MCL1, BCLXL, and BCL2 are critical for cancer cell survival and therefore represent promising therapeutic targets. Both B cells and CLL cells depend primarily on BCL-2 and are thus sensitive to the BCL2 specific inhibitor venetoclax, while plasma cells and multiple myeloma typically depend on Mcl-1 and would therefore be resistant to venetoclax. However, a subset of myeloma is venetoclax sensitive based on recent in vitro and clinical trial data. In preliminary results from a phase I trial of venetoclax in multiple myeloma, 40% of patients positive for t(11;14) had objective responses, while only 6% of t(11;14) negative patients responded. We have made similar observations with in vitro testing of 30 freshly isolated myeloma patient samples, identifying both non-t(11;14) samples sensitive to venetoclax as well as resistant t(11;14) positive samples. Together, these results suggest not only that a subset of multiple myeloma is co-dependent on BCL2 but also that t(11;14) is neither necessary nor sufficient for responding to venetoclax. We therefore set out to identify other factors that may predict BCL2 dependence in multiple myeloma. Previous studies of t(11;14) myeloma have noted increased expression of CD20, CD23, CD79a, and PAX5 which are typically associated with B cells prior to their differentiation into plasma cells. Based on these observations we hypothesized that venetoclax sensitivity in myeloma may be associated with the retention of B cell properties including BCL2 dependence. We probed an online expression database of myeloma cell lines for non-t(11;14) cell lines expressing CD20 and identified two cell lines, OCI-My5 and PCM6, both of which we found to have an IC50 of approximately 50nM when treated with venetoclax. We went on to characterize a panel of 13 cell lines. In addition to OCI-My5 and PCM6, 4 other cell lines were sensitive to venetoclax, all positive for t(11;14). Of the 7 venetoclax resistant cell lines, 2 were t(11;14) positive. Protein levels of MCL1, BCLXL, and BCL2 were comparable among the 13 lines and therefore anti-apoptotic expression is unlikely to be responsible for venetoclax sensitivity. Consistent with our previous co-immunoprecipitation studies, more of the pro-apoptotic BIM was bound to BCL2 in venetoclax sensitive lines compared to resistant lines. In the absence of differences in BCL2 family expression, we next sought to identify other B cell related features correlating with venetoclax sensitivity. We used RNAseq data from our 13 cell lines to compare the expression of 100 genes previously reported to be differentially expressed between normal B cells and plasma cells. Interestingly, unsupervised clustering revealed a group of venetoclax sensitive cells enriched for other B cell associated genes. GSEA revealed enrichment of genes associated with immune system activation at a p < 0.001. We also analyzed the differential expression of genes between our sensitive and resistant lines and again identified overexpression of B cell related genes such as CD20, CD79A, STAT5A, and RASGRP2 in venetoclax sensitive lines, though no single marker was present in all of the venetoclax sensitive lines. We examined the expression of CD20, CD79a, and CD79b in the CoMMpass data set (IA8) as well and found that they were not co-expressed in most patients, again suggesting that no single marker is likely to be predictive. Finally, we created a gene signature from the top differentially expressed genes to predict sensitivity or resistance to venetoclax and used this signature to evaluate a database of 68 myeloma cell lines. One of the top hits predicted to be sensitive by our gene signature is the t(11;14) negative line MOLP2, and indeed this cell line was recently reported to be highly responsive to venetoclax. In conclusion, B cell markers and our gene signature correlate with BCL2 dependence and venetoclax sensitivity independent of t(11;14). Disclosures Bahlis: BMS: Honoraria; Onyx: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria. Nooka:Spectrum, Novartis, Onyx pharmaceuticals: Consultancy. Kaufman:Pharmacyclics: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Lonial:Onyx: Consultancy; Onyx: Consultancy; BMS: Consultancy; Janssen: Consultancy; Merck: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Millenium: Consultancy; BMS: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Janssen: Consultancy.

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CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3457.3457 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3457-3457 ◽

Cited By ~ 3

Author(s):

Eric D. Hsi ◽

Roxanne Steinle ◽

Balaji Balasa ◽

Aparna Draksharapu ◽

Benny Shum ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Target Cells ◽

Dependent Manner ◽

Myeloma Cells ◽

Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

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Frequent Dysregulation of Fc Gamma Receptor IIb (Fc γRIIb) and Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling Occurs in Multiple Myeloma (MM) Patients

Blood ◽

10.1182/blood.v118.21.2917.2917 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2917-2917

Author(s):

Jennifer Li ◽

Andrew Leu ◽

Mingjie Li ◽

Ethan D Hobel ◽

Kevin Delijani ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Critical Role ◽

Rt Pcr ◽

Binding Ability ◽

Downstream Signaling

Abstract Abstract 2917 The inhibitory Fc receptor, Fc γRIIb, is expressed on plasma cells, controls their persistence in the bone marrow (BM) and their ability to produce serum Ig. Activation of Fc γRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing inositol 5'-phosphatase (SHIP) in plasma cells. Immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposing signaling modules that duel for control of plasma cell activation. Fc γRIIb-mediated SHIP phosphorylation activates downstream ITAM or ITIM signaling. To determine whether multiple myeloma (MM) cells express Fc γRIIb, we performed immunohistochemical staining on bone marrow mononuclear cells from MM patients and controls. We found that not only CD20+ B cells expressed Fc γRIIb but more importantly CD138+ cells from MM patients also showed expression of this receptor. Next, we examined whether Fc γRIIb was present and expressed in CD138+ primary MM cells purified from fresh MM BM and the MM cell lines MM1s, RPMI8226, and U266 using PCR and RT-PCR on DNA and mRNA, respectively. We focused on the transmembrane domain of the Fc γRIIb gene with four primers from different parts of this domain since this portion plays a critical role in this receptor's function. The MM cell lines expressed different amounts of Fc γRIIb. Notably, we found that 17% (5/30) of MM patients showed absence of Fc γRIIb both using RT-PCR for mRNA and PCR for DNA. Moreover, use of these same primers on nonmalignant PBMCs from the MM patients also showed absence of this gene in the same five patients. As a result of these findings, we are currently sequencing Fc γRIIb in MM patients to determine if additional patients show mutational changes that affect the function of this receptor. We also further determined SHIP-1 phosphorylation using Western blot analysis since this protein mediates downstream signaling of Fc γRIIb. Following stimulation with Fc complexes, phosphorylation of SHIP-1 was markedly reduced in MM tumor cells compared to normal CD20+ B cells. Interestingly, the patients with missing Fc γRIIb expressed higher levels of SHIP-1 gene expression compared to patients with normal Fc γRIIb expression. We investigated the IgG-binding ability of MM patients (n=33) and normal donors (n=33) to Fc γRIIb. Each serum sample was incubated with cells from MHC1, a cell line that specifically expresses Fc γRIIb but not Fc γRI and Fc γRIIa. The results showed MM patients' serum IgG have much lower Fc γRIIb-binding ability than normal human IgG (P<0.05) by using both flow cytometric and immunofluorescence assays. Our findings suggest that the monoclonal protein produced by MM patients has a very low Fc γRIIb-binding ability and is incapable of signaling through the inhibitory ITIM pathway. Germline loss of Fc γRIIb in MM patients with variation in its expression level and its downstream signaling molecule SHIP and its phosphorylation as well as the inability of MM IgG to bind cells containing this receptor is a potential new mechanism that contributes to the uncontrolled growth of MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

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First Description of B Cell Maturation Antigen Expression in Light Chain Amyloidosis

Blood ◽

10.1182/blood-2019-127332 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5452-5452

Author(s):

Susan Bal ◽

Allison Sigler ◽

Alexander Chan ◽

David J. Chung ◽

Ahmet Dogan ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Plasma Cells ◽

Staining Intensity ◽

Advisory Committees ◽

Light Chain Amyloidosis ◽

B Cell Maturation

Background B-cell maturation antigen (BCMA) is a transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily involved in the regulation of B cell proliferation and survival as well as maturation/differentiation into plasma cells. In multiple myeloma cells, overexpression of BCMA has been shown to activate mitogen activated protein kinase pathways (AKT, ERK1/2, and NF-κB) and upregulates anti-apoptotic proteins (MCL1, BCL2, BCL-xL) resulting in cellular proliferation. Immunotherapeutic strategies targeting BCMA are showing great promise in heavily pre-treated refractory multiple myeloma. Light Chain Amyloidosis (AL) is a multisystem disorder of clonal plasma cells that results in the production of an abnormal light chain which misfolds and deposits in the organs leading to disruption of tissue architecture, cellular stress, dysfunction and eventually, death. The smaller burden and lower proliferative potential of the offending clonal plasma cells in amyloidosis may potentially lend itself favorably to immunotherapeutic strategies targeting BCMA. Given the efficacy of this approach in MM, the evaluation of BCMA expression on the surface of amyloidogenic plasma cells is warranted. Methods All patients diagnosed with Light chain Amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. These unstained BM biopsy samples were prospectively stained for BCMA expression using Immunohistochemistry (IHC). We utilized a clinical-grade assay (clone D6; catalog sc-390147; company Santa-Cruz; monoclonal antibody; dilution 1:400) in a CLIA compliant setting. We scored the biopsies for BCMA expression, intensity, and site of staining. We also obtained their demographic details, staging, and cytogenetic information for the patients with available samples. Results During the queried period, 28 unstained samples were available for testing from the time of disease diagnosis. The median age of the population was 63 years (range 41-73). 64% of patients were male and consistent with the literature; a majority of patients (75%) had lambda-typic clonal plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 36% patients, and chromosome 1q and del 13q were each seen in 32% of patients. No patient had t(4;14) or del 17p. The median clonal PC burden in BM at diagnosis was 10% (range2-80%) and 36% had > 10% plasma cells. In clonal PCs, the median BCMA expression was 80% (range 20-100%). Only one patient had a staining intensity under 50% (20%). Membranous staining was noted in 82% of patients and a Golgi pattern in 11%. The median staining intensity was 2 (range 1-3). Of the patients with baseline diagnostic samples available for testing, six patients had additional unstained bone marrow samples for staining at the time of relapse. The majority of patients (83%) who relapsed had >10% plasma cells with a higher median plasma cell burden of 35% (range 10-80). The median BCMA expression was 65% (range 50-80) with no patient having <50% expression. The staining pattern was membranous in 50%, Golgi in 17%, and Golgi-membranous in 33%. At the time of relapse, the median clonal PC burden was 13% (range 5-30). BCMA expression continued to be present at the time of relapse with a median 75% (range 50-100) with predominantly membranous staining (83%). The median staining intensity in both diagnostic and relapsed tissue within the six samples studied was 1. Conclusions Our study represents the first description of BCMA expression on the surface of amyloidogenic plasma cells to our knowledge. BCMA is uniformly expressed by pathologic PCs in AL amyloidosis both at the time of diagnosis and relapse. Given the efficacy of BCMA directed therapy in multiple myeloma, further investigation of these agents in light-chain amyloidosis are warranted and may provide an effective therapeutic strategy in this devastating disease. Figure Disclosures Dogan: Corvus Pharmaceuticals: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Roche: Consultancy, Research Funding. Giralt:Takeda: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding; Kite: Consultancy; Novartis: Consultancy; Actinium: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Miltenyi: Research Funding; Spectrum Pharmaceuticals: Consultancy. Hassoun:Novartis: Consultancy; Janssen: Research Funding; Celgene: Research Funding. Landau:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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High Dimensional Immune Profiling in Smoldering Multiple Myeloma Identifies Novel Organizing Features of the Tumor Microenvironment

Blood ◽

10.1182/blood-2019-130554 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4384-4384

Author(s):

Hearn Jay Cho ◽

Deepak Perumal ◽

Adeeb H Rahman ◽

Seunghee Kim-Schultze ◽

Jennifer Yesil ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Microenvironment ◽

Tumor Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Plasma Cells ◽

High Dimensional ◽

Integrated Analysis ◽

Symptomatic Disease

Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase lasting months or years before manifesting clinical symptoms warranting therapy. Current research indicates that the tumor microenvironment (TME) in the bone marrow may play a significant role in governing progression to symptomatic disease. Therefore, understanding of the interactions between malignant plasma cells and the TME in early disease states is critical in the pursuit of therapies that will prevent progression to symptomatic disease. We performed high dimensional genomic and immunologic analysis of bone marrow specimens from 73 subjects with SMM. We performed RNA-seq on the malignant plasma cells isolated by anti-CD138 magnetic bead positive selection, mass cytometry (CyTOF) and T cell receptor sequencing (TCR Seq) of CD138-depleted bone marrow mononuclear cells, and proteomics, seromic, and grand serology analysis of bone marrow plasma. These samples and assays provided a broad view of the tumor cells and the cellular and soluble components of the TME. Each of these assays identified self-organizing clusters of subjects, indicating that subgroups of SMM patients shared common characteristics in the tumor or TME populations. We then applied novel bioinformatic methods to compare data from pairs, trios, quartets, and quintets of assays to identify communities of subjects with similar immunologic and genomic characteristics. Integrated analysis of CyTOF, proteomic, and TCR Seq resolved three distinct communities with a high degree of significance. These communities shared distinct cellular and proteomic features that suggested early adaptive, activated adaptive, or innate immune characteristics. These results suggest that the continuum from MGUS to MM does not consist of a single pathway in either the tumor cells or the TME, and that complex interactions ultimately determine progression. This suite of assays (CyTOF, proteomics, and TCR Seq) may be applicable in translational and clinical studies to understand key tumor and immune determinants of SMM and lead to rationally designed therapy to replicate these conditions to prevent progression to symptomatic disease. Disclosures Cho: Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; The Multiple Myeloma Research Foundation: Employment; Takeda: Research Funding; BMS: Consultancy; Agenus: Research Funding; GSK: Consultancy. Adams:Janssen Pharmaceuticals R&D: Employment, Other: Own Stock. Parekh:Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding; Karyopharm Inc.: Research Funding.

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SYK Inhibition Disrupts the Cross-Talk Between B-Cell Activation Factor (BAFF) and B-Cell Receptor (BCR) and Thereby Antagonizes Mcl-1 in Chronic Lymphocytic Leukemia (CLL) B-Cells

Blood ◽

10.1182/blood.v128.22.303.303 ◽

2016 ◽

Vol 128 (22) ◽

pp. 303-303

Author(s):

Cody Paiva ◽

Taylor Rowland ◽

Olga Danilova ◽

Bhargava Sreekantham ◽

Stephen E Spurgeon ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

B Cell ◽

Cell Survival ◽

Research Funding ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Mutational Status ◽

Pathway Gene ◽

Bcr Signaling

Abstract Although small molecule inhibitors of BCR-associated kinases (BCRi) revolutionized therapy in CLL, they provide incomplete responses. Soluble mediators emanating from the tumor microenvironment perpetrate CLL cell survival and may account for resistance to BCRi. Tumor necrosis factor receptor superfamily ligands BAFF and APRIL induce NFκB, which in turn upregulates pro-survival Bcl-2 family proteins and thereby drives anti-apoptotic responses.The exact roles of the individual NFκB pathways, as well as the implications of targeting BCR in context of BAFF signaling in CLL remain understudied. We explored the mechanistic underpinnings of CLL cell survival in response to BAFF signaling, uncovering the functional significance of the BCR-associated kinases and Bcl-2 family proteins in this setting. Peripheral blood mononuclear cells were isolated from patients with CLL. We established a novel BAFF-expressing stromal co-culture model and referenced it to control, CD40L-expressing stroma and soluble BAFF. We employed inhibitors of Bruton tyrosine kinase (BTK, ibrutinib), phosphoinositide-3 kinase (PI3K, idelalisib) and spleen tyrosine kinase (SYK, entospletinib) and measured CLL cell apoptosis, migration, NFκB activity, protein and mRNA expression by flow cytometry, immunoblotting, ELISA, RT-PCR and immunocytochemistry. CLL cells co-cultured with BAFF-expressing stroma were resistant to spontaneous apoptosis (12.3±3.2% after 24 h, vs 34.8±6.2% off stroma) and chemotherapy agents (bendamustine, fludarabine). Gene expression profiling exposed the NFκB pathway gene targets as the most significantly upregulated upon BAFF stimulation (p<0.0001). We and others have shown that CD40L-expressing stroma induces canonical and non-canonical NFκB in CLL. By contrast, while BAFF led to strong activation of the non-canonical NFκB with processing of p100 (to p52) by 4 h and a 5-fold increase in p52 DNA-binding activity by 24 h, canonical NFκB (RelA) activation was less pronounced. BAFF predominantly induced Mcl-1, compared to CD40L which strongly upregulated Bcl-X. BCR is a major driver of canonical NFκB signaling in CLL. Thus, we studied whether BAFF co-opted BCR signaling in CLL. BAFF induced rapid (15 min) phosphorylation of the proximal BCR kinases SYKand LYN, sustained for up to 4 h, as well as ERK, in CLL cells. AKT activation occurred late (>2 h), suggesting that BAFF induced AKT independent of BCR. BAFF-mediated BCR activation did not correlate with IGHV mutational status. Like IgM, BAFF induced CLL cell chemotaxis. SYK inhibition effectively antagonized survival and chemotaxis of BAFF-stimulated CLL cells. By contrast, targeting BTK or PI3K was less effective. All BCRi's fully blocked canonical NFκB activation in BAFF-stimulated CLL cells (suggesting its dependence on BCR signaling), but none inhibited the non-canonical pathway. We found that entospletinib, but not other BCRi's, decreased Mcl-1 expression in CLL cells co-cultured with BAFF-expressing stroma. Unlike in IgM-stimulated cells, entospletinib did not promote Mcl-1 protein degradation. By contrast,, targeting SYK in BAFF-stimulated cells abrogated BAFF-mediated upregulation of pSTAT3, a transcription factor which regulates Mcl-1. This was accompanied by a decrease in Mcl-1 transcript, an effect mimicked by ruxolitinib, a JAK/STAT inhibitor. BAFF receptor signals via the TRAF3/NIK/IKK1 axis to induce non-canonical NFκB activation in neoplastic B-cells. We supposed that NIK (NFκB-inducing kinase) or IKK1 could be directly responsible for SYK activation by BAFF. Indeed, genetic knockdown of NIK resulted in decreased SYK activation, whereas IP experiments demonstrated that NIK directly complexed with SYK in BAFF-stimulated neoplastic B-cells, confirming NIK role in activation of BCR signaling. Thus, BAFF-mediated induction of BCR-associated kinases and Mcl-1 contributes to CLL cell survival. SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and BCR, thereby disrupting the pro-survival microenvironment signaling in CLL. Disclosures Spurgeon: Gilead Sciences: Research Funding; Bristol Myers Squibb: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Danilov:Prime Oncology: Honoraria; Dava Oncology: Honoraria; ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Takeda: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy.

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Multiple Myeloma Includes Phenotypically Defined Subsets of Clonotypic CD20+ B Cells that Persist during Treatment with Rituximab

Clinical medicine Oncology ◽

10.4137/cmo.s615 ◽

2008 ◽

Vol 2 ◽

pp. CMO.S615 ◽

Cited By ~ 1

Author(s):

Linda M. Pilarski ◽

Eva Baigorri ◽

Michael J. Mant ◽

Patrick M. Pilarski ◽

Penelope Adamson ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

B Cell ◽

Plasma Cells ◽

Cell Subset ◽

Light Scatter ◽

Cell Compartments ◽

B Lineage ◽

B Cell Subsets ◽

Anti Cd20

Potential progenitor B cell compartments in multiple myeloma (MM) are clinically important. MM B cells and some circulating MM plasma cells express CD20, predicting their clearance by treatment with anti-CD20. Here we describe two types of clonotypic CD20+ B cell in peripheral blood of myeloma patients, identified by their expression of CD19 and CD20 epitopes, their expression of CD45RA and their light scatter properties. Thus, the circulating component of the MM clone includes at least two distinct CD19+ CD20+ B cell compartments, as well as CD138+CD20+ plasma cells. To determine whether either or both B cell subsets and the CD20+ plasma cell subset were depleted by anti-CD20 therapy, they were evaluated before, during and after treatment of patients with rituximab (anti-CD20), followed by quantifying B cell subsets over a 5 month period during and after treatment. Overall, all three types of circulating B lineage cells persist despite treatment with rituximab. The inability of rituximab to prolong survival in MM may result from this failure to deplete CD20+ B and plasma cells in MM.

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MO251HIGHLY SENSITIVE FLOW CYTOMETRIC DETECTION OF RESIDUAL B-CELLS AFTER RITUXIMAB IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS PATIENTS*

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab104.009 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Y K O Teng ◽

L Van Dam ◽

Jelle Oskam ◽

S W A Kamerling ◽

E J Arends ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Memory B Cells ◽

Cell Depletion ◽

B Cell Depletion ◽

Peripheral Blood Mononuclear

Abstract Background and Aims B-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX. Method EuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA. Results By employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients. Conclusion We demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

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In Multiple Myeloma, Clonal Cells Are Unevenly Distributed among Peripheral Blood B Cell Subpopulations.

Blood ◽

10.1182/blood.v104.11.1426.1426 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1426-1426

Author(s):

Stefanos I. Papadhimitriou ◽

Ioanna Gligori ◽

Elpiniki Kritikou-Griva ◽

Georgios Gortzolidis ◽

Aggeliki Davea ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Plasma Cells ◽

In Situ Hybridisation ◽

Current Evidence ◽

Chain Gene ◽

Virtual Absence ◽

Lineage Differentiation

Abstract BACKGROUND: Current evidence supports the existence of circulating clonal B cells in Multiple Myeloma (MM). However, attempts to enumerate and phenotypically characterise them have so far provided inconsistent results. Most investigators have studied unselected peripheral blood (PB) mononuclear cells, among which the clonal ones make only a small minority. Moreover, in most cases, numerical chromosomal changes were employed as a clonal marker, but aneuploidy is considered a late event in myelomagenesis. To overcome these difficulties, we have followed an alternative approach, by studying purified PB B cells and focusing on chromosomal translocations involving the immunoglobulin heavy chain gene (IGH) on region 14q32, a frequent, early and possibly crucial pathogenetic event in MM. METHODS: The study included 33 MM patients with 14q32 rearrangements, detected by conventional cytogenetics or florescence in-situ hybridisation (FISH) in the bone marrow at diagnosis. PB CD19+ cells were immunomagnetically isolated (>99% purity) and cytocentrifuged on slides. The slides were studied with a FICTION technique, ie a combination of FISH using a “break-apart” IGH probe set (Vysis Inc, Downers Grove, Il, USA) and indirect immunofluorescence for CD34, CD5, CD10, CD23 and CD38. To avoid the possibility of contaminating plasma cells, isolates with >1% CD19+CD38+++ cells on flow cytometry were excluded from FICTION study. RESULTS: “Positive” cells above the cutoff level of false positivity (4%) were detected in 25 cases (75.7%), ranging from 4% to 33% (median 9%) among the total CD19+ population. These cells were found to consistently express CD10 (83% to 100%, median 96%) and CD38 (79% to 100%, median 89%). They less commonly expressed CD23 (39% to 67%, median 46%) and very rarely CD34 (0% to 5%, median 0%) and CD5 (0% to 3%, median 0%). CONCLUSIONS: Our data suggest that circulating cells bearing IGH rearrangements are the rule in MM, making a small but detectable fraction of CD19+ cells. There mmunophenotypic profile supports the concept that clonal B cells represent advanced ontogenetic rather than early stages in B lineage differentiation. Finally, the virtual absence of CD5+ clonal cells is in accord with the view that the high number of PB CD5+ B cells in MM reflects an immunoregulatory network and does not result from clonal expansion.

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