In Multiple Myeloma Clonotypic CD38 − /CD19 + /CD27 + Memory B Cells Recirculate Through Bone Marrow, Peripheral Blood and Lymph Nodes

2004 ◽  
Vol 45 (7) ◽  
pp. 1413-1417 ◽  
Author(s):  
Thomas Rasmussen ◽  
Marianne Lodahl ◽  
Søren Hancke ◽  
Hans Erik Johnsen
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Memory B Cells

Phenotypic Analysis of CD19+ Subsets In Monoclonal Gammopathy Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4998-4998
Author(s):  
Lucie Kovarova ◽  
Pavla Zarbochova ◽  
Tamara Varmuzova ◽  
Ivana Buresova ◽  
Karthick Raja Muthu Raja ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Healthy Volunteers ◽  
Peripheral Blood ◽  
Monoclonal Gammopathy ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Phenotypic Analysis ◽  
Transitional B Cells ◽  
Significant Difference

Abstract Abstract 4998 Background. Monoclonal gammopathy of undetermined significance (MGUS) is the most common plasma cell disorder which can eventually progress into malignant multiple myeloma (MM). Plasma cells (PCs) are the terminal stadium of B cells differentiation, but it is still unclear which population is the source of pathological PCs with malignant transformation and which population is involved in and may give rise to clonogenic myeloma stem cells. Aims. Phenotypic analysis of CD19+ cell subpopulations in monoclonal gammopathy patients and healthy volunteers to asses their frequency and to find differences on cellular level. Methods. Total of 38 samples was analyzed (16 newly diagnosed untreated MM patients, 12 untreated MGUS persons and 10 healthy donors). CD19+ cells were analyzed for surface expression of CD24, CD27, CD38, and IgD by 5-colors immunophenotyping. Subpopulations of pre-plasma cells consist of transitional B cells (CD24+CD38+), naïve B cells (CD38-IgD+), activated B cells (CD38+IgD+), preGC B cells (CD38++IgD+) and memory B cells (CD38-/+IgD-). These were evaluated in whole lysed peripheral blood together with circulating plasmablast/plasma cells (CD38++IgD-). Bone marrow of MGUS and MM patients was analyzed for number of transitional, immature and memory B cells. Results. Flow cytometric analysis shown no statistical difference when compared number of transitional B cells (1.8%; 3.0% and 1.2%) and activated B cells (54.6%; 62.1% and 45.5%) in peripheral blood of healthy volunteers, MGUS and MM patients, respectively. There was found lower number of circulating plasmablast/plasma cells in peripheral blood of healthy volunteers than in MGUS (1.0% vs. 1.7%; p<0.01), but there was no statistically significant difference for MM (1.7%) when compared to others. The highest number of peripheral naive B cells was found in healthy volunteers (21.4%; p<0.001) and the highest number of peripheral memory B cells was found in MM patients (32.9%; p<0.01) when compared to other groups. There was found also higher number of peripheral preGC B cells in MGUS and MM patients (2.7% vs. 1.6% vs. 1.3%; p<0.05) than in healthy volunteers, respectively. Although numbers of transitional and immature B cells in bone marrow were different for MGUS and MM, the only statistically significant difference was found in number of memory B cells (25.4% for MGUS vs. 11.9% for MM; p<0.01). Summary/Conclusions. Our result showed differences in CD19+ subsets when compared peripheral blood of healthy volunteers and monoclonal gammopathy patients as well as in bone marrow of monoclonal gammopathy group. These differences could be a sign of ongoing changes in B cells of monoclonal gammopathy patients. Further analysis will be also focused on changes at DNA level to confirm clonality of selected subpopulations and to find possible myeloma stem cells source. Supported by GACR 301/09/P457, GACR GAP304/10/1395, MSMT LC06027, MSM0021622434, IGA 10408-3, IGA 10406-3. Disclosures: Hajek: Janssen-Cilag: Honoraria; Celgene: Honoraria; Merck, Sharp, and Dohme: Honoraria.

Multiple Myeloma Cancer Stem Cell Animal Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1848-1848
Author(s):  
Christina C.N. Wu ◽  
Daniel Jacob Goff ◽  
Wenxue Ma ◽  
Heather Leu ◽  
Thomas A. Lane ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
The Self ◽  
High Dose ◽  
Self Renewal ◽  
Immunomagnetic Bead

Abstract Abstract 1848 Poster Board I-874 Multiple myeloma (MM) is the second most common hematologic malignancy and characterized by clonal proliferation of CD138+ bone marrow plasma cells. Despite various treatment options few patients with MM have been cured. Furthermore, high relapse rates and recent evidence from xenogeneic transplantation models and primary MM marrow samples indicate that a rare population of cells or MM cancer stem cells (MM CSCs) within the marrow regenerates itself and may be responsible for drug resistance. These MM CSCs are phenotypically similar to memory B cells (CD138- CD34-CD19+) but differ in that they have the capacity to regenerate themselves or self-renewal. However, most of the reports on MM CSC animal models are established in NOD/SCID mice that require a larger number (1 – 10 × 106) of bead sorted cells for each animal. In addition, the latency of MM induction (4 – 6 months) in NOD/SCID mouse models and lack of in vivo tracking of the malignant clone preclude robust pre-clinical testing of novel therapeutic strategies that target MM CSC. Mononuclear cells were isolated from autologous mobilized peripheral blood of at least four primary MM patients after Ficoll gradient centrifugation followed by immunomagnetic bead depletion of CD34+ and CD138+ cells and/or further sorted using a FACSAria. The CD138-CD34- population was transduced with lentiviral luciferase GFP (GLF) and transplanted (10,000 to 106 cells per mouse) intrahepatically into neonatal RAG2-/- gamma chain-/- (RAG2-/-gc-/-) mice. Engraftment was compared to mice transplanted with either CD34+ or CD138+ cells. Mice were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that a relatively rare CD138- CD27+ population, resembling memory B cells (∼1.2%), persists in MM autografts and can engraft immunocompromised mice more rapidly and effectively than the CD138+ (Lin+) population of mature plasma cells. This data supports the persistence of CSCs despite high dose chemotherapy further underscoring the need for CSC targeted therapy. Bioluminescence was detected in live mice transplanted with as little as 60,000 cells of CD138- CD34- population and as soon as 4 weeks after transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+ and CD138+ population in bone marrow, spleen and liver and bioluminescence was also detected in the secondary transplantation of cells from MMCSC primary engraftment demonstrating the self-renewal capacity of this rare CD138- CD27+ population. Our results suggest that by utilizing a lentiviral GFP-luciferase system in a highly immunocompromised mouse strain fewer cells will be required to monitor MM engraftment and perhaps hasten disease development. Further studies to confirm the expression of selected IgG genes from myeloma cells and to characterize the self-renewal capacity with genes involved in developmental signaling such as sonic hedgehog and wnt pathways are underway. Disclosures: Goff: Coronado Biosciences: Research Funding.

scholarly journals Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 446-453 ◽  
Author(s):  
EA Turley ◽  
AJ Belch ◽  
S Poppema ◽  
LM Pilarski
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Lymphocytes ◽  
Transient Expression ◽  
Peripheral Blood ◽  
Pokeweed Mitogen ◽  
Lymphoid Tissues ◽  
Hairy Cells ◽  
Hodgkin's Lymphomas

Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.

Molecular Mechanisms Involved in Homing and Migration of B-Chronic Lymphocytic Leukemia (CLL) in Response to CXCR4 Stimulation and Downstream Activation of the PI3K Pathway.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1909-1909
Author(s):  
Irene M. Ghobrial ◽  
Joanna M. Ghobrial ◽  
Patricia Bramati ◽  
Michael M. Timm ◽  
Ashock Kumar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Confocal Microscopy ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Molecular Mechanisms ◽  
Pi3k Pathway ◽  
Lymphocytic Leukemia ◽  
Cxcr4 Receptor ◽  
And Migration

Abstract Malignant B-cells characteristically home to the bone marrow and lymph nodes. However, the mechanisms by which cells are recruited into and mobilized from the bone marrow/lymph nodes into the peripheral blood are not well understood. Chemokines such as SDF-1 and it receptor CXCR4 play a central role for lymphocyte trafficking and homing. Downstream activation of the PI3K pathway by chemokines has been implicated in the migration of many cell types. We recently demonstrated that CXCR4 expression correlates significantly with disease progression in CLL. Patients with Rai stage 4 had a higher expression of CXCR4 when compared to patients with Rai stage 0. The expression of CXCR4 was not found on CLL B cells resident within the lymph nodes implying that there may have been downregulation of CXCR4. In this study, we explore the molecular mechanisms involved in homing and migration of B-CLL cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of CLL cells in response to SDF-1. Boyden chamber in vitro migration assays were used with primary CLL peripheral blood cells and the MEK1 CLL cell line. In addition, we expressed fluorescent YFP-CXCR4 in the cells and used confocal microscopy to visualize changes in the subcellular location of the fluorescent CXCR4 before and after SDF-1 stimulation. Inhibitors of the PI3K pathway such as the PI3K inhibitor LY294002 (10uM, one hour pretreatment) and the mTOR inhibitor, rapamycin (200nM overnight) were used. SDF-1 induced a dose dependent migration of primary CLL cells and MEK1 cells, indicating functional CXCR4 receptors. MEK 1 cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping and internalization subcellularly. Pretreatment of the cells with PI3K pathway inhibitors led to inhibition of migration with LY294002 but not with rapamycin. Confocal microscopy demonstrated abrogation of subcellular localization in the presence of LY294002 but not with rapamycin with the addition of SDF-1. These results demonstrate that CLL cells express functional CXCR4 receptora and that the PI3K pathway is essential for SDF-1 dependent CLL migration. In addition, we demonstrate that the CXCR4 receptor is internalized in response to SDF-1 stimulation indicating that CLL cells downregulate the CXCR4 receptor once they home to the bone marrow or lymph nodes where there is abundant SDF-1 in the microenvironment. This leads to the confinement of the cells in the microenvironment preventing further migration of the cells outside of the bone marrow/lymph nodes. Supported in part by CA97274

Iterative Germinal Center Re-Entries of Memory B-Cells with t(14;18) Translocation and Early Steps of Follicular Lymphoma Progression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 150-150
Author(s):  
Sandrine Roulland ◽  
Jocelyne Stephanie Sungalee ◽  
Ester Morgado ◽  
Emilie Mamessier ◽  
Emilie Gregoire ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Lymph Nodes ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Systemic Disease ◽  
Memory B Cells ◽  
Lymphoid Organs ◽  
Healthy Humans

Abstract Abstract 150 The recent demonstration that memory B-cells can re-enter germinal centers (GCs) and participate to new rounds of GC reactions has opened the possibility that multi-hit B-cell lymphomagenesis could be a much more dynamic process than initially anticipated, gradually progressing throughout the successive passages of memory B-cells in GCs during a lifetime of successive immunological challenges. Here, we provide evidence for this scenario in follicular lymphoma (FL), a GC derived B-cell malignancy initiated in the bone marrow by the hallmark t(14;18) BCL2/IGH translocation. To address this issue, we engineered an original sporadic BCL2tracer mouse model mimicking the rare occurrence of t(14;18) translocation in humans through V(D)J recombination errors (1 in a million B-cells) allowing to track the resultant BCL2-expressing clones; and underwent a molecular/immunofluorescent tracking of t(14;18)+ clones vs. normal memory B-cells in paired lymphoid tissue samples (spleen, lymph nodes, bone marrow) from healthy individuals. We first show that contrary to the current dogma, ectopic BCL2 expression is not sufficient to provoke the FL's characteristic differentiation arrest of activated B-cells as GC B-cells, thereby suggesting that differentiated BCL2+ memory B-cells must return to the GC to acquire additional oncogenic hits and “fix” in situ growth. Strikingly, we further find that in a small fraction of “healthy” humans, such differentiation arrest already operated, and that a clonally expanded population of t(14;18)+ cells with FL-like features have widely disseminated in blood and in multiple lymphoid organs (spleen, lymph nodes, bone marrow), with unprecedentedly reported frequencies (from 1/million to 1/500 cells in some individuals), shaping the systemic disease presentation observed in FL patients. Using molecular/immunofluorescent backtracking of such clones in various paired and remote lymphoid organs, we further demonstrate that t(14;18)+ clones systematically display an extensive history of AID (activation-induced cytidine deaminase)-mediated events compatible with iterative rounds of GC co-opting, in sharp contrast to single memory B-cell clones from the same individuals. We thus show that BCL2-expressing memory B-cells require multiple GC transits to acquire the distinctive FL-like maturation arrest as centrocyte/centroblasts and to progress to advanced FL precursor stages. Altogether, our data argue for a model of lymphomagenesis, in which progression from precursor stages to FL occurs asymptomatically over an extended period of time by subverting the dynamic and plastic attributes of memory B-cells. This understanding of the pre-clinical phases driving FL development in asymptomatic patients should help rationalize prospective approaches designed to identify biomarkers of risk, and innovative therapeutic targets present in early, potentially more curable phases of the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A novel membrane antigen selectively expressed on terminally differentiated human B cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1922-1930 ◽  
Author(s):  
T Goto ◽  
SJ Kennel ◽  
M Abe ◽  
M Takishita ◽  
M Kosaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Affinity Constant ◽  
Target Antigen ◽  
Specific Marker

Abstract A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

scholarly journals A novel membrane antigen selectively expressed on terminally differentiated human B cells

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1922-1930 ◽  
Author(s):  
T Goto ◽  
SJ Kennel ◽  
M Abe ◽  
M Takishita ◽  
M Kosaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Affinity Constant ◽  
Target Antigen ◽  
Specific Marker

A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

scholarly journals Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 446-453 ◽  
Author(s):  
EA Turley ◽  
AJ Belch ◽  
S Poppema ◽  
LM Pilarski
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Lymphocytes ◽  
Transient Expression ◽  
Peripheral Blood ◽  
Pokeweed Mitogen ◽  
Lymphoid Tissues ◽  
Hairy Cells ◽  
Hodgkin's Lymphomas

Abstract Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.

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