Dominance of Non-CLL Phenotype of Monoclonal B-Cell Lymphocytosis (MBL) in the Middle East and An Overall MBL Prevalence Comparable to Western Countries

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4586-4586
Author(s):  
Maher Albitar ◽  
Faisal Rawas ◽  
Randa Nounou ◽  
Nasir Bakshi ◽  
Fahed Almhareb ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Middle East ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Light Chain ◽  
Peripheral Blood ◽  
Color Flow ◽  
Western Countries ◽  
Two Populations

Abstract Abstract 4586 Chronic lymphocytic leukemia (CLL) is considered more common in Western countries and its incidence is believed to decrease moving east across the globe. CLL is believed to be less common in the Middle East compared to Western countries, although reliable statistical data is not available. Therefore, monoclonal B-cell lymphocytosis (MBL), a pre-CLL condition, is expected to be less common in non-Western countries. In Western countries, MBL prevalence as detected by flow cytometry varies from 0.6% to 14% in healthy individuals older than 40 years, dependent on the level of sensitivity and number of parameters applied. Using 4 to 6 color flow cytometry, most studies report a prevalence of approximately 5 % in the Western population with the CLL-phenotype about 5 times more prevalent than the non-CLL phenotype. MBL incidence and relative proportions of CLL-phenotype versus non-CLL phenotype have not been adequately studied in non-Western countries. We investigated the prevalence and phenotype of MBL in a population sample in the Middle East. Method: 365 individuals, mostly Saudi Arabian nationals and a smaller number of individuals from neighboring countries, aged over 50 years with normal peripheral blood counts and no evidence of hematologic disease. Peripheral blood samples were immunophenotyped by 8-color flow cytometry detecting CD45, CD19, CD20, CD5, CD10, CD3, kappa and lambda light chains, based on acquiring approximately 1 million cells each. Result: Monoclonal B-cells were detected in 21 (6%) individuals (14 male, 7 female, median age 70, range 64–91). However, only 10 cases (48%) displayed the typical CD19+/CD5+ CLL-phenotype. Two cases (9.5%) were CD5-negative clonal B-cells, and 2 (9.5%) were CD10+ clonal B-cells. The remaining 7 cases (33%) showed concomitantly a CD5+ and a CD10+ clonal population, both expressing the same light chain. While we cannot be certain if these CD5+ and CD10+ cell populations represent the same or different clones, the finding that the two populations in all 7 cases showed the same light chain restriction supports the assumption that the two populations represent the same clone. Conclusion: MBL in the Middle Eastern region observed in this study is as common as reported in the Western world. In contrast to Western countries, however, it is the non-CLL phenotype which is more prevelant, comprising 52% of our MBL group and most of these cases show cells expressing CD5 and cells expressing CD10. The exact classification of these cases is difficult. It seems likely that these cases represent marginal zone phenotype, but the possibility of a coexisting follicular lymphoma clone cannot be excluded. Pure follicular lymphoma phenotype is seen in 10% of our MBL cases. Further studies with long follow-up are needed. Disclosures: No relevant conflicts of interest to declare.

scholarly journals THU0040 PROTEINASE 3-REACTIVE B CELL RECONSTITUTION AFTER TREATMENT WITH RITUXIMAB FOR ANCA-ASSOCIATED VASCULITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 233.1-233
Author(s):  
A. Berti ◽  
S. Hillion ◽  
A. Hummel ◽  
E. Carmona ◽  
T. Peikert ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Proteinase 3 ◽  
Research Support ◽  
Color Flow ◽  
Anca Associated Vasculitis ◽  
Cell Repopulation

Background:Proteinase 3 (PR3)-reactive B cells are present in PR3-ANCA-associated vasculitis (AAV) at levels higher than healthy controls.Objectives:To evaluate the dynamics of the PR3-reactive B cell repopulation in patients with PR3-AAV after treatment with rituximab, and to analyze possible associations between these immunological changes and long-lasting remissions.Methods:We analyzed all available frozen peripheral blood mononuclear cells (n=148) from 23 randomly-selected PR3-AAV patients who participated in the RAVE trial and achieved complete remission (BVAS=0, prednisone=0) after treatment with rituximab.We measured PR3-reactive B cells and the relative subsets by a multi-color flow cytometry panel including CD19, IgD, CD27, CD38, CD24, and a biotinylated PR3 revealed by fluorescent streptavidin. The clinical data of the trial were correlated with flow-cytometry data.Results:10/23 (43%) patients relapsed during the follow up, 8/10 relapses were severe. At baseline, clinical features, PR3-ANCA levels, % of total PR3-reactive B cells and PR3-reactive B cell subsets were similar between relapsers and non-relapsers. All patients were followed until the end of the trial, for a mean of 44 months (25-75%IQR 31-54), without difference in follow-up time between relapsers and non-relapsers (p=0.98).The majority of patients had B cell repopulation at 12 (range 12-24) months after rituximab. At the time of B cell repopulation, transitional (CD19+CD24+CD38+) and naïve (CD19+CD27+IgD-) B cells were higher compared to baseline, while total plasmablasts (PB) were unchanged, and mature B cells significantly decreased in both relapsers and non relapsers. PR3-reactive B cells reappeared in all the patients, and the % of PR3-reactive of B cells were higher at the B cell repopulation visit compared to baseline (5.82% vs 4.25%, p<0.05), while total B cells were lower (66/μL vs 151/μL, p<0.01), regardless of future relapse.Within PR3-reactive B cells, only the % of PB (CD19+CD27+CD38+PR3+) were higher in relapsers vs. non-relapsers (median [25-75%IQR]; 1.95% [1.315-3.845] vs 0.84% [0.05-1.66], p=0.022) and severe relapsers vs non-severe relapsers (2.165% [1.66-4.315] vs 0.84% [0.1-1.74], p=0.015). Time-to-relapse and time-to severe-relapse were significantly shorter in patients with circulating PR3-PB higher than the median value of the cohort (1.6%) during B cell reconstitution (Figure 1A-B).Conclusion:In PR3-AAV, during B cell reconstitution after rituximab, the total fraction of PR3-B cells increases, due to the expansion of the transitional and naïve B cell compartments. Circulating PR3-PB within PR3-B cells are enriched in the peripheral blood of relapsing and severely relapsing patients compared to non-relapsing patients. Higher levels of PR3-PB after rituximab during B cell reappearance significantly increased the risk of subsequent relapse and severe relapse.References:[1]Cornec D, Berti A, Hummel A, et al. J Autoimmun. 2017Disclosure of Interests:Alvise Berti: None declared, Sophie Hillion: None declared, Amber Hummel: None declared, Eva Carmona: None declared, Tobias Peikert: None declared, Carol Langford: None declared, Peter A. Merkel: None declared, Paul Monach: None declared, Philip Seo: None declared, Robert Spiera Grant/research support from: Roche-Genetech, GSK, Boehringer Ingelheim, Chemocentryx, Corbus, Forbius, Sanofi, Inflarx, Consultant of: Roche-Genetech, GSK, CSL Behring, Sanofi, Janssen, Chemocentryx, Forbius, Mistubishi Tanabe, E. William St. Clair: None declared, Fernando Fervenza: None declared, Kristina Harris: None declared, John H. Stone Grant/research support from: Roche, Consultant of: Roche, Jacques-Olivier Pers: None declared, Ulrich Specks: None declared, Divi Cornec: None declared

CD5-Negative Monoclonal B-Cell Lymphocytosis (MBL) Is Detectable In 9% of Adults Aged Over 40 but Phenotypes Associated with Clinically Common Non-Hodgkin Lymphomas Are Infrequent (<2%)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2002-2002
Author(s):  
Andy C Rawstron ◽  
Chi Doughty ◽  
Ruth M de Tute ◽  
Fiona Bennett ◽  
Selina Denman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
General Population ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Peripheral Blood ◽  
Marginal Zone ◽  
Residual Disease ◽  
High Sensitivity ◽  
Marginal Zone Lymphoma

Abstract Abstract 2002 Introduction: Monoclonal B-cells with a Chronic Lymphocytic Leukemia (CLL) phenotype are detectable in more than 10% of adults in the general population using high sensitivity flow cytometry assays designed to detect minimal residual disease after treatment. However, the prevalence of MBL with a phenotype corresponding to other B-lymphoproliferative disorders (B-LPD) is less than 2% of the general population even using the most sensitive assays. No studies have reported CD10+ MBL whereas several studies have demonstrated that the t(14;18) is frequently detectable in the general population at a level which should be detectable by flow cytometry. The lack of CD10+ MBL may indicate that the t(14;18) alone rarely results in the expansion of a clonal B-cell population in the blood, or that currently available assays are inadequate for detecting circulating follicular lymphoma. We have previously investigated 66 markers to determine the best candidates for diagnosis and monitoring B-LPD, which were then tested in over 1500 cases. We have developed a single-tube assay to screen for residual disease that can detect lymphoma cells when they represent as few as 1 in 10,000 leucocytes. The aim of this study was to asses the frequency with which lymphoma-phenotype monoclonal B-cells are detected in the general population using a high sensitivity assay. Methods: Cells from 679 individuals (342 male, 337 female, median age 64, range 40–99) with a normal blood count and no current or prior history of cancer were incubated with antibodies to Kappa, Lambda, CD19, CD20, CD5, CD10, LAIR1, CXCR5 and 0.5 million cells were acquired using a BD FACSCanto II cytometer. In cases with detectable MBL further phenotyping was performed and B-cells were selected and stored for FISH and molecular clonality studies. Results: MBL was detected 129/679 cases (19.0%): CLL-type MBL in 86/679 cases (12.7%), non-CLL MBL in 60/679 cases (8.8%) with both CLL-type and non-CLL MBL were present in 17/679 cases (2.5%). Within the non-CLL MBL group, in 21/60 cases the monoclonal B cells had no additional features to confirm a neoplastic population and it was not possible to ascertain whether these were neoplastic cells or a reactive population with a highly skewed kappa/lambda ratio. Of the remaining 39 specimens: none showed evidence of germinal centre differentiation; 12 (1.7% of total) showed a phenotype most consistent with marginal zone lymphoma/lymphoplasmacytic lymphoma; and 27 cases expressed strong levels of LAIR1 coupled with strong CD19 and CD20 and an extended phenotype that is relatively rare in clinical B-LPD, restricted to hairy cell leukemia and a small proportion (<15%) of marginal zone lymphomas. Conclusions: Using an assay designed for detection of residual disease in lymphoma it was possible to detect non-CLL phenotype MBL in 8.8% of individuals over 60 years of age with normal blood counts and no history of cancer. Only a small number of these cases had a phenotype comparable to follicular lymphoma or marginal zone lymphoma, which constitute the majority of clinically significant indolent B-LPD. The complete absence of cases with a germinal centre phenotype contrasts with the high frequency of detection of BCL2-IGH rearrangement by PCR in the peripheral blood of healthy individuals. The flow cytometry assay used in this study readily detects circulating cells with appropriate phenotype in patients known to have follicular lymphoma, diffuse large B-cell lymphoma or marginal zone lymphoma/lymphoplasmacytic lymphoma and the data indicate a high specificity for peripheral blood based diagnosis and monitoring in these conditions. The most clinically prevalent lymphomas are rarely detectable in peripheral blood in individuals without lymphadenopathy but there is a surprisingly frequent development in the general population of both CLL and a restricted subset of marginal zone lymphomas which may indicate a common developmental pathway. Disclosures: No relevant conflicts of interest to declare.

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 ◽  
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.

In Vivo Depletion of Cynomolgus Monkey B Cells Targeted by a Novel Monoclonal Antibody (Xi-20H5) Specific for a Shared Portion of the Human Immunoglobulin Variable Light Chain Lambda 1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2354-2354
Author(s):  
Thierry Sornasse ◽  
Keri Tate ◽  
Kimberly Milner ◽  
Thomas Theriault ◽  
Dan W. Denney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Peripheral Blood ◽  
Cynomolgus Monkey ◽  
Patient Specific ◽  
B Cell Malignancies ◽  
Variable Regions ◽  
Variable Light

Abstract Genitope is developing a novel, personalized treatment for surface immunoglobulin (Ig) positive B cell malignancies. This treatment is based on a panel of monoclonal antibodies (Mabs) directed against shared epitopes expressed on different subsets of the variable regions of human Ig. The concept of targeting surface tumor Ig was explored in a series of clinical trials performed by Dr. Ronald Levy and his colleagues at Stanford. In these studies, Mabs directed against patient specific (idiotypic) determinants of the surface tumor Ig produced significant clinical benefit in relapsed follicular non-Hodgkin’s lymphoma patients. A number of these patients have had long term remissions. In Genitope’s planned clinical use, each patient will receive a single Mab from the panel selected based on its reactivity with the patient’s tumor. The selected Mab will react with the patient’s tumor and a minority of normal B cells, leaving the majority of the normal B cell repertoire intact. The ability of the panel members to provide therapeutic effect requires binding to tumor surface Ig in the presence of serum containing soluble Ig molecules. Mab Xi-20H5, a member of this panel of antibodies, is specific for a shared determinant on the human Ig variable light chain lambda 1. It binds to 25 to 35% of normal human B cells from peripheral blood and to 20 to 30% of normal cynomolgus monkey B cells from peripheral blood. In this study, we sought to demonstrate that, despite the presence of serum Ig, Mab Xi-20H5 would bind to surface Ig expressed on monkey B cells in vivo, resulting in specific depletion of target B cells. Six naïve cynomolgus monkeys received 8 intravenous infusions of the Mab Xi-20H5 at a dose of 40 mg/kg on days 1, 2, 3, 4, 7, 10, 14 & 17. Two naïve control animals received 8 infusions of vehicle only following the same schedule. The frequencies of lymphocyte sub-populations and of target B cells were monitored by flow cytometry on plasma-depleted whole blood samples. Samples were collected 23 hours after each infusion. In addition, two baseline samples were collected prior to treatment. The frequencies of lymphocyte sub-populations and of target B cells were compared to the average of the two baseline measurements. Frequencies of target B cells bound by Mab Xi-20H5 decreased in all treated animals while no significant change was detectable in the control animals. The bulk of the reduction in target B cell frequencies was observed 23 hours after the first infusion (range: 22% – 62%, average 41%). Frequencies of target B cells continued to decrease moderately with additional daily infusions (days 2 – 4), resulting in maximum reduction in target B cell frequency at 23 h post infusion 4 (range: 39% – 78%, average 54%). The frequencies of total B and T lymphocytes did not significantly change during the treatment. In vivo administration of Mab Xi-20H5 results in depletion of target B cells in a manner consistent with the expectation of an immunotherapeutic Mab aimed at treating surface Ig expressing B cell malignancies.

Signaling Diversity in Human Lymphoma B Cells and in Tumor Infiltrating T Cells Correlates with Follicular Lymphoma Patient Clinical Outcomes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 377-377
Author(s):  
Jonathan M. Irish ◽  
Roch Houot ◽  
June H. Myklebust ◽  
Debra K. Czerwinski ◽  
Garry P. Nolan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Bcr Signaling ◽  
Human Lymphoma ◽  
Group 1

Abstract Introduction: Flow cytometry analysis of live cells from cryopreserved tumor samples provides the opportunity to study simultaneously the biology of both cancer cells and the patient’s immune system cells. Furthermore, single cell based approaches can identify and quantify subsets within mixed populations, such as heterogeneous primary tumors, without the need to physically separate cells. We have previously used flow cytometry to identify differences in signaling mechanism between healthy human B cells from two differentiation stages and to compare signaling kinetics of tumor and non-malignant B cells within primary human lymphoma specimens1. Methods: Here we used barcoded phospho-specific flow cytometry to measure signaling events in live lymphoma B cells and tumor-infiltrating T cells from follicular lymphoma (FL) tumor samples obtained before patients received therapy. Patients were then stratified according to signaling and clinical outcome was examined within the resulting groups. We examined response to initial chemotherapy, which was a combination of cyclophosphamide, vincristine, and prednisone (cvp), and overall survival, measured as the time from diagnosis to last follow up or mortality. Results: Differences in B cell receptor (BCR) signaling strength and kinetics characterized previously unappreciated diversity within the lymphoma B cell population. In some cases, BCR crosslinking triggered robust phosphorylation of AKT and ERK in one subset of lymphoma cells, while in another lymphoma subset within the same tumor sample, BCR crosslinking did not lead to phosphorylation of either protein. In these cases, both lymphoma B cell subsets expressed BCL2 and surface immunoglobulin heavy and light chain restricted to the tumor isotype. Patients whose lymphoma tumor contained a BCR insensitive cell subset (Group 2) had significantly worse responses to cvp therapy (p = 0.001) and lower overall survival (p = 0.003) than patients whose lymphoma cells displayed more homogeneous BCR signaling (Group 1). We next examined tumor infiltrating T cell signaling in the FL cases from Group 1, where no significant BCR insensitive subset was observed. Within Group 1, differences in the magnitude of IL-2, IL-7, and IL-15 mediated STAT5 phosphorylation in tumor infiltrating T cells further distinguished a set of patients with significantly higher overall survival (p = 0.04). Conclusions: These results identify BCR signaling in lymphoma B cells and cytokine signaling in tumor infiltrating T cells as clinically relevant biomarkers for tracking and isolating lymphoma cell subsets and for monitoring immune system activity during therapy. By following patients over time, we can now determine whether cell intrinsic signaling diversity enables the emergence of therapy insensitive cancer cell subsets.

Flow Cytometric Expression of bcl-2 by CD10+ B-Cells Reliably Distinguishes Follicular Lymphoma from Follicular Hyperplasia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4551-4551
Author(s):  
Zahid Kaleem ◽  
Ronald L. Leonard
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Light Chain ◽  
Predictive Value ◽  
Expression Patterns ◽  
Follicular Hyperplasia ◽  
Biopsy Specimens ◽  
Normal Expression ◽  
Significant Expression

Abstract The diagnosis of follicular lymphoma (FL) is often straightforward in most biopsy specimens. In a minority of cases, however, a differentiation from follicular hyperplasia (FH) can be challenging, especially in small needle core biopsies. The difficulty can be compounded in cases of FL with non-diagnostic immunoglobulin light chain ratios and cases of lymphoma without any significant surface expression of either immunoglobulin kappa or lambda light chain. The expression of bcl-2 by follicle center B-cells is often used to distinguish FL from FH using immunohistochemistry. The interpretation of bcl-2 expression patterns by immunohistochemistry, however, can be difficult because of normal expression of bcl-2 by T-cells present in reactive lymphoid follicles and by normal expression of bcl-2 by mantle zone B-cells. We have utilized multiparameter flow cytometry to evaluate the expression of bcl-2 in cases of FL and FH with successful results. This study includes consecutive biopsy specimens from well-documented cases of FL (n = 12) and FH (n = 16). Other lymphoma types were not included. Cytoplasmic expression of bcl-2 was evaluated on CD10+CD19+ B-cells using permeabilization techniques on a four-color Beckman-Coulter Epics-XL flow cytometer. Appropriate controls were run in each case. All cases of CD10-expressing FL showed significant expression of bcl-2 by CD10+ B-cells in contrast to no significant expression of bcl-2 by CD10+ follicle center B-cells of reactive follicles (Positive predictive value, 100%; Negative predictive value, 100%). Although our patient population is small, the results indicate that the expression of cytoplasmic bcl-2 on CD10+ B-cells by flow cytometry reliably distingushes FL from FH using CD10 gating. This method is superior to immunohistochemical evaluation of bcl-2 in cases with CD10 expression. A minority of FL, however, do not show bcl-2 expression (not seen in our series) and such cases must be taken into consideration when evaluating a clonal B-cell population with CD10 but no bcl-2 expression. DIAGNOSIS CD45+CD19+(%) CD45+CD19+CD10+(%) bcl2+(%) in CD10+ gate FL grade 1/3 65 65 38 FL grade 1/3 68 44 67 FL grade 1/3 72 72 99 FL grade 1/3 88 62 99 FL grade 2/3 72 68 99 FL grade 1/3 59 59 95 FL grade 1/3 96 84 94 FL grade 1/3 59 59 95 FL grade 1/3 45 38 99 FL grade 2/3 76 74 76 FL grade 1/3 54 47 90 FL grade 2/3 35 34 98

Patients With DLBCL Commonly Have B-Cell Lymphopenia and Circulating Clonal B-Cell Populations With a Phenotype Concordant With The Underlying Tumour

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3013-3013
Author(s):  
Ruth M de Tute ◽  
Sharon Barrans ◽  
Andy C. Rawstron ◽  
Peter W.M. Johnson ◽  
Andrew J Davies ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Peripheral Blood ◽  
Expression Profiling ◽  
Common Finding ◽  
Cell Populations ◽  
The Impact

Abstract Clonal B-cell populations with either a CLL or a non-CLL phenotype are a common finding in normal individuals but uncertainty remains about how this relates to the development of clinically significant disease. The aim of this study was to investigate the frequency of peripheral blood clonal B-cell populations and B-cell subset abnormalities in newly presenting DLBCL patients and to determine whether the incidence of these abnormalities differed between the GCB and ABC subtypes, which are regarded as having distinct pathogenesis. The study was carried out using peripheral blood samples collected from patients entered in the UK-REMoDL-B trial. This trial is testing the hypothesis that the ABC subtype of DLBCL responds preferentially to R-CHOP- Bortezomib. Gene expression profiling is performed on the diagnostic tissue biopsy (FFPE) using the Illumina WG-DASL assay prior to randomisation classified as GCB, ABC or unclassified (UN). The availability of GEP data allows meaningful comparison with the phenotype of clonal populations detected by flow cytometry. Peripheral blood taken prior to first treatment was analysed using multi-colour flow cytometry. Following red cell lysis with ammonium chloride, samples were incubated with a panel of antibodies comprising of a CD19 and CD20 backbone, with Kappa, Lambda, CD5, CD45, CD49d, LAIR-1, CXCR5, CD31, CD95, CD38 and CD10, supplemented in some cases by CD81, CD79b, and CD43. A minimum of 500,000 events were acquired on a FacsCanto II flow cytometer (Becton Dickinson). B-cells were enumerated and any monoclonal populations identified were classified as CLL, germinal centre (GC), non-GC or not otherwise specified (NOS) where the phenotype was indeterminate. 358 samples were eligible for inclusion from patients with an average age of 62.2years (range 22.9-86.1). Abnormalities were detected in 52% of cases (B-lymphopenia ((<0.06 x 109/l) 33%, B-lymphocytosis (>1 x 109/l) 2.8%, CLL clone 3.6%, GC clone 9.8%, non-GC clone 9.8%, clonal population NOS 2.2%). Gene expression profiling results were available for 278 individuals; 51% GCB, 32% ABC and 17% unclassified. The relationship between peripheral blood B-cell findings and the GEP determined phenotype of the tumour is shown in the table:TableB-lymphopeniaCLL CloneMonoclonal GC typeMonoclonalNon-GC typeMonoclonal NOSNormalB-cellGCB n=14241/142 (29%)5/142 (3.5%)21/142 (15%)8/142 (5.6%)2/142 (1%)72/142 (51%)ABC n=8927/89 (30%)2/89 (2%)2/89 (2%)12/89 (13.5%)2/89 (2%)49/89 (55%)Unclassified n=4726/47 (55%)0/50 (0%)2/47 (4%)6/47 (12%)6/47 (5%)14/47 (30%) In patients where clonal populations were detected in the peripheral blood there was striking concordance between the phenotype of the clone and the GEP of the underlying tumour. Presence of a GC-population by flow was highly predictive of GCB GEP (84% GC–type populations detected were in GCB cases). The number of discordant cases and the number of CLL clones detected approximate to the numbers that would be expected in a normal population of a similar age. It is, therefore, likely that in most cases circulating tumour cells or a closely related precursor clone are being detected. The similarity between the results of the ABC and unclassified GEP groups suggest that these are biologically related. An unexpected finding in this study was the high incidence of B-lymphopenia at a level that might be expected to be associated with increased risk of infection. This may reflect suppression of normal B-cells by the neoplastic clone or be a marker of underlying immune dysfunction that may predispose to the development of the tumour. Immuosuppression has a role in the pathogenesis of DLBCL in the elderly and this study suggests that this may also be a factor in the wider patient population. These results may have implications for prognostic assessment and may offer opportunities for early diagnosis and possibly response assessment in some patients. The impact on outcome will be assessed in the course of the trial. Disclosures: Jack: Roche /Genentech: Research Funding.

scholarly journals Identification of circulating MOG-specific B cells in patients with MOG antibodies

2019 ◽  
Vol 6 (6) ◽  
pp. 625 ◽  
Author(s):  
Stephan Winklmeier ◽  
Miriam Schlüter ◽  
Melania Spadaro ◽  
Franziska S. Thaler ◽  
Atay Vural ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Myelin Oligodendrocyte Glycoprotein ◽  
Epitope Specificity ◽  
Mog Antibodies ◽  
Different Sources ◽  
Cells Cultured

ObjectiveTo identify circulating myelin oligodendrocyte glycoprotein (MOG)-specific B cells in the blood of patients with MOG antibodies (Abs) and to determine whether circulating MOG-specific B cells are linked to levels and epitope specificity of serum anti-MOG-Abs.MethodsWe compared peripheral blood from 21 patients with MOG-Abs and 26 controls for the presence of MOG-specific B cells. We differentiated blood-derived B cells in vitro in separate culture wells to Ab-producing cells via engagement of Toll-like receptors 7 and 8. We quantified the anti-MOG reactivity with a live cell–based assay by flow cytometry. We determined the recognition of MOG epitopes with a panel of mutated variants of MOG.ResultsMOG-Ab–positive patients had a higher frequency of MOG-specific B cells in blood than controls, but MOG-specific B cells were only detected in about 60% of these patients. MOG-specific B cells in blood showed no correlation with anti-MOG Ab levels in serum, neither in the whole group nor in the untreated patients. Epitope analysis of MOG-Abs secreted from MOG-specific B cells cultured in different wells revealed an intraindividual heterogeneity of the anti-MOG autoimmunity.ConclusionsThis study shows that patients with MOG-Abs greatly differ in the abundance of circulating MOG-specific B cells, which are not linked to levels of MOG-Abs in serum suggesting different sources of MOG-Abs. Identification of MOG-specific B cells in blood could be of future relevance for selecting patients with MOG-Abs for B cell–directed therapy.

scholarly journals Human Immunoglobulin (Ig)M+IgD+ Peripheral Blood B Cells Expressing the CD27 Cell Surface Antigen Carry Somatically Mutated Variable Region Genes: CD27 as a General Marker for Somatically Mutated (Memory) B Cells

1998 ◽  
Vol 188 (9) ◽  
pp. 1679-1689 ◽  
Author(s):  
Ulf Klein ◽  
Klaus Rajewsky ◽  
Ralf Küppers
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Surface Antigen ◽  
Light Chain ◽  
Peripheral Blood ◽  
Memory B Cells ◽  
Cell Surface Antigen ◽  
Cell Phenotype ◽  
Light Chain Gene

Immunoglobulin (Ig)M+IgD+ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM+IgD+ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM+IgD+ B cells. IgM+IgD+CD27+ B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise ∼15% of PB B lymphocytes in healthy adults. Moreover, a very small population (&lt;1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of ∼40% mutated memory B cells and 60% unmutated, naive IgD+CD27− B cells (including CD5+ B cells). In the somatically mutated B cells, VH region genes carry a two- to threefold higher load of somatic mutation than rearranged Vκ genes. This might be due to an intrinsically lower mutation rate in κ light chain genes compared with heavy chain genes and/or result from κ light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.

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