Circulating B Cell Clones in Familial Waldenström Macroglobulinemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2703-2703
Author(s):  
Mark C. Lanasa ◽  
Sallie D. Allgood ◽  
Lynn R. Goldin ◽  
Danielle M. Brander ◽  
Mary L. McMaster
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Family Members ◽  
Similar Proportion ◽  
Cell Populations ◽  
Color Flow ◽  
Flow Cytometric ◽  
Cell Clones ◽  
Genomic Studies

Abstract Abstract 2703 Background and Significance: Lymphoplasmacytic lymphoma (LPL) is an indolent but incurable B cell lymphoproliferative characterized by the clonal expansion of plasmacytoid lymphocytes in the lymph nodes and bone marrow. The majority of cases are associated with an IgM isotype paraprotein, and when present, is termed Waldenström macroglobulinemia (WM). WM shows familial aggregation suggesting an inherited risk for disease, and also co-aggregates with CLL in families. In our previous work, we have shown that clonal populations of peripherally circulating B cells can be identified in 18% of unaffected family members from CLL kindreds. Most of these clonal populations have a typical CLL immunophenotype and have been termed CLL-like monoclonal B cell lymphocytosis (MBL). Because CLL and WM have related gene expression profiles and appear to have shared genetic risk, we hypothesized that unaffected family members of WM kindreds would have detectable circulating clonal B cell populations. Further, we undertook systematic flow cytometric screening of familial WM and IgM MGUS cases to determine the prevalence, immunphenotype, and biologic characteristics of circulating malignant B cells. Methods: A diagnosis of LPL / WM or IgM MGUS was determined using standard WHO criteria. All patients and unaffected family members were ascertained at the National Cancer Institute and provided informed consent. Peripheral blood mononuclear cells were isolated using density centrifugation and viably frozen in DMSO containing media. We developed a two tube, nine color flow cytometric assay: the first tube allowed for detection of CLL-like clones based upon co-expression of CD5, CD20, and CD23; the second tube targeted WM populations based upon expression on CD19, CD20, CD25, CD38, and surface IgM. Cell populations were considered clonally restricted if the κ: λ was > 3.0 or < 0.3. Clonal populations were then isolated using fluorescence activated cell sorting (FACS). RNA and genomic DNA were extracted for genetic and genomic studies using phenol: chloroform purification. Results: A total of 155 individuals were analyzed: 54 WM / LPL, 17 IgM MGUS, 1 IgG MGUS, 1 IgA MGUS, 1 NHL, and 81 unaffected family members. Twenty of 54 WM patients had detectable peripherally circulating populations. Thirteen WM patients had no detectable B cells, of these, 11 patients had prior treatment. As such, among the 41 WM patients with a B cell compartment that was analyzable by flow cytometry, 49% (20 of 41) had peripherally circulating B cell clones detected. Four of these 20 cases showed two immunophenotypically distinct clonal B cell populations. The immunophenotype was somewhat heterogenenous: 18 cases expressed surface IgM, CD38 was variable but expressed in most cases, CD25 was not detected in any case, and 4 cases showed a CLL like (CD5+CD20dimCD23+) immunophenotype. Interestingly, we detected peripherally circulating B cell clones in 9 of 17 cases (53%) of IgM MGUS, a proportion nearly identical to that identified in WM / LPL. Three of 9 were “CLL-like” with co-expression of CD5 and CD23, while the majority of clones were CD5negIgM+CD38+. Among unaffected family members, we identified B cell clones in only 4 of 81 (5%). All 4 cases expressed CD5, and 3 showed a CLL-like phenotype, consistent with these individuals having MBL. Among all study subjects, 20 clonal B cell populations of > 104 B cells were FACS purified from 18 different cases: 12 WM, 4 IgM MGUS, 1 IgA MGUS, and 1 unaffected family member. Conclusions: Peripherally circulating B cell clones with an immunophenotype similar to that of LPL can be identified in approximately half of patients with WM / LPL. We observed for the first time that a similar proportion of patients with IgM MGUS have detectable clonal populations with an immunophenotype similar to that observed in WM patients. Genetic and genomic studies to determine the lineage of these populations are currently underway. The frequency of MBL among unaffected family members with WM is lower than that observed in CLL kindreds. CLL-like MBL can be detected at very low numbers because the cell population is immunophenotypically abnormal. The peripherally circulating clones identified in WM / LPL patients have an otherwise normal B cell immunophenotype and can only be detected by light chain restriction. This likely significantly limits the ability of flow cytometry to detect pre-clinical CD5neg IgM expressing clonal populations. Disclosures: No relevant conflicts of interest to declare.

Identifying Candidate Normal and Leukemic B Cell Progenitor Populations with Hierarchical Clustering of 6-Color Flow Cytometry Data - A Better View.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1428-1428 ◽  
Author(s):  
Karel Fišer ◽  
Tomáš Sieger ◽  
Josef H. Vormoor
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Hierarchical Clustering ◽  
Mahalanobis Distance ◽  
Cell Populations ◽  
Flow Data ◽  
Color Flow ◽  
Two Parameter

Abstract 6-color flow cytometry allows multiparameter analysis of high numbers of single cells. It is an excellent tool for the characterization of a wide range of hematopoietic populations and for monitoring minimal residual disease. However, analysis of complex flow data is challenging. Gating populations on 28 two-parameter plots is extremely tedious and does not reflect the multidimensionality of the data. Here, we describe a novel approach, employing hierarchical clustering (HCA) and support vector machine (SVM) learning in analyzing flow data. This approach provides a new perspective for looking at flow data and promises better identification of rare and novel subpopulations that escape classic analysis. Our aim was to identify normal and leukemic B cell progenitor/stem cell populations in normal (n=6) and ALL (n=10) bone marrow. Samples were labelled with fluorochrome-conjugated antibodies to 6 CD markers (CD 10, 19, 22, 34, 38, 117) and 104 to 106 events were acquired (FACSCanto, BD Biosciences). To analyze flow data with HCA we developed a new algorithm, better suited for the ellipsoid nature of cell populations than other current HCA metrics. Data exported from DiVa software were externally compensated and Hyperlog transformed to achieve a logarithmic-like scale that displayed zero and negative values. Normalized data were then subjected to HCA employing a scale-invariant Mahalanobis distance measurement for merging clusters. This reflects the extended ellipsoid shape of the populations (here: 8 dimensional ellipsoids). We developed a new adaptive linkage algorithm that smoothly shifts from the Euclidean distance (when clusters are too small to compute Mahalanobis distance) to Mahalanobis distance measurement. This allowed us to build the hierarchy from single events, yet to retain the advantage of Mahalanobis measurement for larger clusters. To build classifiers we used SVM employing polynomial kernel. All work was carried out in MATLAB (MathWorks, Inc.). The resulting hierarchical tree combined with the heatmap of the CD marker expression allows visualization of hierarchically clustered data with all 8 parameters displayed in a single plot (!) as compared to 28 traditional two-parameter plots. HCA has big advantage of providing populations homogenous in their expression pattern of all parameters (without the need for complex sub or back gating). We were able to identify populations corresponding to the different stages of B-cell development. In a normal control bone marrow we could detect the following candidate B-lineage progenitor populations: CD34+117+38+10−22−19− (0.94% of total) progenitor/stem cells, CD34+117−38+10+22+19med (0.26% of total) pro-B cells, CD34−117−38+10+22+19+ (2.77% of total) small pre-B cells (lower FCS values), CD34−117−38+10+22+19+ (1.09% of total) large pre-B cells (higher FCS values) and CD34−117−38lo10−22+19+ (5.94% of total) (immature) B cells. In 10 diagnostic or relapse samples HCA clearly identified the main leukemic population. HCA is able to visualize otherwise “hidden” populations. This was exemplified by a distinct CD38+B-lin− population that overlapped with other populations in all 28 two-parameter plots (most likely T cells). We have built a classifier able to find established populations across samples and in large datasets (106 events) for which HCA would be computationally too demanding. In summary, we show the advantages of using hierarchical clustering analysis for large complex multiparameter flow cytometry datasets.

Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2321-2326 ◽  
Author(s):  
D van der Harst ◽  
D de Jong ◽  
J Limpens ◽  
PM Kluin ◽  
Y Rozier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Malignant Lymphoma ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Clinical Manifestations ◽  
Cell Populations ◽  
Dna Rearrangement ◽  
Thrombocytopenic Purpura

Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sjogren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.

Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4771-4771
Author(s):  
Magali Le Garff-Tavernier ◽  
Michel Ticchioni ◽  
Rémi Letestu ◽  
Martine Brissard ◽  
Frédéric Davi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Protein Detection ◽  
Flow Cytometric ◽  
Mutational Status ◽  
Healthy Donors

Abstract Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and &gt; 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells &gt; 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 &gt; 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 &lt; 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.

ROR-1 Is a Highly Discriminative Marker in Flow Cytometric Minimal Residual Disease (MRD) Detection in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3197-3197 ◽  
Author(s):  
Michaela Patz ◽  
Barbara Pentok ◽  
Kathrin Cremer ◽  
Stefanie Linnartz ◽  
Esther Lilienweiss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Cell Populations ◽  
Discrimination Analysis ◽  
Flow Cytometric ◽  
Correct Discrimination ◽  
Minimal Residual

Abstract Introduction:With the advent of new potent therapies for chronic lymphocytic leukemia (CLL) minimal residual disease (MRD) detection becomes increasingly important to assess remission depth. While molecular MRD detection for CLL remains laborious and time consuming flow cytometry is a fast, economic and sensitive method in detecting low frequencies of CLL cells. The usefulness of the antigens CD81, CD5, CD20, CD43 and CD79b has been previously described for this purpose. ROR-1 has recently been identified as a signature gene in CLL and mantle cell lymphoma. The potential utility of ROR-1 in flow cytometric minimal residual cell analysis has not been evaluated yet. Methods: 10 normal samples and 77 remnants of randomly selected samples from diagnosed patients undergoing CLL therapy were analyzed by flow cytometry. A customized dry formulation of an antibody panel was used, comprising antibodies directed against CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 (DuraClone RE CLB). Linearity, repeatability and inter-operator variability of data analysis of the method were examined. B cell populations comprising at least 50 positive events (46 normal B cell populations, 25 CLL populations, paired and unpaired) were analyzed for their expression profile as assessed by respective mean fluorescence intensities of the antibody labels within classified populations. The expression profiles were subject to supervised discrimination analysis (DA). Results: Between124,000 and 2,122,000 (683,000 ± 450,000) CD45+ events were acquired from the 87 samples. The background of cells with a CLL-like phenotype in the normal samples was determined as <0.001% of CD45+ events. Linearity was confirmed in the range from 1% to 0.0025%. The Repeatability analysis and the inter-operator variability showed concordance with typical Poisson distribution characteristics. The 46 populations with a typical normal B cell phenotype ranged from 0.014% to 9.592% with an average of 2.45% ± 2.75 of CD45+ events. The 25 populations with a classical or non-classical CLL phenotype ranged from 0.007% to 5.459% with an average of 1.41% ± 1.65 of CD45+ events. Posterior discrimination analysis revealed 100% correct discrimination for CLL populations and 96% correct discrimination for normal populations when relying on ROR-1 expression alone in CD19+CD45+ B cells. This result was only surpassed by the complete antibody combination (100% / 100%) but not by any other of the markers, neither in single use nor in combination Conclusion: The 8-color dry flow cytometry panel comprising CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 demonstrated sensitive, linear and specific detection of residual CLL cells in a relevant low range of frequency. ROR-1 revealed to be a highly discriminative marker in the analysis of residual CLL cells by flow cytometry. Utilizing this flow cytometry approach, MRD detection showing sensitivity comparable to molecular techniques can be achieved in CLL. Disclosures Hallek: AbbVIe: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria; Glaxo-SmithKline: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria; Roche: Consultancy, Research Funding, Speakers Bureau. Kreuzer:Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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

Hematopoietic Stem Cells Are Primarily Involved in Pathogenesis of Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 29-29
Author(s):  
YoshiKane KiKushige ◽  
Toshihiro Miyamoto ◽  
Tadafumi Iino ◽  
Fumihiko Ishikawa ◽  
Koichi Akashi
Keyword(s):  
B Cells ◽  
B Cell ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Cell Populations ◽  
Vdj Recombination ◽  
Hematopoietic Stem ◽  
Cell Clones ◽  
Nog Mice ◽  
Igh Rearrangement

Abstract Chronic lymphocytic leukemia (CLL) is characterized by consistent expansion of B cells in peripheral lymphoid organs. CLL B cells frequently express CD5 antigen, and have clonal rearrangement of immunoglobulin heavy chain (IGH) gene with restricted usage of V1, V3 and V4 of the variant region. CLL has thus been believed to represent retention or proliferation of abnormal B cell clones presumably with anti-apoptotic potential, or with deregulated response to auto-antigens. In this study, we extensively search for CLL-initiating cells by utilizing the NOD/SCID/IL2rgnull (NOG) xenogeneic transplantation system, in which human hematopoietic stem cells (HSCs) can normally develop multi-lineage cells including polyclonal B and T cells. In the NOG xenotransplant system, neither CD34-CD19+ circulating B cells, nor CD34+CD38+ bone marrow (BM) progenitor populations from 12 CLL patients engrafted even after injection of &gt;106 cells. We then transplanted CD34+CD38− BM HSC population from 7 CLL patients into 13 NOG mice. Injection of as few as 103 cells of the CD34+CD38− BM population resulted in multi-lineage reconstitution. Most of these mice, however, died within 12–24 weeks after xenotransplantation. In 9 mice analyzed by multi-color FACS, 7 mice possessed both CD5+ and CD5− B cell populations, and the remaining 2 mice had only CD5− B cells. These CD5+ or CD5− human B cell populations were purified separately by FACS, and tested for the IGH gene rearrangement. Strikingly, 16 out of 20 B cell populations were clonal with single IGH rearrangement irrespective of their CD5 expression, by multiplex PCR analysis. In contrast, CD34+CD38− HSC populations in CLL patients never had IGH rearrangement. We then directly sequenced PCR products of IGH gene in each B cell clones as well as those in the original CLL cells purified directly from patients’ blood. Surprisingly, VDJ recombination in B cell clones developed in NOG mice were different from that of the original CLL clones in all 7 CLL cases. Interestingly, all of these clones used only V1, V3 and V4 regions for their VDJ recombination like primary CLL cells. Furthermore, when the CD34+CD38− BM HSC fraction from single CLL patients was transplanted into a set of 3 mice simultaneously, each mouse developed independent B cell clones with different VDJ recombination in all 3 experiments. The fact that CD34+CD38− HSCs from CLL patients but not those from normal individuals give rise to clonal B cell population in our xenograft model strongly suggests that some genetic abnormality for CLL progression is acquired already at the HSC level in CLL patients. HSCs in CLL patients are multipotent, but once they commit to the B cell lineage, they use preferentially the V1, V3 and V4 regions for IGH recombination. Our hypothesis is that such B cell clones may already be abnormal in that they clonally expand in response, for example, to auto-antigens (xeno-antigens in NOG mice), and they may possibly sequentially receive additional mutations to become clinical CLL. Although this xenograft model may not recapitulate full picture of CLL progression, our data clearly show that primary leukemogenic event occurs at the multipotent HSC stage in human CLL.

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 Cytometric detection of DNA amplified with fluorescent primers: applications to analysis of clonal bcl-2 and IgH gene rearrangements in malignant lymphomas

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1079-1085 ◽  
Author(s):  
RL Barker ◽  
CA Worth ◽  
SC Peiper
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Gel Electrophoresis ◽  
Flow Cytometric Analysis ◽  
Dna Amplification ◽  
Cell Populations ◽  
Gene Rearrangements ◽  
Flow Cytometric ◽  
Hodgkin's Lymphomas ◽  
Follicular Lymphomas

Abstract Follicular lymphomas comprise almost two thirds of the US adult non- Hodgkin's lymphomas (NHL) and are the most common malignancy of B- lineage lymphocytes. Polymerase chain reaction (PCR) protocols have been developed to detect the t(14;18) translocation, which juxtaposes the bcl-2 proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular lymphomas and monoclonal rearrangements of the IgH gene in B- cell NHL that lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH rearrangements in DNA from patients with lymphoproliferative disorders and analyzed the products in parallel by gel electrophoresis and flow cytometry, which detected PCR products incorporating fluoresceinated oligonucleotide primers by sequence-specific capture to oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior to electrophoresis of ethidium-bromide-stained agarose gels for detection of products of nested PCR to detect intergenic rearrangements involving bcl-2 and single primer-pair amplification of clonal rearrangement of IgH. Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+ B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal B-cell populations. In contrast, analysis by gel electrophoresis detected bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene rearrangements in only 9 of 17 monoclonal B-cell populations. Flow cytometric analysis was more sensitive than gel electrophoresis and could detect a 16-fold greater dilution of a bcl-2-amplified product than gel electrophoresis. Similarly, flow cytometry could detect an amplification product when template DNA was diluted 10,000-fold, whereas gel electrophoresis only detected amplification products when template was subjected to dilution between 100- and 1,000-fold. This shows the utility of flow cytometry for the analysis of DNA amplification products incorporating fluorochrome-labeled primers as a rapid, objective alternative to conventional strategies. Because current-generation clinical laboratories emphasize automation, flow cytometric analysis of PCR- amplified products shows increased analytic sensitivity and offers a vehicle for automation of DNA amplification tests.

Patients with Malignant Hyperthermia Demonstrate an Altered Calcium Control Mechanism in B Lymphocytes

2002 ◽  
Vol 97 (5) ◽  
pp. 1052-1058 ◽  
Author(s):  
Yoshitatsu Sei ◽  
Barbara W. Brandom ◽  
Saiid Bina ◽  
Eiji Hosoi ◽  
Kathleen L. Gallagher ◽  
...  
Keyword(s):  
B Cells ◽  
Malignant Hyperthermia ◽  
B Cell ◽  
B Lymphocytes ◽  
Molecular Mechanisms ◽  
Cell Phenotype ◽  
Color Flow ◽  
Flow Cytometric ◽  
Malignant Hyperthermia Susceptible ◽  
And Control

Background Altered Ca2+ homeostasis in skeletal muscle is a key molecular event triggering malignant hyperthermia (MH) in malignant hyperthermia-susceptible (MHS) individuals. Genetic studies have shown that mutations in the type 1 ryanodine receptor (RYR1) are associated with MH susceptibility. Because human B lymphocytes express the RYR1, it is hypothesized that Ca2+ homeostasis in B lymphocytes is altered in MHS individuals. Methods This study investigated the Ca2+ response of B cells to caffeine and 4-chloro-m-cresol in 13 MHS and 21 MH-negative (MHN) individuals who had been diagnosed by caffeine halothane contracture test (CHCT) and 18 healthy volunteers. Changes in [Ca2+]i in B cells were measured directly in fluo-3 loaded cells using a dual-color flow cytometric technique. Further, B cell phenotype was correlated with CHCT results in a family with the Val2168Met (G6502A) mutation. Results Caffeine-induced (50 mm) increases in [Ca2+]i in B cells were significantly greater in MHS than in MHN (P = 0.0004), control (P = 0.0001) or non-MHS (MHN and control) individuals (P &lt; 0.0001). The 4-chloro-m-cresol-induced (400 microm) increases in [Ca2+]i were also significantly different between MHS and controls (P = 0.003) or between MHS and non-MHS (MHN and control) individuals (P = 0.0078). A study of a family with the Val2168Met mutation demonstrated expression of the RYR1 mRNA mutant in B cells from the family members with MHS phenotype and a clear segregation of genotype with B-cell phenotype. Conclusion The Ca2+ responses to caffeine or 4-chloro-m-cresol in B lymphocytes showed significant differences between MHS and MHN (or control) individuals. Although the molecular mechanisms of these alterations are currently undetermined, the results suggest that the enhanced Ca2+ responses are associated with mutations in the RYR1 gene in some MHS individuals.

scholarly journals Cytometric detection of DNA amplified with fluorescent primers: applications to analysis of clonal bcl-2 and IgH gene rearrangements in malignant lymphomas

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1079-1085
Author(s):  
RL Barker ◽  
CA Worth ◽  
SC Peiper
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Gel Electrophoresis ◽  
Flow Cytometric Analysis ◽  
Dna Amplification ◽  
Cell Populations ◽  
Gene Rearrangements ◽  
Flow Cytometric ◽  
Hodgkin's Lymphomas ◽  
Follicular Lymphomas

Follicular lymphomas comprise almost two thirds of the US adult non- Hodgkin's lymphomas (NHL) and are the most common malignancy of B- lineage lymphocytes. Polymerase chain reaction (PCR) protocols have been developed to detect the t(14;18) translocation, which juxtaposes the bcl-2 proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular lymphomas and monoclonal rearrangements of the IgH gene in B- cell NHL that lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH rearrangements in DNA from patients with lymphoproliferative disorders and analyzed the products in parallel by gel electrophoresis and flow cytometry, which detected PCR products incorporating fluoresceinated oligonucleotide primers by sequence-specific capture to oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior to electrophoresis of ethidium-bromide-stained agarose gels for detection of products of nested PCR to detect intergenic rearrangements involving bcl-2 and single primer-pair amplification of clonal rearrangement of IgH. Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+ B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal B-cell populations. In contrast, analysis by gel electrophoresis detected bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene rearrangements in only 9 of 17 monoclonal B-cell populations. Flow cytometric analysis was more sensitive than gel electrophoresis and could detect a 16-fold greater dilution of a bcl-2-amplified product than gel electrophoresis. Similarly, flow cytometry could detect an amplification product when template DNA was diluted 10,000-fold, whereas gel electrophoresis only detected amplification products when template was subjected to dilution between 100- and 1,000-fold. This shows the utility of flow cytometry for the analysis of DNA amplification products incorporating fluorochrome-labeled primers as a rapid, objective alternative to conventional strategies. Because current-generation clinical laboratories emphasize automation, flow cytometric analysis of PCR- amplified products shows increased analytic sensitivity and offers a vehicle for automation of DNA amplification tests.

Sign in / Sign up

Export Citation Format

Share Document