Diagnostic Usefulness of Aberrant CD22 Expression in Differentiating Neoplastic Cells of B-Cell Chronic Lymphoproliferative Disorders From Admixed Benign B Cells in Four-Color Multiparameter Flow Cytometry

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

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Lymphoproliferative disorders (LPD) involving lungs: T and B cell subsets within pulmonary infiltrates and in peripheral blood

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010011338x ◽

1984 ◽

Vol 42 ◽

pp. 700-701

Author(s):

Irene Stachura ◽

Milton H. Dalbow ◽

Michael J. Niemiec ◽

Matias Pardo ◽

Gurmukh Singh ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Lymphoproliferative Disorders ◽

Mixed Population ◽

Lymphoid Cells ◽

Lymphomatoid Granulomatosis ◽

Cell Type ◽

Cell Surface Antigens ◽

Pulmonary Infiltrates ◽

B Cell Subsets

Lymphoid cells were analyzed within pulmonary infiltrates of six patients with lymphoproliferative disorders involving lungs by immunofluorescence and immunoperoxidase techniques utilizing monoclonal antibodies to cell surface antigens T11 (total T), T4 (inducer/helper T), T8 (cytotoxic/suppressor T) and B1 (B cells) and the antisera against heavy (G,A,M) and light (kappa, lambda) immunoglobulin chains. Three patients had pseudolymphoma, two patients had lymphoma and one patient had lymphomatoid granulomatosis.A mixed population of cells was present in tissue infiltrates from the three patients with pseudolymphoma, IgM-kappa producing cells constituted the main B cell type in one patient. In two patients with lymphoma pattern the infiltrates were composed exclusively of T4+ cells and IgG-lambda B cells predominated slightly in the patient with lymphomatoid granulomatosis.

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OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.908 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 112.2-113

Author(s):

M. Gatto ◽

S. Bjursten ◽

C. Jonell ◽

C. Jonsson ◽

S. Mcgrath ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Adverse Events ◽

B Cell ◽

Mononuclear Cells ◽

Checkpoint Inhibitors ◽

Therapy Monitoring ◽

Cell Subset ◽

Peripheral Blood Mononuclear ◽

Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

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Association between the proliferative rate of neoplastic B cells, their maturation stage, and underlying cytogenetic abnormalities in B-cell chronic lymphoproliferative disorders: analysis of a series of 432 patients

Blood ◽

10.1182/blood-2007-10-119289 ◽

2008 ◽

Vol 111 (10) ◽

pp. 5130-5141 ◽

Cited By ~ 17

Author(s):

Sandra Quijano ◽

Antonio López ◽

Ana Rasillo ◽

Susana Barrena ◽

Maria Luz Sánchez ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoproliferative Disorders ◽

Lymphocytic Leukemia ◽

Maturation Stage ◽

Cytogenetic Abnormalities ◽

M Phase ◽

The Impact

Abstract Limited knowledge exists about the impact of specific genetic abnormalities on the proliferation of neoplastic B cells from chronic lymphoproliferative disorders (B-CLPDs). Here we analyze the impact of cytogenetic abnormalities on the proliferation of neoplastic B cells in 432 B-CLPD patients, grouped according to diagnosis and site of sampling, versus their normal counterparts. Overall, proliferation of neoplastic B cells highly varied among the different B-CLPD subtypes, the greatest numbers of proliferating cells being identified in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Compared with normal B cells, neoplastic B-CLPD cells showed significantly increased S + G2/M-phase values in mantle cell lymphoma (MCL), B-chronic lymphocytic leukemia (B-CLL), BL, and some DLBCL cases. Conversely, decreased proliferation was observed in follicular lymphoma, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM), and some DLBCL patients; hairy cell leukemia, splenic marginal zone, and MALT-lymphoma patients showed S + G2/M phase values similar to normal mature B lymphocytes from LN. Interestingly, in B-CLL and MCL significantly higher percentages of S + G2/M cells were detected in BM versus PB and in LN versus BM and PB samples, respectively. In turn, presence of 14q32.3 gene rearrangements and DNA aneuploidy, was associated with a higher percentage of S + G2/M-phase cells among LPL/WM and B-CLL cases, respectively.

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Comparison of Multiparameter Flow Cytometry with Cluster Analysis and Immunohistochemistry for the Detection of CD10 in Diffuse Large B-Cell Lymphomas

Modern Pathology ◽

10.1038/modpathol.3880539 ◽

2002 ◽

Vol 15 (4) ◽

pp. 413-419 ◽

Cited By ~ 24

Author(s):

Yin Xu ◽

Robert W McKenna ◽

Steven H Kroft

Keyword(s):

Flow Cytometry ◽

Cluster Analysis ◽

B Cell ◽

Multiparameter Flow Cytometry ◽

B Cell Lymphomas ◽

Large B Cell

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Detection of t(14;18)(q32;q21) in B-Cell Chronic Lymphocytic Leukemia

Archives of Pathology & Laboratory Medicine ◽

10.5858/2005-129-410-doqibc ◽

2005 ◽

Vol 129 (3) ◽

pp. 410-411

Author(s):

Wolfgang Kern ◽

Torsten Haferlach ◽

Susanne Schnittger ◽

Claudia Schoch

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

Chronic Lymphocytic Leukemia ◽

Follicular Lymphoma ◽

B Cell ◽

Lymphocytic Leukemia ◽

Multiparameter Flow Cytometry ◽

Lymphoma Therapy ◽

Cell Chronic Lymphocytic Leukemia

Abstract Cytomorphologic testing and multiparameter flow cytometry are the mainstays in diagnosing B-cell chronic lymphocytic leukemia, whereas fluorescence in situ hybridization that targets the translocation t(14;18)(q32;q21) often is used to identify follicular lymphoma. Therapy is highly diverse between both diseases. We describe a case with cytomorphologically and immunologically proven B-cell chronic lymphocytic leukemia in which t(14;18)(q32;q21) was found.

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Multiparameter Flow Cytometry Reveals Differences in B Cells Subsets among Infants Born to Mothers being Treated for IBD

Gastroenterology ◽

10.1016/s0016-5085(17)30635-2 ◽

2017 ◽

Vol 152 (5) ◽

pp. S86 ◽

Cited By ~ 1

Author(s):

Michael Kattah ◽

Jeffrey Milush ◽

Trevor Burt ◽

Uma Mahadevan

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Multiparameter Flow Cytometry

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Hematologist‐Level Classification of Mature B‐Cell Neoplasm Using Deep Learning on Multiparameter Flow Cytometry Data

Cytometry Part A ◽

10.1002/cyto.a.24159 ◽

2020 ◽

Vol 97 (10) ◽

pp. 1073-1080

Author(s):

Max Zhao ◽

Nanditha Mallesh ◽

Alexander Höllein ◽

Richard Schabath ◽

Claudia Haferlach ◽

...

Keyword(s):

Flow Cytometry ◽

Deep Learning ◽

B Cell ◽

Multiparameter Flow Cytometry ◽

Flow Cytometry Data ◽

Cell Neoplasm

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The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Blood ◽

10.1182/blood-2020-142107 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 5-5

Author(s):

Ling Tian ◽

Monique Chavez ◽

Lukas D Wartman

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Plasma Cells ◽

Cell Development ◽

Young Female ◽

B Cell Development ◽

Cell Immune Response ◽

Germinal Center Hyperplasia

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

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