Original Study Analysis of B-Cell Subpopulations in Monoclonal Gammopathies Pavla V sianská, 1,2,3 Lucie Ríhová, 1,2 Tamara Varmu zová, 1,2 Renata Suská, 1 Fedor Kryukov, 2,4,5 Aneta Mikulá sová, 1,2,3 Renata Kupská, 2 Miroslav Penka, 1 Lud ek Pour, 2,6 Zden ek Adam, 6 Roman Hájek 1,2,4,5 Abstract To clarify the pathophysiology of multiple myeloma (MM) it is necessary to investigate the various develop- mental stages of B-cells. We provide a detailed phenotypic profile and enumeration of B-cell subpopulations in monoclonal gammopathy patients. A reduction of B-cells and differentiation shift to more numerous antigen- stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in one of these subpopulations. Background: Multiple myeloma (MM) is characterized by accumulation of pathological plasma cells (PCs) in bone marrow (BM) as a result of deregulation of B-cell development. To clarify its pathophysiology it is necessary to investigate in detail the developmental stages of B-cells. Materials and Methods: Enumeration of total CD19-positive (CD19 þ ) cells and their subpopulations together with PCs was done in peripheral blood (PB) and BM of newly diagnosed monoclonal gammopathy patients and control subjects. Representation of subsets was compared among groups and relationships between subset percentage and cytogenetic/biochemical findings were analyzed. Results: A lower number of total CD19 þ cells was found in MM, particularly in advanced stages of disease. Reduction of naive (P < .01) and transitional B-cells (P < .05) and increase of switched memory and switched CD27 B-cells and germinal center founder cells were detected in PB of MM compared with controls (P < .01). Similar results were found in BM. b2 microglobulin level in MM positively correlated with the number of PCs and negatively with percentage of naive B-cells (P < .05). Conclusion: Our results provided a detailed phenotypic profile and enumeration of B and PC sub- populations in monoclonal gammopathy patients. A reduced number of B-cells and particularly a differentiation shift to more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma- initiating cells in one of these subpopulations. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: B-cell, Flow Cytometry, Multiple Myeloma, Monoclonal Gammopathy, Plasma Cell Introduction B-cells are cells of specific humoral immunity. Similar to other blood cells, B-cells develop from a common stem cell. Their development may be divided into 2 phases: in the first phase, independently of the antigen, precursors of B-cells in the bone marrow (BM) mature—there are 5 main developmental stages: pro B, pre B I, pre B II, immature B, and mature naive B-cells. 1,2 During this phase of development, the rearrangement of genes coding for light and heavy immunoglobulin (Ig) chains (Variable, diversity, joining gene segments recombination) occurs as a part of the antigen-specific B-cell receptor. 3 Next, the B-cell undergoes several check points to generate only functional B-cells, and nonfunctional or strongly autoreactive B-cells undergo apoptosis because they represent a threat to the organism. A mature naive B-cell leaves the BM and enters the blood stream where the second phase of development occurs, this time antigen-dependent. During the antigen-dependent phase, the mature naive B-cell (CD19 þ CD38 þ/CD20 þ CD27 IgM þ /IgD þ ) enters the lymph node. After antigen recognition presented by the follicular dendritic 1 Department of Clinical Haematology, University Hospital Brno, Brno, Czech Republic 2 Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 3 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 4 Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic 5 Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic 6 Department of Internal Medicine, Haematology and Oncology, University Hospital Brno, Brno, Czech Republic Submitted: Oct 16, 2014; Revised: Nov 26, 2014; Accepted: Dec 6, 2014 Address for correspondence: Lucie Ríhová, PhD, Department of Clinical Haematology, University Hospital Brno, Jihlavska 20, 625 00 Brno, Czech Republic E-mail contact: [email protected]2152-2650/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2014.12.003 Clinical Lymphoma, Myeloma & Leukemia Month 2015 - 1
Transcript
Original Study
Analysis of B-Cell Subpopulations in MonoclonalGammopathies
Pavla V�sianská,1,2,3 Lucie �Ríhová,1,2 Tamara Varmu�zová,1,2 Renata Suská,1
Fedor Kryukov,2,4,5 Aneta Mikulá�sová,1,2,3 Renata Kupská,2 Miroslav Penka,1
Lud�ek Pour,2,6 Zden�ek Adam,6 Roman Hájek1,2,4,5
AbstractTo clarify the pathophysiology of multiple myeloma (MM) it is necessary to investigate the various develop-mental stages of B-cells. We provide a detailed phenotypic profile and enumeration of B-cell subpopulations inmonoclonal gammopathy patients. A reduction of B-cells and differentiation shift to more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in oneof these subpopulations.Background: Multiple myeloma (MM) is characterized by accumulation of pathological plasma cells (PCs) in bonemarrow (BM) as a result of deregulation of B-cell development. To clarify its pathophysiology it is necessary toinvestigate in detail the developmental stages of B-cells.Materials and Methods: Enumeration of total CD19-positive(CD19þ) cells and their subpopulations together with PCs was done in peripheral blood (PB) and BM of newlydiagnosed monoclonal gammopathy patients and control subjects. Representation of subsets was compared amonggroups and relationships between subset percentage and cytogenetic/biochemical findings were analyzed. Results:A lower number of total CD19þ cells was found in MM, particularly in advanced stages of disease. Reduction of naive(P < .01) and transitional B-cells (P < .05) and increase of switched memory and switched CD27� B-cells and germinalcenter founder cells were detected in PB of MM compared with controls (P < .01). Similar results were found in BM. b2microglobulin level in MM positively correlated with the number of PCs and negatively with percentage of naive B-cells(P < .05). Conclusion: Our results provided a detailed phenotypic profile and enumeration of B and PC sub-populations in monoclonal gammopathy patients. A reduced number of B-cells and particularly a differentiation shiftto more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in one of these subpopulations.
Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved.Keywords: B-cell, Flow Cytometry, Multiple Myeloma, Monoclonal Gammopathy, Plasma Cell
IntroductionB-cells are cells of specific humoral immunity. Similar to other
blood cells, B-cells develop from a common stem cell. Their
1Department of Clinical Haematology, University Hospital Brno, Brno, CzechRepublic2Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine,Masaryk University, Brno, Czech Republic3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno,Czech Republic4Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic5Department of Haematooncology, University Hospital Ostrava, Ostrava, CzechRepublic6Department of Internal Medicine, Haematology and Oncology, University HospitalBrno, Brno, Czech Republic
Submitted: Oct 16, 2014; Revised: Nov 26, 2014; Accepted: Dec 6, 2014
Address for correspondence: Lucie �Ríhová, PhD, Department of Clinical Haematology,University Hospital Brno, Jihlavska 20, 625 00 Brno, Czech RepublicE-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.clml.2014.12.003
development may be divided into 2 phases: in the first phase,independently of the antigen, precursors of B-cells in the bonemarrow (BM) mature—there are 5 main developmental stages: proB, pre B I, pre B II, immature B, and mature naive B-cells.1,2
During this phase of development, the rearrangement of genescoding for light and heavy immunoglobulin (Ig) chains (Variable,diversity, joining gene segments recombination) occurs as a part ofthe antigen-specific B-cell receptor.3 Next, the B-cell undergoesseveral check points to generate only functional B-cells, andnonfunctional or strongly autoreactive B-cells undergo apoptosisbecause they represent a threat to the organism. A mature naiveB-cell leaves the BM and enters the blood stream where the secondphase of development occurs, this time antigen-dependent.
During the antigen-dependent phase, the mature naive B-cell(CD19þCD38þ/�CD20þCD27�IgMþ/IgDþ) enters the lymphnode. After antigen recognition presented by the follicular dendritic
cell and receipt of the costimulatory signal from the specific antigen-activated T-cell, the B-cell becomes activated.3,4 The activated B-cellthen enters the extrafollicular space where it differentiates into ashort-term plasma cell (PC) or migrates into the lymphoid folliclewhere it starts the germinal center (GC).5,6 In the GC, the B-cellproliferates, undergoes somatic hypermutations of variable regions ofthe Ig chains and isotype switch followed by affinity maturation.3,6-8
The aim of these processes is to generate B-cells capable of binding aspecific antigen with high affinity. A part of these cells differentiatesinto plasmablasts (CD19þCD38þþCD20�CD138�CD27þ) thatmigrate into the BM where they mature into long-term PCs(CD38þCD138þ) producing high affinity antibodies while anotherpart differentiates into long-term memory B-cells (CD19þCD38þ/�
CD20þCD27þIgM�/þIgD�/þ).9-11 Next to these memory B-cellsoriginating from GC, there are also memory B-cells that do notexpress the typical CD27 marker (CD19þCD38þ/�CD20þ
CD27�IgMþ/�IgD�).12,13 These memory cells probably originateindependently of the GC.14 Besides these already mentioned stagesof the antigen-dependent phase, there are also GC founder cells(CD38þþIgDþCD27�)15,16 in peripheral blood (PB).
Not only completely matured B-cells, but also transitional B-cellsare present in PB. These are defined as immature B-cells circulatingin the periphery (CD38þþCD24þIgDþIgMþCD27�).17
Monoclonal gammopathies (MGs) are characterized by thepresence of a clone of PCs that lose their physiological function andstart production of monoclonal Ig that damage the organism.Monoclonal gammopathy of undetermined significance (MGUS) isa benign disease that is typically with the presence of physiologicaland pathological PCs in various ratios.18-20 It is a precancerosis thatcan give rise to a malignant tumor—multiple myeloma (MM)characterized by accumulation of pathological PCs in the BM.20,21
Pathological PCs are derived from malignant transformation in theB-cell development that most probably happens in cells that wentthrough the GC22,23; however, this has not been confirmed.
In this work, we identified individual subpopulations of B-cells inPB and BM and compared their percentages in healthy donors andMM and MGUS patients with the aim to map and describe sub-population profiles in PC development in MGs. Finding the dif-ferences in representations of subpopulations between MG andhealthy control subjects could be helpful in further analysis aimed atclarification of MG pathophysiology and finding of myeloma-initiating cells.
Materials and MethodsClinical Samples
Peripheral blood and BM samples from 38 patients with newlydiagnosed MM and 18 MGUS patients were used. As control, 8samples of PB and BM of patients with Ph� myeloproliferativedisease (MPD) and 10 samples of healthy donor PB were used. Thedata set characteristics and clinical data of patients are presented inTable 1. PB and BM of MM and MGUS patients were obtained atthe Department of Internal Medicine, Hematology and Oncologyat the University Hospital Brno. All samples were included onlyafter the patients signed the informed consent form approved by theethical committee of the hospital. PB samples were collected intoethylenediaminetetraacetic acid. Heparinized BM was mixed withethylenediaminetetraacetic acid after sampling.
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Table 2 Phenotype of B and Plasma Cell Subpopulations
Polychromatic Flow Cytometric AnalysisAppropriate volumes of PB and/or BM were lysed using NH4Cl
to destroy erythrocytes and washed with phosphate-buffered saline(PBS). Then, they were incubated with monoclonal antibodies(MoAb) against individual markers on the surface of B-cells andPCs. For PB, an 8-color combination of MoAb was used:CD38-Pacific Blue (Exbio, clone HIT2), CD45-Krome Orange(Beckman Coulter, clone J.33), IgD-fluorescein isothiocyanate(FITC) (Becton Dickinson, clone IA6-2), CD24-phycoerythrin(PE) (Exbio, clone SN3), CD20-peridinin chlorophyll (PerCP;Exbio, clone LT20), CD19-phycoerythrin-cyanine 7 (PC7; Beck-man Coulter, clone J3-119), IgM-allophycocyanin (APC; BectonDickinson, clone G20-127), CD27-allophycocyanin-cyanine 7(APC-H7; Becton Dickinson, clone M-T271). BM was incubatedwith 8-color combination of MoAb: CD38-Pacific Blue (Exbio,
Figure 1 B-Cell Subpopulations and PCs in BM of MM Patient: CD4Naive (Green), Isotype-Switched CD27L B (Light Blue), Isot(Light Purple), Plasmablasts (Orange), and Plasmocytes (R
Abbreviations: BM ¼ bone marrow; MM ¼ multiple myeloma; PC ¼ plasma cell.
After incubation, the samples were washed with PBS stabilizedwith sodium azide and then analyzed using a flow cytometer, BDFACSCanto II (Becton Dickinson), equipped with 3 lasers (violet:405 nm; blue: 488 nm; red: 633 nm). Reanalysis of data wereperformed using software Infinicyt version 1.3.0 (Cytognos).
Gating Strategy and Subpopulation IdentificationIn mononuclear cells (MNC) fraction, identified based on
forward scatter versus side scatter, the B-cells were identified asCD19þ cells. This population was further divided based ondifferent expression of surface markers of individual subpopulations.In PB, the following subpopulations were found: naive B-cells,transitional B-cells, GC founder cells (preGC), memory B-cells withisotype switch, isotype-unswitched memory B-cells, isotype-switched CD27� B-cells, and plasmablasts.
In the BM, PCs were excluded from whole leukocytes first, thenidentification CD19þ B-cells in the population of MNC was doneas described herein previously. The same subpopulations wereidentified in BM as in PB; however, instead of transitional B-cells,we identified immature CD45þ B-cells and immature CD45dimþ
B-cells. The phenotypes of individual subpopulations are shown inTable 2 and Figure 1.
The percentage of the individual subpopulations were comparedbetween groups based on diagnosis—MM versus MGUS versushealthy controls, and in MM patients, based on individual stages ofthe disease based on Durie Salmon staging (D-S) as well.
Molecular CytogeneticsOnly data of MM patients were used for this work. First, using
MNC from BM of M patients, CD138þ PCs were isolated using
5dimD Immature B (Yellow), CD45D Immature B (Dark Blue),ype-Switched Memory B (Purple), Isotype-Unswitched Memory Bed)
magnetic and/or fluorescence activated cell sorting.24,25 Then, sepa-rated PCs were used for detection of chromosomal aberrations usinginterphase fluorescent in situ hybridization (I-FISH) analysis.26,27
For I-FISH, the following commercial DNA probes were used:LSI 13 (13q14) SpectrumGreen Probe for del(13)(q14), LSI p53(17p13.1) Spectrum Orange Probe for del(17)(p13) (both fromAbbott Molecular) and immunoglobulin heavy chain (IGH) rear-rangement was detected using XL IGH break apart probe (Meta-Systems). For gain (1)(q21), XL 1p32/1q21 locus-specific probe(MetaSystems) was used. Hyperdiploidy was detected using a VysisD5S23, D5S721/CEP 9/CEP 15 FISH Probe Kit and CEP9SpectrumAqua Probe (both from Abbott Molecular). For deletions,numerical aberrations and IGH rearrangement, the cutoff values of20% recommended by the European Myeloma Network wereused.28,29
StatisticsThe results were analyzed using Statistica 9.1 (StatSoft, Inc)
and SPSS 20.0 (IBM) programs. Differences in subpopulationsbetween groups of patients were analyzed using the nonpara-metric KruskaleWallis test or ManneWhitney U test. On theset of MM patients, the correlation between individual sub-populations and clinical data was calculated using Spearmancorrelation coefficient. The value for statistical significance was setat P ¼ .05.
ResultsB-Cell Subsets in PB
Compared with MG patients, data of healthy subjects were usedas controls. However, no statistically significant difference wasfound when healthy subjects and patients with MPD werecompared, which was used in subsequent analyses.
No statistical significant difference was found in percentage oftotal CD19þ cells when MGUS patients and healthy controls werecompared, and no difference was found between MGUS and MMpatients (Table 3). In MM patients, percentage of total CD19þ cellswas statistically significantly decreased compared with healthycontrols (median, 3.9%; range, 0.4-8.9 vs. 6.8%; range, 3.8-12.2;P < .005).
Between MGUS patients and healthy controls, no statisticalsignificant difference was found in percentage of any of analyzedsubsets; thus, all changes are related only to patients with MM.
When analyzing subpopulations, a lower number of transitionalB was found in MM compared with controls (0.9%; range, 0.0-12.2vs. 2.5%; range, 0.8-5.8; P < .05). Furthermore, patients with MMhad a statistically significantly reduced amount of naive B-cellscompared with MGUS patients (36.4%; range, 2.4-70.9 vs.56.7%; range, 7.7-77.2; P < .05) and healthy controls (56.0%;range, 45.2-70.6; P < .01). In contrast, the total number ofmemory B-cells was significantly increased in MM patientscompared with MGUS patients (43.0%; range, 11.9-82.6 vs.25.8%; 9.3-60.5; P < .05), and healthy controls (32.7%; range,11.1-42.5; P < .05). An increased number of memory B in pa-tients with MM was due to the higher proportion of isotype-switched memory B-cells in MM patients compared withMGUS patients (20.5%; range, 4.9-67.2 vs. 11.3%; 4.2-41.3;P < .01) and controls (12.5%; range, 6.5-17.1; P < .01), while
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no statistically significant differences were found in percentage ofisotype-unswitched memory B-cells. In percentage of transitional,preGC and isotype-switched CD27� B-cells, a statistically signif-icant difference was found only when MM patients and controlswere compared. Transitional B-cells were reduced in MMcompared with controls (0.9%; range, 0.0-12.2 vs. 2.5%; 0.8-5.8;P < .05), preGC B-cells were increased in MM patients comparedwith controls (0.7%; range, 0.0-2.4 vs. 0.3%; range, 0.0-0.8;P < .01), and percentage of isotype-switched CD27� B-cells wassignificantly greater in MM patients than in controls (10.0%;range, 2.6-38.0 vs. 5.2%; range, 2.4-9.0; P < .01). No differencewas found when plasmablasts were compared in all 3 groups.Representation of individual subpopulations is shown in Figure 2.
B-Cell Subsets in BMIn the case of BM, samples from healthy people were not avail-
able; therefore, BM samples from patients with MPD were used ascontrols.
Percentage of total CD19þ cells was significantly lower in MMpatients compared with controls (4.2%; range, 0.6-50.9 vs. 11.3%;range, 3.5-20.9; P < .05) and MGUS patients (7.9%; range, 3.9-21.1; P < .005); (Table 4). Detailed analysis and stratification ofMM patients into groups according to the D-S staging systemshowed that patients in stage I had a significantly increased numberof total CD19þ cells compared with patients in stage II (10.9%;range, 4.8-12.5 vs. 2.3%; range, 1.2-8.4; P < .005) and in stage III(3.8%; range, 0.6-50.9; P < .05).
Figure 2 Flow Chart of B-Cell Development: B-Cell Subpopulations Pin Healthy Control Subjects (Green), MGUS (Pink), and MM
Abbreviations: MGUS ¼ monoclonal gammopathy of undetermined significance; PC ¼ plasma cell.
No significant difference was found in percentage of immatureCD45dimþ B and immature CD45þ B-cells between observedgroups, although in MM patients we found the lowest percentage ofthese cells. Percentage of naive B-cells was significantly decreased inMM patients compared with control and MGUS patients (37.0%;range, 0.6-67.9 vs. 48.0%; range, 27.3-86.3; P ¼ .05 and 37.0%;range, 0.6-67.9 vs. 50.1%; range, 13.8-74.9; P < .05). An increasednumber of total memory B-cells was found in MM patientscompared with controls (32.4%; range, 2.9-68.8 vs. 16.0%; range,7.8-26.1; P < .05); however, compared with MGUS patients, nosignificant difference was found (32.4%; range, 2.9-68.8 vs. 19.4%;range, 2.2-49.7; not significant [n.s.]). The difference was againcaused particularly by isotype-switched memory B-cells, which weresignificantly increased in MM patients compared with controls(14.0%; range, 1.4-41.3 vs. 5.7%; range, 1.7-11.5; P < .05), andMGUS patients (6.0%; range, 0.5-21.3; P < .05). Regardingisotype-unswitched memory B-cells, no difference was found whenall 3 groups were compared, similar as in the subpopulation ofisotype-switched CD27� B-cells. Percentage of plasmablastsdiffered only when MM and MGUS patients cases werecompared—a significantly greater proportion was found in MM(2.6%; range, 0.1-18.0 vs. 1.2%; range, 0.4-31.6; P < .05).
Comparison of B-Cell Subsets in PB Versus BMIn control samples (MPD), there was approximately the same
percentage of all subpopulations in PB compared with BM(immature CD45þ B-cells [2.2; range, 0.9-5.2 vs. 3.6; range,
resent in Peripheral Blood With Medians of Their Representation(Red)
1.7-8.1; n.s.], naïve B-cells (51.9; range, 24.4-86.3 vs. 48.0; range,27.3-86.3; n.s.), total memory B-cells (34.1; range, 6.9-54.5 vs.15.9; range, 7.8-26.1; n.s.), isotype-switched CD27� B-cells (8.5;range, 3.0-17.1 vs. 5.9; range, 1.4-13.7; n.s.), and plasmablasts (1.8;range, 0.4-5.3 vs. 1.4; range, 0.4-3.8; n.s.)). Equally in MGUSpatients, no significant difference was found when PB and BM werecompared (immature CD45þ B-cells, (1.9; range, 0.0-19.0 vs. 2.6;range, 0.1-8.7; n.s.), naïve B-cells, (56.7; range, 7.7-77.2 vs. 50.1;range, 13.8-75.0; n.s.), total memory B-cells, (25.8; range, 9.3-60.5vs. 19.4; range, 2.2-49.7; n.s.), isotype-switched CD27� B-cells,(6.5; range, 1.8-28.9 vs. 4.9; range, 2.1-15.5; n.s.), plasmablasts,1.4; range, (0.2-30.8 vs. 1.2; range, 0.4-31.6; n.s.)). In MM pa-tients, there was a significantly greater percentage of immatureCD45þ B-cells in the BM compared with PB (2.4; range, 0.0-13.1vs. 0.9; range, 0.0-12.2; P < .01). In contrast, the number of totalmemory B-cells and isotype-switched CD27� B-cells was increasedin PB compared with BM (43.0; range, 11.9-82.6 vs. 32.4; range,2.9-68.8; P < .01 and 10.0; range, 2.6-38.0 vs. 8.1; range,1.3-96.4; P < .05). No difference was found in percentage of naiveB-cells and plasmablasts when PB and BM were compared (36.4;range, 2.4-70.9 vs. 37.0; range, 0.6-67.9; n.s. and 2.3; range,0.3-26.7 vs. 2.4; range, 0.0-13.1; n.s.).
Bone Marrow PCsAs expected, the highest number of PCs was found in MM
patients compared with the MGUS group (19.4%; range, 0.4-63.0vs. 0.6%; range, 0.1-1.6; P < .005) and the control group (0.4%;range, 0.0-1.1; P < .005). Also, the percentage of PCs was signif-icantly greater in MGUS patients compared with controls (0.6%;range, 0.1-1.6 vs. 0.4%; range, 0.0-1.1; P < .05).
Comparison of B-Cell Subsets With Cytogenetic Findingsin MM Patients
Analysis of chromosomal aberrations of MM patients showedthat patients with RB1 (locus 13q14) deletion had a decreasednumber of isotype-switched memory B-cells and increased numberof naive B-cells in PB compared with those without this deletion(14.3%; range, 4.9-33.2 vs. 27.3%; range, 8.5-67.2; P < .05 and51.2%; range, 14.9-70.9 vs. 27.3%; range, 2.4-70.3; P < .05).There was a greater percentage of immature CD45þ B-cells in theBM of these patients compared with patients without deletion(3.2%; range, 1.0-6.6 vs. 0.9%; range, 0.0-4.3; P < .01). Also alower percentage of plasmablasts was found in PB of patients withIGH disruption compared with patients without this aberration(2.0%; range, 0.3-26.7 vs. 3.8%; range, 1.7-15.3; P < .05).
Correlations Between B-Cell and PC Subsets in BM andSelected Biochemical Parameters
There was a negative correlation between percentage of PCs andtotal percentage of CD19þ B-cells (rS ¼ �0.38; P < .05), andbetween percentage of PCs and percentage of naive B-cells(rS ¼ �0.37; P < .05; Table 5). A positive correlation betweenthe percentage of total memory and isotype-switched memoryB-cells and isotype-switched CD27� B-cells was found (rS ¼ 0.47;P < .005 and rS ¼ .57; P < .001). Regarding the relationshipbetween the percentage of subpopulations and biochemical pa-rameters, in MM patients, there was a positive correlation between
Table 5 Correlations Between Individual Subpopulation Percentages in Bone Marrow of Multiple Myeloma Patients Calculated Using Spearman Correlation Coefficient (rs)
the serum level of C-reactive protein (CRP) and plasmablasts inthe BM (rS ¼ 0.35; P < .05), and between percentage of isotype-switched memory B-cells and the amount of platelets (rS ¼ 0.33;P < .05) and between the relative number of naive B-cells andlevel of hemoglobin (rS ¼ 0.38; P < .05; Table 6). On thecontrary, between the percentage of naive B-cells and level ofb2 microglobulin (b2m), there was a negative correlation(rS ¼ �0.35; P < .05), while between the level of b2m andpercentage of PCs in the BM, there was a positive correlation(rS ¼ 0.43; P < .05).
It was also found that the percentage of PCs and isotype-switchedCD27� B-cells in the BM correlated with the level of calcium(rS ¼ 0.35 and rS ¼ 0.36; P < .05), and level of serum creatinine(rS ¼ 0.35 and rS ¼ 0.35; P < .05).
DiscussionThe main aim of this work was to determine the phenotype
profile of B-cells and compare individual subpopulations presentin PB and BM of individuals with MG and healthy donors. Itcan be assumed that in the era of polychromatic cytometry,the classification of mature B-cell subpopulations based on anolder system does not have to include the whole phenotypicrange and differentiation variability. More detailed analysismight provide the basis for clarification of MG pathophysiology.PB B-cells and their subpopulations have been studied in detailin healthy donors and patients with autoimmune dis-eases,3,15,16,30 although there is not enough information aboutphenotype and percentage of individual B subpopulations in theBM of MG.
One of the reasons is decreased percentage of total B-cells inMM patients compared with healthy donors,31,32 which has beenproven in this study as well. We also found that a decreasednumber of B-cells is connected to advanced stages of the disease,which might be caused by insufficient hematopoiesis and/or moreprobably by early B-cell development failure because of increasedinfiltration of PCs.
The amount of used markers and software used for analysis iscrucial for identification of amount and percentage of differentsubpopulations. Older analyses used especially CD38, IgD, andCD27, which allow for identification of B-cell subpopulationsfrom naive to memory cells; however, certain B-cells might beleft out.15
In PB, naive and memory cells are the main subpopulations ofB-cells.3,30 Our analysis showed that the population of memoryB-cells form the majority of CD19þ cells in PB of MM. It isknown that a part of memory B-cells do not undergo isotypeswitch and remain IgDþ.16 Polychromatic analyses using IgD andIgM indicate that these isotype-unswitched memory B-cells mightreceive the IgMþIgD�/þ phenotype; which is why the olderclassification based only on IgD is not sufficient for identificationof the whole-memory B population. Our finding that morethan half of CD27þ memory B-cells (17% from 30% of thewhole-memory B-cell population)—which usually carry somatichypermutation—are formed by isotype-switched B-cells is inconcordance with the literature.3,10
Next to these subpopulations, we also identified a subpopulationof CD27� B-cells that underwent isotype switch (IgM�IgD�) in
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PB and BM. The subpopulation with a similar phenotype(CD27�IgD�) was previously described in PB as a part of thememory B-cell compartment.12,13 Moreover, the finding of a pos-itive correlation between the number of total and isotype-switchedmemory B-cells and isotype-switched CD27� B-cells in the BMof MM patients contributes to confirmation of the connection ofthis population with memory B-cells.
The finding of a low percentage of total B-cells in BM and PBof MM patients compared with MGUS and healthy donors ismost probably caused by a lower percentage of naive B-cells inMM. In contrast, an increased number of memory B-cells in MMpatients might suggest that the precursors of malignant PCs mightoriginate from the population of memory cells, which has beenpublished previously.33 Isotype-unswitched memory B-cells thatmight develop independently of GC34 were not significantlyincreased in MM patients compared with isotype-switchedmemory B-cells, which were significantly increased in MM pa-tients in PB and BM. Based on this, we can hypothesize that thesource population of malignant PC precursors are only memoryB-cells that underwent isotype switch, which corresponds to anoccurrence of malignant transformation in GC.35 Nevertheless, inPB of MM patients, we found an increased percentage of a sub-population of isotype-switched CD27� B-cells that representanother population of memory B-cells which do not arise fromGC.14 It was also found that PC percentage, and percentage ofisotype-switched CD27� B-cells in the BM in MM patientscorrelates with the level of calcium and creatinine in the serum,suggesting a connection between isotype-switched CD27� B-cellsand pathological PCs.
The plasmablast subpopulation was more abundant in BM ofMM patients only compared with MGUS. Plasmablasts might ormight not be the source population of precursors of pathologicalPCs, however, in contrast to PCs, is not possible to infer clonalaffiliation of plasmablasts only according to surface immunophe-notype. One of the reasons that there were no differences foundin plasmablasts in PB might be age differences in healthy donors(42 years) and MGUS (69 years) and/or MM (67 years) because apercentage of circulating memory B-cells and plasmablasts decreasewith age, especially in people older than 60 years.30,36 Nevertheless,we found an increased abundance of memory B-cells in MMpatients.
In MM patients, we found a positive correlation between CRP inserum and plasmablast percentage in BM. It has been reported thatCRP sustains proliferation of myeloma cells and protects them fromapoptosis.37 An increased abundance of plasmablasts in the BM ofMM patients could be connected to their increased survival becauseof CRP.
Serum b2m has been described as a marker related to the size ofMM tumor mass.38 In our work, a positive correlation betweenthe percentage of PCs in BM and the level of b2m in the serumwas found. A negative correlation between the percentage of naiveB-cells in the BM and the level of b2m in the serum and a negativecorrelation between the number of naive B-cells and PCs in the BMsuggest that naive B-cells decrease in MM patients, possibly becauseof the size of tumor mass.
Although in healthy donors and people with MGUS, all sub-populations were approximately similarly represented in PB and
Table 6 Correlations Between Individual Subpopulation Percentages in Bone Marrow of Multiple Myeloma Patients and Biochemical Parameters Calculated Using Spearman CorrelationCoefficient (rs)
BM, in MM patients, the subpopulations of memory and isotype-switched CD27� B-cells were found to be more abundant in PBthan in BM. This difference, which is characterized by increaseddrift of more mature forms of B-cells into the periphery, could bebecause of a breakdown in regulation of B-cell development in MMpatients.
In total, there is a decrease in the number of B-cells in MM. Therewas also a differentiation shift to more antigen-stimulated forms ofB-cells (from preGC including plasmablasts and PCs), which canbe explained by an increased survival of antigen-stimulated formsof B-cells or various processes leading to perpetual renewal of PCs(such as antigen stimulation, BM microenvironment, cytokine pro-duction, etc).
ConclusionWe provided a detailed phenotypic profile and enumeration
of B-cell subpopulations in monoclonal gammopathy patients.This analysis of B-cell differentiation and representation ofindividual subsets is intended as a first step to identification ofa myeloma-initiating cell. Despite the robustness of poly-chromatic flow cytometry, it is not possible to identify thesource population of myeloma-initiating cells based only onsurface phenotype. This area requires a deeper investigationusing an analysis on the molecular level. Analysis of specific geneaberrations using next-generation sequencing could be possibleto identify a subpopulation belonging to a myeloma clone, andevaluation of stem cell functions could identify myeloma-initiating cells.
Clinical Practice Points
� B-cell subsets in MG patients have not been described in detail.The identification and enumeration of 8 different B-cell sub-populations together with a detailed phenotypic profile of themwas done in this study.
� A decreasing trend in number of total B-cells from non-malignantMGUS to MM could be a marker of more advanced stages ofMM.
� An increased number of switched-memory B-cells could indicatea potential source of myeloma-initiating cells within thissubpopulation.
AcknowledgmentsThis work was supported by grant of The Czech Science
Foundation (GAP304/10/1395), a research project of The Ministryof Education, Youth and Sports (MSM0021622434), grant IGA ofThe Ministry of Health (NT12425), and grant of the Ministry ofHealth, Czech Republic conceptual development of research orga-nization (FNBr, 65269705).
DisclosureThe authors have stated that they have no conflicts of interest.
References1. van Zelm MC, van der Burg M, de Ridder D, et al. Ig gene rearrangement steps are
initiated in early human precursor B cell subsets and correlate with specific tran-scription factor expression. J Immunol 2005; 175:5912-22.
Clinical Lymphoma, Myeloma & Leukemia Month 2015
2. Espeli M, Rossi B, Mancini SJ, et al. Initiation of pre-B cell receptor signaling:common and distinctive features in human and mouse. Semin Immunol 2006; 18:56-66.
3. Perez-Andres M, Paiva B, Nieto WG, et al. Human peripheral blood B-cellcompartments: a crossroad in B-cell traffic. Cytometry B Clin Cytom 2010; 78:S47-60.
4. Okada T, Miller MJ, Parker I, et al. Antigen-engaged B cells undergo chemotaxistoward the T zone and form motile conjugates with helper T cells. PLoS Biol 2005;3:1047-61.
5. Liu YJ, Zhang J, Lane PJ, Chan EY, MacLennan IC. Sites of specific B cellactivation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur J Immunol 1991; 21:2951-62.
6. Allen CD, Okada T, Cyster JG. Germinal-center organization and cellulardynamics. Immunity 2007; 27:190-202.
7. Benson MJ, Erickson LD, Gleeson MW, Noelle RJ. Affinity of antigen encounterand other early B-cell signals determine B-cell fate. Curr Opin Immunol 2007; 19:275-80.
8. Dal Porto JM, Haberman AM, Shlomchik MJ, Kelsoe G. Antigen drives very lowaffinity B cells to become plasmacytes and enter germinal centers. J Immunol 1998;161:5373-81.
9. Agematsu K, Hokibara S, Nagumo H, Komiyama A. CD27: a memory B-cellmarker. Immunol Today 2000; 21:204-6.
10. Klein U, Rajewsky K, Küppers R. Human immunoglobulin (Ig)MþIgDþ pe-ripheral blood B cells expressing the CD27 cell surface antigen carry somaticallymutated variable region genes: CD27 as a general marker for somatically mutated(memory) B cells. J Exp Med 1998; 188:1679-89.
11. Blink EJ, Light A, Kallies A, Nutt SL, Hodgkin PD, Tarlinton DM. Earlyappearance of germinal center-derived memory B cells and plasma cells in bloodafter primary immunization. J Exp Med 2005; 201:545-54.
12. Fecteau JF, Côté G, Néron S. A new memory CD27-IgGþ B cell population inperipheral blood expressing VH genes with low frequency of somatic mutation.J Immunol 2006; 177:3728-36.
13. Wei C, Anolik J, Cappione A, et al. A new population of cells lacking expression ofCD27 represents a notable component of the B cell memory compartment insystemic lupus erythematosus. J Immunol 2007; 178:6624-33.
14. Taylor JJ, Pape KA, Jenkins MK. A germinal center-independent pathway gen-erates unswitched memory B cells early in the primary response. J Exp Med 2012;209:597-606.
15. Bohnhorst JØ, Bjørgan MB, Thoen JE, Natvig JB, Thompson KM. Bm1-Bm5classification of peripheral blood B cells reveals circulating germinal center foundercells in healthy individuals and disturbance in the B cell subpopulations in patientswith primary Sjögren’s syndrome. J Immunol 2001; 167:3610-8.
16. Sanz I, Wei C, Lee FE, Anolik J. Phenotypic and functional heterogeneity ofhuman memory B cells. Semin Immunol 2008; 20:67-82.
17. Carsetti R, Rosado MM, Wardmann H. Peripheral development of B cells inmouse and man. Immunol Rev 2004; 197:179-91.
18. Ocqueteau M, Orfao A, Almeida J, et al. Immunophenotypic characterization ofplasma cells from monoclonal gammopathy of undetermined significance patients.Implications for the differential diagnosis between MGUS and multiple myeloma.Am J Pathol 1998; 152:1655-65.
19. Harada H, Kawano MM, Juany N, et al. Phenotypic difference of normal plasmacells from mature myeloma cells. Blood 1993; 81:2658-63.
20. Sezer O, Heider U, Zavrski I, Possinger K. Differentiation of monoclonalgammopathy of undetermined significance and multiple myeloma using flowcytometric characteristics of plasma cells. Haematologica 2001; 86:837-43.
21. Kovarova L, Buresova I, Buchler T, et al. Phenotype of plasma cells in multiplemyeloma and monoclonal gammopathy of undetermined significance. Neoplasma2009; 56:526-32.
22. Bakkus MH, Heirman C, Van Riet I, Van Camp B, Thielemans K. Evidence thatmultiple myeloma Ig heavy chain VDJ genes contain somatic mutations but showno intraclonal variation. Blood 1992; 80:2326-35.
23. Arpin C, de Bouteiller O, Razanajaona D, et al. The normal counterpart ofIgD myeloma cells in germinal center displays extensively mutated IgVH gene,Cmu-Cdelta switch, and lambda light chain expression. J Exp Med 1998; 187:1169-78.
24. Cumova J, Kovarova L, Potacova A, et al. Optimization of immunomagnetic se-lection of myeloma cells from bone marrow using magnetic activated cell sorting.Int J Hematol 2010; 92:314-9.
25. Buresova I, Cumova J, Kovarova L, et al. Bone marrow plasma cell separation -validation of separation algorithm. Clin Chem Lab Med 2012; 50:1139-40.
26. Ahmann GJ, Jalal SM, Juneau AL, et al. A novel three-color, clone-specific fluo-rescence in situ hybridization procedure for monoclonal gammopathies. CancerGenet Cytogenet 1998; 101:7-11.
27. Greslikova H, Zaoralova R, Filkova H, et al. Negative prognostic significance oftwo or more cytogenetic abnormalities in multiple myeloma patients treated withautologous stem cell transplantation. Neoplasma 2010; 57:111-7.
28. Ross FM, Avet-Loiseau H, Ameye G, et al. Report from the European MyelomaNetwork on interphase FISH in multiple myeloma and related disorders. Hae-matologica 2012; 97:1272-7.
29. Mikulá�sová A, Kuglík P, Smetana J, et al. The role of chromosomal aberrations inpathogenesis of monoclonal gammopathy of undetermined significance [Czech].Klin Biochem Metab 2012; 20:91-6.
30. Caraux A, Klein B, Paiva B, et al. Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normalCD138- and CD138þ plasma cells. Haematologica 2010; 95:1016-20.
31. Rawstron AC, Davies FE, Owen RG, et al. B-lymphocyte suppression in multiple
myeloma is a reversible phenomenon specific to normal B-cell progenitors andplasma cell precursors. Br J Haematol 1998; 100:176-83.
32. Luque R, Brieva JA, Moreno A, et al. Normal and clonal B lineage cells can bedistinguished by their differential expression of B cell antigens and adhesionmolecules in peripheral blood from multiple myeloma (MM) patientsediagnosticand clinical implications. Clin Exp Immunol 1998; 112:410-8.
33. Matsui W, Wang Q, Barber JP, et al. Clonogenic multiple myeloma progenitors,stem cell properties, and drug resistance. Cancer Res 2008; 68:190-7.
34. Weller S, Faili A, Garcia C, et al. CD40-CD40L independent Ig gene hyper-mutation suggests a second B cell diversification pathway in humans. Proc NatlAcad Sci U S A 2001; 98:1166-70.
35. Kuehl WM, Bergsagel PL. Early genetic events provide the basis for a clinicalclassification of multiple myeloma. Hematology Am Soc Hematol Educ Program2005; 2005:346-52.
36. Frasca D, Landin AM, Riley RL, Blomberg BB. Mechanisms for decreasedfunction of B cells in aged mice and humans. J Immunol 2008; 180:2741-6.
37. Yang J, Wezeman M, Zhang X, et al. Human C-reactive protein binds activatingFcgamma receptors and protects myeloma tumor cells from apoptosis. Cancer Cell2007; 12:252-65.
38. Bataille R, Magub M, Grenier J, Donnadio D, Sany J. Serum beta-2-microglobulinin multiple myeloma: relation to presenting features and clinical status. Eur JCancer Clin Oncol 1982; 18:59-66.
