Blood, 15 December 2000, Vol. 96, No. 13, pp. 4357-4359
BRIEF REPORT
Frequency and kinetics of polyclonal and clonal B cells in the
peripheral blood of patients being treated for multiple
myeloma
Thomas Rasmussen,
Linda Jensen,
Lone Honoré, and
Hans Erik Johnsen
From the Department of Hematology L, Herlev Hospital,
University of Copenhagen, Herlev, Denmark.
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Abstract |
Recent studies concerning the numbers of circulating clonal B cells
in patients with multiple myeloma (MM) have reported conflicting data
regarding the exact level and phenotype of clonal B cells and their
response to treatment. In this report we document that the peripheral
blood tumor burden at presentation was reduced by induction therapy to
a low level, regardless of the initial tumor burden. However, the
residual clonal compartment persisted before and after transplant.
The level of clonal cells showed no correlation with CD19+
cell levels. In a single patient with MM, high numbers of
phenotypically aberrant clonal cells with altered CD19 expression were identified.
(Blood. 2000;96:4357-4359)
© 2000 by The American Society of Hematology.
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Introduction |
Studies reporting the phenotype and frequency
of clonally related B cells and their role in the pathogenesis of
multiple myeloma (MM) represent an ongoing controversy in myeloma
research. It has been reported that the peripheral blood (PB) of
patients with MM has CD19+ cell levels significantly higher
than those in healthy donors1 and that the majority of
these cells were aberrant lymphocytes clonally related to bone marrow
(BM)-localized myeloma plasma cells.2 In contrast,
others have found that CD19+ cell levels in patients with
MM were not significantly different from healthy donors, although a
pronounced heterogeneity was seen in the patients with
MM,3 and that only a minor fraction of the
CD19+ cells were clonal.4,5 The
CD19+ PB-localized clonal cells have also been reported to
be insensitive to chemotherapy and thus hypothesized to be responsible
for relapse.6,7 To address this controversial area of
myeloma research, the level of circulating clonal cells and their
responses to treatment were monitored using quantitative real time and
single-cell reverse transcriptase-polymerase chain reaction
(RT-PCR) assays.
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Study design |
A total of 9 diagnostic BM aspirates and PB samples from 27 patients with MM and 5 healthy donors were obtained at multiple time
points after informed consent. PB samples obtained at diagnosis from
the 27 patients were simultaneously analyzed by flow cytometry and CD19
real-time RT-PCR. Further, 5 patients were analyzed by single-cell CD19
RT-PCR.
The isolation of BM mononuclear cells (BMMNCs), PB mononuclear
cells (PBMNCs), DNA, RNA, and flow-sorting of single cells was
performed as recently reported.8 All patients were treated as previously described.8 PBMNCs were stained with the
CD19 monoclonal antibodies (MoAbs) Leu-12 fluorescein
isothiocyanate/phycoerythrin (FITC/PE) (Becton Dickinson
Immunocytometry Systems [BDIS], San Jose, CA) and B4 FITC/PE (Coulter
Clone, Miami, FL) as previously described.9 Generation of
allele-specific oligonucleotides (ASOs) IgH PCR assays were performed
as previously described.8 For real-time ASO PCR, a forward
CDR3 primer was designed to span the V-D junction, and the CDR3 TaqMan
probe was designed to span the D-J junction, giving maximum specificity
of the assays (Table 1). Real-time ASO
IgH PCR and CD19 single-cell RT-PCR assays were performed as previously
described.10-12
[View Larger Version of this Image (22K GIF file)]
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Results and discussion |
Kinetics of polyclonal and clonal cells during treatment
The level of clonal cells in PBMNCs was determined in a cohort of
patients with MM at the time of diagnosis and during treatment by a
quantitative real-time ASO IgH PCR assay10 (Figure
1). At the time of diagnosis, the level
of clonally related cells in PB ranged from less than 0.00002 to 2020 clonal cells per microliter blood (n = 9, mean 282, median 15). These
findings are very similar to those reported by Billadeau et
al13 and Kay et al.3 The proportion of clonal
cells (less than 0.00001% to 30.55% of PBMNCs) was reduced to less
than 0.5% of PBMNCs in all patients tested (n = 7) after induction
therapy, regardless of the initial level of clonal cells (Figure 1).
However, the level of clonal cells after induction therapy appeared to
be stable before and after PB stem cell (PBSC) transplantation. This is
in accordance with the findings of Billadeau et al.14
However, the observed initial reduction and low numbers of residual
PB-localized clonal cells after chemotherapy is in conflict with recent
reports.6,15 The numbers of CD19+ cells
differed greatly both between patients at diagnosis (4 to 1790 cells/µL blood, mean 323, n = 27) and in individual patients (0.08 to 324 cells/µL blood, Figure 1, MM-4). In general, low numbers of
CD19+ cells were seen during induction and high-dose
treatment, followed by a significant increase in CD19+
cells after transplant (Figure 1).
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| Figure 1.
Levels of CD19+ and clonal cells among
PBMNCs.
The level of CD19+ ( ) and clonal cells
( ) among PBMNCs as determined by flow cytometry and real-time ASO
IgH PCR. In patient MM-1, the level of CD19 mRNA ( ) was measured by
quantitative real-time CD19 RT-PCR. The amount of CD19 mRNA is given as
the fraction of  -actin mRNA. Serial PB samples were obtained at
diagnosis, after 3 cycles of VAD, after cyclophosphamide (Cy), before
(pre-Tx) and after PBSC transplantation.
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Clonal cells with aberrant CD19 expression
In patient MM-1, the numbers of CD19+ cells (170 cells/µL blood) and myeloma plasma cells (PCS) (10 cells/µL blood)
at the time of diagnosis was far below the level of clonal cells
detected in PB (2020 cells/µL blood). By following the levels of CD19
messenger RNA (mRNA), using real-time CD19 RT-PCR (Figure 1), we
observed a high level of CD19 mRNA at diagnosis that was markedly
reduced after induction therapy, reaching a minimum level at 3 months after transplant. This correlated with the clonal cell levels. In
general, numbers of CD19+ cells were not at variance with
the level of CD19 mRNA at the time of diagnosis. However, when looking
at patient MM-1, a clear discrepancy was observed between the number of
CD19+ cells and the level of CD19 mRNA, at the time of
diagnosis. After transplant, a simultaneously rise in CD19+
cells and CD19 mRNA was observed, and at this time, the frequency of
CD19+ cells correlated with the level of CD19 mRNA. To
assess whether these cells had cytoplasmic CD19 protein, surface
staining was performed with B4 FITC, followed by intracellular staining
with Leu-12 PE. At the time of diagnosis both a population of cells with high-scatter properties, having only cytoplasmic CD19 protein (610 cells/µL blood), and a population of lymphoid cells (170 cells/µL
blood), having both surface and cytoplasmic CD19 protein, were present.
After VAD treatment, no cells with only cytoplasmic CD19 protein was
observed. The correct cellular localization of the CD19 MoAbs tested
was confirmed by confocal microscopy. In addition, the frequency of
CD19 mRNA+ cells in the monocyte gate was determined using
a single-cell CD19 RT-PCR assay.12 In both healthy donors
and patients with MM (5 each), the frequency of CD19+
(mRNA+) cells was between 1:100 to 1:1000 (data not shown),
probably due to the presence of activated B cells. Although we had
previously observed a patient with MM similar to MM-1 with abundant
CD19
/CD19 mRNA+ clonal cells,12
the frequency of these patients seems low. Thus, their rare existence
does not fully explain the conflicting reports regarding the consistent
findings of the high numbers of aberrant CD19 clonal
cells.16
In contrast to normal plasma cells, the myeloma cells at
one stage lose CD19 surface expression before final differentiation into mature myeloma PCS.17 It has been shown in
transfection experiments with myeloma cell lines that surface
expression of CD19 introduces growth regulation in myeloma cell
lines.18 It is tempting to speculate that the position in
the clonal hierarchy of the CD19/CD19 mRNA+
cells is at the point where the myeloma cells select against the CD19
surface expression to gain a growth advantage. These cells may be
transient or present in all patients with MM in low numbers, making
them difficult to detect.
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Footnotes |
Submitted October 1, 1999; accepted July 31, 2000.
Supported by the Danish Cancer Society, The Meyer Foundation, and the
Desirè & Niels Ydes Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Thomas Rasmussen, Department of Hematology L 54P4,
Herlev Hospital, University of Copenhagen, DK-2730 Herlev, Denmark;
email: thra{at}herlevhosp.kbhamt.dk.
| |
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Abstract
Introduction
