Blood, 1 May 2001, Vol. 97, No. 9, pp. 2913-2914
CORRESPONDENCE
To the editor:
Clonotypic B cells in the peripheral blood of patients with
multiple myeloma
The frequency and the biologic significance of circulating
clonal cells expressing CD19 in patients with multiple myeloma (MM)
remain an issue of controversy. So far, in a small number of patients,
the proportion of circulating clonotypic cells has been determined
using different methods based on IgH-specific primers, and highly
divergent results were reported.1,2
Recently, a brief report from Rasmussen et al also addressed this
question.3 In this paper, the numbers of clonal cells in
the peripheral blood (PB) from patients with MM were investigated by
quantitative real-time polymerase chain reaction (PCR). The levels of
CD19+ cells in PB were assessed by flow cytometry,
and the CD19 mRNA was quantitated by real-time RT-PCR.
With this approach, the frequency of clonotypic B cells cannot be
determined. Thus, it remains unclear whether
and if so, in
what proportionthe CD19+ or the CD19
CD19-mRNA-positive cells described by Rasmussen et
al3 belong to the malignant clone. To address this
question, an assessment of clonality in sorted CD19+ and
CD19 cell fractions is necessary.
Using a quantitative PCR assay with allele-specific oligonucleotides
(ASOs) based on the method of limiting dilutions,4 we
investigated the kinetics of clonotypic cells in the PB in the course
of a sequential high-dose therapy (HDT) regimen. The absolute levels of
clonotypic cells were found to be very low prior to HDT (median,
0.004%; n = 20). But we observed a significant decrease in the
proportion of circulating clonotypic cells after the first cycle of HDT
(median, from 65 to 2.7 cells/mL PB; n = 20). The second cycle did
not lead to a further reduction (median, 3.5/mL;
n = 20).5 Upon analyzing highly purified
CD19+ and CD19 fractions of PB, a significant
reduction of clonotypic cells was observed after one cycle of HDT only
in the CD19 cell fractions (53/mL, n = 8,
before, compared with numbers below the detection limit, n = 10,
afterward), while the median number of CD19+ clonotypic
cells remained stable (0.92/mL before, compared with 1.05/mL
afterward). With disease progression, an expansion of CD19 and CD19+ cells occurred (63/mL and
21/mL, respectively; n = 7).6 Because different
properties of the antibodies used against CD19 are discussed to explain
the heterogenous findings regarding the frequency of circulating
clonotypic B cells, highly purified B-cell fractions were obtained
using CD20 as a second pan-B-cell marker. The proportions of
clonotypic cells found did not differ significantly from those found in
CD19-sorted cell fractions.7
Taking these results together, we conclude that the absolute numbers of
circulating clonotypic cells in MM are small and that, prior to
HDT, most of these cells are CD19. Our results reveal
direct evidence that circulating CD19+/CD20+
clonotypic B cells seem to be resistant even to sequential HDT and
comprise the vast majority of clonotypic cells in the PB of patients in
remission after HDT. An expansion of CD19+
clonotypic cells and an even more pronounced expansion of
CD19 clonotypic cells occur with disease progression.
These findings were the rationale for a B-cell-directed consolidation
therapy for patients in remission after autologous
transplantation.8
Friedrich W. Cremer, Hartmut Goldschmidt, and Marion Moos
Department of Internal Medicine V University of
Heidelberg Heidelberg, Germany
References
1.
Szczepek AJ, Seeberger K, Wizniak K, et al.
A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.
Blood.
1998;92:2844-2855[Abstract/Free Full Text].
2.
Chen BJ, Epstein J.
Circulating clonal lymphocytes in myeloma constitute only a minor subpopulation of B cells.
Blood.
1996;87:1972-1976[Abstract/Free Full Text].
3.
Rasmussen T, Jensen L, Honoré L, Johnsen HE.
Frequency and kinetics of clonal and polyclonal B cells in the peripheral blood of patients being treated for multiple myeloma.
Blood.
2000;96:4357-4359[Abstract/Free Full Text].
4.
Cremer FW, Kiel K, Wallmeier M, Goldschmidt H, Moos M.
A quantitative PCR assay for the detection of low amounts of malignant cells in multiple myeloma.
Ann Oncol.
1997;8:633-636[Abstract/Free Full Text].
5.
Cremer FW, Ehrbrecht E, Benner A, et al.
Evaluation of the kinetics of bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as predictive factor in patients with multiple myeloma.
Bone Marrow Transplant.
2000;26:851-858[CrossRef][Medline]
[Order article via Infotrieve].
6.
Kiel K, Cremer FW, Rottenburger C, et al.
Analysis of circulating tumor cells in patients with multiple myeloma in the course of high-dose therapy with peripheral blood stem cell transplantation.
Bone Marrow Transplant.
1999;23:1019-1027[CrossRef][Medline]
[Order article via Infotrieve].
7.
Rottenburger C, Kiel K, Bösing T, et al.
Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation.
Br J Haematol.
1999;106:545-552[CrossRef][Medline]
[Order article via Infotrieve].
8.
Cremer FW, Gemmel C, Witzens M, et al.
Treatment with the anti-CD20 antibody rituximab as consolidation therapy for patients with multiple myeloma after peripheral blood (PB) stem cell transplantation (SCT) [abstract].
Blood.
2000;96:298b.
Response:
Controversies surrounding the circulating clonotypic B
lymphocytes in multiple myeloma
Since the article from Pilarski's laboratory in 1995 reporting
the presence of high numbers of clonotypic CD19+ B
lymphocytes in the peripheral blood of patients with
myeloma,1 numerous groups have reported conflicting data
on the frequency and characteristics of circulating clonotypic B
lymphocytes.2,3 Recently, we published a paper dealing
with the frequency and response to treatment of circulating clonotypic
cells in myeloma.4 In their letter, Cremer et al question
the rationale for this study, since no data were reported on the
frequency of clonotypic cells within a purified CD19+
population of B lymphocytes.
We fully agree with Cremer et al that, in order to determine the
frequency of CD19+ clonotypic cells, it is necessary to
perform allele-specific oligonucleotide (ASO) polymerase chain reaction
(PCR) on purified CD19+ cells. Actually, we did determine
the frequency of clonal cells in the CD19 compartment in a cohort of
patients with myeloma, including all patients in the reported study, by
combining single-cell ASO IgH RT-PCR with flow sorting and Poisson
statistics. These data were obtained to verify a rationale for CD19
depletion of stem cell products from patients with myeloma. But the
data were not included in the paper since they did not contain novel
information concerning the controversial aspects addressed in the paper.
We determined the proportion of clonal CD19+ cells
after peripheral blood stem cell (PBSC) transplantation in 10 patients
and found variable levels of clonal cells in the CD19 compartment after
transplantation, ranging from 0.101% to 6.103% (mean, 1.019%) of
CD19+ cells. This is comparable to the clonal cell levels
reported by Chen and Epstein,2 who found levels from
0.04% to 5.00% (mean, 1.60%). Rottenburger et al5
reported PCR negativity in CD19+-enriched fractions in
approximately 50% of patients with multiple myeloma (MM) in remission.
In contrast, we found clonal cells in the CD19+ compartment
in all 10 patients with MM observed in continuous remission for at
least 3 months. Pilarski's group reported that cells originating from
the lymphocyte gate contained a polyclonal B-lymphocyte population with
a minor fraction of clonotypic CD19+ cells.1
In general, nearly all CD19+ cells that we have identified
in patients with myeloma originate from the lymphocyte gate. The
frequency of clonotypic cells that we identified within this
CD19+ lymphocyte gate was comparable to that found both by
Pilarski's group1,3 and Chen and Epstein2;
thus, on this point, there is no controversy.
Pilarski's group reported the controversial finding that most
cells originating from the monocyte gate were clonotypic, expressing CD34 and CD19 but with an altered CD19 epitope. We have addressed these
issues previously using ASO IgH RT-PCR on cells originating from both
the lymphocyte and monocyte gate.6,7 In a single case, we
identified a high frequency of clonal cells, phenotypically distinct
from the myeloma plasma cells. These clonal cells expressed CD19 mRNA
and lacked or had an altered CD19 surface expression, obscuring their
detection by flow cytometry.8 For further investigation of
this observation, we used quantitative real-time CD19 RT-PCR in
combination with flow cytometry, as reported.4
The high numbers of circulating clonal cells found by Pilarski's group
were also reported to be insensitive to chemotherapy.9,10 In the published study, we showed that in general, there is a low
frequency of circulating clonal cells in patients with myeloma, and most importantly, we showed that circulating clonal cells respond
well to induction therapy, even in situations where high numbers of clonal cells were present. Although most circulating clonal
cells responded to chemotherapy, we have (like Cremer et al) also found
a small proportion of clonal CD19+ cells that are
resistant to high-dose chemotherapy. But whether there is a
rationale for consolidation therapy using anti-CD20 antibodies is
questionable, since there is no proof that the clonotypic B lymphocytes
are involved in the pathogenesis of the disease. Additionally, our
ongoing studies on the nature of the circulating CD19+
clonotypic B lymphocytes have resulted in identification of a CD19+/CD20 subset (Figure
1).
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| Figure 1.
ASO IgH RT-PCRs were performed on a serial dilution of
CD19+/CD20+ and
CD19+/CD20 cells (performed by a cell
sorter).
The result shown illustrates that therapies using anti-CD20
antibodies only target a subset of the clonotypic B lymphocytes.
|
|
Thomas Rasmussen and Hans Erik Johnsen
Department of Hematology L Herlev Hospital
University of Copenhagen Herlev, Denmark
References
1.
Bergsagel PL, Smith AM, Szczepek A, et al.
In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain.
Blood.
1995;85:436-447[Abstract/Free Full Text].
2.
Chen BJ, Epstein J.
Circulating clonal lymphocytes in myeloma constitute a minor subpopulation of B cells.
Blood.
1996;87:1972-1976.
3.
Szczepek AJ, Seeberger K, Wizniak J, et al.
A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.
Blood.
1998;92:2844-2855.
4.
Rasmussen T, Jensen L, Honoré L, Johnsen HE.
Frequency and kinetics of polyclonal and clonal B cells in the peripheral blood of patients being treated for multiple myeloma.
Blood.
2000;96:4357-4359.
5.
Rottenburger C, Kiel K, Bösing T, Cremer FW.
Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation.
Br J Haematol.
1999;106:545-552.
6.
Rasmussen T, Jensen L, Honoré L, Andersen H, Johnsen HE.
Circulating clonal cells in multiple myeloma do not express CD34 mRNA, as measured by single-cell and real-time RT-PCR assays.
Br J Haematol.
1999;107:818-824[CrossRef][Medline]
[Order article via Infotrieve].
7.
Rasmussen T, Jensen L, Johnsen HE.
Levels of circulating CD19+ cells in patients with multiple myeloma.
Blood.
2000;95:4020-4021[Free Full Text].
8.
Rasmussen T, Kastrup J, Knudsen LM, Johnsen HE.
High numbers of clonal CD19+ cells in the peripheral blood of a patient with multiple myeloma.
Br J Haematol.
1999;105:265-267[Medline]
[Order article via Infotrieve].
9.
Pilarski LM, Belch AR.
Intrinsic expression of the multidrug transporter, P-glycoprotein 170, in multiple myeloma: implications for treatment.
Leukem Lymphom.
1995;17:367-374[Medline]
[Order article via Infotrieve].
10.
Pilarski LM, Belch AR.
Circulating monoclonal B cells expressing P glycoprotein may be a reservoir of multidrug-resistant disease in multiple myeloma.
Blood.
1994;83:724-736[Abstract/Free Full Text].
