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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2844-2855
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
Agnieszka J. Szczepek,
Karen Seeberger,
Juanita Wizniak,
Michael
J. Mant,
Andrew R. Belch, and
Linda M. Pilarski
From the Departments of Oncology and Medicine, University of Alberta
and the Cross Cancer Institute, Edmonton, Alberta T6G1Z2 Canada.
In multiple myeloma (MM), the VDJ rearrangement of the
immunoglobulin heavy chain expressed by MM plasma cells provides a unique clonotypic marker. Although clonotypic MM cells have been found
in the circulation, their number has been controversial. Our objective
was to provide direct evidence, using single-cell assays, for the
frequency of clonotypic cells in blood of 18 MM patients, and to
confirm their identity as B cells. The clonotypic Ig heavy-chain
(IgH) VDJ was determined from single plasma cells using
consensus reverse transcriptase-polymerase chain reaction (RT-PCR),
subcloning, and sequencing. For all patients, using patient-specific
primers, clonotypic transcripts were amplified from 10 or more
individual plasma cells. Using in situ RT-PCR, for all patients greater
than 80% of plasma cells were found to be clonotypic. Three separate
methods, RT-PCR, single-cell RT-PCR, and in situ RT-PCR, were used to
analyze clonotypic cells in peripheral blood mononuclear cells (PBMC)
from MM patients. Sequencing of the IgH transcripts expressed by
individual cells obtained by limiting dilution of freshly isolated PBMC
from a MM patient showed that all B cells expressed an identical CDR3.
This intraclonal homogeneity indicates an escape from
antigenic-selection, characteristic of malignant B cells. For this
patient, the frequency of clonotypic PBMC, about 25%, was comparable
to the number of PBMC B cells (34%). Because the PBMC included less
than 1% plasma cells, virtually all clonotypic PBMC must be B cells.
Using single-cell RT-PCR, clonotypic IgH transcripts were identified in
individual sorted B cells from blood. To accurately quantify the number
of clonotypic B cells, sorted B cells derived from 18 MM patients (36 samples) and 18 healthy donors (53 samples) were analyzed using in situ RT-PCR with patient-specific primers. Clonotypic transcripts were not
detectable among normal B cells. For the 18 MM patients, a mean of 66% ± 4% (SE) of blood B cells were clonotypic (range, 9% to 95%),
with mean absolute number of 0.15 ± .02 × 109/L blood.
Over time in individual patients, conventional chemotherapy transiently
decreased circulating clonotypic B cells. Their numbers were increased
in granulocyte colony-stimulating factor (G-CSF)- mobilized
blood of one patient. However, clonotypic B cells of a one patient
became undetectable after allogeneic transplant, correlating with
complete remission. Although contributions to MM spread and progression
is likely, their malignant status and impact has yet to be clarified.
Their high frequency in the blood, and their resistence to conventional
chemotherapy suggests that the number of circulating clonotypic cells
should be clinically monitored, and that therapeutic targeting of these
B cells may benefit myeloma patients.
© 1998 by The American Society of Hematology.
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