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.
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 x 10(9)/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.
Departments of Oncology and Medicine, University of Alberta and the Cross Cancer Institute, Edmonton, Alberta T6G1Z2 Canada.
A J
AJ
Szczepek
K
K
Seeberger
J
J
Wizniak
M J
MJ
Mant
A R
AR
Belch
L M
LM
Pilarski
D016175:B-Lymphocyte Subsets / Q000276:immunology*; D001853:Bone Marrow / Q000473:pathology*; D016026:Bone Marrow Transplantation; D002999:Clone Cells / Q000276:immunology; D003114:Colony-Forming Units Assay; D004273:DNA, Neoplasm / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D017403:In Situ Hybridization; D009101:Multiple Myeloma / Q000276:immunology* / Q000473:pathology; D009194:Myeloma Proteins / Q000235:genetics*; D009360:Neoplastic Cells, Circulating / Q000276:immunology*; D010950:Plasma Cells / Q000276:immunology*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004273:DNA, Neoplasm; D007143:Immunoglobulin Heavy Chains; D009194:Myeloma Proteins; D016179:Granulocyte Colony-Stimulating Factor