Quantitation of minimal residual disease in multiple myeloma using an allele-specific real-time PCR assay.
OBJECTIVE
To develop a real-time PCR method, based on the 5'nuclease TaqMan technology, for quantitation of clonal cells in multiple myeloma (MM).


METHODS
The real-time quantitative PCR method incorporates both an allele-specific oligonucleotides (ASO) primer and an ASO dual-labeled fluorogenic probe (ASO TaqMan probe). The ASO primer and probe corresponded to the complementary determining region 3 (CDR3) of the rearranged immunoglobulin heavy chain gene (IgH). With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-time PCR method was compared with flow cytometric quantitation of myeloma plasma cells.


RESULTS
The application of the real-time quantitative ASO IgH PCR method is illustrated by a sequential analysis of minimal residual disease (MRD) in bone marrow (BM) samples from myeloma patients undergoing peripheral blood stem cell (PBSC) transplantation. The real-time PCR method was able to quantitate residual malignant cells in BM samples from patients who were considered to be in complete remission. Further, it was illustrated that a potential problem in determining tumor cell content in myeloma BM samples is the heterogeneous infiltration of the marrow.


CONCLUSIONS
The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells.
Rasmussen
T
T
Department of Hematology L, Herlev Hospital, University of Copenhagen, Herlev, Denmark. thra@herlevhosp.kbhamt.dk
Poulsen
T S
TS
Honoré
L
L
Johnsen
H E
HE
D000328:Adult; D000483:Alleles; D001853:Bone Marrow / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002999:Clone Cells; D005434:Flow Cytometry; D005500:Follow-Up Studies; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis / Q000235:genetics; D008875:Middle Aged; D009101:Multiple Myeloma / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007136:Immunoglobulins; D009363:Neoplasm Proteins; D011933:Reagent Kits, Diagnostic