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), Materials and 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 realtime 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. Conclusion, The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.