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 TaqM an technology, for quantitation of clonal cells in multiple myeloma (MM), Materials and Methods. The real-time quantitative PCR method incorporates b oth an allele-specific oligonucleotides (ASO) primer and an ASO dual-labele d fluorogenic probe (ASO TaqMan probe). The ASO primer and probe correspond ed to the complementary determining region 3 (CDR3) of the rearranged immun oglobulin heavy chain gene (IgH), With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-ti me PCR method was compared with flow cytometric quantitation of myeloma pla sma cells. Results. The application of the real-time quantitative ASO IgH PCR method i s 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 quan titate residual malignant cells in BM samples from patients who were consid ered to be in complete remission, Further, it was illustrated that a potent ial 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 sensitiv e, highly specific, and reproducible quantitation of myeloma cells. (C) 200 0 International Society for Experimental Hematology. Published by Elsevier Science Inc.