title: Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards.
abstract: The complementarity determining region III of the rearranged immunoglobulin heavy chain gene has been the target for tumor-specific PCR assays for the detection and follow-up of B-cell malignancies. Previously, these assays have relied on gel-based end point data collection methods (i.e., band densitometry) and, thus, have provided at best a semiquantitative assessment of tumor levels. We show the development of a novel, real-time TaqMan PCR assay to quantitate residual multiple myeloma cells in clinical samples after high-dose chemotherapy and autologous stem cell transplantation. We provide evidence that real-time PCR is reproducible, sensitive, and quantitative. In a 40-replicate PCR experiment targeting the beta-actin gene, the coefficient of variation for threshold cycle data was 1.6%, whereas it increased to 13.6% and 31%, respectively, for end point fluorescence and gel densitometry. Moreover, in an experiment directly comparing standard curves obtained from band densitometry and threshold cycle data, the standard curve constructed from threshold cycle data had a multiple R2 value of 1.00 and demonstrated a dynamic range >4 logs, compared with the 2-log linear range of gel densitometry. Finally, we show that when a complementarity determining region III-specific PCR primer is used in conjunction with a consensus primer for the immunoglobulin heavy chain joining gene, plasmid DNA can be used as a readily available and effective substitute for clonal plasma-cell genomic DNA when preparing standards. By applying real-time PCR to the analysis of clinical samples, we are able to quantitate levels of tumor involvement with unparalleled reproducibility and statistical confidence. Real-time PCR technology may well provide the accuracy and reliability necessary for minimal residual disease detection to have real prognostic significance.
authors.affiliation: SyStemix, Inc., Palo Alto, California 94304, USA. cgerard@diadexus.com
authors.forename: C J
authors.initials: CJ
authors.lastname: Gerard
authors.forename: K
authors.initials: K
authors.lastname: Olsson
authors.forename: R
authors.initials: R
authors.lastname: Ramanathan
authors.forename: C
authors.initials: C
authors.lastname: Reading
authors.forename: E G
authors.initials: EG
authors.lastname: Hanania
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000175:diagnosis*; D018365:Neoplasm, Residual; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D004247:DNA