Real-time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma

The majority of patients with multiple myeloma (il IM) have persistence of minimal residual disease (MRD), as determined by polymerase chain reaction (PCR) detection of clonal immunoglobulin H (IgH) gene rearrangements. As a result, PCR analysis has not provided clinically useful prognostic informat ion in myeloma patients. Instead, quantitative PCR approaches are required to predict patient outcomes and assess response to novel treatment strategi es. We adapted real-time PCR technology to quantify myeloma cells using the IgH rearrangement and then assessed the utility of this approach in 29 pat ients with myeloma who had undergone autologous stem cell transplantation. Because of the high cost of producing a specific reporting probe for each p atient, H-chain V-region family-specific consensus probes were used in asso ciation with allele-specific oligonucleotides for PCR amplification. Becaus e of the high frequency with which somatic hypermutation at the immunoglobu lin locus occurs in MM, a number of mismatches occurred between the patient sequences and the consensus probe. However, construction of a limited numb er of probes allowed real-time PCR with a sensitivity of 10(-4) to 10(-5). To validate this method, we extensively evaluated assay accuracy and reprod ucibility. Results indicate that real-time PCR using consensus probes provi des a feasible, accurate, and reproducible method for evaluating MRD in MM and possibly in other differentiated B-cell malignancies, and one that is l ess expensive than the use of patient-specific probes. This technique is be ing used to assess tumor depletion after immunologic purging and changes in tumor burden in patients undergoing stem cell transplantation and novel tr eatment approaches.