A QUANTITATIVE PCR ASSAY FOR THE DETECTION OF LOW AMOUNTS OF MALIGNANT-CELLS IN MULTIPLE-MYELOMA

Background: High-dose chemotherapy (HDT) with autografting of hematopo ietic stem cells induces up to 50% of complete remissions in patients with multiple myeloma. Cases of molecular remissions have been reporte d. However, qualitative assays determine only the absence or presence of a monoclonal population depending on their sensitivity. Therefore r eliable and sensitive methods to quantitate tumor loads are necessary. Materials ann methods: We have established a quantitative PCR assay ( qPCR) with allele-specific primers complementary to hypervariable CDR3 regions. Sample DNA was serially diluted in 0.5 log steps and amplifi ed in 10 replicates. PCR results were analysed by likelihood maximizat ion and chi(2) minimization to calculate the tumor load. Results: Thre e approaches were taken to validate the qPCR. 1) Single copies of the CDR3 region of U266 cells could be detected. 2) Analysis of a bone mar row sample by FAGS for CD 38++ and kappa/lambda restricted plasma cell s and by qPCR yielded results of 1.4 and 2.5% respectively. 3) qPCR re sults with plasmids carrying CDR3 regions simulating different tumor l oads diverged by no more than a factor of 1.6 from the expected values . Conclusion: We consider the qPCR to be an accurate method for assess ing samples with low amounts of malignant cells.