Quantitative Detection Using SYBR Green of Residual Disease in Myeloma Patients Treated with Revlimid or Velcade.

Abstract The treatment of multiple myeloma (MM) with new drugs like Velcade and Revlimid shows promise in the clinic. These two drugs give excellent partial to complete remissions, but the disease invariably returns indicating escape of the malignant clone. The current clinical practices for quantifying minimal residual disease (MRD), such as plasma cell counts, lack needed sensitivity. PCR based assays amplify the clonotypic IgH VDJ gene rearrangement in myeloma and identify all compartments of the malignant clone, providing a unique molecular signature to monitor disease. Every myeloma cell carries one copy of the specific IgH VDJ rearrangement and provides a quantitative measure of the number of malignant cells remaining in each patient. No previous work has quantified MRD after treatment with either Revlimid or Velcade. TaqMan real-time quantitative PCR (rqPCR) assays are able to quantify the level of disease burden but probes are expensive and difficult to design, making this assay unsuitable for routine clinical use. SYBR green, however, is an inexpensive alternative to probes and utilizes the same unique molecular VDJ signature with consistency and reliability (r2=0.9997). PCR product specificity is confirmed using melting curve analysis. This is the first report of the successful use of SYBR green in quantifying the level of malignant disease in MM. The specific clonal rearrangement is identified for each patient. Patient specific primers are designed and verified as being present in the majority of plasma cells using single cell PCR. A cloned IgH VDJ product for each patient provides a standard curve for each patient-specific rqPCR. A cloned B2Microglobulin PCR product provides an independent standard curve to quantify cell number. After correcting for copy number differences, the percentage of malignant cells is calculated by dividing the number of VDJ molecules by the number of B2M molecules. For all tests, the level of residual disease in bone marrow was measured using 150ng of DNA. In patients achieving complete clinical remission during treatment with Revlimid, the SYBR green method detected 0.104% clonal cells with a range of 0.014%–0.26%. In patients with a partial remission, 3.034% clonal cells were detected, with a range of 0.62%–7.79%. A subsequent bone marrow from one Revlimid treated patient revealed no clonal cells this using SYBR Green method. In patients treated with Velcade at the time of relapse, the remaining clonal cells averaged 1.042% with a range of 0.00075%–3.11%. When Velcade was used as a frontline therapy followed by a stem cell transplant, 0.10% clonal cells remained with a range of 0%–0.287%. In one dexamethasone treated patient who achieved a complete remission, 0.018% clonal cells were found. The SYBR green rqPCR assay for patient specific clonal VDJ was shown to reliably quantify residual disease. To date, our results suggest that frontline treatment with Velcade may lead to lower levels of residual disease than treatment at the time of relapse. In Revlimid treated patients, the extent of residual disease correlates with clinical classifications of complete or partial remission. The significance of these low numbers of malignant cells remains to be established, but the persistent occurrence of relapse suggests they are clinically relevant.

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