The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45- CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).
Mutations of members of the ras family are among the most common oncogene mutations found in multiple myeloma (MM). We have examined the mutational status of the N- and K-ras genes at codons 12, 13, and 61 in 160 newly diagnosed MM patients enrolled on the Eastern Cooperative Oncology Group (ECOG) phase III clinical trial E9486. The total incidence of ras mutations was found to be 39% of the samples analyzed. Five patients showed evidence of more than one mutation. We obtained 22 marrow samples from patients at the time of disease progression or relapse, for whom a ras mutation was identified at diagnosis. In all cases, the ras mutation of the disease progression sample was identical to that found at diagnosis. In contrast, three of 25 patients who did not show any ras mutation at diagnosis acquired a ras mutation at the time of disease progression. No significant association was observed between any ras mutation and stage of disease, beta 2-microglobulin levels, labelling index, or protein type. The mean tumor burden and median survival for patients with mutations of N-ras was indistinguishable from patients with no ras mutations. However, patients with K-ras mutations had a significantly higher mean bone marrow tumor burden at diagnosis than patients with no ras mutations (57% v 36%, P ‼ .02); and the median survival of patients with a K-ras mutation was significantly shorter (2.0 v 3.7 years, P ‼ .02). To determine if the status of ras mutations could affect treatment response, we examined patient survival on the three treatment arms of E9486. Although the presence of a ras mutation in the multidrug treatment, VBMCP alone, showed a marginal significance, neither the VBMCP, nor the addition of interferon-alpha showed statistically significant survival differences between mutant and wildtype ras status. Interestingly, there appeared to be a statistically significant difference in survival of patients treated with VBMCP and alternating high doses of cyclophosphamide + prednisone. Patients with ras mutations had a median survival of 2.1 years; patients with wild-type ras had a median survival of 4.0 years (P ‼ .01).
The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45- CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).
The junctional sequences corresponding to the complementarity determining region 3 (CDR3) of rearranged heavy chain Ig genes can provide allele-specific markers in the detection of B-lymphoid malignancies. Consensus oligonucleotide primers were used to amplify CDR3 regions of rearranged heavy chain alleles in clinical samples from myeloma, acute lymphocytic leukemia, and chronic lymphocytic leukemia patients. From the sequence of the amplified products, allele-specific primers were synthesized and used directly in polymerase chain reaction (PCR) amplification to detect the malignant clone. This method was both highly specific and sensitive to 1 malignant B-cell in a background of 10(5) normal cells. In addition, parameters that affect the linearity of PCR detection were determined and, by using titrations of malignant target cells to generate standard curves, quantitations of residual malignancies were determined. The application of this method is shown in an analysis of myeloma patients whose marrows were analyzed sequentially during therapy. Allele-specific oligonucleotide-PCR provided a rapid, highly specific and quantitative measure of residual disease, even in patients with clinical parameters indicating complete remission.
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