Clonotypic B cells in the peripheral blood of patients with multiple myeloma.

The frequency and the biologic significance of circulating clonal cells expressing CD19 in patients with multiple myeloma (MM) remain an issue of controversy. So far, in a small number of patients, the proportion of circulating clonotypic cells has been determined using different methods based on IgH-specific primers, and highly divergent results were reported. 1,2 Recently, a brief report from Rasmussen et al also addressed this question. 3 In this paper, the numbers of clonal cells in the peripheral blood (PB) from patients with MM were investigated by quantitative real-time polymerase chain reaction (PCR). The levels of CD19 ϩ cells in PB were assessed by flow cytometry, and the CD19 mRNA was quantitated by real-time RT-PCR. With this approach, the frequency of clonotypic B cells cannot be determined. Thus, it remains unclear whether—and if so, in what proportion—the CD19 ϩ or the CD19 Ϫ CD19-mRNA– positive cells described by Rasmussen et al 3 belong to the malignant clone. To address this question, an assessment of clonality in sorted CD19 ϩ and CD19 Ϫ cell fractions is necessary. Using a quantitative PCR assay with allele-specific oligonucle-otides (ASOs) based on the method of limiting dilutions, 4 we investigated the kinetics of clonotypic cells in the PB in the course of a sequential high-dose therapy (HDT) regimen. The absolute levels of clonotypic cells were found to be very low prior to HDT (median, 0.004%; n ϭ 20). But we observed a significant decrease in the proportion of circulating clonotypic cells after the first cycle of HDT (median, from 65 to 2.7 cells/mL PB; n ϭ 20). The second cycle did not lead to a further reduction (median, 3.5/mL; n ϭ 20). 5 Upon analyzing highly purified CD19 ϩ and CD19 Ϫ fractions of PB, a significant reduction of clonotypic cells was observed after one cycle of HDT only in the CD19 Ϫ cell fractions (53/mL, n ϭ 8, before, compared with numbers below the detection limit, n ϭ 10, afterward), while the median number of CD19 ϩ clonotypic cells remained stable (0.92/mL before, compared with 1.05/mL after-ward). With disease progression, an expansion of CD19 Ϫ and CD19 ϩ cells occurred (63/mL and 21/mL, respectively; n ϭ 7). 6 Because different properties of the antibodies used against CD19 are discussed to explain the heterogenous findings regarding the frequency of circulating clonotypic B cells, highly purified B-cell fractions were obtained using CD20 as a second pan–B-cell …