In Multiple Myeloma, Clonal Cells Are Unevenly Distributed among Peripheral Blood B Cell Subpopulations.

Abstract

BACKGROUND: Current evidence supports the existence of circulating clonal B cells in Multiple Myeloma (MM). However, attempts to enumerate and phenotypically characterise them have so far provided inconsistent results. Most investigators have studied unselected peripheral blood (PB) mononuclear cells, among which the clonal ones make only a small minority. Moreover, in most cases, numerical chromosomal changes were employed as a clonal marker, but aneuploidy is considered a late event in myelomagenesis. To overcome these difficulties, we have followed an alternative approach, by studying purified PB B cells and focusing on chromosomal translocations involving the immunoglobulin heavy chain gene (IGH) on region 14q32, a frequent, early and possibly crucial pathogenetic event in MM. METHODS: The study included 33 MM patients with 14q32 rearrangements, detected by conventional cytogenetics or florescence in-situ hybridisation (FISH) in the bone marrow at diagnosis. PB CD19+ cells were immunomagnetically isolated (>99% purity) and cytocentrifuged on slides. The slides were studied with a FICTION technique, ie a combination of FISH using a “break-apart” IGH probe set (Vysis Inc, Downers Grove, Il, USA) and indirect immunofluorescence for CD34, CD5, CD10, CD23 and CD38. To avoid the possibility of contaminating plasma cells, isolates with >1% CD19+CD38+++ cells on flow cytometry were excluded from FICTION study. RESULTS: “Positive” cells above the cutoff level of false positivity (4%) were detected in 25 cases (75.7%), ranging from 4% to 33% (median 9%) among the total CD19+ population. These cells were found to consistently express CD10 (83% to 100%, median 96%) and CD38 (79% to 100%, median 89%). They less commonly expressed CD23 (39% to 67%, median 46%) and very rarely CD34 (0% to 5%, median 0%) and CD5 (0% to 3%, median 0%). CONCLUSIONS: Our data suggest that circulating cells bearing IGH rearrangements are the rule in MM, making a small but detectable fraction of CD19+ cells. There mmunophenotypic profile supports the concept that clonal B cells represent advanced ontogenetic rather than early stages in B lineage differentiation. Finally, the virtual absence of CD5+ clonal cells is in accord with the view that the high number of PB CD5+ B cells in MM reflects an immunoregulatory network and does not result from clonal expansion.