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Controversies surrounding the clonogenic origin of multiple myeloma

2000; Wiley; Volume: 110; Issue: 1 Linguagem: Inglês

10.1046/j.1365-2141.2000.02072-6.x

1365-2141

Faith E. Davies, Andy C. Rawstron, Roger G. Owen, Gareth J. Morgan,

Monoclonal and Polyclonal Antibodies Research

The involvement of the B-cell compartment in multiple myeloma has been the subject of intense debate over the last decade. During this time, it has largely been of theoretical interest, however, following the description of the beneficial clinical effects of directed therapy using anti-CD2O in non-Hodgkin's lymphoma, it is now of practical interest. The important question is whether these cells are clonogenic, part of a premyeloma phase or clonally related memory B cells that share identical V gene sequences. In recent issues of this journal, two papers have addressed this question and correlated the presence of clonogenic B cells in cell harvests and peripheral blood with outcome after autologous transplantation. Mitterer et al (1999) suggest that the presence of clonally related B cells in peripheral blood stem cell harvests correlates with outcome after high-dose chemotherapy. Peripheral blood stem cell harvests were purged with a B-cell antibody panel and the ‘waste’ B-cell fraction examined for the presence of clonogenic cells. The cases were separated into three groups dependent upon the immunoglobulin heavy chain polymerase chain reaction (PCR) pattern observed (clonal, oligoclonal and polyclonal) and the cases that displayed a monoclonal pattern were associated with early disease relapse. The numbers in the study were very small and the interpretation of the PCR results is open to question. Specifically, the authors define an oligoclonal group where determining the nature of a clonal peak within a skewed distribution of B cells is not possible. We have previously shown, using the same technique, that it is only possible to detect a clonal population of cells when they represent 2% or more of a polyclonal B-cell background and that the sensitivity is equivalent to three-colour flow cytometry at 1–104−6 ( Rawstron et al, 1997 ). In this study, only 32% of the cases demonstrated a true clonal rearrangement consistent with clonally related cells being present at a level of > 2% of the B cells. The results of the second B-cell purge were also informative; a background pattern of V gene rearrangements was present after the first purge within which a clonal rearrangement was present, this background pattern was not present after the second cycle and a small clonal band was seen. A plausible explanation for this is that the B cells are removed in the first purging cycle and that the small band represents contaminating myeloma plasma cells. The purity of the ‘B-cell-enriched fraction’ is not truly known as flow cytometry after the release of bound antibody is difficult and no morphological data were presented, making the differentiation of clonal B cells from contaminating plasma cells difficult. These results therefore cannot be interpreted as a convincing demonstration that the presence of B cells clonally related to the myeloma plasma cell lead to relapse. A more likely explanation is that the clonal pattern seen represented contamination by myeloma plasma cells and that the poor disease outcome relates to disease bulk at the time of transplantation. In the second paper, Rottenburger et al (1999) used magnetic beads and flow cytometry to enrich for CD19+ and CD2O+ cells in the peripheral blood of patients after high-dose therapy. The B-cell subsets were then examined for the presence of clonotypic cells using a semiquantitative ASO-PCR and the results were related to disease outcome. The calculation of tumour load was based on the assumption that a single clonal cell would yield a positive PCR result. Unfortunately, the purity of the populations examined ranged from 78.3% to 98.4%, implying that contamination by clonal plasma cells is a likely event, again raising the possibility that the clonogenic cells detected using the very sensitive PCR were, in fact, contaminating plasma cells and not clonogenic B cells. This conclusion was supported by their results, which showed that the number of clonally related cells was not significantly different between the CD19- and CD2O-positive and -negative fractions from cases in remission. This contrasts with the patients at relapse who had significantly more clonally related cells in the CD19- and CD2O-negative fraction. Previous studies have demonstrated that clonally related cells are present in the B-cell fraction, but that these are at low levels ( Chen & Epstein, 1996). These data taken together with the results of the two studies described above illustrate the technical problems associated with obtaining a 100% pure B-cell population with no tumour cell contamination. Therefore, the only acceptable approach to studying such cells is by simultaneous determination of phenotype and clonal identity. We have previously reported a slide-based method of directly examining the bone marrow for clonogenic B cells ( Davies et al, 1999 ). In this technique, we combined morphology with immunophenotyping and fluorescence in situ hybridization (FISH) to examine for clonal genetic abnormalities. In the 13 cases examined, although myeloma plasma cells showed aneuploidy, no abnormalities were demonstrated within the B-cell compartment. We concluded that if the B-cell compartment did contain clonogenic cells then these were at a low level (95% confidence interval 3.03%) and unlikely to contribute significantly to the disease course. This is in agreement with the data from Chen & Epstein (1996), who demonstrated that clonally related B cells account for 0.04–5% of total CDl9 cells. The most important question, regardless of the controversy over the exact nature of the clonally related cells within the B-cell compartment, is whether such cells are truly clonogenic and can contribute to disease relapse. There are few experimental data that directly address this question, however a recent report of a myeloma SCID-hu murine model showed that, although myeloma plasma cells were able to produce disease within the host, B cells isolated from patients and injected into the same model were unable to produce such an effect ( Yaccoby & Epstein, 1999). This suggests that if clonogenic cells are present within the B-cell compartment they are not capable of proliferation and self-renewal and therefore are not an appropriate target for B-cell antibody-directed therapy. Such cells may in fact represent memory B cells carrying the same clonal rearrangement as the myeloma plasma cell.