Summary. There is a wide variation in the degree of marrow and blood involvement between patients with multiple myeloma. Both of these parameters are known to be highly significant prognostic factors, and the differences between patients may be due to variable expression of adhesion molecules. To test this we used three-colour flow cytometry to study three adhesion molecules associated with myeloma, namely CD38, CD56 and CD138. The level of expression of these molecules was compared with the distribution of myeloma plasma cells in bone marrow (n¼59) and peripheral blood (n¼26) in patients at presentation or relapse. The extent of marrow infiltration on the trephine biopsy correlated inversely with CD56 expression (MannWhitney U Test, P¼0·022); there was no difference in CD38 or in CD138 expression. CD56 expression also correlated inversely with the number of circulating plasma cells (linear regression, R 2 ¼0·4268, slope¼¹0·58, P¼0·0003). Peripheral blood plasma cell numbers correlated weakly with bone marrow plasmacytosis, and inversely with CD38 expression. The level of CD56 expression by neoplastic plasma cells was assessed in 37 patients over a median of 11 months (range 6-25). There was no significant change in expression (Wilcoxon Signed Rank, P¼0·6271). We conclude that plasma cell CD56 expression is constant over the course of the disease; unlike CD138 expression, it is significantly linked to the degree of both bone marrow and peripheral blood involvement.

Summary. The reduced levels of normal immunoglobulin in patients with myeloma may be due to suppression of normal B-cell differentiation. However, reports on the numbers of B cells vary, with some finding decreases consistent with immunoparesis, and others reporting expansions of phenotypically aberrant cells. We have therefore assessed the phenotype and levels of B lymphocytes in patients at presentation (n ¼ 23), in plateau or complete remission (PB n ¼ 42, BM n ¼ 18), and in relapse (PB n ¼ 17, BM n ¼ 14), in comparison to normal individuals (n ¼ 10). Phenotypic analysis was performed using five-parameter flow cytometry, with CD14 used to exclude monocytes where necessary.We found no evidence of a phenotypically distinctive blood or marrow B-cell population in patients with myeloma, nor of an increase in the levels of any B-cell subset. Numbers of blood CD19 þ 38 þ normal plasma cell precursors were significantly reduced in presentation/relapse patients, but not in patients in plateau/remission. Total CD19 þ cells were significantly reduced only in patients with circulating myeloma cells, detected by IgH-PCR. In the marrow, CD19þ B cells expressing CD5, CD10, CD34, CD38, CD45 low and Syndecan-1 were significantly decreased at presentation/relapse, but not in patients in plateau/remission. The majority of these antigens are expressed by normal B-cell progenitors, indicating that myeloma also affects the early stages of B-cell development. The suppression of progenitor cells was not restricted to B-lymphoid differentiation, as total CD34 þ cells were also significantly reduced in the marrow of myeloma patients at presentation.These results indicate that, if neoplastic B cells are present in myeloma, they are low in number and have a phenotype similar to their normal counterparts. Furthermore, there is a reversible suppression of CD19 þ B lymphocytes that correlates inversely with disease stage, and specifically affects the early and late stages of normal B-cell differentiation.

Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti–IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5− “immature” plasma cells showed the highest levels of CD126 expression, but “mature” VLA-5+ myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS.

Summary. The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA-5 has been reported to identify proliferating precursor' plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA-5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45 1 and CD45 2 cells, however, cycling plasma cells were consistently VLA-5-. There was close correlation between the expression of VLA-5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin-associated molecules CD79a/b (Spearman, n 10, P , 0´0001). In short-term culture, cells that were initially VLA-5-showed increasing VLA-5 expression with time. However, simultaneous analysis of the DNA-binding dye 7-aminoactinomycin D demonstrated that this was not as a result of differentiation, as VLA-51 plasma cells were all non-viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA-5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38 1 CD138 1 VLA-5 2 . These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self-replenishing population.

In peripheral blood stem cell transplantation (PBSCT), the number of CD34+ cells transplanted has been shown to correlate well with both rapidity and durability of engraftment. However, it is clear that engraftment does not necessarily correlate with total CD34+ cell numbers in some patients. Consequently, there is increasing interest in evaluating the role of CD34+ subsets in haemopoietic recovery as a more accurate marker of harvest quality. We analysed the numbers of CD34+ cell subsets, namely Thy‐1+, L‐Selectin+ and CD38−, and correlated this with engraftment in 86 patients undergoing PBSCT. Adequate engraftment was defined as being a platelet count greater than 50 × 109/l and a neutrophil count greater than 1·0 × 109/l. CD34+L‐Selectin+ provided the best prediction of engraftment rapidity, although the improvement over total CD34+ cell dose was minor. Only the dose of CD34+Thy‐1+ cells transplanted correlated with durable engraftment. The probability of adequate 3‐month engraftment increased with the dose of CD34+ cells transplanted, but 10% of patients receiving > 5 × 106/kg still showed poor engraftment at 3 months. However, all patients receiving > 2·5 × 105/kg CD34+Thy‐1+ showed adequate engraftment at this time point. We also demonstrated that CD34+Thy‐1+ progenitors were restricted to the bone marrow under normal conditions and, during stem cell mobilization, their kinetics generally paralleled total CD34+ numbers.