Expression of multiple β1 integrins on circulating monoclonal B cells in patients with multiple myeloma

We have previously reported the presence of monoclonal, tumor-related B lineage cells in the blood of myeloma patients. The cells are continuously differentiating, and the majority are at a very late stage of B cell differentiation into plasma cells, consistent with the hypothesis that they comprise a precursor cell subset responsible for disseminating and possibly for relapse of the disease. The pattern of β1 integrin expression on monoclonal B lineage cells from blood and bone marrow of myeloma patients was evaluated using multiparameter flow cytometry in comparison to normal blood or tissue B cells and malignant B cells from B-CLL, B lymphoma, or plasma cell leukemia. The α4 and β1 chains were found on the majority of normal B cells, usually with a higher expression of α4 compared to β1. α5 was detectable at low density on B cells from lymph node, bone marrow, and lamina propria. The α2 and α6 chains are absent on B cells localized in normal lymphoid tissues as well as on normal blood B cells and in vitro activated B cells. In myeloma, the blood B cells express α2, α5 and α6, suggesting important functional differences between these tumor-related B cells and their normal counterparts. The plasma cells located in myeloma bone marrow express no α2, and almost no α6, although they have variable expression of α4, α5, and β1. Thus the end-stage plasma cells appear to lack receptors that would support a propensity for invasion of basement membranes and exit to extravascular spaces. In contrast, the circulating plasmablasts in a patient with plasma cell leukemia make up a large subset of early plasma cells expressing all integrin receptors analyzed, including α2 and α6. Malignant cells from B-CLL and B lymphoma express only the α4 and β integrins, and some B-CLL have very low levels of α3, but no α2, α5, or α6, suggesting that they may be limited to the vascular spaces and do not extravasate, at least for the stages of disease analyzed here. Our results are therefore consistent with a working hypothesis that invasive capacity in myeloma is to be found within the abnormal monoclonal B cells in peripheral blood, which alone among B cells or plasma cells, and like plasma cell leukemia, express multiple β1 integrins, including the α2 and α6 integrin receptors, necessary for cellular translocation across the endothelial basement membranes and into extravascular spaces. This study raises the possibility that expression of, in particular, the α2 and α6 integrins may underlie the invasiveness of these diseases and offers a new perspective for future avenues of clinical intervention.     

MPC-1-CD49e- immature myeloma cells include CD45+ subpopulations that can proliferate in response to IL-6 in human myelomas

Using three-colour phenotypic analysis, we detected five subpopulations of myeloma cells (CD38++) in the bone marrow mononuclear cells of human myeloma patients: MPC-1-CD45-CD49e-, MPC-1-CD45+CD49e-, MPC-1+CD45-CD49e-, MPC-1+CD45+CD49e- and MPC-1+CD45+CD49e+. Most of the myeloma cells did not express CD45 but a few MPC-1- immature myeloma cells and some MPC-1+ myeloma cells expressed CD45 and CD45RO but not CD45RA, whereas all of normal early plasma cells in the peripheral blood, lymph node plasma cells and bone marrow plasma cells expressed CD45 and CD45RA, CD45RB but not CD45RO. In order to clarify the biological character of these myeloma subpopulations, we examined the expression of Ki-67 antigen. Proliferating myeloma cells (Ki-67+) were found in the MPC-1- fractions and the MPC-1-CD45+ fractions rather than MPC-1-CD45- fractions. Next, in order to further clarify the biological difference of two immature subpopulations (MPC-1-CD45-CD49e- and MPC-1- CD45+CD49e-), determined cell viability and phenotypic change after culturing with interleukin 6 (IL-6) in vitro. In the presence of IL-6, MPC-1-CD45+ cells kept their viability more than MPC-1-CD45- cells and some MPC-1-CD45- cells could be converted to MPC-1-CD45+ cells. In conclusion, these data suggest that human myeloma cells are phenotypically subdivided into five subpopulations, and among these subpopulations MPC-1-CD45+CD49e- but not MPC-1-CD45-CD49e- immature cells contain proliferating cells in response to IL-6, and IL-6 can also induce expression of CD45 on MPC-1-CD45- subpopulation of immature myeloma cells.     

CD117 expression in gammopathies is associated with an altered maturation of the myeloid and lymphoid hematopoietic cell compartments and favorable disease features

Aberrant CD117 expression is associated with a favorable outcome in multiple myeloma. We analyzed 106 patients with symptomatic multiple myeloma (n=50), smoldering multiple myeloma (n=38) and monoclonal gammopathy of undetermined significance (n=18) to elucidate biological features of CD117⁺ versus CD117⁻ monoclonal gammopathies. CD117⁺ (mono)clonal plasma cells were detected in 30% symptomatic multiple myeloma, 45% smoldering multiple myeloma and 72% monoclonal gammopathy of undetermined significance patients. CD117 expression was associated with higher percentages of normal bone marrow plasma cells, CD117⁺ myeloid precursors and CD38⁺ B lymphocytes in all monoclonal gammopathies. Conversely, the number of bone marrow CD34⁺ myeloid cells and peripheral blood neutrophils was reduced among CD117⁺ multiple myeloma but not monoclonal gammopathy of undetermined significance patients.CD117 expression by (mono)clonal plasma cells is associated with uniquely altered patterns of production of hematopoietic bone marrow cells with decreased peripheral blood neutrophil counts and persistence of normal residual bone marrow plasma cells.     

In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA-5 and CD45 expression

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+ and CD45- 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-amino-actinomycin D demonstrated that this was not as a result of differentiation, as VLA-5+ 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+CD138+VLA-5-. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self-replenishing population.     

Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.     

An Increase in MPC-1- and MPC-1-CD45+ Immature Myeloma Cells in the Progressive States of Bone Marrow Plasmacytosis: The Revised Phenotypic Classification of Monoclonal Marrow Plasmacytosis (MOMP-2005)

The heterogeneity of bone marrow plasmacytosis is clearly analyzed by multicolor staining with anti-CD38 antibody. To date, at least 5 subpopulations of plasma cells have been identified in the bone marrow of multiple myeloma (MM) patients with regard to the expression of MPC-1, CD49e (VLA-5), and CD45: MPC-1(-)CD49e(-)CD45(+) proliferative immature cells, MPC-1(-)CD49e(-)CD45(-) immature myeloma cells, MPC-1(+)CD49e(-)CD45(-) and MPC-1(+)CD49e(-)CD45(+) intermediate myeloma cells, and MPC-1(+)CD49e(+)CD45(+) mature myeloma cells. We performed phenotypic analyses in 75 cases of monoclonal bone marrow plasmacytosis, including 46 cases of MM and 29 cases of monoclonal gammopathy of undetermined significance (MGUS). In 31 cases of progressive MM disease, MPC-1(-) immature and MPC-1(-)CD45(+) proliferative immature myeloma cells were significantly increased up to >25% and >10%, respectively, of the plasma cell fractions (CD38(++) cells), whereas there were no increases in MPC-1(-) or MPC-1(-)CD45(+) proliferative immature myeloma cells in 15 cases of stable disease. Interestingly, the proportions of MPC-1(-) and MPC-1(-)CD45(+) immature monoclonal plasma cells also increased in the 7 progressive cases of MGUS. Finally, we present the revised (2005) phenotypic classification of monoclonal marrow plasmacytosis (MOMP-2005).     

Normal and malignant human plasma cells: proliferation, differentiation, and expansions in relation to CD45 expression

In this study, we have evaluated the proliferation and the phenotype of human plasma cells of different origins, i.e., from tonsil, peripheral blood, bone marrow as well as plasma cells generated in vitro from memory B cells. We have demonstrated that plasma cells from tonsil, peripheral blood, as well as those generated in vitro, were highly proliferating and presented a homogeneous CD45bright phenotype. In contrast, bone marrow plasma cells were heterogeneous for CD45 expression but their proliferation was restricted to the CD45bright compartment. Subsequently, their CD45 expression decreased with proliferation arrest and final maturation. We also studied the proliferation of abnormal plasma cells, i.e., peripheral blood reactive plasmacytoses and multiple myeloma (MM). All reactive plasmacytoses turned out to be homogeneous expansions of CD45bright plasma cells with unusually high labeling index. In contrast, CD45 expression was heterogeneous in MM as in normal bone marrow. However, a minor CD45bright population was also always the most proliferating one as opposed to a major population of less or non-proliferating myeloma cells characterized by a weaker or a lack of CD45 expression. In conclusion, proliferation is linked to plasma-cell generation and a CD45bright phenotype is the hallmark of the most proliferating normal, reactive as well as malignant plasma cells.     

Relationship between CD45 antigen expression and putative stages of differentiation in B-cell malignancies.

The cell-surface antigen CD45 is a complex family of high-molecular-weight glycoproteins expressed on all lymphohematopoietic cells, but not in the same molecular isoform. This antigen complex is known to exhibit protein tyrosine phosphatase (PTPase) activity and appears to have a role in regulation of cell differentiation. In that CD45 expression parallels stages of differentiation in normal bone marrow B cells, it was of interest to evaluate this process in malignant B cells. Monoclonal antibodies (MoAbs) were used to investigate the quantitative expression of CD45 and CD45RA on the B cells of lymphoid leukemias. Employing standardized flow cytometric methods, it was found that the fluorescence intensity (FI) of immunostained malignant B cells, as a reflection of the antigen content, demonstrated correlations with the putative stage of cell differentiation for malignancies at the earlier stages, but at the later stages, a progressive loss of CD45 was observed. Since this antigen family has been found to display PTPase activity, further investigation of CD45 alterations in malignancies may provide insight into potential regulatory disturbances.     

Extensive flow cytometric characterization of plasmacytoid dendritic cell leukemia cells

OBJECTIVES: Accumulating evidence suggests that non-T, non-B cell CD4+CD56+ neoplasms with lymphoblastic morphology include clinically and immunophenotypically diverse entities. Although their cells of origin or classification are still controversial several entities clearly represent a distinct type of neoplasms that are clinically aggressive. METHODS: In this work we present the immunophenotypic and genotypic features of bone marrow (BM), peripheral blood (PB), lymph node and skin lymphocytes from a patient diagnosed as plasmacytoid dendritic cell leukemia involving the skin, BM, PB, lymph nodes, liver and spleen. For determination of immunophenotypic characteristics of malignant plasmacytoid dendritic cells 73 monoclonal antibodies detecting lineage markers, chemokine receptors, cytokine receptors, activation, and co-stimulatory molecules were used. RESULTS AND CONCLUSION: The malignant cells proved to express CD4+, CD56+ lineage negative leukemia phenotype characteristically positive for CD36, CD38, CD40, CD45, CD45RA, CD68, CD123, CD184, HLA-DR, BDCA2, and granzyme-B corresponding to the preplasmacytoid dendritic cell developmental stage. The presence of CD11a/CD18, CD84, CD91, CD95, alphavbeta5, CDw197, and the absence of CD52 and CD133 in this case can be regarded as additional features of malignant cells. Completing the immunophenotypes with multidrug resistance function can provide additional information for characterizing pDC leukemia.     

Multiple myeloma tumor progression in the 5T2MM murine model is a multistage and dynamic process of differentiation,proliferation, invasion,and apoptosis

At clinical presentation, multiple myeloma (MM) is already a well-established disease. The processes involved in earlier stages are, however, unknown. Here the 5T2MM murine model was used to analyze differentiation, proliferation, invasion, and apoptosis of MM cells during disease progression. Naive mice were injected with 5T2MM cells and from the onset of the experiment 3 mice were killed each week until the end stage. Myeloma cells were isolated from the bone marrow and selected by sequential gating of 5T2MM idiotype(+) cells by flow cytometry. Microscopic analysis of these sorted 5T2MM idiotype(+) cells confirmed their identity as true myeloma cells. Based on serum paraprotein concentration and bone marrow tumor load, 3 disease stages were distinguished: a quiescent stage, an intermediate stage, and an end stage, of slow, moderate, and accelerated tumor progression, respectively. In the quiescent stage, the majority of the myeloma cells were CD45(+)CD138(-)IL-6R alpha(+), corresponding to an immature, invasive, and apoptosis-resistant phenotype. In the end stage the majority of the myeloma cells had differentiated into CD45(-)CD138(+)IL-6R alpha(-) cells, corresponding to a mature, less invasive, and apoptosis-sensitive phenotype. In the intermediate stage a gradual transition from the quiescent toward the end stage was observed. In line with these data, analysis of sorted 5T2MM cells demonstrated a significant decrease in invasive capacity and a significant increase in (dexamethasone-induced) apoptosis sensitivity and in proliferation during the disease progression. These data suggest that myeloma disease progression is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis.     

Galectin-1 supports the survival of CD45RA(-) primary myeloma cells in vitro

The survival and proliferation of human myeloma cells are considered to be heavily dependent on the microenvironment of bone marrow (BM). This study confirmed that galectin-1 (Gal-1) and SDF-1α were produced by bone marrow mononuclear cells of myeloma patients. The addition of Gal-1 and SDF-1α to a serum-free synthetic medium, maintained the viability of primary myeloma cells for 2 weeks similar to that before culture. While Gal-1 reduced the viable cell number in CD45RA(+) B cell lines, it maintained the viability of CD45(−) U266 and CD45RA(−)RO(+) ILKM3 myeloma cell lines in the synthetic medium. This was confirmed with the transfection of the PTPRC (CD45) RA, -RB, or -RO gene into CD45(−) U266 cells. The combination of Gal-1 and SDF-1α significantly induced phosphorylation of Akt and IkB, while the phosphorylation of ERK1/2 was significantly reduced in CD45RA(+) U266 and Raji cells but not CD45(−) or CD45RA(−) U266 cells. Furthermore, we confirmed that Gal-1 bound to CD45RA in CD45RA(+) Raji cells, and also physically interacted with β1-integrin by immunoprecipitation followed by Western blotting and confocal microscopy. The results suggest that Gal-1 has two different actions depending on its binding partner, and supports the survival of CD45RA(−) myeloma cells.     

The proliferative potential of myeloma plasma cells manifest in the SCID-hu host.

The low proliferative activity of myeloma plasma cells prompted the notion that the clonotypic B cells that exist in the blood and bone marrow of all myeloma patients contain the proliferative myeloma cells (stem cell). We have exploited our severe combined immunodeficiency (SCID)-hu host system for primary myeloma to investigate whether myeloma plasma cells are capable of sustained proliferation. Purified CD38(++)CD45(-) plasma cells consistently grew and produced myeloma and its manifestations in SCID-hu hosts (8 of 9 experiments). In contrast, the plasma cell-depleted bone marrow cells from 6 patients did not grow or produce myeloma in SCID-hu hosts. Similarly, whereas plasma-cell containing blood cells from 4 patients grew and produced myeloma in hosts, neither the PC-depleted blood cells from 3 of the patients nor a blood specimen that did not contain plasma cells grew in SCID-hu hosts, regardless of their CD19-expressing cell contents. Also, in hosts injected with blood cells, although the myeloma cells were able to disseminate through the murine host system, they were only able to grow in the human bones within a human microenvironment and were not detectable in the murine blood or other organs. Interestingly, the circulating plasma cells appear to grow more avidly in the SCID-hu hosts than their bone marrow counterparts, suggesting that they represent a subpopulation of the plasma cells in the bone marrow. Although our studies clearly demonstrate the proliferative potential of myeloma plasma cells, they are suggestive, not conclusive, as to the existence of a preplasmacytic myeloma progenitor cell.     

Multiple myeloma: circulating lymphocytes that express plasma cell antigens

The bone marrow and peripheral blood of 14 patients with multiple myeloma were studied with murine monoclonal antibodies that identify antigens on plasma cells (R1-3 and OKT10). Peripheral blood lymphocytes expressing plasma cell antigens were found in six cases. Five of these cases expressed the same antigens that were present on the plasma cells in the bone marrow. Patients that showed such peripheral blood involvement were found to have a larger tumor burden and higher bone marrow plasma cell proliferative activity. In some patients, antigens normally found at earlier stages of B cell differentiation (B1, B2, and J5) were expressed by peripheral blood lymphocytes and/or bone marrow plasma cells.     

In vivo homing and differentiation characteristics of mature (CD45-) and immature (CD45+) 5T multiple myeloma cells

Multiple myeloma, a plasma cell malignancy, is predominantly localized in the bone marrow. These tumoral cells display a heterogeneous expression of CD45. It is, however, unclear which subpopulation is responsible for the homing and outgrowth of the myeloma cells. In this work, we investigated the in vivo homing, proliferation, and differentiation of both CD45+ and CD45- cells in two murine myeloma models.5T2MM and 5T33MM in vivo lines of murine multiple myeloma were used. CD45 and IGF-I receptor expression was analyzed by FACS. Proliferative capacity was assessed by in vivo bromodeoxyuridine incorporation. 5TMM cells were separated into CD45+ and CD45- fractions by MACS. Initial homing was investigated in vivo by tracing of radioactively labeled cells. Myeloma cells were detected by FACS and histology. Osteolytic lesions were analyzed by radiography. Both CD45+ and CD45- 5TMM cells were able to home to the bone marrow, although the migration of the latter subset was lower, which was related to a low IGF-I receptor expression. Recipients of both fractions developed myeloma as evidenced by the presence of serum paraprotein, osteolytic lesions, and bone marrow infiltration by myeloma cells. The tumor load in the recipients of CD45- cells was higher than the CD45+ cells, which could be explained by a lower proliferation rate of the latter population. While the separated cells before injection had a homogenous expression of CD45, cells isolated from the bone marrow of these terminally diseased mice had a heterogeneous expression pattern, indicating an in vivo differentiation pattern of CD45- to CD45+ cells and vice versa. We conclude that both CD45+ and CD45- 5TMM subpopulations contain clonogenic myeloma cells with bone marrow homing and proliferative capacity.     

Selective expression of CD45 isoforms defines CALLA+ monoclonal B-lineage cells in peripheral blood from myeloma patients as late stage B cells.

The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B-lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.     

Transitions in CD45 isoform expression indicate continuous differentiation of a monoclonal CD5+ CD11b+ B lineage in Waldenstrom's macroglobulinemia.

Waldenstrom's macroglobulinemia (WM) has been hypothesized to be a pleomorphic B-cell malignancy with persistent maturation towards plasma cells in all lymphoid tissue. This proposal is based on detection of a heterogeneous density of monoclonal Ig on peripheral blood B-cells in patients with WM. We now present data derived from 2- and 3-color immunofluorescence and flow cytometric analysis that strongly supports this hypothesis. Abnormally high numbers of B lineage cells, defined by expression of CD19, CD20, and CD24, were found among peripheral blood mononuclear cells (PBMC). These B-cells are monoclonal as defined by light chain expression and by the existence of rearranged Ig genes (Southern blot analysis), although they exhibit heterogeneity in the density of surface light chain. Unlike normal PBMC B-cells, the monoclonal B-cells bear CD5 and CD10 (CALLA), express adhesion and adhesion-related molecules (CD11b, CD9), and appear to be actively differentiating during the course of the disease, based on the pattern of CD45 isoform expression. At any given point in time, the population of monoclonal B-cells is heterogeneous in differentiation stage based on transitions in the expression of CD45 isoforms from expression of CD45RA, the high molecular mass isoforms of CD45, to the low molecular mass isoform CD45R0 which appears only on very late stage B-cells and early plasma cells. For one patient, analysis of CD45 isoform expression over 2 years showed that the monoclonal B-cell population as a whole progressed towards terminal differentiation as defined by loss of CD45RA and acquisition of CD45R0. This indicates a continuously differentiating lineage of an unusual B-cell phenotype, and/or malignant transformation of a distinct lineage of B-cells in WM.     

B-lymphocyte suppression in multiple myeloma is a reversible phenomenon specific to normal B-cell progenitors and plasma cell precursors

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 = 23), in plateau or complete remission (PB n = 42, BM n = 18), and in relapse (PB n = 17, BM n = 14), in comparison to normal individuals (n = 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⁺38⁺ normal plasma cell precursors were significantly reduced in presentation/relapse patients, but not in patients in plateau/remission. Total CD19⁺ cells were significantly reduced only in patients with circulating myeloma cells, detected by IgH-PCR. In the marrow, CD19⁺ 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⁺ 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⁺ B lymphocytes that correlates inversely with disease stage, and specifically affects the early and late stages of normal B-cell differentiation.     

Stromal-mediated down-regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients

The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down-regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation-dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.     

B-cell surface phenotypes of proliferating myeloma cells: target antigens for immunotherapy

Dual-parameter flow cytometric analysis of B-cell antigens and DNA content was used to determine the phenotypes of proliferating tumor cells (S-phase cells) from 30 patients with multiple myeloma. B4 (CD19), J5 (CALLA, CD10), B1 (CD20), and monotypic surface immunoglobulin (Slg) were expressed heterogeneously in 24 patients. J5 and monotypic Slg were found most frequently but were always expressed on a significantly lower percentage of cells than the antigens typically associated with plasma cells, cytoplasmic immunoglobulin (Clg) and T10 (CD38). S-phase cells were found in each antigen(+) subset. B antigen(+) cycling cells were demonstrated in 16 patients whose marrow or blood cells expressed B antigens exclusively in the hyperdiploid fraction and therefore were certainly part of the myeloma clone. Similar to the low level of proliferative activity of the T10(+), Clg(+), and PCA1(+) subsets, the percentages of cycling cells of the preplasma cell B-antigen-bearing myeloma subsets ranged from less than 1% to 12%. The tumor cells of four patients were also studied with dual-color surface antigen analysis and demonstrated independent expression of B antigens, with only rare coexpression of T10 and monotypic Slg, J5, or B4. These findings are consistent with the presence of distinct myeloma subsets bearing differing B phenotypes in the same tumor and provide evidence that the proliferation in myeloma is occurring at various developmental stages in the malignant B lineage. These antigens may be important targets for immunologic therapy aimed at eliminating the entire proliferating compartment of this B-cell tumor.     

Analysis of surface antigen expression of human immunoglobulin-secreting cells: phenotypic heterogeneity in normal counterparts of myeloma cells

Human myeloma cells are malignant counterparts of plasma cells which represent the most differentiated B cells. Myeloma cells are, however, heterogeneous in their surface antigen expression (Katagiri et al, 1984, 1985), which may reflect that normal plasma cells have a spectrum of differentiation. To test this hypothesis, immunoglobulin-secreting cells (ISC) of non-neoplastic nature were studied with regard to their surface antigen expression by using a combination of reverse haemolytic plaque assay and complement-dependent cytolysis. Non-neoplastic ISC were found to have a broad spectrum of differentiation stages from the immature type of CD20+, HLA-DR+, CD38+ in the peripheral blood to the mature type of CD20-, HLA-DR-, CD38+ in the bone marrow. In patients with polyclonal B cell activation (PBA), ISC showed a more immature antigen expression in comparison with ISC in normal controls or patients without PBA. The surface antigen development of ISC was clearly demonstrated throughout the stages in the analysis of mitogen-induced ISC in vitro. No significant difference in the surface phenotype of ISC was found among heavy chain classes. Thus, non-neoplastic ISC show a spectrum of differentiation similar to that of myeloma cells, depending on the site where ISC are located, and on the degree of PBA in vivo.