Functional B7.2 and B7-H2 molecules on myeloma cells are associated with a growth advantage.

PURPOSE: B7 family molecules expressed on antigen-presenting cells stimulate or inhibit normal immune responses. The aim of this study was to investigate whether functional B7.2 and B7-H2 molecules are expressed on myeloma cells and, if so, whether they are associated with pathophysiology in myeloma. EXPERIMENTAL DESIGN: The expression of B7.2 and B7-H2 molecules on normal plasma and neoplastic (myeloma) plasma cells was analyzed. The cell proliferation and immunomodulatory function of myeloma cells related to B7.2 and B7-H2 expression were examined. RESULTS: Human myeloma cell lines commonly expressed B7.2 and B7-H2 molecules. B7.2 expression on plasma cells was more common in myeloma patients (n = 35) compared with that in patients with monoclonal gammopathy of unknown significance (n = 12) or hematologically normal individuals (n = 10). Plasma cells expressing B7-H2 were observed in myeloma patients alone, although rarely. Patients whose myeloma cells showed high B7.2 expression were more anemic and thrombocytopenic than other myeloma patients. The expression of these molecules was induced or augmented by cultivating myeloma cells with autologous stroma cells or tumor necrosis factor-alpha, a key cytokine in myeloma biology. Cell proliferation was more rapid in the B7.2+ and B7-H2+ populations compared with the B7.2(-) and B7-H2(-) populations, respectively, in the human myeloma cell lines examined. B7.2 and B7-H2 molecules on myeloma cells induced normal CD4+ T cells to proliferate and produce soluble factors, including interleukin-10 that stimulate myeloma cell proliferation. CONCLUSIONS: Functional B7.2 and B7-H2 molecules detected on myeloma cells may be involved in the pathophysiology of myeloma.

Aggressive characteristics of myeloblasts expressing CD7 in myelodysplastic syndromes.

Clinical data suggest that CD7+ myeloblasts are linked with poor prognosis in myeloid malignancies including myelodysplastic syndromes (MDS). To explore the biology behind this, we compared cell characteristics between CD34+CD7+ and CD34+CD7- myeloblasts from an MDS cell line and fresh samples from MDS patients. Compared with CD34+CD7- myeloblasts, CD34+CD7+ myeloblasts showed greater proliferative capacity, more active cell cycling, and less apoptosis. In analyses of a cell line, CD34+CD7+ myeloblasts produced CD34+CD7- myeloblasts and showed lower expressions of interleukin-8 and chemokine (C-C motif) ligand 2 genes, suggesting immaturity of these cells. These findings might underlie the clinical aggressiveness in CD7+ MDS.

Flow cytometric parameters with little interexaminer variability for diagnosing low-grade myelodysplastic syndromes.

Recent studies have suggested that flow cytometry (FCM) helps diagnose myelodysplastic syndromes (MDS). However, appropriate FCM diagnostic parameters that are easily reproducible by different examiners remain unclarified. We found that "the Ly/Mbl CD45 ratio (mean fluorescence intensity [MFI] of CD45 on lymphocytes/MFI of CD45 on CD34+ myeloblasts)," "the percentage of CD34+ myeloblasts among all nucleated cells," and "the percentage of CD34+ B-cell precursors among all CD34+ cells" had little interexaminer variability. These parameters can be analyzed from one test tube for three-color FCM, and their analysis in combination can diagnose a certain percentage of low-grade MDS patients.

Increased apoptosis of circulating T cells in myelodysplastic syndromes.

The mechanism of T cell lymphopenia in myelodysplastic syndromes (MDS) is unknown. We investigated apoptosis in freshly isolated and cultured lymphocytes; the latter were used to detect cells not yet apoptotic but destined for apoptosis. Apoptosis increased in both fresh and cultured T cells in MDS compared with those from healthy controls. Furthermore, in lymphopenic MDS patients the lymphocyte count correlated negatively with the degree of T cell apoptosis. MDS T cells showed increased Fas expression. However, in MDS but not in controls, the degree of T cell apoptosis was independent of the Fas expression level, and exogenous anti-Fas antibodies did not modulate T cell apoptosis. Mechanisms other than the Fas-Fas ligand pathway may induce T cell apoptosis in MDS.

Diagnostic application of flow cytometric characteristics of CD34+ cells in low-grade myelodysplastic syndromes.

The diagnosis of myelodysplastic syndromes (MDS) without an increase in blasts and ringed sideroblasts (low-grade MDS without ringed sideroblasts [LGw/oRS]) may be problematic because dysplastic features are not specific to MDS and approximately 50% of patients with LGw/oRS lack chromosomal aberrations. Here, we report the usefulness of flow cytometric characteristics of CD34(+) cells for LGw/oRS diagnosis. Bone marrow cells from LGw/oRS patients and controls (eg, cytopenic individuals without MDS) were analyzed using 4-color flow cytometry (FCM). We objectively determined reference ranges of 13 parameters related to CD34(+) cells with data from controls. In LGw/oRS patients, various abnormalities of CD34(+) cells-eg, decrease in CD34(+) B-cell precursors, aberrant expression or overexpression of various antigens on CD34(+) myeloblasts-were observed. We constructed a reproducible, flow cytometric scoring system for LGw/oRS diagnosis. High scores were observed in 16 of 27 LGw/oRS patients, regardless of the presence or absence of chromosomal aberrations, but not in any of the 90 controls. Among LGw/oRS patients with chromosomal aberrations, patients with trisomy 8 or del20(q) had low FCM scores (P = .002). As a result, most LGw/oRS patients were identified based on high FCM score, chromosomal aberration, or both.

Hypothesis: myeloid-restricted hematopoietic stem cells with self-renewal capacity may be the transformation site in acute myeloid leukemia.

The transformation site in acute myeloid leukemia (AML) has been proposed to be pluripotent hematopoietic stem cells (PHSCs), and the lymphoid development of leukemic PHSCs may be suppressed by leukemogenic events. Recent data from multiple laboratories have contradicted the current hierarchical model of hematopoiesis and indicated the presence of myeloid HSCs with minimal lymphopoietic potential (MyHSCs) in mice. Based on these findings and re-evaluating the published data on AML stem cells, we hypothesize that MyHSCs may be the transformation site in AML. If so, therapy targeting leukemic MyHSCs but sparing PHSCs is worth investigating.

Identification and hematopoietic potential of CD45- clonal cells with very immature phenotype (CD45-CD34-CD38-Lin-) in patients with myelodysplastic syndromes.

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38-Lin- cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34-CD38-Lin- cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site is a matter of debate, and CD45+CD34+CD38-Lin- HSCs were recently reported to be clonal. In the study reported here, we detected CD45-CD34-CD38-Lin- cells in the peripheral blood and bone marrow of patients with MDS and isolated them by successive application of density centrifugation, magnetic cell sorting, and fluorescence-activated cell sorting. Fluorescence in situ hybridization showed that CD45-CD34-CD38-Lin- cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-, they lacked CD117 and CD133 expression. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some patients. Freshly isolated CD45-CD34-CD38-Lin- cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When cocultured with stroma cells, CD45-CD34-CD38-Lin- cells showed only weak potential for proliferation and differentiation, yet they differentiated into CD34+ cells and then mature myeloid cells. This newly identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.

Association between phenotypic features of blasts and the blast percentage in bone marrow of patients with myelodysplastic syndromes.

Although the blast percentage in the bone marrow (BM) is a key parameter for the classification of myelodysplastic syndromes (MDS), the current blast percentages used to define MDS subtypes have not been shown to have strong biological relevance. We determined the blast phenotypes and examined their relationship with the BM blast percentage in 90 MDS cases. When the BM blast percentage increased, cases whose blasts expressed CD7, CD56 and CD117 increased whereas cases whose blasts expressed CD10, CD11b and CD15 decreased. The BM blast percentages where the blast immunophenotype changed were 5, 10, 20 and 25%. Blast immunophenotypes have the potential to provide a biological basis for and refine the present MDS classifications.