Toshiya Sumikuma

Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor

OBJECTIVE One of the mechanisms for mobilization of hematopoietic stem cells and progenitor cells is alternation of adhesion molecules. We investigated the mobilization of hematopoietic progenitor cells in blood by administration of anti-vascular cell adhesion molecule (VCAM)-1 antibody (Ab) in mice. MATERIALS AND METHODS Twelve- to 14-week old C57BL/6J mice were injected intravenously with anti-VCAM-1 Ab and anti-very late antigen (VLA)-4 Ab at a dose of 5 mg/kg for 2 days. RESULTS The number of colony-forming cells (CFCs) in blood was increased 11.4-fold after anti-VCAM-1 Ab treatment, but the number of CFCs was not increased after treatment with anti-VLA-4 Ab. The number of colony-forming unit spleen (CFU-S) also was increased 21.6-fold in the peripheral blood by administration of anti-VCAM-1 Ab. The number of CFCs and CFU-S in the bone marrow of mice treated with anti-VCAM-1 Ab was decreased and that in the spleen also was decreased. On administration of recombinant human granulocyte colony-stimulating factor (125 microg/kg twice daily) with anti-VCAM-1 Ab, the numbers of CFCs and CFU-S were increased 141.8-fold and 439-fold, respectively. CONCLUSIONS These observations demonstrated that administration of anti-VCAM-1 Ab induced mobilization of hematopoietic progenitor cells into blood from bone marrow and spleen and that granulocyte colony-stimulating factor has synergistic effects on anti-VCAM-1 Ab-induced mobilization.

CD34 + /CD41a + cells best predict platelet recovery after autologous peripheral blood stem cell transplantation

Reliable markers for megakaryocytic reconstitution after peripheral blood stem cell transplantation (PBSCT) have not been established. To determine a convenient and reliable predictor, we measured the number of megakaryocyte progenitor cells in PBSC grafts by clonogenic and flow cytometric assays. Seventeen patients with hematological and solid malignancies were included in this study. For the clonogenic assay, we used thrombopoietin (TPO) as a growth factor to evaluate the maximum number of megakaryocyte progenitor cells. Using a flow cytometric assay, we examined the expression of platelet glycoproteins on CD34+ cells to count the number of megakaryocyte progenitor cells. We used buffer containing EDTA to prevent platelet adhesion to CD34+ cells and selected CD34+ cells by immunomagnetic beads. The best correlation was observed between the number of CD34+/CD41a+ cells and the time to platelet recovery (P = 0.0205), rather than the total number of CD34+ cells. In addition, a close correlation was observed between the number of CD34+/CD41a+ cells and colony-forming unit megakaryocyte (CFU-MK) (P = 0.0018). These observations suggest that the number of CD34+/CD41a+ cells is the best predictor for platelet reconstitution after PBSCT.

Humanized anti-interleukin-6 receptor monoclonal antibody induced apoptosis of fresh and cloned human myeloma cells in vitro

We investigated the effect of anti-IL-6 receptor monoclonal antibody (hPM1) on the in vitro proliferation of cloned and freshly isolated myeloma cells from 20 patients with advanced stage multiple myeloma (MM). Humanized PM1 significantly inhibited the growth of a myeloma cell line in a dose-dependent manner and inhibited more than 30% of the proliferation of fresh myeloma cells in 10 of the 19 cases. Flow cytometric analysis using annexin V and 7AAD showed that hPM1 induced apoptosis of myeloma cells. These observations suggest the possibility of using hPM1 for treating some patients with MM whose growth depends on IL-6.

Successful non-T cell-depleted HLA haplo-identical three-loci mismatched hematopoietic stem cell transplantation from mother to son based on the feto-maternal microchimerism in chronic myelogenous leukemia

A 17-year-old male with chronic myelogenous leukemia in blast crisis received a non-T cell-depleted (TCD) HLA haplo-identical three-loci mismatched hematopoietic stem cell transplant (HSCT) from his mother. Long-term feto-maternal microchimerism was detected by nested polymerase chain reaction with sequence-specific primer typing. The post-transplantation prophylaxis against graft-versus-host disease (GVHD) was tacrolimus with minidose methotrexate. Sustained engraftment was obtained. Acute GVHD (grade 2) developed, but improved rapidly. Bone marrow aspiration on day 120 showed complete remission. Non-TCD HLA haplo-identical HSCT based on feto-maternal microchimerism might be feasible and has important implications in the selection of alternative family donors in HSCT.

Expression of T‐cell‐associated antigens in B‐cell non‐Hodgkin's lymphoma

We performed the immunophenotyping of 101 patients with B‐cell non‐Hodgkins lymphoma (B‐NHL) using two‐colour flow cytometry (FCM) and found that lymphoma cells coexpressed at least one kind of T‐cell‐associated antigen (T‐Ag; CD2, CD5, CD7) in 25 patients (24·8%). Among these three T‐Ags, CD5 was the most frequently expressed, in 21 patients (20·8%), followed by CD7, expressed in five patients (5·0%), and CD2, which was expressed in two patients (2·0%). Two kinds of T‐Ag were simultaneusly expressed in three patients (CD2/CD5, CD2/CD7, and CD5/CD7, each expressed in one patient). Concerning the expression pattern of T‐Ag, there were no significant differences between lymph nodes and extranodal organs in the three patients with T‐Ag‐positive B‐NHL (T‐Ag(+) B‐NHL) who were analysed. When comparing the clinical features between T‐Ag(+) B‐NHL and T‐Ag‐negative B‐NHL (T‐Ag(–) B‐NHL), extranodal involvement and higher International Prognostic Index (H and H.I.) were significantly frequent in the former subgroup (P = 0·0119 and P = 0·0302 respectively).

Effects of thrombopoietin (c-mpl ligand) on growth of blast cells from patients with transient abnormal myelopoiesis and acute myeloblastic leukemia

Abstract: Thrombopoietin (TPO) is a ligand for c‐mpl that promotes both proliferation and differentiation of megakaryocytes in vivo and in vitro. We investigated the expression of c‐mpl transcripts and the effects of recombinant human TPO (rhTPO) on the proliferation and differentiation of human leukemic cell lines or fresh samples obtained from 32 patients with transient abnormal myelopoiesis (TAM) or acute myeloblastic leukemia (AML). Cells were cultured with TPO alone or combined with rh interleukin‐3 (IL‐3) or stem cell factor (SCF). Expression of c‐mpl was verified in 6 of 13 cases tested. All but one of the cases that showed c‐mpl expression responded to TPO. Blasts from all cases of TAM or French–American–British (FAB) subtype M7 showed growth responses to TPO with higher sensitivity than cells of other FAB subtypes and these responses were increased by addition of rhIL‐3 or rhSCF in some cases. Responses of cells of other FAB subtypes varied. In addition, increased expression of platelet‐specific surface antigens on MO7E cells after incubation with rhTPO was observed. These data suggest that TPO may be involved in the abnormal proliferation and differentiation of human leukemic cells, especially of M7 and TAM cells, considered to be of megakaryocytic lineage.

Successful peripheral blood stem cell transplantation for myelodysplastic syndrome

Wilms’ tumor (WT1) gene expression is increased in patients with leukemia as well as myelodysplastic syndrome (MDS) and is useful for detection of minimal residual disease (MRD). A 47-year-old man given a diagnosis of refractory anemia with excess of blasts in transformation (RAEB-T) received myeloablative therapy followed by autologous peripheral blood stem cell transplantation (PBSCT). MRD by WT1 expression was not detected in the graft. The patient has been in CR for 25 months after PBSCT. These observations suggest that PBSCT is feasible for patients with RAEB-T and analysis of WT1 expression can be applied for patients with high risk MDS.

CD34 + /CD90 + cells infused best predict late haematopoietic reconstitution following autologous peripheral blood stem cell transplantation

Summary.  This study aimed to identify which graft product subset of cells might be the most predictive of late haematopoietic recovery (three to 12 months) following autologous peripheral blood stem cell transplantation (PBSCT). The relationships between the numbers of reinfused CD34+ cells and their immature subsets such as CD34+/CD90+, CD34+/AC133+, CD34+/CD38– and CD34+/HLA‐DR– cells, and haemoglobin, white blood cell (WBC) and platelet counts at 3, 6, 9 and 12 months after PBSCT, were studied in 25 patients with haematological and solid malignancies. The total CD34+ cell number, as well as CD34+/CD90+ and CD34+/AC133+ cell numbers, correlated with platelet counts at 3, 6, 9 and 12 months after PBSCT, but the CD34+/CD90+ cells infused best predicted platelet recovery during the first 12 months after PBSCT (P < 0·0238 at any time‐point). The CD34+/AC133+ cell dose also correlated with WBC counts at 3 months post PBSCT. In addition, all patients receiving more than 80 × 104 CD34+/CD90+ cells/kg showed platelet counts greater than 100 × 109/l at all points after PBSCT, suggesting that this value of the CD34+/CD90+ cells infused was a threshold dose for durable haematopoietic engraftment after PBSCT.

Clinicopathological features of myeloid/natural killer (NK) cell precursor acute leukemia

Four patients (three males and one female) were diagnosed as myeloid/natural killer (NK) cell precursor acute leukemia in our department. Two patients showed the extramedullary involvement at initial presentation. Leukemic cells expressed CD7, CD33, CD45 and CD56 in all patients. Additionally, CD13, CD34, HLA-DR, cytoplasmic CD3 and myeloperoxidase were expressed in some patients. Trisomy 10 was found in two patients, which has not been reported in this disease. Therefore, myeloid/NK cell precursor acute leukemia might be rather heterogeneous especially in chromosomal abnormality though it seemed to constitute the distinct clinical entity among acute myeloid leukemia of M0 subtype.

T-cell associated antigen-positive B-cell lymphoma.

We immunophenotyped 128 patients with B-cell non-Hodgkins lymphoma (B-NHL) of various histological subtypes using two-color flow cytometry (FCM), and found that lymphoma cells obtained from 31 patients (24.2%) coexpressed at least one of the following T-cell associated antigens (T-Ag); CD2 (2.3%), CD5 (18.0%) or CD7 (6.3%). Moreover, 3 patients expressed two kinds of T-Ag (CD2/CD5, CD2/CD7 or CD5/CD7) as reported by other investigators. Though we could not find coexpression of CD3, CD4 or CD8 antigen in any patients analyzed in our study, such T-Ag(+) B-NHL have also been reported in the literature. As clinical features, extranodular involvement and higher International Prognostic Index (high and high intermediate) seemed more frequent in T-Ag(+) B-NHL than T-Ag(-) B-NHL in our study. Such prognostic significance of T-Ag expression is also reported by other investigators especially in CD5(+) diffuse large B-cell lymphoma. In addition, two-color FCM for detecting such aberrant T-Ag expression in B-NHL is useful for monitoring the minimal residual disease in the subgroup with T-Ag(+) B-NHL.