Kazuhiro Nagai

In Vivo Comparison of the Bone Regeneration Capability of Human Bone Marrow Concentrates vs. Platelet-Rich Plasma

Background Bone marrow aspirate concentrate (BMAC) including high densities of stem cells and progenitor cells may possess a stronger bone regenerative capability compared with Platelet-rich plasma (PRP), which contains enriched growth factors. The objective of this study was to evaluate the effects of human BMAC and PRP in combination with β-tricalcium phosphate (β-TCP) on promoting initial bone augmentation in an immunodeficient mouse model. Methodology/Principal Findings BMAC and PRP were concentrated with an automated blood separator from the bone marrow and peripheral blood aspirates. β-TCP particles were employed as a scaffold to carry cells. After cell counting and FACS characterization, three groups of nude mice (BMAC+TCP, PRP+TCP, and a TCP control) were implanted with graft materials for onlay placement on the cranium. Samples were harvested after 4 weeks, and serial sections were prepared. We observed the new bone on light microscopy and performed histomorphometric analysis. After centrifugation, the concentrations of nucleated cells and platelets in BMAC were increased by factors of 2.8±0.8 and 5.3±2.4, respectively, whereas leucocytes and platelets in PRP were increased by factors of 4.1±1.8 and 4.4±1.9, respectively. The concentrations of CD34-, CD271-, CD90-, CD105-, and CD146-positive cells were markedly increased in both BMAC and PRP. The percentage of new bone in the BMAC group (7.6±3.9%) and the PRP group (7.2±3.8%) were significantly higher than that of TCP group (2.7±1.4%). Significantly more bone cells in the new bone occurred in sites transplanted with BMAC (552±257) and PRP (491±211) compared to TCP alone (187±94). But the difference between the treatment groups was not significant. Conclusions/Significance Both human BMACs and PRP may provide therapeutic benefits in bone tissue engineering applications. These fractions possess a similar ability to enhance early-phase bone regeneration.

Activation of p53 by Nutlin-3a, an antagonist of MDM2, induces apoptosis and cellular senescence in adult T-cell leukemia cells

It has been reported that the induction of cellular senescence through p53 activation is an effective strategy in tumor regression. Unfortunately, however, tumors including adult T-cell leukemia/lymphoma (ATL) have disadvantages such as p53 mutations and a lack of p16INK4a and/or p14ARF. In this study we characterized Nutlin-3a-induced cell death in 16 leukemia/lymphoma cell lines. Eight cell lines, including six ATL-related cell lines, had wild-type p53 and Nutlin-3a-activated p53, and the cell lines underwent apoptosis or cell-cycle arrest, whereas eight cell lines with mutated p53 were resistant. Interestingly, senescence-associated-β-galactosidase (SA-β-gal) staining revealed that only ATL-related cell lines with wild-type p53 showed cellular senescence, although they lack both p16INK4a and p14ARF. These results indicate that cellular senescence is an important event in p53-dependent cell death in ATL cells and is inducible without p16INK4a and p14ARF. Furthermore, knockdown of Tp53-induced glycolysis and apoptosis regulator (TIGAR), a novel target gene of p53, by small interfering RNA(siRNA) indicated its important role in the induction of cellular senescence. As many patients with ATL carry wild-type p53, our study suggests that p53 activation by Nutlin-3a is a promising strategy in ATL. We also found synergism with a combination of Nutlin-3a and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), suggesting the application of Nutlin-3a-based therapy to be broader than expected.

Impact of miR-155 and miR-126 as novel biomarkers on the assessment of disease progression and prognosis in adult T-cell leukemia

OBJECTIVE Micro RNAs (miRNAs) provide new insight in the development of cancer, but little is known about their clinical relevance as biomarkers in the assessment of diagnosis, classification, progression and prognosis of various cancers. To explore a potential novel biomarker, we examined the cellular and plasma miRNA profiles in adult T-cell leukemia (ATL) characterized by diverse clinical features. METHODS AND RESULTS Using CD4-positive cells isolated from 2 non-infected healthy individuals, 3 chronic ATL patients and 3 acute ATL patients, cellular miRNAs were profiled by microarray. The microarray screened 5 miRNAs namely miR-155, let-7g, miR-126, miR-130a and let-7b because of the large difference in their expression in diseased vs. that of healthy controls. The expression levels of before 5 miRNAs re-quantified by reverse transcription quantifiable polymerase chain reaction (RT-qPCR) were not always accordant in cells and plasma. The high and low plasma levels of miR-155 and miR-126 changed with ATL stage. CONCLUSION The present study revealed that there is a quantitative discrepancy between cellular and plasma miRNAs. The elevation of plasma miR-155 and the reduction in miR-126 correlated with poor prognosis, indicating their usefulness as a novel biomarker for the assessment of disease stage.

Long-term efficacy of imatinib in a practical setting is correlated with imatinib trough concentration that is influenced by body size: a report by the Nagasaki CML Study Group

Imatinib has dramatically improved long-term survival of chronic myelogenous leukemia (CML) patients. To analyze its efficacy in a practical setting, we registered most of CML patients in Nagasaki Prefecture of Japan. Of these, 73 patients received imatinib as an initial therapy. The overall survival rate of these patients was 88.7% at 6 years, and the cumulative complete cytogenetic response rate was 82.5% at 18 months. These results are comparable with the data of other reports including the IRIS study; however, the administered imatinib dose was smaller in our study than that in other reports. To address these discrepancies, we measured the trough concentration of imatinib among 35 patients. Although 39% of the patients were administered less than 400 mg/day, the trough level was comparable to those of previous reports. The trough level of imatinib showed a significant relationship with its efficacy, and was clearly related to dose of imatinib administrated and dose of imatinib divided by body surface area (BSA). Considering the smaller BSA of Japanese patients as compared to those of foreign origin, the results suggest that a lower dose of imatinib could maintain enough trough level and provided excellent results for the treatment of CML in our registry.

Identifying progression-associated genes in adult T-cell leukemia/lymphoma by using oligonucleotide microarrays

Adult T‐cell leukemia/lymphoma (ATL) is associated with human T‐lymphotropic virus type‐1 (HTLV‐1). To understand the changes in expression that occur in the progression of chronic phase of ATL to acute crisis, the gene expression profiles of fresh ATL cells were compared in 4 pairs of samples (progression of chronic to acute phase in 3 patients, as well as 1 typical chronic phase sample vs. 1 typical acute phase sample) using high‐density oligonucleotide DNA arrays. We identified 203 genes that were commonly upregulated in acute vs. chronic phase samples including ribosomal proteins, proteosome subunits, eukaryotic translation factors, immunophilins, heat shock proteins and genes important for DNA replication. Additionally, we identified 91 commonly downregulated genes including immune molecules related to MHC and a phosphatase. Several of the genes were previously identified to be associated with the Tax protein of HTLV‐1. Some of the upregulated genes were located in amplified regions identified by comparative genomic hybridization in the corresponding chronic/acute ATL sample. Using real‐time quantitative PCR, we confirmed the array‐results in those specimens analyzed by microarray. These results demonstrated that distinct sets of genes that are known to be critical in cellular transformation and/or activation are up‐ or down‐regulated during the transition to the acute phase of ATL.

A unique case of t(15;17) acute promyelocytic leukaemia (M3) developing into acute myeloblastic leukaemia (M1) with t(7;21) at relapse

Summary. This report describes a case of t(15;17) acute promyelcytic leukaemia (APL, FAB subtype M3) with dysgranulopoiesis at diagnosis in a patient who developed myelodysplasia (MDS) and then a second phenotype of t(7;21) acute myeloblastic leukaemia (AML, FAB subtype M1) at the time of relapse. To our knowledge, there is no report of a second phenotype of AML occurring after complete remission (CR) of APL. Furthermore, this is the first report of chromosomal abnormality t(7;21) in a case of AML. Several hypotheses for this unusual course of APL are discussed.

Diagnostic criteria for hypocellular acute leukemia: A clinical entity distinct from overt acute leukemia and myelodysplastic syndrome

In order to establish diagnostic criteria for hypocellular acute leukemia (HL), we have reviewed 32 cases selected on the basis of hypothetical 40% or less cellularity, by focusing on morphology, immunophenotype, karyotype and response to low dose Ara-C (LDAC) regimen and compared them with 40 cases of myelodysplastic syndrome (MDS) and 66 cases of overt acute myeloid leukemia (AML). The onset age ranged from 44 to 75 years (median 67 years). Bone marrow (BM) cellularity ranged from 12.4 to 39.8% (mean 29.8%) in HL, being significantly lower than in MDS (mean 80.7%) or AML (mean 86.4%) (P < 0.001). All reviewed cases characteristically showed smoldering clinical course, bi- or pancytopenia with rare leukemic blasts in the peripheral blood (PB), proliferation of type I leukemic blasts in the BM and markedly reduced background hematopoietic cells with some dysplastic changes in 12/32 cases (37.50/6). Blast percentage (blast %) in the BM ranged from 38.2 to 93.7% (mean 57.3%) in all nucleated cells (ANC). Although a considerable number of cases had blasts with negative or very low myeloperoxidase activity, immunophenotyping revealed that the leukemic blasts in HL had only myeloid markers. Karyotyping revealed non-random chromosome abnormalities in 30% of cases analyzed, which were considerably different from those seen in MDS. With LDAC regimen, a significantly higher CR rate (13/20 cases: 65.0%) was gained in HL than in RAEB/RAEB-t (0%) and overt AML in the elderly cases (27.3%) (P < 0.05). In CR, most cases showed recovery to normocellular BM with an apparent normalization of PB parameters. However, 12 CR cases relapsed 4-12 months later; most of which again showed hypocellular BM. These results indicate that HL is a distinct subtype of AML characterized by slow but distinct proliferation of immature myeloid blasts and by unique hematological features distinct from MDS or overt AML in the elderly. We propose the following diagnostic criteria: (1) pancytopenia with rare appearance of blasts in PB; (2) less than 40% BM hypocellularity; (3) more than 30% blasts in BM-ANC; and (4) myeloid phenotypes of leukemic blasts by MPO staining and/or immunophenotyping.

Durable remission by sobuzoxane in an hiv-seronegative patient with human herpesvirus 8-negative primary effusion lymphoma

Primary effusion lymphoma (PEL) is recognized as a unique clinicopathological entity associated with human herpesvirus 8 (HHV-8), and it occurs almost exclusively in human immunodeficiency virus (HIV)-infected individuals. In the majority of PEL cases, Epstein-Barr virus (EBV) has been found in the tumor cells as well.We describe here an elderly HIV seronegative female patient with PEL in the pleura and pericardium not associated with HHV-8 or EBV. Cytologic examinations of the pleural effusion revealed large lymphoma cells with immunophenotypes positive for CD8, CD10, CD19, CD20, CD22, CD24, CD45, and HLA-DR but negative for CD30 and surface immunoglobulin. Chromosome analysis showed complicated abnormalities including add(3)(q27). Immunoglobulin gene rearrangement was detected by Southern blotting; however, c-myc, Bcl-2, and Bcl-6 genes were not rearranged.The patient was treated with a modified CHOP (cyclophosphamide, hydroxydoxorubicin, oncovine, and prednisolone) regimen, and achieved remission. Recurrence of PEL in the pericardium as effusion lymphoma was found 3 months after the discontinuation of CHOP. After approximately 1 year of intermittent multiagent salvage therapy for pericardial recurrences, a treatment that resulted in a partial response, 3 cycles of monotherapy with sobuzoxane were administered. At the time of this report the patient had been free from PEL for more than 18 months without chemotherapy.

Absolute number of circulating CD34+ cells is abnormally low in refractory anemias and extremely high in RAEB and RAEB-t; novel pathologic features of myelodysplastic syndromes identified by highly sensitive flow cytometry

We scored absolute numbers of circulating CD34+ cells by a highly sensitive triple-color flow cytometric analysis using CD45 monoclonal antibody, CD34 monoclonal antibody and propidium iodide. Forty-one patients with MDS (RA: 27, RARS: 1, RAEB: 6, RAEB-t: 3,CMML: 4), 12 patients with aplastic anemia (AA) and 36 age-adjusted normal subjects were studied. RA had significantly decreased numbers of cells expressing CD34 (0.21 +/- 0.29 x 10(6)/l) compared with normal subjects (0.81 +/- 0.36 x 10(6)/l)(P < 0.001). This low number of CD34+ cells in RA resembles the case of AA (0.39 +/- 0.73 x 10(6)/l). In light-scatter analysis, the CD34+ cells of RA patients were distributed mainly in low forward scatter (FSC) (lymphocyte region). In contrast, the CD34+ cell counts were extremely high in patients with RAEB (46.54 +/- 71.37 x 10(6)/l) and RAEB-t (57.00 +/- 52.36 x 10(6)/l) (P < 0.001) and the CD34+ cells were observed in high FSC (blast region).CMML patients showed moderately increased numbers of CD34+ cells (3.69 +/- 4.64 x 10(6)/l). Thus, there was a distinct difference in cell size and number of circulating CD34+ cells between RA and RAEB/RAEB-t. In univariate and multivariate analysis, a high CD34+ cell count (> or = 1.0 x 10(6)/l) was a poor prognostic factor. This method allows one to distinguish RA from other MDS subtypes more reliably than by morphology alone and provides early signs of progression to acute leukemia.

Effect of a selective Abl tyrosine kinase inhibitor, STI571, on in vitro growth of BCR-ABL-positive acute lymphoblastic leukemia cells

By employing a new semi-quantitative assay system that includes co-culturing leukemia cells with the mouse bone marrow-derived stromal cell line MS-5, we examined the suppressive effect of a selective inhibitor of ABL tyrosine kinase, STI571, on acute lymphoblastic leukemia (ALL) cells with BCR-ABL fusion. Leukemic blast cells from eight patients with B-precursor ALL, including three patients with BCR-ABL-positive ALL, were cultured on monolayers of MS-5 cells for 3 weeks with or without addition of variable amounts of STI571. In all cases, cobblestone areas (CAs) were formed, showing clear linear cell dose-dependent curves, allowing quantitative assessment of blast cell growth. The progenitor frequencies obtained by this direct CA-forming cell (CAFC) assay were equivalent to ALL progenitor frequencies assessed by the standard limiting dilution assay. The number of CAFCs ranged from 12.3 to 140.3/104 cells. In BCR-ABL-positive ALL patients, CA-containing cells were examined by FISH, and all contained BCR-ABL fusion genes. STI571 inhibited CA formation of BCR-ABL-positive ALL cells virtually 100% at 0.1–1.0 μmol/l. None of the five BCR-ABL-negative ALL patients showed this growth inhibition by STI571 at 0.1–1.0 μmol/l. Our results indicate that STI571 selectively inhibits in vitro growth of BCR-ABL-positive ALL cells.