Hideo Hyodo

Transforming growth factor-β regulates growth as well as collagen and fibronectin synthesis of human marrow fibroblasts

Summary Three growth factors present in platelets, namely platelet‐derived growth factor (PDGF), transforming growth factor‐β (TGF‐β) and epidermal growth factor (EGF), have been implicated in the pathogenesis of bone marrow fibrosis frequently associated with myeloproliferative disorders. In this study, regulation of the proliferation, as well as collagen and fibronectin synthesis from marrow fibroblasts by TGF‐β was investigated. TGF‐β alone at high plating density stimulated the proliferation of cells at low concentrations, but rather showed inhibition at high concentrations in both MPD patients and control subjects. In the presence of PDGF, which has been confirmed to be a main growth factor for marrow fibroblasts, low concentration of TGF‐β inhibited the proliferation at low cell density, but there was no inhibition at high cell density. The synthesis of both type I and type III procollagen was enhanced by high concentrations of TGF‐β in both MPD patients and control subjects, while PDGF or EGF showed no effect. The fibronectin synthesis was also enhanced by TGF‐β, but not by PDGF or EGF. These results suggest that growth and stromal protein synthesis of fibroblasts causing marrow fibrosis are regulated by TGF‐β as well as PDGF and EGF, when these factors are released or leaked from platelets or megakaryocytes into marrow environment in MPD patients.

c-kit point mutation of extracellular domain in patients with myeloproliferative disorders.

Summary. c‐kit is a tyrosine kinase receptor whose ligand is stem cell factor (SCF). Gene alteration of the c‐kit extracellular domain was analysed by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) in 25 patients with myeloproliferative disorders (MPD). In the N‐terminal part of the domain, mobility shifts indicating sequence alteration were detected in three of the patients, two primary myelofibrosis (PMF) and one chronic myelogenous leukaemia (CML). The subsequent sequencing revealed the same point mutations at codon 52 causing amino acid substitution (Asp → Asn). To our knowledge this is the first report with a c‐kit point mutation found in human fresh tumour cells.

c-Kit Point Mutation in Patients with Myeloproliferative Disorders

Myeloproliferative disorders (MPD) constitute a group of hematopoietic neoplasms at the myeloid stem cell level. Myeloid stem cells and/or progenitor cells from MPD have been considered sensitive to hematopoietic growth factors, including erythropoietin, thrombopoietin and stem cell factor (SCF). SCF is a ligand for c-kit receptor with tyrosine kinase. We analysed the gene alteration of the c-kit extracellular domain in MPD patients by PCR-SSCP and subsequent nucleotide sequencing. The point mutation in the N-terminal part of the domain, codon 52 (Asp-->Asn), was found in two patients with primary myelofibrosis and one with chronic myelogenous leukemia. We review the literature regarding the role of SCF/c-kit system in the oncogenesis of leukemia and MPD, and then discuss the significance of our finding in the context of growth advantage of the mutated clones over the normal clones.

Platelet‐derived growth factor expression in accelerated and blastic phase of chronic myelogenous leukaemia with myelofibrosis

Summary Myelofibrosis is sometimes associated with accelerated and blastic phase of chronic myelogenous leukaemia (CML). In order to investigate the role of platelet derived growth factor (PDGF) in this pathogenesis, expression and production of PDGF was studied in the blast cells from 11 patients. Five patients had myelofibrosis with myeloid blasts, while six patients did not show fibrosis, including three with myeloid blasts and three with lymphoid blasts. PDGF‐A chain transcript was expressed in most of the patients. On the other hand, PDGF‐B chain transcript was detected in all of the five patients with myeloid blasts and with fibrosis, in one of the three patients with myeloid blasts and without fibrosis, and in none of the three lymphoid crisis patients without fibrosis. In the patients with myeloid blasts and with fibrosis, PDGF protein. PDGF‐AB and/or PDGF‐BB, was found to be secreted from blast cells. In addition, the PDGF activity in the culture of myeloid blasts from two patients with fibrosis was also growth stimulatory for human marrow fibroblasts. These results suggest that expression and secretion of PDGF‐AB or PDGF‐BB in blast cells play an important role in the pathogenesis of marrow fibrosis associated with accelerated and blastic phase of CML.

Platelet Derived Growth Factor Expression, Myelofibrosis and Chronic Myelogenous Leukemia

CML is often associated with myelofibrosis, and fibrosis in the accelerated phase is one of the diagnostic criteria for this accelerated phase. In this review, the mechanism of myelofibrosis associated with CML is discussed with emphasis on the cell origin of the production and release of platelet derived growth factor (PDGF) and its interaction with marrow fibroblasts. In the initial stage of myelofibrosis in chronic phase CML, atypical small megakaryocytes might leak PDGF, possibly PDGF-AB, together with other growth factors. As the clinical phase of the disease progresses to accelerated or blastic phase, a larger quantity of PDGF-AB or PDGF-BB might be secreted from blastic cells with myeloid phenotype. In addition some fibroblasts may be attracted by the PDGF and proliferate, and deposit collagen as well as fibronectin in the bone marrow stroma.

Clinical and genetic features of therapy-related myeloid neoplasms after chemotherapy for acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a highly curable disease with excellent complete remission and long-term survival rates. However, the development of therapy-related myeloid neoplasms (t-MN) is being reported with increasing frequency in patients successfully treated for APL. We attempted to clarify the different clinical features and hematologic findings between t-MN and relapse cases, and to identify gene alterations involved in t-MN. We compared 10 relapse and 11 t-MN cases that developed in 108 patients during their first complete remission from APL. At APL diagnosis, t-MN patients had lower white blood cell counts than did relapse patients (P = .048). Overall survival starting from chemotherapy was significantly worse in t-MN patients than in relapse patients (P = .022). The t-MN cases were characterized as CD34(+)/HLA-DR(+) and PML-RARA(-), and 4 RUNX1/AML1 mutations were detected. T-MN is easily distinguished from APL relapse by evaluating these hematologic features, and it may originate from primitive myeloid cells by chemotherapy-induced RUNX1 mutations.

Acute myelogenous leukemia with PIG-A gene mutation evolved from aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome.

We report a patient with aplastic anemia (AA)—paroxysmal nocturnal hemoglobinuria (PNH) syndrome who developed acute myelogenous leukemia (AML). Flow cytometric analysis showed that the leukemic cells in the bone marrow lacked CD59 antigen on their surface and were positive for P-glycoprotein. Heteroduplex and single-strand conformation polymorphism analysis followed by sequencing of the leukemic cells in the bone marrow disclosed 1 frameshift-type mutation in exon 2 of the phosphatidylinositol glycan-class A (PIG-A) gene, which deductively produces truncated PIG-A protein. These findings provide direct evidence that the leukemic cells evolved from the affected PNH clone. Cytogenetic analysis in the bone marrow in each stage of AA-PNH, AML, and at relapse of AML showed normal, −7, and −7 plus −20, respectively, showing evidence of a clonal evolution. Because complete remission of AML was not achieved by intensive chemotherapies, allogeneic peripheral blood stem cell transplantation (PBSCT) from the patient’s HLA-matched sister was performed successfully with recovery of CD59 antigen on bone marrow hematopoietic cells; however, leukemia relapsed 4 months after PBSCT. Leukemia derived from PNH may be resistant to intensive chemotherapy, and a highly myeloablative regimen may be required for stem cell transplantation to eradicate the PNH-derived leukemia clone.

Autocrine and/or paracrine mechanism operate during the growth of human bone marrow fibroblasts

Summary The growth of human marrow fibroblasts was studied at various plating cell densities. The growth rate of fibroblasts cultured with human plasma derived serum (PDS) increased depending on the cell density plated, whereas fibroblasts cultured with human serum proliferated almost independent of the cell density. Conditioned medium obtained from the culture with PDS at high cell density enhanced the growth of fibroblasts plated at low density, as well as that of fibroblast colony forming cells. Characterization of this conditioned medium showed heat unstable and acid stable peptide factor(s) with high molecular weight (∼106) may be responsible for this activity. These results suggest that autocrine and/or paracrine mechanism can operate during the proliferation of marrow fibroblasts, a process thought to be involved in the progression of marrow fibrosis.

Leukemic Transformation of Primary Myelofibrosis: Immunophenotype, Genotype and Growth Characteristics of Blast Cells

Blast cells from six patients with leukemic transformation of primary myelofibrosis (PMF) were studied by morphology, immunophenotype and genotype as well as response to hematopoietic growth factors. The majority of the patients showed granulocytic or granulo-monocytic blasts, and only one had T lymphoid-monocytic blasts. None of the patients showed rearrangement of Ig or TCR genes, or the existence of the bcr-abl fused gene. A prominent growth response to GM-CSF and IL-3 was evident in all of the patients examined in liquid as well as semisolid cultures. The response to G-CSF was observed in four of the six patients in suspension culture, and in two of three patients in the clonogenic assay. Stem cell factor (SCF) was a weak growth stimulant, however the combination of this factor with GM-CSF or IL-3 was synergistically stimulatory. These results suggest that leukemic transformation of PMF occurs mainly at the level of myeloid stem cell, and that GM-CSF, IL-3, G-CSF and SCF are major growth factors for the blast cells in these cases.

L-selectin expression in CD34 positive cells in chronic myeloid leukemia.

L-selectin is a cell adhesion molecule, expressed on leukocytes and involved in the regulation of leukocyte traffic. This adhesion receptor is implicated in hematopoiesis by the interaction of hematopoietic stem cells and progenitors to stroma in the bone marrow microenvironment. We found that L-selectin expression on CD34++ cells from patients with chronic myelogenous leukemia (CML) is decreased or deficient, reflecting one of the features of malignant CML progenitors. In this review, we briefly describe the structure and function of L-selectin, and its role in hematopoiesis and its expression in leukemia and lymphoma. Finally, we discuss the abnormal adhesiveness of CML progenitor cells, and the role of L-selectin in this defect.