Keiko Fujii

Elevation of serum hepatocyte growth factor during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization.

We examined serum levels of the angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. Granulocyte colony‐stimulating factor (G‐CSF) (filgrastim 400 μg/m2/d) was administered to 23 donors for 5 d and aphereses were performed on days 4 and 5. Although bFGF remained at similar levels after G‐CSF treatment, serum VEGF and HGF levels increased 1·5‐fold (n = 13; P = 0·02) and 6·8‐fold (n = 23; P < 0·0001) respectively. The serum HGF level before G‐CSF administration on day 1 correlated inversely with mobilized CD34+ cell numbers. Time course kinetics of HGF showed that on the day after G‐CSF administration (day 2), serum HGF levels increased to 3678 pg/ml. For auto PBSC mobilization with chemotherapy and G‐CSF 200 μg/m2/d (n = 8), we observed similar HGF elevation, which appeared to be dose‐dependent on the G‐CSF administered.

Overexpression of novel short isoforms of Helios in a patient with T-cell acute lymphoblastic leukemia.

OBJECTIVE In previous studies, we demonstrated overexpression of the dominant-negative isoform of the transcription factor Ikaros, Ik-6, in patients with blast crisis of chronic myelogenous leukemia and B-cell acute lymphoblastic leukemia. In the present study, we analyzed expression of the Ikaros family genes Ikaros, Aiolos, and Helios in a panel of human T-cell leukemia/lymphoma cell lines and bone marrow samples of patients with T-cell acute lymphoblastic leukemia. MATERIALS AND METHODS We performed reverse transcriptase polymerase chain reaction, sequencing analysis, immunoblotting, and Southern blotting. RESULTS We found overexpression of novel short isoforms of Helios (Hel-5 and Hel-6) in the HD-Mar cell line. Southern blot analysis suggested that there might be a small deletion in the Helios locus of HD-Mar. In addition, we observed decreased expression of more than one Ikaros family gene in 3 of 9 patients with T-cell acute lymphoblastic leukemia. Moreover, one of the patients overexpressed novel short isoforms of Helios (Hel-7 and Hel-8). CONCLUSION This study provides the first evidence of an Ikaros family member (other than Ikaros) of which novel short isoforms become overexpressed in human leukemia.

The type 1 CD10/neutral endopeptidase 24.11 promoter: functional characterization of the 5¢-untranslated region

Summary. The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes and a variety of epithelial cells. Because CD10/NEP functions as part of a regulatory loop that controls local concentrations of peptide substrates and associated peptide‐mediated signal transduction, its role in each tissue is different depending on the availability of substrate. To characterize further how this widely distributed molecule is regulated differentially in each tissue, we analysed the major type 2 CD10/NEP promoter and found three functionally important transcription factor binding sites, one of which was identical to CCAAT‐binding transcription factor/nuclear transcription factor Y. In this report, we analyse the type 1 CD10/NEP promoter and found a functionally important transcription factor binding site in the 5′‐untranslated region. The results of the competition and supershift experiments demonstrated that the functionally important transcription factor was identical to Sp1. Our results suggest that ubiquitously expressed Sp1 may play an important role in differentiation stage‐specific regulation of CD10/NEP expression in lymphoid lineage.

Over-expression of short isoforms of Helios in patients with adult T-cell leukaemia/lymphoma

Summary. In previous studies, we demonstrated an over‐expression of the dominant‐negative isoform of the transcription factor Ikaros in patients with blast crisis of both chronic myelogenous leukaemia and B‐cell acute lymphoblastic leukaemia (ALL). Recently, we reported an over‐expression of the short isoforms of Helios, which is one of the members of the Ikaros gene family, in a patient with T‐cell ALL. In the present study, we found over‐expression of short isoforms of Helios in human T lymphotropic virus‐I (HTLV1)‐infected patients who had developed chronic and acute forms of adult T‐cell leukaemia/lymphoma. In contrast, we could not detect any over‐expression of short isoforms of Helios in healthy HTLV1 carriers. By Southern blotting, we detected a small deletion in the Helios gene locus of adult T‐cell leukaemia/lymphoma patients. The present results suggest that Helios gene abnormalities might be one of the important mechanisms in the disease progression of HTLV1 infection.

Over-expression of the dominant-negative isoform of Ikaros confers resistance to dexamethasone-induced and anti-IgM-induced apoptosis

Summary. In previous studies, we demonstrated an over‐expression of the dominant‐negative isoform of the transcription factor Ikaros, Ik‐6, in patients with B‐cell malignancies, including blast crisis of chronic myelogenous leukaemia and acute lymphoblastic leukaemia. To investigate the consequence of over‐expression of Ik‐6 in B cells, we constructed Ik‐6 transfectants of the FDH‐1 and Ramos cell lines. FDH‐1, which was established from a patient with early pre‐B acute lymphoblastic leukaemia, undergoes apoptosis with dexamethasone treatment, whereas Ramos undergoes apoptosis following anti‐IgM antibody treatment. Compared with the wild type, the over‐expression of Ik‐6 rendered the FDH‐1 and Ramos transfectants resistant to dexamethasone‐induced and anti‐IgM‐induced apoptosis respectively. An immunoblotting study demonstrated bcl‐2 upregulation in anti‐IgM‐induced Ramos Ik‐6 transfectants, but not in FDH‐1 Ik‐6 transfectants. Further investigations of the mechanism of leukaemogenesis associated with the over‐expression of Ik‐6 are warranted.

Plasma stromal cell-derived factor-1 during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

Summary:In this report, we examined plasma stromal cell-derived factor-1 levels in normal healthy donors for allogeneic peripheral blood stem cell transplantation (PBSCT) and in patients for autologous PBSCT using an enzyme-linked immunosorbent assay. The average level of plasma stromal cell-derived factor-1 was 2197 pg/ml before granulocyte colony-stimulating factor administration and 1899 pg/ml on day 4, demonstrating a significant decrease in the peripheral blood of healthy donors (P=0.0003). In patients for autologous PBSCT, a significant decrease of plasma stromal cell-derived factor-1 in the peripheral blood was also observed (P=0.0464). However, the physiologic gradient of stromal cell-derived factor-1 between peripheral blood and bone marrow was never inverted in normal healthy donors or in autologous PBSCT patients. Our results suggest that stromal cell-derived factor-1 may not be involved in the granulocyte colony-stimulating factor-induced release of CD34+ cells to the peripheral blood. Further studies of a possible additive effect of granulocyte colony-stimulating factor and stromal cell-derived factor-1 are warranted.

Risk of Neutropenic Fever and Early Infectious Complications after Autologous Peripheral Blood Stem Cell Transplantation for Malignant Diseases

Autologous peripheral blood stem cell transplantation (auto-PBSCT) has facilitated high-dose chemotherapy for the treatment of various types of malignancy, but the factors affecting the treatment outcome have not been well defined. We evaluated patients who underwent auto-PBSCT (46 patients with hematological malignancies and 39 with solid tumors) to elucidate the risks of background factors, including age, in association with infectious complications. In contrast to former reports, faster engraftment did not influence the incidence of documented infection or neutropenic fever, whereas high age (age ≥50 years old) and delayed platelet recovery (≥18 days) were demonstrated to be positively involved. The odds ratio (OR) for documented infection in elderly patients was 4.94 (95% confidence interval, 1.22-15.8). Another risk factor of infection was the HD-ICE regimen (ifosfamide, carboplatin, etoposide) given to patients with solid tumors (OR, 8.00; 95% confidence interval, 1.61-39.7). In conclusion, we found that elderly patients and patients on the HD-ICE regimen have a higher risk of infectious complications even after auto-PBSCT. Although the clinical indications for auto-PBSCT can be extended to elderly patients, thorough precautions should be taken against infectious complications during the pre-engraftment phase.

Graft-versus-leukemia effect with a WT1-specific T-cell response induced by azacitidine and donor lymphocyte infusions after allogeneic hematopoietic stem cell transplantation

BACKGROUND Azacitidine (Aza) and donor lymphocyte infusion (DLI) therapy has recently been reported as an effective salvage therapy for relapsed acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Despite the high response rate and relatively long period of remission, most patients relapse again. The immunologic mechanism of the response and limited efficacy remain unknown. CASE REPORT Aza + DLI therapy was performed for a patient with therapy-related MDS (t-MDS), who had relapsed after allogeneic peripheral blood stem cell transplantation. We observed a powerful graft-versus-leukemia (GVL) effect accompanied by an evident Wilms tumor antigen 1 (WT1)-specific CD8 T-cell response. Remission continued for 15 months, but finally the patient relapsed. The kinetics of the WT1-specific CD8 T cells were inversely associated with WT1 messenger RNA (mRNA), suggesting a WT1-driven GVL effect. DISCUSSION A difference of T-cell phenotype between the whole T cells and the WT1-specific CD8 T cells was observed. It is of note that the memory phenotype of the WT1-specific T cell was limited and decreased early. The immunoescape mechanism was partly supported by loss of the memory phenotype due to failure of expansion and differentiation. CONCLUSION Our data suggested that a WT1-specific T-cell response at least partly contributes to the GVL effect induced by Aza + DLI. A strategy for maximizing and maintaining the memory phenotype of the CTL may be required for durable remission.

Complete resolution of steroid-resistant organizing pneumonia associated with myelodysplastic syndrome following allogeneic hematopoietic cell transplantation

Pulmonary complications in patients with hematological malignancies are often caused by infection but are sometimes associated with an underlying disease such as organizing pneumonia (OP). Here, we report a case of life-threatening steroid-resistant OP associated with myelodysplastic syndrome (MDS) and successfully performed allogeneic hematopoietic cell transplantation (HSCT). A 33-year-old female with refractory anemia with excess blasts-1 that had progressed from refractory anemia with ringed sideroblasts and concomitant Sweet’s syndrome was admitted. Multiple pulmonary infiltrates were revealed on a chest computed tomography scan, which progressively worsened even after chemotherapy and corticosteroid therapy. No evidence of infection was observed in bronchoalveolar lavage fluid. A histological examination of a transbronchial lung biopsy specimen showed lymphocyte invasion with fibrosis, indicating that the pulmonary infiltrates were OP associated with MDS. Before transplantation, she suffered from respiratory failure and required oxygen supplementation. She developed idiopathic pneumonitis syndrome on day 61 that responded well to corticosteroid therapy, and the OP pulmonary infiltrates improved gradually after HSCT, She was discharged on day 104 and is well without recurrence of OP or MDS 2 years after HSCT.