Toyoshi Tatekawa

The Wilms’ tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes

The Wilms’ tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.

Combined spectral karyotyping and DAPI banding analysis of chromosome abnormalities in myelodysplastic syndrome.

Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy‐related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G‐banding. To locate the chromosomal breakpoints, DAPI‐counterstained band images from all metaphases were transformed to G‐band–like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G‐banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G‐banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of ‐5/5q‐ and ‐7/7q‐) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with ‐5/5q‐ and in 4 of 8 with ‐7/7q‐, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G‐banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336–345, 1999.

Combination of tacrolimus, methotrexate, and methylprednisolone prevents acute but not chronic Graft-Versus-Host disease in unrelated bone marrow transplantation

Background. Graft-versus-host disease (GVHD) is still a major problem in allogeneic bone marrow transplantation (BMT). Prophylactic regimens used against GVHD in unrelated BMT, including cyclosporine (CsA)-plus-methotrexate (MTX), CsA-plus-MTX-plus-prednisone, and tacrolimus (FK506)-plus-MTX, are still unsatisfactory (34–70% occurrence of grades II–IV GVHD). To address this problem, we examined the efficacy of FK506-plus-MTX-plus-methylprednisolone (mPSL) in 20 patients who underwent BMT from unrelated donors. Methods. All patients received FK506 beginning the day before transplantation at a dose of 0.03 mg/kg per day by continuous intravenous (IV) infusion. MTX was administered at a dose of 10 mg/m2 IV on day 1, and 7 mg/m2 on days 3, 6, and 11. Intravenous administration of mPSL was started at a dose of 2 mg/kg per day on day 1. In the absence of acute GVHD, mPSL was gradually tapered from day 29. Results. Development of acute GVHD was almost completely suppressed (one patient with grade I, none with grades II–IV). However, the incidence and severity of chronic GVHD did not decrease. Eight of 12 patients with extensive chronic GVHD died of thrombotic microangiopathy or infection. A vigorous fluctuation (>100 U/mL per 10 days) of the soluble interleukin 2 receptor level in the serum after engraftment was highly related to the occurrence of chronic GVHD. Conclusions. An FK506-plus(+)-MTX-plus(+)-mPSL prophylactic regimen could almost completely suppress acute GVHD but not chronic GVHD in unrelated BMT. In this GVHD prophylactic system, the extent of the change of soluble interleukin 2 receptor level may be a good predictor of development of chronic GVHD.

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34 + CD38 − cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38− cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38− cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38− and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38− CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.

High Incidence of Chemotherapy-Induced Acral Erythema in Female Patients with non-Hodgkin's Lymphoma Treated with the Vacop-B Regimen

Seven patients, all females out of 29 with non-Hodgkins lymphoma (NHL) (16 males and 13 females) treated with the VACOP-B regimen utilizing granulocyte-colony-stimulating factor (G-CSF) support developed chemotherapy-induced acral erythema (CAE). In contrast, none of 32 patients with NHL who were treated with CHOP, MACOP-B, or biweekly CHOP regimens without G-CSF developed CAE. Total dose intensities of VACOP-B regimen were higher than those of the three other regimens. However, no significant difference in dose intensities of each drug in the patients treated with the VACOP-B regimen was found between male and female patients and between female patients with or without CAE. The cause of the high incidence of CAE (7/13) in the female patients treated with VACOP-B regimen remains unknown. However, female sex hormones may increase susceptibility to CAE. Since the occurrence of CAE interrupts intensive chemotherapy and reduces the cure rate, high risk patients for CAE should be carefully monitored for early symptoms and signs of CAE and should be treated early and appropriately.

A case of immune recovery vitritis induced by donor leukocyte infusion for the treatment of cytomegalovirus retinitis

Abstract:  Donor leukocyte infusion (DLI) has been successfully used for some life‐threatening viral infections after stem cell transplantation (SCT). We describe here the first case of DLI treatment for cytomegalovirus (CMV) retinitis. A 49‐year‐old female patient with AML, M1 underwent SCT with a reduced‐intensity conditioning regimen from HLA‐haploidentical son. On day +140, the patient developed CMV retinitis of her left eye despite the continuing antiviral therapy. DLI at a dose of 1 × 105 CD3+ cells/kg was added to ganciclovir and foscarnet therapy. Eighteen days after the DLI, the funduscopic findings revealed improvement of the retinitis and the development of vitreous inflammation. Simultaneously, the number of CD4+ cells in the peripheral blood rapidly increased. Thus, we consider it likely that DLI induced a local immune response against CMV antigens, which resulted in the immune recovery vitritis. This case suggested the potentiality of DLI for the treatment of CMV retinitis.

A novel direct competitive repopulation assay for human hematopoietic stem cells using NOD/SCID mice

BACKGROUND The major problem in cord blood (CB) transplantation for adult patients is shortage of stem cell number. To overcome this disadvantage, several studies on ex vivo expansion have been performed. However, such efforts are always troubled by the lack of a reliable and simple assay system for stem cells. Our aim was to establish an in vivo assay system to compare the directly repopulating ability of two populations of human hematopoietic stem cells using a xenogeneic transplant system. METHODS Thirty CB samples from infants of each sex were pooled and enriched for CD34(+) progenitor cells. Enriched CD34(+) cells were transplanted into irradiated NOD/SCID mice at different male to female ratios, and human hematopoietic cells recovered 7 weeks after transplantation were analyzed by a quantitative DNA sex test using competitive PCR for the amelogenin gene. Using this assay system, ex vivo cultured and non-cultured CB cells were compared for repopulating ability. RESULTS The sex ratio of human CB cells transplanted was found to be maintained for 7 weeks in matured and progenitor cells. The competitive repopulation assay of cultured and non-cultured CB cells showed a marked defect in the repopulating ability of cultured cells, although the LTCIC count was maintained during cultivation. DISCUSSION Our assay system is a simple and reliable quantitative method that permits direct comparison of two stem cell compartments. The assay system will be useful for the assessment of the functional abilities of various human hematopoietic stem cells.