Kunitake Hirashima

Fusion of TEL/ETV6 to a Novel ACS2 in Myelodysplastic Syndrome and Acute Myelogenous Leukemia With t(5;12)(q31;p13)

We identified a novel human long fatty acyl CoA synthetase 2 gene, ACS2, as a new ETV6 fusion partner gene in a recurrent t(5;12)(q31;p13) translocation in a patient with refractory anemia with excess blasts (RAEB) with basophilia, a patient with acute myelogenous leukemia (AML) with eosinophilia, and a patient with acute eosinophilic leukemia (AEL). ACS2 is expressed in the brain and bone marrow and is highly conserved in man and rats. The resulting ETV6/ACS2 fusion transcripts showed an out‐frame fusion of exon 1 of ETV6 to exon 1 of ACS2 in the AEL case, an out‐frame fusion of exon 1 of ETV6 to exon 11 of ACS2 in the AML case, and a short in‐frame fusion of ETV6 exon 1 to the 3′ untranslated region of ACS2 in the RAEB case. Reciprocal ACS2/ETV6 transcripts were identified in two of the cases. Fluorescence in situ hybridization (FISH) analysis with ETV6 cosmids on 12p13, and BACs and P1s on 5q31, demonstrated that the 5q31 breakpoints of the AML and AEL cases involved the 5′ portion of the ACS2 gene, and that the 5q31, breakpoint of the RAEB case involved the 3′ portion of the ACS2 gene. None of the resulting chimeric transcripts except for the ACS2/ETV6 transcript in the RAEB case led to a fusion protein. Disruption of the second ETV6 allele by t(12;19) was detected in the AML case by FISH analysis. These observations suggest that the disruption of ETV6 and/or ACS2 may lead to the pathogenesis of hematologic malignancies with t(5;12)(q31;p13). Genes Chromosomes Cancer 26:192–202, 1999.

A phase II trial of recombinant human granulocyte colony-stimulating factor in the myelodysplastic syndromes

Summary. We conducted a phase II study of the intravenous administration of a glycosylated recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) for 7–14 d in 41 patients with the myelodysplastic syndromes (MDS). Administration of rhG‐CSF elicited striking rises in both leucocyte and neutrophil counts in the majority of the patients irrespective of the FAB subtypes of MDS. The rises in neutrophil counts were dose dependent and 5 μg/kg/d of rhG‐CSF yielded approximately an 8‐fold increase in neutrophil counts. Leucocytes and neutrophil counts started to increase shortly after the first injection of 5 μg/kg, was maintained at significantly elevated levels during 14 d of treatment, and returned to the pretreatment levels within several days following discontinuation of rhG‐CSF. The action of rhG‐CSF was specific for neutrophils since leucocytosis was due exclusively to neutrophilic increase associated with an increased marrow myeloid maturation. There were no consistent changes in the monocyte, eosinophil, lymphocyte, platelet or reticulocyte counts. After treatment, the percentage of marrow blast cells was reduced in eight of 13 evaluable patients with refractory anaemia with an excess of blasts (RAEB) or RAEB in transformation (RAEB‐t). No patients developed acute leukaemia during the treatment or in the immediate follow‐up period. The treatment was well tolerated with only minimal toxicity. The results suggest that rhG‐CSF is a safe and effective way to promptly improve neutropenia in MDS patients.

Hair dye use and occupational exposure to organic solvents as risk factors for myelodysplastic syndrome

To investigate the relationships of personal hair dye use and environmental factors to myelodysplastic syndromes (MDS), we conducted a case-control study in Japan. A total of 111 MDS cases and 830 controls randomly selected from the residents in the same prefecture of cases using telephone directories responded to a health questionnaire. The odds ratio (OR) for ever having used hair dye was 1.99 (95% confidence interval (CI) 1.17-3.38) and there were statistically significant trends in risk with increasing duration and number of hair dye use. Occupational exposure to organic solvents was marginally associated with the risk of MDS (OR = 1.99; 95% CI 0.97-4.10).

Treatment of the anemia of aplastic anemia patients with recombinant human erythropoietin in combination with granulocyte colony - stimulating factor: a multicenter randomized controlled study

Abstract:  A multicenter randomized controlled study was undertaken in order to determine whether epoetin beta (EPO) ameliorates the anemia in aplastic anemia (AA) patients treated with granulocyte colony‐stimulating factor (G–CSF). Enrolled patients were randomized into 3 groups: group C receiving G–CSF alone as the control; group L receiving G–CSF and 200 IU/kg of EPO; group H receiving G–CSF and 400 IU/kg of EPO. Throughout the study, the dose and the administration interval of G–CSF were adjusted to maintain neutrophil counts between 1000 and 5000 μl. EPO was administered subcutaneously for 12 wk as the first step in treatment and when favorable effects were observed over this period, treatment was continued for another 12 wk as the second step in treatment. Significant erythroid responses were defined as increases in untransfused hemoglobin values >1.0 g/dl or >50% decreases in RBC transfusion requirements over the treatment period. Of 131 patients enrolled, 88 patients allocated to groups L and H were evaluated for toxicity to EPO and 110 were evaluated for erythroid responses. Four of the 31 patients (12.9%) in group C, 6 of the 41 patients (14.6%) of group L, and 14 of the 38 patients (36.8%) of group H showed erythroid responses in the first step in treatment. The erythroid responses of group H were significantly higher than those of the other 2 groups (p<0.05). The significant effects of EPO were due to erythroid responses in non‐severe AA. Responding patients were significantly different from non‐responders with regard to disease severity, hemoglobin concentration, reticulocyte count, serum endogenous erythropoietin levels and serum transferrin receptors; non‐severe AA patients were more likely to respond to EPO, and responding patients had lower serum EPO and higher hemoglobin concentration, reticulocyte count and serum transferrin receptors than non‐responders. The response rate increased in the second step in treatment, suggesting that long‐term treatment improved the efficacy of EPO. No serious side‐effects were observed. From these results, we conclude that EPO given in combination with G–CSF is a safe and effective alternative for the treatment of anemia of a subset of AA patients.

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations ‘RA with severe dysplasia (RASD)’ and ‘RA with minimal dysplasia (RAminiD)’. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger–Huet anomalies of mature neutrophils ⩾3% and/or micromegakaryocytes (mMgk) of megakaryocytes ⩾10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P = 0.06; RASD vs RAEB, P = 0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.

Effects of Interleukin‐6 and Granulocyte Colony‐stimulating Factor on the Proliferation of Leukemic Blast Progenitors from Acute Myeloblastic Leukemia Patients

The effects of recombinant human interleukin‐6 (rh IL‐6), which has homology with rh granulocyte colony‐stimulating factor (rh G‐CSF) at the amino acid sequence level, and rh G‐CSF on normal human bone marrow cells, fresh leukemic blast progenitors from 16 acute myeloblastic leukemia (AML) patients, and G‐CSF‐dependent human AML cell line (OCI/AML 1a) were investigated. rh G‐CSF stimulated the proliferation of leukemic blast progenitors from 13 out of 16 AML patients tested. rh IL‐6 stimulated the proliferation of blasts from eight AML patients and enhanced the G‐CSF‐dependent proliferation of the fresh AML blasts from two out of eight patients tested. On the other hand, rh IL‐6 suppressed the blast colony formation from two AML patients and OCI/AML 1a cells and also reduced the G‐CSF‐dependent proliferation of the blast progenitors from one of the two patients and the cell line. rh IL‐6 had no effect on the colony formation of normal granulocyte‐macrophage colony‐forming units (CFU‐GM) with or without rh G‐CSF. Differentiation‐induction by rh IL‐6 was not observed in the fresh AML blasts but was observed in OCI/AML 1a. The effect of IL‐6 on the blast colony formation and G‐CSF‐dependent blast cell growth was complicated and heterogenous among the AML cases; IL‐6 stimulated blast colony formation in some cases and suppressed it in others. The heterogeneity of the response was supposed to be derived from the heterogeneity of the characteristics of AML cells. Although G‐CSF simply stimulated the blast colony formation, IL‐6 had a bimodulatory effect on the proliferation of leukemic blast progenitors from AML patients. IL‐6 might be involved in the regulation of the proliferation of AML cells in vivo as well as in vitro.

New system for assessing the prognosis of refractory anemia patients

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies ⩾3% were classified as high risk (12 patients); of patients without pseudo-Pelger–Huët anomalies ⩾3%, those with intermediate/poor karyotype according to IPSS, Hb ⩽6 g/dl or mMgk ⩾10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

A long term follow-up of a randomized trial comparing interferon-α with busulfan for chronic myelogenous leukemia

Abstract To evaluate the long-term effectiveness of interferon- α (IFN- α ) therapy in patients with chronic myelogenous leukemia (CML) in chronic phase, we examined the updated outcomes of 159 patients who had been enrolled between 1988 and 1991 into a randomized trial comparing IFN- α with busulfan. At a median follow-up of 73 months, the median survival was 71 months in the IFN- α group and 55 months in the busulfan group ( P =0.0563), and the median time of remaining in chronic phase was 58 months in the IFN- α group and 39 months in the busulfan group ( P =0.4676). Landmark analysis showed a significant advantage in survival ( P =0.009) and duration of chronic phase ( P =0.0001) in patients with any cytogenetic response among the IFN- α group. About half patients were discontinued IFN- α administration in spite of cytogenetic response in this study. It appears that continuation of IFN- α might possibly confer a survival advantage. Pretreatment factors associated with cytogenetic response included high hemoglobin level, low percentage of peripheral basophils and low leukocyte counts. Multivariate analysis identified lower percentage of bone marrow basophilia ( P =0.007) for survival advantage. If a group with a very good prognosis is predicted by a new prognostic model, it might be an option to wait for bone marrow transplantation.

Diagnostic significance of serum soluble transferrin receptors in various anemic diseases: The first multi-institutional joint study in Japan

Serum soluble transferrin receptor (sTfR) has been reported to be higher in patients with iron deficiency or with elevated erythropoiesis. In the present study, serum sTfR was measured in various anemic diseases and their clinical significance was examined in a multi-institutional joint study. Serum sTfRs in patients with the following anemic diseases were markedly higher than those in normal healthy adults: non-treated iron deficiency anemia (IDA) (9.13 +/- 7.04 mg/l, n = 52, p < 0.0001), anemia of chronic disorders (ACD) (3.45 +/- 1.38 mg/l, n = 20, p < 0.0001), hemolytic anemia (HA) (5.57 +/- 3.26 mg/l, n = 17, p < 0.0001), and myelodysplastic syndrome (MDS) (4.03 +/- 2.83 mg/l, n = 20, p < 0.0001). There were significant differences between IDA and ACD (p < 0.0001), between aplastic anemia (AA) (1.58 +/- 1.26 mg/l, n = 16) and MDS (p < 0.001), and between AA and MDS with refractory anemia (MDS-RA) (4.16 +/- 3.40 mg/l, n = 9) (p < 0.02). In patients with chronic renal failure (CRF), serum sTfR levels and serum sTfR/log serum ferritin ratios (sTfR/F index) were compared in the two classified groups according to Muirheads criteria, as IDA and non-IDA groups with or without recombinant human erythropoietin (rHuEPO) treatment. Significantly high levels of both serum sTfR (p < 0.0001) and the sTfR/F index (p < 0.0001) were observed in IDA without rHuEPO treatment. Especially in CRF with rHuEPO treatment, the sTfR/F index showed marked elevation in the IDA group (p < 0.0001) compared with serum sTfR (p < 0.001), indicating more diagnostic efficacy of the sTfR/F index for CRF with IDA. In conclusion, the serum sTfR concentration is a useful diagnostic tool for discrimination between IDA and ACD, and between AA and MDS-RA, and for the detection of iron deficiency in CRF patients in the Japanese population.

Effects of recombinant human erythropoietin on anaemic W/Wv and Sl/Sld mice.

Summary. The effects of recombinant human erythropoietin (rHuEPO) on anaemic W/Wv and SI/SId mice were investigated. rHuEPO was injected every day for a week in doses up to 86000 iu/kg. Wv/+ and SId+ mice, which have genetically a weak anaemia, received 17 or 86 iu/kg of rHuEPO and showed dose‐dependent increases in haemoglobin, PCV, RBC and reticulocytes to the same extent as that in normal mice. W/Wv mice also showed increases in the haematological parameters in response to 8600 iu/kg of rHuEPO but the dose was much higher than that for normal mice. A reticulocyte increase in W/Wv mice appeared later than in normal mice and was not sustained for 2 weeks even though the rHuEPO treatment was continued. SI/SId mice, however, did not show any significant haematological effect from doses up to 86 000 iu/kg. In both W/Wv and SI/SId mice receiving 8600 and 86000 iu/kg of rHuEPO, respectively, an increase in splenic or bone marrow CFU‐E was observed regardless of the defect in their haemopoietic systems. The plasma erythropoietin (EPO) level in W/Wv and SI/SId mice was inversely correlated with the haemoglobin, indicating that EPO production was not influenced by the haemopoietic defect and was regulated by the hypoxic properties of the anaemia. These results indicate that a large dose of exogenous rHuEPO is effective for the anaemia in W/Wv mice caused by a stem cell defect but not for the anaemia in SI/SId mice caused by a defective microenvironment.