Seiji Gomi

Effects of interleukin-1 and tumor necrosis factor on megakaryocytopoiesis: mechanism of reactive thrombocytosis.

We studied the effects of interleukin-1 (IL-1) and tumor necrosis factor (TNF) on mouse megakaryocytopoiesis to evaluate the role of these cytokines in reactive thrombocytosis associated with inflammation. Injections of IL-1 or TNF to mice induced a significant increase in the megakaryocyte progenitor cell (CFU-Meg) count in the spleen. When IL-1 and TNF were injected simultaneously, the splenic CFU-Meg count was remarkably increased compared with mice injected with either IL-1 (p < 0.003) or TNF (p < 0.001) alone. On the other hand, neither IL-1 nor TNF showed any megakaryocyte-potentiating or -stimulating effects in vitro. In the sera obtained 4 h after administration of IL-1, TNF or both, high megakaryocyte potentiating activities were found. Furthermore, an extremely high level of IL-6 was detected in the serum after administration of both IL-1 and TNF. These results strongly suggest that IL-1 and TNF stimulate megakaryocytopoiesis indirectly via other cytokine(s) induced from accessory cells, and that increased levels of IL-1 and TNF play important roles in the development of reactive thrombocytosis caused by inflammation.

Identification and functional characterization of novel telomerase variant alleles in Japanese patients with bone-marrow failure syndromes.

As the incidence of bone-marrow failure syndromes (BMFS) is 2-3x higher in East Asia than in the West, we examined peripheral blood or marrow cells of 100 Japanese patients for possible pathogenic mutations in the two main components of the telomere-synthesizing enzyme telomerase (hTERC RNA and hTERT protein) that have recently been implicated in the disease pathogenesis. We analyzed samples collected from 34 patients with acquired aplastic anemia (AA), 66 patients with myelodysplastic syndromes (MDS) and 120 healthy controls. In addition to two polymorphic germ-line sequence changes (n-771A/G and n-714 C insertion) in the promoter region of hTERC and eleven hTERT polymorphisms that were identified in both patients and healthy individuals, we found a novel germ-line C323T mutation in the hTERC RNA in an MDS patient only. This heterozygous C323T mutation abolished telomerase enzymatic activity and functioned in a haploinsufficiency manner to modulate telomerase activity in cells. In summary, this study reports a novel telomerase natural variant that abolishes telomerase function, which may lead to telomere shortening and marrow hypocellularity in patients with BMFS. This study also highlights the rarity of genetic alterations in BMFS patients in Japan, which suggests that other factors may play a more prominent role in the disease pathogenesis in East Asia.

Megakaryocyte, Erythroid and Granulocyte-Macrophage Colony Formation in Myelodysplastic Syndromes

Bone marrow progenitor cell assays of three cell lineages, i.e., colony-forming unit megakaryocytes (CFU-Meg), burst-forming unit erythrocytes (BFU-E) and colony-forming unit granulocyte-macrophages (CFU-GM), were performed for 21 patients with myelodysplastic syndromes (MDS). Markedly reduced or absent colony formation was found in 67% of the patients for CFU-Meg and all patients except 2 with refractory anemia (RA) for BFU-E. Abnormal CFU-GM colony formation was found in only 5 of 12 patients with RA and RA with ring sideroblasts, in contrast to all of the RA patients with excess of blasts and excess of blasts in transformation. Defective colony formation of all three cell lineages was seen in 63% of the MDS patients. The colony number of CFU-Meg correlated significantly with the numbers of both BFU-E and CFU-GM. These findings indicate that hematopoiesis in MDS patients is disturbed due to a qualitative or quantitative defect at the multipotent stem cell level.

Chemotherapy for minimally differentiated acute myeloid leukemia (AML-M0) : a report on five cases and review of the literature

SummaryWith the objective of establishing the optimal therapy for minimally differentiated acute myeloid leukemia (AML-M0), we examined the therapeutic results of five AML-M0 cases and reviewed the literature. In a series of 63 patients with newly diagnosed acute leukemia who were admitted to the Main Hospital of Nippon Medical School, five patients fit the criteria for AML-M0: negative myeloperoxidase (MPO) and Sudan black B reaction by light microscopy, negative for B- and T-lineage markers, and positive for myeloid markers. They were treated by means of AdVP [adriamycin, vincristine, and prednisolone (PSL)] therapy and/or BHAC-DMP [behenoylcytosine arabinoside (BHAC), daunorubicin (DNR), 6-mercaptopurine (6-MP), and PSL] therapy. The AdVP therapy was unsuccessful in the two patients who received it, while a complete remission (CR) was achieved with the BHAC-DMP therapy in three of four patients. Although one patient treated with BHAC-DMP did not achieve CR, his blasts were apparently sensitive to the therapy. In assessable cases in the literature where leukemic blasts were MPO-negative, myeloid marker-positive and B- and T-lineage marker-negative, CR was achieved in 54.5% and 44.4% with anti-acute myeloid leukemia therapy and anti-acute lymphocytic leukemia therapy, respectively. Five cases in the literature were treated with a chemotherapeutic regimen containing BHAC [or cytosine arabinoside (Ara-C)], DNR, and 6-MP, and all achieved CR. The regimen containing BHAC (or Ara-C), DNR, and 6-MP may be useful as induction chemotherapy for AML-MO.

Low-dose etoposide: a potential therapy for myelodysplastic syndromes

Summary. Four patients with refractory anaemia with excess blasts in transformation (RAEB‐t) and seven patients with acute leukaemia (ALL transformed from myelodysplastic syndromes (MDS) were treated with etoposide (50 mg, 2 h infusion, two to seven times per week) for at least 4 weeks. Of 10 assessable patients, three RAEB‐t patients achieved partial response and one AL patient achieved complete remission. Three of the four responders were resistant to prior repeated low‐dose cytarabine therapy. The responders did not require transfusions for 2–9 months while continuing on etoposide therapy. The side‐effects were mild and well tolerated. Three possible mechanisms, i.e. a cytotoxic effect, differentiation‐induction of malignant cells, and prolongation of blood cell survival by destroying the reticuloendotheliai system, may explain the effects of etoposide. We conclude that low‐dose etoposide is a potential therapy for MDS and atypical leukaemia.

Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and ‘simplified analysis of co-mutations’ using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

Clinical features of adult acute leukemia with 11q23 abnormalities in Japan: a co-operative multicenter study

To clarify the clinical features of adult patients with acute leukemia (AL) with 11q23 abnormalities, we performed a retrospective analysis of data from 58 adult Japanese patients: 51 with acute myeloid leukemia (AML), and 7 with acute lymphoblastic leukemia (ALL). The incidences according to fusion partners in AML were: t(9;11), 31.3%; t(11;19), 27.4%; t(6;11), 21.5%. The incidence of patients with t(11;19) was higher than those in the US and Europe, and the incidence of t(4;11) was lower than that in childhood. The results indicated the poor prognosis of AML with 11q23 abnormalities regardless of the fusion partners. AML patients with 11q23 aged <60 years in the first CR who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) showed a more favorable outcome than those treated without allo-HSCT, although the differences were not statistically significant (P = 0.322 for DFS, P = 0.138 for OS). This result suggests that treatment strategies including allo-HSCT may be considered in the first CR in cases of AML with 11q23 abnormalities. However, further studies involving a large number of cases are required to assess the effect of allo-HSCT on adult AL with 11q23 abnormalities.

Successful treatment of acute promyelocytic leukemia in pregnancy with all-trans retinoic acid.

We report a case of acute promyelocytic leukemia (APL) in pregnancy which was treated successfully with all-trans retinoic acid (ATRA). A 30-year-old woman in the 30th week of pregnancy was admitted to the Nippon Medical School Hospital because of shortness of breath and fever. The blood count showed hemoglobin 6.4 g/dl, hematocrit 17.6%, red cell count 1.78 x 106/0,1, reticulocytes 1.7%, platelets 2.7 x 103/ixl, and white cell count (WBC) 1000/txl, with 38% atypical promyelocytes. Sternal bone marrow aspiration revealed extreme hypercellularity with 93% atypical promyelocytes. These atypical promyeloeytes had irregularly shaped nuclei and coarse azurophilic granules with frequent Auer rods, and they were positive for myeloperoxidase. Cytogenetic analysis revealed 46,XX,t(15;17) in 100% of the bone marrow cells. The reverse transcription-polymerase chain reaction (RTPCR) was performed, and a PML-RAR o~ fusion mRNA product was strongly detected. Coagulation studies revealed FDP 63.3 lxg/ml (normal < 10 txg/ml), D-dimer 8-16 pog/ml (normal <0.5 txg/ml), thrombinantithrombin III complex 51.9 ixg/dl (normal < 3 p,g/1), plasmin-a 2 plasmin inhibitor complex 5.6 ixg/dl (normal < 0.8 Ixg/1), and fibrinogen 216 mg/dl. A diagnosis of acute promyelocytic leukemia complicated with DIC was made. At the time of her admission, the fetus was well with normal development. After obtaining informed consent, treatment with ATRA (70 mg/day) was started on the day of admission (day 1) (Fig. 1). On day 9, the general condition of the fetus worsened, compatible with fetal distress syndrome. On day 12, when the mothers plasma FDP level decreased and the

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations.

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future.

Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations.

In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations – mutations of the genes that regulate gene expression through DNA methylation – is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (P<0.0001) and in patients with intermediate cytogenetic risk (P<0.0001) accompanied by a high white blood cell count (P=0.0032). DNA-methylation regulatory gene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.