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Abstract: A 64-year-old man in a severely immunocompromised state due to acute myelogenous leukemia died, respirator-unaided, about 10 h after the abrupt onset of coma. An earlier blood culture had yielded Bacillus cereus. The autopsy, performed 2 h after death, demonstrated diffuse subarachnoid hemorrhage without berry aneurysms, and the formalin-fixed brain was tinged with gray-brownish discoloration. The sections of the brain presented a whitish tint of the surface layer of all portion of the cerebral cortices, even those in the sulci. Histological examination of the brain revealed leptomeningeal B. cereus dissemination, and widespread necrosis of the leptomeninges and arachnoid vessels without inflammatory cell reaction. The grossly recognizable whitish surface layer of the cerebral cortex showed overt hyperchromatism, and contained neurons more degenerative than those located in the deeper cortical layer. The total absence of inflammatory reaction may be explained by a combination of the immunocompromised state of the patient and the character of B. cereus infection, which in itself induces little inflammatory reaction. The prominent lesions were confined to the cerebral surface layer and leptomeningeal tissue including the arachnoid vessels, which were all bathed in the cerebrospinal fluid, suggesting that some necrotizing toxins had been secreted into the fluid by the B. cereus. The necrosis of arachnoid vessels is thought to have in turn caused diffuse subarachnoid hemorrhage and marked disturbance of the cerebral blood flow, resulting in the terminal coma.

Many amplified-chromosome regions in human cancers have been identified, while there are only a limited number of reports for amplified genes in a single amplicon. We recently isolated four cDNA clones, A39, B47, C51, andCAB1,from 500 kilobases of yeast artificial chromosome DNA containing the c-ERBB-2gene. B47 consisted of 615 base pairs and had about 85% homology with the corresponding sequence of mouseGrb-7.We report here the structure of humanGRB-7cDNA, and we report that the amounts of mRNA for c-ERBB-2, CAB1,andGRB-7were elevated in concordance with the amplification.

In the IDSA guideline published in 2010 on the use of antimicrobial agents in neutropenic cancer patients, monotherapy with an anti-pseudomonal beta-lactam agent, such as cefepime (CFPM), carbapenem [meropenem (MEPM) or imipenem-cilastatin (IPM)], or piperacillin-tazobactam, is recommended as an empiric therapy for high risk febrile neutropenia (FN) patients. There have been few reports on the efficacy of cefozopran (CZOP), one of the 4thgeneration beta-lactams, in the treatment of such patients. We conducted an open-label randomized controlled study to evaluate the clinical efficacy of CZOP, MEPM, or IPM in high-risk FN adult patients with hematologic malignancies and solid tumors, using CFPM as a control.In this trial, 386 patients registered from 23 centers were randomized to receive either 4thgeneration beta-lactam (CFPM 2g, q12h IV or CZOP 2g, q12h IV) or carbapenem (MEPM 1g, q12h IV or IPM 1g, q12h IV). The primary endpoint was response to treatment defined as complete defervescence by day 7 with improvement in infection-related symptoms and laboratory findings. The clinical data were analyzed both by intention to treat and per protocol. We also evaluated the efficacy of each of the initial four drugs at days 3 to 5as a secondary endpoint.386 patients received the assigned antibiotic (CFPM: n=95, CZOP: n=98, MEPM: n=96, IPM: n=97) and 377 were evaluated for efficacy (CFPM: n=94, CZOP: n=95, MEPM: n=94, IPM: n=94). The 4 groups were comparable in terms of the baseline characteristics, including as age (median: 59 years (range: 17–87)), sex (male: n=215, female: n=171), body weight (median: 55 kg (range: 32–100)), underlying malignancy (leukemia: n=212 (54.9%), lymphoma: n=122 (31.6%), multiple myeloma: n=37 (9.6%), myelodysplastic syndrome: n=6 (1.6%), and other malignancy: n=9 (2.3%)), duration of initial antibiotic therapy (median: 7 days (range: 1–29)), median duration of severe neutropenia (neutrophil count < 100×106/L) (median: 6 days (range: 0–56)) and MASCC score (median: 21 (range:3–26)). Only neutrophil count at onset of IPM was significantly lower compared to the other treatments (CFPM: 38×106/L, CZOP: 35 x106/L, MEPM: 18×106/L, and IPM: 3×106/L, p=0.003). In intention-to-treat analysis, the response rates at day 7 were not significantly different among the four arms (CFPM: 63%, CZOP: 59%, MEPM: 61%, IPM: 69% (P=0.52)). The three antibiotics investigated in this trial were as effective as the reference agent, CFPM, in both the intention-to-treat and per-protocol populations. In both the intention-to-treat and per protocol analyses, success rates of the initial therapy at days 3 to 5 were not significantly different among four drugs [CFPM: 52 and 54%, CZOP: 46 and 47%, MEPM: 54 and 55%, IPM: 49 and 53% (P=0.70 and 0.68, respectively)].CZOP, MEPM, and IPM were as effective as CFPM for adult FN patients with hematological malignancies and solid tumors as an empiric therapy.Tamura: Kyowa Hakko Kirin Co., Ltd,: Consultancy.

We recently reported that human interleukin 1 beta (IL-1 beta) stimulates bone-marrow stromal myofibroblasts to express a hematopoietic stem cell marker, CD34 (16th EHA, and 53rdASH meeting). To characterize precisely the effects of IL-1 beta to the stromal cells, adult human dermal fibroblasts (HDF) were cultured with IL-1 beta, and the morphological changes and molecular expressions were observed.HDF were purchased, and cultured in knockout DMEM medium with 15% KSR with or without recombinant human IL-1 beta for 3 weeks. The morphological changes and the expression of specific hematopoiesis-related genes were analyzed time-dependently. The concentration of hematopoietic growth factors in the culturing supernatants was also measured.When HDF were cultured with human IL-1 beta for 3 weeks, the cellular morphology changed to the filamentous appearance. RT-PCR analyses revealed that cells expressed Prox-1, a master molecule for lymphatic duct neogenesis, and also vascular endothelial growth factor receptor (VEGFR) type-3. Smad7 and PAI1 were also expressed; however, LYVE1, a marker for lymphatic vascular endothelial cell, was not induced. VEGF-C production was also demonstrated at RNA and protein levels, and VEGF-C and VEGFR type-3 system played an important role in morphological changes and cell-proliferation.We previously reported that when HDF were cultured in DMEM/F12 medium with IL-1 beta and EPO, hematopoietic markers were induced. When HDF were cultured in knockout DMEM, a condition for immature cell-growth at ES cell levels, and IL-1 beta, lymphatic duct neogenesis-related genes were activated, and HDF converted their morphology without an introduction of any genes externally. We are now making clear the precise action of IL-1 beta to HDF using microarray analysis.No relevant conflicts of interest to declare.

Abstract 4738

Abstract 1037This icon denotes a clinically relevant abstract

Epstein-Barr-virus- (EBV-) associated lymphoproliferative disorder (LPD) after immunosuppressive therapy for aplastic anemia (AA), in a nontransplant setting, has not been well described. We report one case of colonic EBV-LPD after a single course of immunosuppressive therapy for AA. The patient developed multiple colonic tumors 3 months after receiving immunosuppressive therapy, which consisted of rabbit antithymocyte globulin (ATG), cyclosporine, and methyl-predonisolone. The histological findings of biopsy specimens revealed that atypical lymphocytes had infiltrated colonic glands. Immunohistochemical staining for CD20 was positive, and in situ hybridization for EBV-encoded small RNAs was also positive. The EBV viral load in peripheral blood was slightly increased to 140/106 white blood cells. After the cessation of immunosuppressant, the colonic tumors spontaneously regressed, and the EBV viral load decreased to undetectable levels. This is the first report of the single use of rabbit ATG inducing colonic EBV-LPD. Because a single use of immunosuppressive therapy containing rabbit ATG can cause EBV-LPD, we should carefully observe patients receiving rabbit ATG for AA.

Takeshita: Novartis Pharma: Research Funding; Takeda Pharma: Research Funding. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

No relevant conflicts of interest to declare.

We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF).cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed.Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo.No relevant conflicts of interest to declare.

We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and in vivo. Recently, we also reported that myelogenous leukemia-derived myofibroblasts formed blastomas in non-obese diabetes severe combined immunodeficiency (NOD/SCID) mice, in which the CD56-positive cell-fraction was selectively proliferated (16th EHA). To ascertain whether normal human bone marrow-stroma-derived cells also behave like leukemia-derived cells, stromal cells were injected to NOD/SCID mice, and tumor-formation was observed.Bone marrow cells were collected from informed normal individuals, whose adherent cells were separated and were cultured for one month to eliminate monocyte/macrophage, vascular cell, and pericyte to prepare stromal myofibroblast-rich fraction. Cells were injected to NOD/SCID mice intra-venously, peritoneally, and subcutaneously (3 × 106/mice). When the tumor formation was observed or mice were dead, they were sacrificed and the engrafted cells were analyzed.Between at day 70 and 90 after injection mice were dead. Autopsy findings revealed tumor formation at subcutaneous injection sites, and cells were also infiltrated to the liver and ascitic fluid. These cells expressed CD56, CD146, and Nestin strongly, but not other lineage-specific markers including CD133; however, the ratio of CD56(+)CD146(+) fraction in the injected stromal cells was below 0.1%. When the tumor cells were cultured in vitro, they exhibited spindle-shaped appearance, and doubling time was 12 to 16 hours. They expressed c-Myc, Klf4, Nanog, Sox2, and other molecules that are expressed in an immature somatic stem cell. These observations indicate that CD56(+)CD146(+) cell-fraction in bone marrow-stroma is very immature and highly proliferative. We are now analyzing biological characteristics of this specific cell-fraction, and determining the contribution to normal hematopoiesis.No relevant conflicts of interest to declare.

Abstract 4815

Abstract 1532

Abstract 1487

Abstract 1328

Bone marrow cells were collected from informed AML (M2) patients who had chromosomal translocation of RUNX1 and ETO, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, the non-adherent mononuclear cells were injected to the 3.0 Gray-irradiated non-obese diabetes (NOD)/SCID mouse intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Runx1 and ETO fusion transcript, and mice were sacrificed after chimeric mRNA was observed. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select AML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed.When non-adherent AML (M2) blast cells were transplanted to NOD/SCID mice, cells were engrafted after 10 weeks. In murine bone marrow cells human stromal cells were identified, in which RUNX1 and ETO gene was fused with FISH analysis. When the parental AML blast cells were cultured on the expanded AML-derived myofibroblasts, AML cells grew extensively. These results indicate that AML cells can create their own microenvironment for proliferation in vivo.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 1621

In acute promyelocytic leukemia (M3), a prominent production of vascular endothelial growth factor (VEGF) is reported, and the obvious angiogenesis is observed in bone marrow specimens; however, VEGF receptors (VEGFRs) are not expressed in M3 cells in general. We analyzed VEGF systems in M3 blasts.Bone marrow cells were obtained from informed M3 patients, whose mononuclear cells were prepared with density-sedimentation method. Cells were cultured for one day for the elimination of an adherent cell-fraction. RNA was extracted from the non-adherent mononuclear cell-fraction, and cDNA was synthesized. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of VEGF and VEGFRs. When the expression of VEGFR type-1 and -2 was demonstrated with RT-PCR, cells were analyzed on the protein level with FACS. VEGF-A levels in sera and in the conditioned media from the cultured M3 cells were determined with ELISA kit. When M3 blasts expressed VEGFR type-1, -2, and also secreted VEGF-A, a growth-inhibition by anti-VEGF antibody was assayed in in vitro cultures.In all 25 cases examined VEGF-A production was observed, in which VEGFR type-3 was not expressed in any cases. VEGFR type-1 and -2 were expressed in 3 cases, in all of which WBC count at the onset of the disease was above 20,000/μl. When VEGF antibody was added to the blast cell-cultures, the cell-growth was inhibited significantly. These observations indicate that VEGF system works on proliferation in a few of M3 cases with hyper-leukocytosis.No relevant conflicts of interest to declare.

Abstract 4127

Abstract 2191

Objective: A chromosomal translocation plays an important role on the leukemia-generation from the normal hematopoietic cells. Various kinds of translocation-patterns and -partners have been reported; however, a typical translocation has been reported like AML-1 and MTG8 in acute myelogenous leukemia (AML) (M2), and PML and RARα in AML (M3). This implies that some common mechanism exists on the chromosomal double strand breaks and the generation of translocations. We make a hypothesis that, when normal immature cells come into a maturation process, a specific event may occur on DNA level to induce irreversible changes toward cell-death. And a target region for the irreversible change may be the near site to chromosomal translocations. We compared the digested fragment-size for restriction-endonucleases using southern blotting among hematopoietic cell lines, normal bone marrow immature progenitor cells and normal blood mature neutrophils.

The internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3/ITD) is now accounted as one of important risk factors for the prognosis in acute myelogenous leukemia (AML) patients with normal karyotype. In all-transretinoic acid era, acute promyelocytic leukemia (APL) has become the most curative subtype of adult AML, and usually hematopoietic stem cell transplantation is not selected for APL patients in 1stcomplete remission (CR) or 2ndCR without minimal residual disease (MRD) at the polymerase chain-reaction level; however, relapses are still reported to occur in 15–25% of cases after achieving 1stCR without MRD, and some risk factors are proposed including the difference of breakpoint region of the translocation, and leukocyte count at the onset. The incidence of FLT3/ITD in APL has been reported 17–35%; however, it is still a matter of debate that the presence of FLT3/ITD is associated with poor prognosis in APL patients. Thus, we focused on the incidence, clinical features, and prognostic implications of FLT3/ITD in adult newly diagnosed 17 APL patients (median age of 45 years, range 22–67 years, 11 males and 6 females, and median total leukocyte count (TLC) of 2.0 × 109/L, range 0.4– 156.1 × 109/L). All patients achieved CR. The incidence of the presence of FLT3/ITD was 29.4%. The median TLC was significantly higher in FLT3/ITD positive group (median TLC of 9.4 × 109/L) as compared in the negative group (median TLC of 1.2 × 109/L). Higher LDH level in blood was also observed in FLT3/ITD positive group. The rate of central nerves system (CNS) involvement was significantly higher in FLT3/ITD positive group than that in the negative group (60 % vs. 0%). Relapse rate was also significantly higher in FLT3/ITD group (100 % vs. 0%). These data indicate that the presence of FLT3/ITD is associated with the high relapse rate and CNS involvement in APL patients, and implies one of poor prognostic factors.

Objective: A chromosomal translocation plays an important role on the leukemia-generation from the normal hematopoietic cells. Various kinds of translocation-patterns and -partners have been reported; however, a typical translocation has been reported like AML-1 and MTG8 in acute myelogenous leukemia (AML) (M2), and PML and RARα in AML (M3). This implies that some common mechanism exists on the chromosomal double strand breaks and the generation of translocations. We make a hypothesis that, when normal immature cells come into a maturation process, a specific event may occur on DNA level to induce irreversible changes toward cell-death. And a target region for the irreversible change may be the near site to chromosomal translocations. We compared the digested fragment-size for restriction-endonucleases using southern blotting among hematopoietic cell lines, normal bone marrow immature progenitor cells and normal blood mature neutrophils. Materials and Methods: Bone marrows and bloods were obtained from informed healthy persons, and mononuclear cells were prepared after Ficoll-Paque sedimentation method, and neutrophil-rich fractions were prepared with density gravity centrifugation and hypotonic RBC lyses method. Various kinds of human cell lines, cultured in DMEM with 10% FCS, were also prepared. High molecular weight DNA was extracted from these cells, and they were digested with several kinds of restriction endonucleases. After electrophoresis, the digested DNA was transferred to a nitrocellulose membrane, and southern blot-hybridization was examined. The probe used was AML-1, MTG-8, PML, and RARα, in which the 5′ and 3′ breakpoint regions observed in chromosomal translocations were selected, respectively. Results and Discussion: No changes on the blotted fragment-size were observed among in the cultured cell lines, bone marrow immature mononuclear cells, and blood mononuclear cells; however, when 5′-PML probe was hybridized after DNA was digested with HindIII, the observed blot size was changed between normal immature myeloid progenitor cells and normal blood mature neutrophilic granulocytes. This change was not due to the personal polymorphism. These observations indicate that when myeloid cells become mature to neutrophilic granulocytes, some change occurs at DNA level. PML works on the maintenance of nucleobody for cell division, and it is very important to determine whether PML gene is maintained in neutrophilic granulocytes. We now prepare a genomic library from blood neutrophils to isolate the target clone, and determine DNA sequence near at the HindIII site to identify the modification of DNA during maturation of myeloid cells.

The internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3/ITD) is now accounted as one of important risk factors for the prognosis in acute myelogenous leukemia (AML) patients with normal karyotype. In all-trans retinoic acid era, acute promyelocytic leukemia (APL) has become the most curative subtype of adult AML, and usually hematopoietic stem cell transplantation is not selected for APL patients in 1st complete remission (CR) or 2nd CR without minimal residual disease (MRD) at the polymerase chain-reaction level; however, relapses are still reported to occur in 15–25% of cases after achieving 1st CR without MRD, and some risk factors are proposed including the difference of breakpoint region of the translocation, and leukocyte count at the onset. The incidence of FLT3/ITD in APL has been reported 17–35%; however, it is still a matter of debate that the presence of FLT3/ITD is associated with poor prognosis in APL patients. Thus, we focused on the incidence, clinical features, and prognostic implications of FLT3/ITD in adult newly diagnosed 17 APL patients (median age of 45 years, range 22–67 years, 11 males and 6 females, and median total leukocyte count (TLC) of 2.0 × 109/L, range 0.4– 156.1 × 109/L). All patients achieved CR. The incidence of the presence of FLT3/ITD was 29.4%. The median TLC was significantly higher in FLT3/ITD positive group (median TLC of 9.4 × 109/L) as compared in the negative group (median TLC of 1.2 × 109/L). Higher LDH level in blood was also observed in FLT3/ITD positive group. The rate of central nerves system (CNS) involvement was significantly higher in FLT3/ITD positive group than that in the negative group (60 % vs. 0%). Relapse rate was also significantly higher in FLT3/ITD group (100 % vs. 0%). These data indicate that the presence of FLT3/ITD is associated with the high relapse rate and CNS involvement in APL patients, and implies one of poor prognostic factors.

Upshaw-Schulman syndrome (USS) is a congenital deficiency of the activity of von Willebrand factor (VWF)-cleaving protease or ADAMTS13 due to its gene mutations. USS is a complex thrombo-hemorrhagic disease, but its hallmark is severe neonatal jaundice soon after birth that often requires for exchange blood transfusion, and subsequently during childhood they have repeated episodes of chronic thrombocytopenia and hemolytic anemia that are reversed by infusions of fresh frozen plasma. However, after a discovery of ADAMTS13, the presence of two phenotype expression on USS-patients was described; one is the early-onset type as abovementioned, and the other is the late-onset type which is asymptomatic during childhood and the first bout develops after adolescent or during adulthood, in association with infections or pregnancy. During the past 8 years, we diagnosed 33 patients with USS. Through analyzing the natural history and the phenotype-genotype expression in these patients, and more specifically to 9 USS-patients who had their first bout at pregnancy, we found that two clinical phenotypes of USS are mostly attributable to misdiagnosis or overlook of thrombocytopenia during childhood, and excluded a possibility of the concern on a trace amount of ADAMTS13 activity which has not been evaluated by the previous methods. Further, in vitro studies we have clearly shown that platelet aggregation or thrombi formation under high shear stress is tremendously up-regulated in the absence of ADAMTS13, that occurs proportionally to the amount of high VWF multimers, that in part is assumed to be an in vitro reflection of the circulation in USS-gravida. Figure Figure

Upshaw-Schulman syndrome (USS) is a congenital deficiency of the activity of von Willebrand factor (VWF)-cleaving protease or ADAMTS13 due to its gene mutations. USS is a complex thrombo-hemorrhagic disease, but its hallmark is severe neonatal jaundice soon after birth that often requires for exchange blood transfusion, and subsequently during childhood they have repeated episodes of chronic thrombocytopenia and hemolytic anemia that are reversed by infusions of fresh frozen plasma. However, after a discovery of ADAMTS13, the presence of two phenotype expression on USS-patients was described; one is the early-onset type as abovementioned, and the other is the late-onset type which is asymptomatic during childhood and the first bout develops after adolescent or during adulthood, in association with infections or pregnancy. During the past 8 years, we diagnosed 33 patients with USS. Through analyzing the natural history and the phenotype-genotype expression in these patients, and more specifically to 9 USS-patients who had their first bout at pregnancy, we found that two clinical phenotypes of USS are mostly attributable to misdiagnosis or overlook of thrombocytopenia during childhood, and excluded a possibility of the concern on a trace amount of ADAMTS13 activity which has not been evaluated by the previous methods. Further, in vitro studies we have clearly shown that platelet aggregation or thrombi formation under high shear stress is tremendously up-regulated in the absence of ADAMTS13, that occurs proportionally to the amount of high VWF multimers, that in part is assumed to be an in vitro reflection of the circulation in USS-gravida.

Vitamin K2 (VK2) analogs, including menaquinone-4 (MK4) but not vitamin K1, effectively induce apoptosis in acute myeloid leukemia cells (AML). Apoptosis induction by VK2 is specific toward leukemia cells, and almost no effect is observed in normal hematopoietic progenitor cells (Miyazawa K, Leukemia 1999, 2000, 2001). Combined treatment with VK2 plus 1α, 25-dihydroxy-vitamin D3 (D3) dramatically enhanced differentiation of leukemia cells as compared to the cells treated with D3 alone. Concomitant with the induction of differentiation and final maturation by VK2 plus D3, the cells became resistant to various apoptotic stimuli (Funato K, Leukemia, 2002). VK2 has been shown to improve the supportive hematopoietic functions of bone marrow stromal cells (Miyazawa K, Stem Cell Dev. 2004). Since VK2 (MK4, GlakayR) has been used to treat patients with osteoporosis in Japan, its non-toxicity and safety for long-term daily administration have already been well established. Case reports demonstrating the clinical benefits of using VK2 with or without D3 for treating patients with myelodysplastic syndrome (MDS) and AML have been accumulating, i.e. improving cytopenias in refractory anemia (RA) and reduction of blast cells in AML. We performed a multicenter prospective trial to determine the therapeutic benefit of VK2 with or without D3 in MDS. Patients with RA/RCMD with low/ int-1 by IPSS received VK2 (Glakay, 45 mg/day, po) for 16 weeks. The therapeutic response was assessed according to the International Working Group Criteria. Patients who did not respond to VK2 were sequentially re-registered and received a combination of VK2 plus D3 (alfacalcidol, AlfarolR, 0.75 microgram/day, po) for 16 weeks. Patients showing response to VK2 alone or VK2 plus D3 after 16 weeks continued to receive therapy for a total of 12 months. Forty-three patients have been enrolled and 29 patients were evaluable for their response to VK2. Five patients (17.2 %) showed improvement of cytopenia (major HI-E (1), minor HI-E (2), major HI-P (3), and minor HI-P (1)). Twenty out of 24 patients who showed no response to VK2 alone subsequently received VK2 plus D3. Eleven patients were eligible for evaluation and 4 patients (36.4%) showed improvement of cytopenia in response to VK2 plus D3 (major HI-E (3), minor HI-E (1) and major HI-P (1)). Four out of 7 patients (57 %) with anemia showed hematologic response. No adverse events were observed, except for one case of skin rash (Grade I) during VK2 plus D3 therapy. These data suggest that the therapeutic response rate is improved by combined treatment with VK2 plus D3 as compared with that by VK2 monothrapy. Safety and low price also support the therapeutic benefit of VK2 with D3 for improving cytopenia in RA.

Upshaw-Schulman syndrome (USS) is a congenital thrombotic thrombocytopenic purpura (TTP) characterized by a deficiency of ADAMTS13 activity due to its mutations. More than half of USS patients have an episode of severe jaundice during the newborn period and require exchange blood transfusions. Beyond this period, however, the clinical manifestations of USS vary significantly from patient to patient, and do not usually include Moschowitz’s pentad, a hallmark of TTP. However, a consistent clinical sign in these patients is an occasional or chronic thrombocytopenia that is enhanced by precipitating factors such as viral infections. Therefore, USS patients are often incorrectly diagnosed with idiopathic thrombocytopenic purpura (ITP) or Evans syndrome during childhood, and the underlying ADAMTS13 gene defects are overlooked. Here, we report on 7 female USS patients belonging to 4 different families, including 3 pairs of siblings who were uniformly diagnosed by episodes of thrombocytopenia or prevalence of TTP at 5~6 months of pregnancy. Plasma ADAMTS13 activity was measured with a highly sensitive ELISA using a monoclonal antibody directed against the VWF-A2 domain developed in our laboratory. The ADAMTS13 activity and gene mutations in these patients are described: Family K: an elder sister K1 (activity 0.69%; gene mutation Y304C/G525D) and a younger sister K2 (activity <0.5%; gene mutation Y304C/G525D), Family L: an elder sister L1 (activity 1.3%; gene mutation 372insGT/Q1302X) and a younger sister L2 (activity 0.96%; gene mutation 372insGT/Q1302X), Family M: an elder sister M1 (activity <0.5%; gene mutation R193W/R349C) and a younger sister M2 (activity <0.5%; gene mutation R193W/R349C), and Family O: a patient (activity <0.5%;gene mutation I178T/Q929X). Figure indicates family pedigree of USS involved in this study. Fig. Family pedigree of Upshaw-Schulman syndrome Fig. Family pedigree of Upshaw-Schulman syndrome

Vitamin K2(VK2) analogs, including menaquinone-4 (MK4) but not vitamin K1, effectively induce apoptosis in acute myeloid leukemia cells (AML). Apoptosis induction by VK2 is specific toward leukemia cells, and almost no effect is observed in normal hematopoietic progenitor cells (Miyazawa K, Leukemia 1999, 2000, 2001). Combined treatment with VK2 plus 1α, 25-dihydroxy-vitamin D3(D3) dramatically enhanced differentiation of leukemia cells as compared to the cells treated with D3 alone. Concomitant with the induction of differentiation and final maturation by VK2 plus D3, the cells became resistant to various apoptotic stimuli (Funato K, Leukemia, 2002). VK2 has been shown to improve the supportive hematopoietic functions of bone marrow stromal cells (Miyazawa K, Stem Cell Dev. 2004). Since VK2 (MK4, GlakayR) has been used to treat patients with osteoporosis in Japan, its non-toxicity and safety for long-term daily administration have already been well established. Case reports demonstrating the clinical benefits of using VK2 with or without D3 for treating patients with myelodysplastic syndrome (MDS) and AML have been accumulating, i.e. improving cytopenias in refractory anemia (RA) and reduction of blast cells in AML. We performed a multicenter prospective trial to determine the therapeutic benefit of VK2 with or without D3 in MDS. Patients with RA/RCMD with low/ int-1 by IPSS received VK2 (Glakay, 45 mg/day, po) for 16 weeks. The therapeutic response was assessed according to the International Working Group Criteria. Patients who did not respond to VK2 were sequentially re-registered and received a combination of VK2 plus D3 (alfacalcidol, AlfarolR, 0.75 microgram/day, po) for 16 weeks. Patients showing response to VK2 alone or VK2 plus D3 after 16 weeks continued to receive therapy for a total of 12 months. Forty-three patients have been enrolled and 29 patients were evaluable for their response to VK2. Five patients (17.2 %) showed improvement of cytopenia (major HI-E (1), minor HI-E (2), major HI-P (3), and minor HI-P (1)). Twenty out of 24 patients who showed no response to VK2 alone subsequently received VK2 plus D3. Eleven patients were eligible for evaluation and 4 patients (36.4%) showed improvement of cytopenia in response to VK2 plus D3 (major HI-E (3), minor HI-E (1) and major HI-P (1)). Four out of 7 patients (57 %) with anemia showed hematologic response. No adverse events were observed, except for one case of skin rash (Grade I) during VK2 plus D3 therapy. These data suggest that the therapeutic response rate is improved by combined treatment with VK2 plus D3 as compared with that by VK2 monothrapy. Safety and low price also support the therapeutic benefit of VK2 with D3 for improving cytopenia in RA.

Upshaw-Schulman syndrome (USS) is a congenital thrombotic thrombocytopenic purpura (TTP) characterized by a deficiency of ADAMTS13 activity due to its mutations. More than half of USS patients have an episode of severe jaundice during the newborn period and require exchange blood transfusions. Beyond this period, however, the clinical manifestations of USS vary significantly from patient to patient, and do not usually include Moschowitz's pentad, a hallmark of TTP. However, a consistent clinical sign in these patients is an occasional or chronic thrombocytopenia that is enhanced by precipitating factors such as viral infections. Therefore, USS patients are often incorrectly diagnosed with idiopathic thrombocytopenic purpura (ITP) or Evans syndrome during childhood, and the underlying ADAMTS13 gene defects are overlooked.