Search

Background Paroxysmal nocturnal hemoglobinuria (PNH) often affects young adults, including women of childbearing age. Because PNH hemolysis increases the risk of complications such as thrombosis during pregnancy and the postpartum period, proper management should be provided for pregnant patients with PNH. Eculizumab, a C5 complement inhibitor, could be a therapeutic option for such patients. Aim/methods Japan PNH Study Group aimed to survey the current status of management of pregnant PNH patients and to propose a consensus recommendation for such a challenging situation in the treatment of PNH. Clinical data were collected from 10 pregnant PNH patients from 7 institutes and analyzed. We had consensus meetings 5 times since January 2012. Results Summary of patients' characteristics are shown in Table. Age of the patients at the time of conception ranged from 29 to 40 years old (median 30). Plasma levels of LDH at the baseline assessment were 200-2300 IU/L (median 1900). PNH clone sizes in granulocytes were 1.6-96.0% (median 81). Prophylactic anticoagulation with heparin was employed in all patients except one who showed little hemolysis due to a small size of PNH clone. Four patients received eculizumab during pregnancy with favorable efficacy. Complications including preeclampsia, hemolytic episodes and deep venous thrombosis were manageable. Newborn babies were all healthy. A negligible amount of eculizumab was detected in cord blood of three babies. Discussion Recently successful pregnancy outcomes have been reported in patients with PNH on eculizumab therapy during pregnancy, and we also experienced additional 4 cases. As eculizumab inhibits intravascular hemolysis itself, it can be expected to lower the risk of complications during pregnancy and postpartum period in women with PNH. Eculizumab is a hybrid of IgG4 and IgG2, and the latter is known to be less prone to cross the placenta. In fact eculizumab concentrations in the cord blood was much lower than therapeutic levels. Importance of anticoagulation therapy has been emphasized for the management of pregnancy with PNH. Theoretically, if intravascular hemolysis is completely inhibited by eculizumab, the risk of thromboembolic complication would become as low as that in non-PNH pregnancies. It is not clear whether the use of anticoagulant is mandatory for the pregnant patients who are receiving eculizumab, although the use of anticoagulant should not be hesitated. Conclusion Eculizumab could be safely and effectively used in pregnant patients with PNH. Taking these findings into account, we propose the following protocol for the management of a pregnancy with PNH, as shown in Figure. The patients are classified into 4 categories based on eculizumab use and the history of venous thromboembolisms or anticoagulant use. Disclosures: Usuki: Alexion Pharmaceuticals: Speakers Bureau. Urabe:Alexion Pharmaceuticals: Speakers Bureau. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Miura:Alexion Pharmaceuticals: Research Funding. Morita:Alexion Pharmaceuticals: Research Funding. Nishiwaki:Alexion Pharmaceuticals: Research Funding. Ninomiya:Alexion Pharmaceuticals: Honoraria. Gotoh:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Nishimura:Alexion Pharma: Research Funding, Speakers Bureau. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau.

Abstract 1037 Background: 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. Methods: 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. Results: 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)]. Conclusions: CZOP, MEPM, and IPM were as effective as CFPM for adult FN patients with hematological malignancies and solid tumors as an empiric therapy. Disclosures: Tamura: Kyowa Hakko Kirin Co., Ltd,: Consultancy.

Abstract 5019 Background: MDS patients receiving chronic transfusions can develop significant iron accumulation in key organs such as the liver following 10–20 transfusions (Porter et al. BJH 2001). The diagnosis and monitoring of iron overload, as well as the effect of iron chelation therapy in MDS patients, is often based on serum ferritin (SF), with limited data on liver iron concentration (LIC), primarily due to the biopsy-related increased risk of bleeding and infections in these patients. However, LIC is clinically a more robust and direct measure of body iron burden and with the availability of non-invasive determination of LIC by MRI, LIC assessment has become more practical in MDS patients. This pooled analysis focuses on LIC assessments from a population of 71 MDS patients who completed 1 year of treatment with deferasirox (Exjade®), including assessment of the relationship between LIC vs SF and alanine aminotransferase (ALT). Methods: Analysis is based on 1-year pooled data from iron-overloaded patients with MDS who were enrolled in 4 open-label single-arm deferasirox studies: US02 (Low/Int-1 MDS patients, starting dose 20 mg/kg/day); 2409 (MDS patients with life expectancy >1 yr, starting dose 10–30 mg/kg/day); 108 (MDS patients with life expectancy >1 yr, dosing 5–40 mg/kg/day), and 2204 (Low/Int-1 MDS patients, starting dose 10–30 mg/kg/day). LIC was assessed in the US02, 2409 and 2204 studies using R2 MRI (St Pierre et al. Blood 2004). In the 108 study, LIC was assessed by magnetic liver susceptometry using a superconducting quantum interference device (SQUID) or ultrasound-guided percutaneous liver biopsy; LIC values obtained by SQUID were multiplied by a factor of 2 to correct for the underestimation of LIC by SQUID compared to biopsy (Porter et al. EJH 2008). Datasets were pooled for baseline (BL) characteristics, as well as LIC, SF and ALT at BL and end of study (EOS). Correlations were evaluated on a Pearson's correlation coefficient. Results: 71 patients (56.3% male) were assessed with a mean age of 65 years (range 16.5–82.0). Mean transfusional iron intake ± SD was 0.31 ± 0.12 mg/kg/day. Mean actual deferasirox dose was 19.6 ± 6.5 mg/kg/day. At BL, mean ± SD LIC was 20.5 ± 14.6 mg Fe/g dw (BL LIC 15 mg Fe/g dw, 54.9%). Median BL SF was 2620 ng/mL (range 538–12,639) (BL SF ≤2500 ng/mL, 47.9%; >2500–≤5000 ng/mL, 32.4%; and >5000 ng/mL, 15.5%). With 1 year of DFX, mean ± SD LIC decreased to 13.9 ± 13.1 mg Fe/g dw (mean absolute change –6.6 mg Fe/g dw). In patients with BL LIC Median SF decreased to 2035 ng/mL (range 158–10520 ng/mL) with median absolute change from baseline of –630 ng/mL. There was a significant correlation between BL LIC and SF (R=0.548; P Conclusions: This pooled analysis in a large cohort of transfusion-dependent MDS patients with LIC assessment shows significantly elevated LIC, with a high proportion of patients (55%) having severely elevated LIC of >15 mg Fe/g dw, a level known to markedly increase liver dysfunction and other iron overload-related complications. One year of treatment with deferasirox produced relevant reductions in LIC, an outcome possibly indicative of a clinical benefit. SF and ALT (an important indicator of liver function) also decreased, with reductions correlating with those of LIC. These findings indicate that correction of moderate-to-severe iron overload in MDS patients is associated with a parallel improvement in liver function. Disclosures: Gattermann: Novartis Pharma: Honoraria, Research Funding. Greenberg:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Habr:Novartis Pharma: Employment. Kpamegan:Novartis Pharma: Employment. Porter:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 4838 Background: Regularly transfused patients including those with MDS and AA inevitably accumulate iron in various tissues, hence the importance of iron chelation to prevent end organ dysfunction. Studies of iron chelation in MDS and AA have mostly used serum ferritin as an efficacy indicator; LIC data, a more direct measure of clinical benefit are limited in these patients. Here, we report results from a 1-year Phase II, multicenter study evaluating the effect of deferasirox (DFX) on LIC in iron-overloaded patients with MDS, AA, and other rare anemias. Methods: Patients aged ≥2 yrs with transfusional iron overload due to low/intermediate (Int-1) risk MDS, AA and other congenital or acquired anemias were enrolled. Patients with thalassemia and SCD were excluded. Patients required a lifetime transfusion history of ≥20 units of packed red blood cells (RBC) or serum ferritin >1000 ng/mL. DFX was administered at a starting dose of 20 mg/kg/day; some patients started on 10 or 30 mg/kg/day based on transfusion requirements and therapeutic goals. Dose adjustments were based on serum ferritin trends and safety parameters. Primary endpoint was absolute change in LIC assessed by R2 MRI (Ferriscan®) from baseline (BL) to 1 year. Secondary objectives included analyses of change in serum ferritin, iron balance and safety. Results: 102 patients (MDS n=42 [41.2%], AA n=29 [28.4%] and other rare anemias n=31 [30.4%]) were enrolled. Median age was 56.5 yrs (range 2–85 yrs). 68 (67%) patients completed 1 year of treatment. Average actual dose (mean ± SD) was 18.5 ± 5.6 mg/kg/day; 64 patients (62.7%) received an average dose of 15– Mean ± SD BL LIC was 24.5 ± 15.6 mg Fe/g dw (n=102); with 23.5% (n=24) and 56.9% (n=58) of patients having LIC >7– Most frequent (≥5%) adverse events (AE) suspected by the investigator to be drug-related were increased blood creatinine (n=19, 18.6%), skin rash (n=9, 8.8%), renal impairment (n=8, 7.8%), diarrhea (n=7, 6.9%), nausea (n=7, 6.9%), abdominal pain (n=6, 5.9%) and constipation (n=6, 5.9%). 14 severe AEs suspected to be drug-related were reported in 10 patients, the most frequent (≥2) being rash (n=2) and hypersensitivity (n=2). Five patients died (general physical health deterioration [n=2], cerebellar hemorrhage, disseminated intravascular coagulation and traumatic hemorrhage [n=1 for each]); none were considered drug-related. Cataract was reported in 4 patients (3.9%); 1 suspected as drug-related. Auditory AEs were reported in 7 patients; aggravated deafness (n=1) and tinnitus (n=1) suspected to be drug-related. Of 19 patients treated with concomitant cyclosporine (CyA), 11 (57.9%) had increases in serum creatinine (>33% above BL and >ULN at 2 consecutive visits) compared with 18 of the remaining 83 patients (21.7%) who did not receive CyA. Conclusions: A large proportion of patients in this study with transfusion-dependent Low/Int-1 risk MDS, AA and other rare anemias had severe liver iron overload (LIC ≥15 mg Fe/g dw). 1 year of DFX treatment significantly reduced iron burden, as assessed by both LIC and serum ferritin; with iron excretion 2–3 times higher than iron intake. Furthermore, change in LIC correlated with change in serum ferritin and ALT, a clinically relevant indicator of liver function. Overall the DFX safety profile was consistent with that from previous studies. Renal function in patients receiving DFX and concomitant CyA should be closely monitored as previously noted in the EPIC trial. Disclosures: Kohgo: Kyorin Pharma: Research Funding; Sapporo Brewery: Research Funding; Asahikasei Kurare Medical: Research Funding; Chugai Roche: Research Funding; Novartis: Research Funding, Speakers Bureau. Sanz:Novartis: Speakers Bureau. Helou:Novartis: Employment. Habr:Novartis: Employment. Malet:Novartis: Employment. Glaser:Novartis: Employment. Wiktor-Jedrzejczak:Novartis: Honoraria, Research Funding; Janssen-Cilag: Honoraria.

Pure red cell aplasia (PRCA) has been reported in association with lymphoma as one of the autoimmune diseases seen during the course of lymphoid malignancies. However, the relation of PRCA with the underlying lymphomas remains unclear. The aim of this study was to clarify the histologic subtypes of lymphomas, the chronological sequence of anemia and lymphoma, and the response to treatment, and eventually to establish the treatment guideline for lymphoma-associated PRCA. We conducted a nationwide survey in Japan. From a cohort of 185 patients with PRCA, 8 patients with lymphoma were evaluated. Complete response (CR), partial response (PR) and no response (NR) were defined as the achievement of normal hemoglobin levels without transfusion, the presence of anemia but without transfusion dependence, and the continued presence of transfusion-dependence, respectively. Patient age at the onset of PRCA ranged from 48 to 82 years (median age, 68 years) with an equal male to female ratio. Histologic subtypes varied and the lymphoma was of the B-cell type in four cases and of the T-cell type in four. Four patients had coexisting anemia and lymphoma. One patient developed PRCA before the onset of lymphoma. Three other patients developed PRCA following lymphoma, two of whom developed anemia during remission. Anemia responded to chemotherapy and/or immunosuppressive therapy in seven of eight patients. In four responding patients, PRCA remained in durable remission without maintenance immunosuppressive therapy (27, 76, 97 and 127 months), which is a different feature from that of idiopathic PRCA (Sawada K, et al. Haematologica 2007;92:1021). Four patients were alive, and two of these four remained in remission for both lymphoma and PRCA. Effective chemotherapy was associated with remission of anemia in patients with concurrent lymphoma and PRCA. In patients with PRCA presenting before or after the onset of lymphoma, immunosuppressive therapy was effective for improving anemia. In conclusion, chemotherapy should be introduced for patients with coexisting lymphoma and PRCA. Additional immunosuppressive therapy may be necessary for PRCA that has failed to respond to chemotherapy. Durable maintenance-free remission of anemia may be obtained in lymphoma-associated PRCA. Table 1. Patient characteristics and induction therapy for PRCA | Age/Sex | Histologic subtypes | Days from lymphoma to PRCA | Disease status of lymphoma | Induction therapy for PRCA * | Response of PRCA | Response of lymphoma | |:--------------------------------------------------------:| ------------------- | -------------------------- | -------------------------- | ---------------------------- | ---------------- | -------------------- | | *(/), in sequential administration; (-), in combination. | | 76/F | DLBCL | −114 | - | Steroid | CR | - | | 75/M | DLBCL | −13 | On disease | R-CHOP/CsA | NR/CR | PR | | 62/M | ATLL | | On disease | CHOP-CsA | CR | PD | | 82/F | MZL | | On disease | Steroid | NR | NR | | 58/F | AILT | 35 | On disease | CHOP | CR | CR | | 48/M | Follicular | 720 | On disease | Steroid/CHOP | NR/CR | CR | | 64/M | T-LBL | 205 | CR | Steroid | PR | - | | 71/F | AILT | 801 | CR | Steroid/CsA | PR/PR | -

Background: Invasive fungal infections (IFIs) are of serious concern in the management of immunocompromised patients (pts) with hematological disorders. Empiric antifungal therapy is recommended for neutropenic pts with persistent fever, because treatment after confirmation of fungal infection often produces poor outcomes. Micafungin (MCFG), one of the echinocandin families, was launched first in Japan in 2002, and has now been approved in more than 11 countries and areas including the USA and the EU. Although the efficacy and safety of MCFG against both Candida and Aspergillus infections has been shown in many clinical trials, there are few clinical study reports on the empiric therapy of a suspected fungal infection. Here, we report the multi-center study results of MCFG for the empiric antifungal therapy, which were conducted from April 2005 to September 2006 in Japan. Objective: This prospective study was performed to clarify the efficacy and safety of MCFG for the empirical antifungal therapy on suspected fungal infection in pts with hematological disorders and neutropenia. Methods: Study design: A multiple-center, open, uncontrolled study. The investigator registered pts with neutropenia (< 1,000/μl) who met the following criteria to the Subject Registration Center. Suspected fungal infections were divided into two categories: possible fungal infection defined by positive clinical symptoms/findings and serological testing (beta-D-glucan or galactomannan) or diagnostic imaging (chest X-ray or CT scan), refractory fever defined by unexplained persistent fever (an axillary temperature higher than 37.5 °C) after the antibacterial treatment over 2 days and by positive clinical symptoms/findings. IFIs categorized as proven or probable were not included in this study. Efficacy evaluation was performed using an algorithm based on each of the evaluation of clinical symptoms/findings, imaging study findings, and serological tests. Results: 388 pts (M:234, F:154, mean age:57.8 years old) were registered. The mean dosage and duration of treatment with MCFG were 154.6±55.3 mg/day and 14.0±6.9 days, respectively. The main underlying hematological disorders were acute leukemia (61.3%), non-Hodgkin’s lymphoma (18.3%) and myelodysplastic syndrome (10.8%). The number of pts with hematopoietic stem cell transplantation (HSCT) was 76 (19.6%). The clinical response rate (CRR), excluding 4 non-evaluable pts was 63.3% (243/384): 60.1% (89/148) for pts with possible fungal infection and 65.3% (154/236) for pts with refractory fever, respectively. Even in persistent neutropenic pts whose neutrophil counts were < 500/μL throughout the treatment with MCFG, the CRR was high enough: 46.9% (61/130). No difference was observed in the CRR among the main underlying hematological disorders. The CRR in pts with HSCT and other conditions were 63.2% (48/76) and 63.3% (195/308), respectively. Drug-related adverse events (DAEs) were observed in 16.8% (65/388). Serious DAEs such as elevation of serum bilirubin and renal dysfunction was observed in 0.52% (2/388). Conclusion: MCFG was confirmed to have high clinical efficacy and be safe for the treatment of suspected fungal infection in pts with hematological disorders and neutropenia.

Mesenchymal stem cells (MSCs) are known to have a tendency to accumulate at the site of tumors, and therefore can be utilized as a platform for targeted delivery of anti-cancer agents. The MSC-based targeted cancer gene therapy can enhance the therapeutic efficacy, because MSCs are considered to reach tumors including metastatic lesions and to deliver therapeutic molecules in a concentrated fashion. This targeted therapy can also reduce systemic adverse side effects, because the anti-cancer agents act locally at the site of tumors without elevating their systemic concentrations. In the present study, we developed genetically-modified MSCs that produce retroviral vectors encoding HSVtk, aiming at augmenting therapeutic efficacy of systemic suicide cancer gene therapy. The tumor tropism and anti-tumor effects of vector-producing MSCs (VP-MSCs) were examined by intravascular injection in tumor-bearing nude mice. MSCs isolated from the bone marrow of SD rats were transfected with plasmid DNA expressing luciferase alone (=non-VP-MSCs) or whole retroviral vector components (LTR-Luc or LTR-HSVtk with Gag-pol and VSV-G) (=VP-MSCs) by nucleofection. To assess tumor tropism of MSCs, nude mice were subcutaneously inoculated with 9L rat glioma cells or Rat-1 fibroblasts, and were subsequently injected with luciferase-expressing MSCs through the left ventricular cavity. The transgene expression was periodically traced by using an in vivo imaging system. As a result, the transgene expression accumulated at the site of subcutaneous 9L tumors, but undetectable at the site of Rat-1 fibroblasts. In addition, the injection of luciferase-expressing VP-MSCs caused much stronger signal of bioluminescence at the site of 9L tumors compared with luciferease-expressing non-VP-MSCs. Immunostaining study showed that luciferase-positive cells (injected MSCs and transduced glioma cells) were detected at the periphery of tumors. To evaluate the therapeutic efficacy, tumor-bearing nude mice were treated with non-VP-MSCs or VP-MSCs combined with HSVtk/GCV system and then the size of subcutaneous tumors was periodically measured. In this model experiments, tumor growth was more efficiently suppressed by injecting VP-MSCs compared with non-VP-MSCs. The present study suggests the effectiveness of VP-MSCs in suicide cancer gene therapy. The therapeutic benefit of this strategy should be further examined in orthotopic and metastatic tumor models.

Mesenchymal stem cells (MSCs), which are key elements of hematopoietic microenvironment in bone marrow, are known to play a critical role in supporting hematopoiesis. A variety of hematopoietic growth factors are produced from MSCs, and cell-to-cell contact is also believed to be crucial in the interaction between hematopoietic stem cells (HSCs) and MSCs. However, the molecular mechanisms of hematopoiesis-supporting ability of MSCs are still unclear. In particular, there is little information regarding the effects of HSCs on MSC function. In the present study, we investigated the cellular and molecular events in the interactive communication between HSCs and MSCs using a differentiation-inducible MSC model; i.e. parent C3H10T1/2 cells and 10T1/2-derived cell lines, A54 preadipocytes and M1601 myoblasts. These cells were co-cultured with murine HSCs (Lin-Sca1+) isolated from bone marrow. There was 9-fold increase in the number of hematopoietic progenitors after co-culture with A54 preadipocytes, whereas there was no increase when co-cultured with parent 10T1/2 or M1601 cells. More intriguingly, cobblestone areas were observed only when HSCs were co-cultured with A54 cells. Quantitative RT-PCR showed that A54 cells express significantly higher levels of SCF, SDF-1, and angiopoietin-1 (Ang-1) compared with parent 10T1/2 cells and M1601 cells, although these cytokines were not up-regulated when co-cultured with HSCs. To search for the genes involved in the interaction between HSCs and MSCs, we compared gene expression profiles before and after the co-culture by using a microarray analysis. Among the candidate genes with up-regulation after the co-culture, we paid attention to the Notch system, because Notch ligands are considered to play an important role in nurturing HSCs within the hematopoietic microenvironment. As a result, the expression of Notch ligands, Jagged1 and Dll3, increased in A54 cells after the coculture with HSCs. On the other hand, the expression of Notch1 and Hes-1 also increased in HSCs upon co-culture with A54 cells. These data were confirmed by quantitative RT-PCR. Moreover, when HSCs were co-cultured with A54 cells without cell-to-cell contact using Transwell permeable supports, there was neither increase in the number of progenitors in the upper wells, nor the up-regulation of Notch ligands in A54 cells in the lower wells. These findings support the idea that HSCs act on MSCs to induce the expression of Notch ligands via direct cell-to-cell contact and that the Notch ligands derived from MSCs act on HSCs in turn to activate Notch signaling pathway, possibly leading to the cobblestone formation with the maintenance of immature state of HSCs. The Notch system may be one of the critical elements in the interactive communication between HSCs and MSCs.

Background: Large granular lymphocyte (LGL) leukemia accounts for a significant part of secondary PRCA. Cyclophosphamide (CPA) therapy has been reported as being useful in the literature. However, because of its rarity, the long-term response and relapse rate after immunosuppressive therapy including CPA in LGL leukemia-associated PRCA are largely unknown, and optimal management of this disorder remains unclear. Objectives: We conducted a nationwide survey of immunosuppressive therapy for PRCA in Japan to elucidate the long-term response and overall survival (OS) of this disorder. This report is a summary focusing on CPA therapy for LGL leukemia-associated PRCA. Methods: The questionnaires were sent to 109 medical institutions in Japan to estimate the number of patients with newly diagnosed acquired PRCA. From a total of 185 patients consisting of 73 idiopathic and 112 secondary PRCA cases, we evaluated 14 patients with LGL leukemia for this report. Endpoints of this study were the response rate and the duration of the response to immunosuppressive therapy and overall survival. Results. Efficacy of the first remission induction therapy was seen in 6 out of 8 patients treated with CPA (75%), 1 out of 4 patients with cyclosporine (CsA) (25%), and 0 of 2 with corticosteroid (0%), respectively. The median initial dose of CPA for the responding patients (n=6) was 100 mg. The initial dose of CsA for the responding patient was 3.7 mg/kg. Two patients, who failed to respond to the initial CPA therapy, received CsA therapy resulting in the response in one patient. Three patients, who failed to respond to the initial CsA therapy, received CPA therapy resulting in the response in all of three patients. Two patients, who failed to respond to the initial prednisolone therapy, received CsA therapy resulting in the response in one patient. We classified the patients with LGL-associated PRCA into the CPA-group (n=9) and the CsA-group (n=3) according to the response to initial or salvage therapy. Relapse was seen in two CPA-responders following discontinuation of maintenance therapy (23, 45 months), whereas no patients relapsed on maintenance CPA therapy. The estimated median duration of the response to CPA was 125 months. The median OS has yet been reached with the median observation period of 89 months. Conclusion: CPA showed an excellent response in LGL leukemia-associated PRCA and CPA-containing regimens were effective to prevent relapse of anemia. However, most patients are still receiving CPA for maintenance therapy, and it remains uncertain whether CPA can induce maintenance-free hematological response.

Background: The efficacy of corticosteroids (CS), cyclophosphamide (CY) and cyclosporine (CsA) for the patients with primary or secondary pure red cell aplasia (PRCA) is between 30–56%, 7–20% and 75–87%, respectively. Although it is evident that the efficacy of CsA is the highest amongst these three drugs, CsA has remained second to third-line therapy largely because the long-term relapse-free survival (RFS) and overall survival (OS) after CsA therapy are unknown. Objectives: We conducted a nationwide survey of immunosuppressive therapy for PRCA in Japan to elucidate the long-term RFS and OS of CsA therapy for acquired primary idiopathic (API-) PRCA. Methods: Questionnaires were sent to 109 medical centers. From 1990 through 2006, we identified 174 adult patients with acquired PRCA. The underlying diseases could affect RFS and OS of immunosuppressive therapy. We, therefore, selected 64 patients (38%, median age, 56 years; range, 18 to 89 years) with API-PRCA according to the classification proposed by Dessypris and Lipton. Complete remission (CR), partial remission (PR) and no response (NR) was defined as the achievement of normal hemoglobin levels, the presence of anemia but with transfusion independence, and the continued presence of transfusion dependence, respectively. RFS was estimated as transfusion free survival without the dose escalation more than 50% of maintenance-dose. Survival was estimated by the Kaplan-Meier method and statistical difference was calculated by the generalized Wilcoxon test and qui square test. Results: The efficacy (CR+PR) of primary treatment was seen in 24 of 32 patients treated with CsA (75%), 16 of 26 patients with PSL (62%), 0 of 3 patients with CY (0%), 0 of 1 patient with anabolic steroid and was undetermined in 2 patients because of short observation periods ( Conclusions: We, for the first time, demonstrated that CsA ensures prolonged PFS than CS in API-PRCA. However, most patients are still receiving CsA for maintenance therapy. Therefore, other therapeutic modalities may be required to cure API-PRCA.

Introduction: In Japan, myelodysplastic syndromes (MDS) and aplastic anemia (AA) are the most common transfusion-dependent anemias. Deferasirox (Exjade®, ICL670) is a once-daily, oral iron chelator for the treatment of transfusional iron overload; its efficacy and tolerability have been established in adults and children with a range of transfusion-dependent anemias, including MDS. Here, deferasirox has been evaluated in Japanese patients and the pharmacokinetics (PK) and pharmacodynamics (PD) compared with those seen in a US study. Methods: The tolerability, PK and PD of deferasirox have been assessed in a Phase I, open-label, unblinded, dose-escalation trial (1101) in Japanese patients with MDS and AA in 9 centers. Deferasirox was administered as a single dose of 5 (n=6), 10 (n=7), 20 (n=6) or 30 mg/kg (n=7). After a 7-day break, daily doses were administered (same doses/same patients) for 7 consecutive days. Data were compared with a study (0104) in 24 Caucasian β-thalassemia patients (age range 18–39 years) who were administered doses of deferasirox of 10, 20 or 40 mg/kg/day for 12 days. Throughout the study, iron intake and excretion were rigorously measured. Results: A total of 26 Japanese patients (8 male, 18 female; mean age 65.6 years; 8 aged Figure Figure Dose-dependent iron excretion (range 0.07–0.61 mg iron/kg/day) was observed, similar to that in the Caucasian patients (range 0.12–0.45 mg iron/kg/day). A linear relationship (PK/PD) was noted between AUC and iron excretion. Deferasirox was well tolerated, with generally infrequent and mild adverse events (AEs). The most common AEs related to deferasirox were diarrhea (n=2 each after single and multiple doses of 30 mg/kg/day) and nausea (n=1 each after multiple doses at 10 and 30 mg/kg/day). One patient had two serious AEs, pyrexia and duodenal ulcer, both suspected to be related to study drug, after multiple doses of 30 mg/kg/day. Conclusions: Deferasirox is well tolerated both after single and multiple doses in Japanese MDS/AA patients, with similar PK/PD parameters to those in the Caucasian β-thalassemia cohort. Exposure to deferasirox in the Japanese patient cohort was dose dependent with a linear PK/PD relationship resulting in dose-dependent iron excretion. These data suggest that deferasirox is effective and well tolerated regardless of underlying disease, race or age.

Background and Purpose: Severe infections in patients (pts) with hematological disorders (HD) are mostly the result of a compromised immune system by the underlying disease itself including neutropenia aggravated by chemotherapy and conditioning treatment for hematopoietic stem cell transplantation (HSCT). Systemic fungal infections (SFIs) particularly are often encountered in this field associated with morbidity and mortality. It is deemed crucial to initiate an early introduction of antifungal therapy by using criteria for an early diagnosis. Micafungin (MCFG) that was launched in Japan in 2002 and approved in another 3 countries is shown to be safe and efficacious for SFIs. Since there are few clinical reports on the effects of MCFG in a large number of patients, a multicenter study (87 institutions) was initiated to see its efficacy and safety. Methods: The study was conducted from Apr. 03 to Mar. 05 on pts diagnosed with HD, who met any of the following criteria: ptswho are found to have a causative fungus identified by mycological or pathological testing (proven),with a SFI defined by clinical symptoms/findings and serological testing or diagnostic imaging (probable/possible), orwith a suspected SFI identified by unexplained persistent fever (an axillary temperature higher than 37.5 °C) and clinical symptoms/findings (refractory to antibacterial treatment). Efficacy was evaluated by the degree of improvement in clinical symptoms/findings, mycological findings, imaging study findings such as chest X-ray or CT, and fungal serological tests. The overall effects were rated as either “effective” or “ineffective”, based on an algorithm combining these indices. Results: A total of 277 pts were registered to the central office and 276 were evaluable for safety assessment. Eighty-one pts were not evaluable for efficacy due to a violation of entry criteria, etc. Thus 196 pts were assessed for clinical efficacy by the steering committee (118 males and 78 females, the mean age: 56.8). There were 62 pts with HSCT and 134 with chemotherapy in whom 67 had acute leukemia. The mean dosage and duration of MCFG were 170.8mg per day and 21.8days, respectively. Overall clinical effects were achieved in 134 out of 196 pts (68.4%). Clinical response was seen in 72.6% (45/62) for those with HSCT and 66.4% (89/134) for those of chemotherapy and particularly 68.7% (46/67) of the pts with chemotherapy for acute leukemia, respectively. The success rates were 87.5% (7/8) for pts with proven SFIs like candidemia, 44.7% (17/38) for probable SFIs, 62.9% (39/62) for possible SFIs, and 80.7% (71/88) for those who failed to respond to antibacterial treatment. The success rate for 13 pts with persistent fever on antibacterials and neutrophil counts below 500/μL before and after MCFG treatment was 69.2%. The incidence of adverse events related to MCFG was 30.1% (83/276), and the most common one was elevation of liver transaminase levels. Conclusions: MCFG demonstrated excellent clinical efficacy and safety when used to treat possible to proven SFIs in pts with HD, indicating its usefulness as a novel therapeutic drug for both empirical and targeted therapy for deep-seeded fungal infection.

Background: Secondary pure red cell aplasia (PRCA) is associated with various underlying diseases and thymoma accounts for a significant part of secondary PRCA. Thymoma-associated PRCA is generally believed to be an organ-specific autoimmune disease as well as idiopathic form, and immunosuppressive therapy including corticosteroids (CS), cyclophosphamide (CY) and cyclosporine A (CsA) have been proven useful. However, efficacy and long-term outcome of immunosuppressive therapy for secondary PRCA could be distinct among its underlying diseases. Up to the present, the overall long-term response and relapse rates after immunosuppressive therapy in thymoma-associated PRCA are largely unknown. Objectives: We conducted a nationwide survey of immunosuppressive therapy for PRCA in Japan to elucidate the long-term relapse-free survival (RFS) and overall survival (OS) of this disorder. This report is a summary focusing on CsA therapy for thymoma-associated PRCA. Methods: The first questionnaires were sent to 109 medical centers. 273 patients were enrolled from 45 institutions. Secondary questionnaires were sent and the data on 185 patients were collected. 174 patients were eligible for further analysis and we selected 42 of 174 patients (24%) (median age, 64.5 years; range, 26 to 82 years) as thymoma-associated PRCA. Complete remission (CR), partial remission (PR) and no response (NR) were defined as the achievement of normal hemoglobin levels, the presence of anemia but with transfusion-independence and the continued presence of transfusion-dependence, respectively. RFS was estimated as transfusion-free survival without the dose escalation more than 50% of maintenance-dose. Survival was estimated by the Kaplan-Meier method and statistical difference was calculated by the generalized Wilcoxon test. Results. Surgical removal of thymoma was reported in 31 patients, and 17 patients developed PRCA after thymectomy with a median interval of 77 months (range; 1 to 366 months). Five patients underwent surgery without any immunosuppressive therapy after the diagnosis of PRCA, and no patients who achieved response were recognized. The efficacy (CR+PR) of primary treatment was seen in 19 out of 20 patients treated with CsA (95%), 6 out of 14 patients with CS (43%) and 1 of 1 with CY (100%), respectively. Relapse rates in CsA-responders and CS-responders were 0% (median observation period, 18 months) and 50% (34 months), respectively. The RFS in CsA- and CS-responders were significantly different (p=0.018). CsA-responders became independent of blood transfusion within two weeks after starting treatment. The OS did not differ substantially between the CsA- and CS-responders (p=0.663). Most CsA-responders were still on maintenance therapy, and only two patients were alive in CR after stopping therapy. Conclusion: CsA showed an excellent response in thymoma-associated PRCA. However, most patients are still receiving CsA for maintenance therapy. Therefore, other therapeutic modalities may be required to cure this disorder as is the case with idiopathic PRCA.

We have conducted a nationwide survey to define the first-line immunosuppressive treatment for idiopathic acquired pure red cell aplasia (PRCA) in Japanese adult patients diagnosed from January 1990 to December 2004. Questionnaires were sent to forty-seven medical centers and eighty-seven patients with acquired chronic PRCA from forty-seven institutions were registered in this retrospective study. Thirty-four patients (39%) were classified into idiopathic PRCA. Cyclosporine A (CsA), prednisolone (PSL) and cyclophosphamide (CY) were initially given to 20, 10 and 3 patients with idiopathic PRCA, respectively. One patient was treated with anabolic steroid as first-line therapy. The response rates with CsA and PSL were 80 and 60 %, respectively. All patients who had been resistant to PSL and two out of three CY-resistant patients were responsive to CsA. An anabolic steroid resistant patient also responded to CsA. Thus, CsA was used in twenty nine patients as a form of primary or salvage treatment with an 86 % clinical response rate. Nine out of 25 (36%) CsA-responders eventually relapsed and 12.5% of the patients relapsed during therapy. The median remission duration in the CsA responders was seven years. All PSL-responders were predicted to eventually relapse within 2 years, and four out of seven patients on prednisolone relapsed during maintenance therapy. All patients responded to CsA during their second and/or third remission induction with or without PSL. Overall survival was not different between the CsA- and PSL-responders. Although the difference in remission duration between both responders was not statistically significant (p

After a considerable dispute, it was shown that cells with hematopoietic activity in muscles are derived from hematopoietic organ. Transdifferentiation is not a frequent event, if any, and such a phenomenon cannot be applied to tissue regeneration easily. To achieve a breakthrough, we explored the possibility that genetic manipulation may enhance the efficiency of transdifferentiation. In this regard, there is a notable report that transient overexpression of a homeobox-containing transcriptional repressor Msx1 in muscles generated abundant mononuclear cells (MNCs) capable of differentiating into myotubes, chondrocytes, adipocytes and osteocytes. That is, enforced Msx1 expression caused dedifferentiation of myotubes, and subsequent Msx1 suppression induced redifferentiation (Odelberg SJ et al, Cell103:1099). Recently, we found that similar dedifferentiation-redifferentiation events also occur in vivo after transient Msx1 expression in muscles using adeno-associated virus (AAV) vectors (Endo T et al, manuscript in preparation). Since virtually all of AAV vector-mediated transgenes exist as episomes, they gradually disappear as the host cells divide. In the present study, we took advantage of this feature of AAV vectors; muscle-derived MNCs would lose Msx1 transgene through cell divisions after dedifferentiation, and a proper differentiation cue might redirect these undifferentiated cells into the hematopoietic lineage as well. AAV vector expressing Msx1 (AAV/msx1) was injected into tibialis anterior muscles of C57BL/6 mice. Flow cytometric analysis revealed that MNCs from AAV/msx1-treated muscles contained a considerable number of cells expressing hematopoietic stem cell markers. CD34−/c-Kit+ cells (1.3±0.9% of MNCs in control muscles) were increased in AAV/msx1-treated muscles (13.6±6.0% at 4 weeks). CD45+/Sca-1+ cells (2.4±0.4% in control muscles) were also increased in the AAV/msx1-treated muscle, peaking at 2 weeks (36.0±8.1%; P =.01). To evaluate hematopoietic activity, MNCs were cultured in methylcellulose medium containing stem cell factor, IL-3, IL-6 and erythropoietin. After AAV/msx1 injection, colony-forming cells in the muscles were gradually increased, reaching a peak at 3 weeks (48.9±24.1/muscle), in contrast to very few progenitors detected in control muscles (1.4±3.0/muscle; P

A human lung cancer (OTUK-tumor) was transplanted serially to nude mice, which invariably developed a marked neutrophilia. In order to analyze this phenomenon, in vitro agar culture studies were carried out. A three- to fourfold increase of colony-forming units in culture was observed in the femurs of these nude mice. Moreover, the plasma of nude mice bearing this tumor, as well as the tumor extract, had a significantly higher colony-stimulating activity on mouse bone marrow cells than appropriate controls. This activity had the property to stimulate granulocyte and/or mixed cell type colonies rather than mononuclear cells. This activity was also demonstrated, in a dose- dependent way, on normal human bone marrow cells. These findings indicated that the OTUK-tumor produced human granulopoietic colony- stimulating activity, which may have stimulated granulopoiesis in vivo as well.

We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide release and membrane depolarization in parallel in human granulocytes stimulated by the receptor-mediated agonists, N-formyl-methionyl-leucyl-phenylalanine and wheat germ agglutinin, but not by the Ca2+ ionophore ionomycin and phorbol myristate acetate, which bypass the receptors to stimulate the cells. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL (1.3 to 2.6 nmol/L) rhG-CSF for 10 minutes at 37 degrees C. rhG-CSF produced by bacteria and mammalian cells had identical biological effects on a molar basis. rhG-CSF neither affected stimulus-induced increase in cytoplasmic free Ca2+ nor changed the number and affinity of N-formyl-methionyl-leucyl-phenylalanine receptors. The priming effect of rhG-CSF was temperature dependent and did not require new protein synthesis. rhG-CSF increased the expression of C3bi receptors on human granulocytes and enhanced granulocyte adherence to nylon fiber. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL rhG-CSF for 30 minutes at 37 degrees C. rhG-CSF had no effect on human monocytes. These findings demonstrate that rhG-CSF can selectively stimulate mature granulocyte functions.