Abstract: Abstract 3197 Paroxysmal nocturnal hemoglobinuria (PNH) is a consequence of clonal expansion of hematopoietic stem cells that have acquired a somatic mutation in PIGA. The resulting hematopoietic cells are deficient in glycosylphosphatidylinositol(GPI)-anchored proteins. Deficiency in the GPI-anchored complement regulatory proteins CD55 and CD59 accounts for the intravascular hemolysis which is the primary clinical manifestation of PNH. Eculizumab is a humanized monoclonal antibody that specifically targets the terminal complement protein C5, thereby inhibiting terminal complement-mediated hemolysis. PNH patients treated with eculizumab exhibit, significantly reduced hemolysis and thrombotic events, and improved renal impairment and QoL. Of note, in the Japanese AEGIS PNH-eculizumab study, 2 poor-responders (UPN1 and 2) were identified out of 29 cases. Currently, more than 250 patients have been treated with eculizumab, and a total of 9 poor-responders (UPN1 to 9) were identified all of whom are Japanese. To clarify the mechanism of difference in the responsiveness of eculizumab, blood samples from poor responders were analyzed. In UPN1 and 2, the levels of lactate dehydrogenase were markedly elevated before eculizumab treatment, and were not decreased during the 12 weeks AEGIS study. From the pharmacokinetic analysis, peak and trough levels of eculizumab during the study were well above the minimal level required to completely inhibit complement-mediated hemolysis in PNH patients. The pharmacodynamics of eculizumab were determined by measuring the capacity of the patients' serum to lyse chicken erythrocytes in a standard hemolytic assay. Serum samples analysed from these two patients failed over the entire treatment period, to show the typical strong inhibition of hemolysis, prompting further study of the effect of exogenous eculizumab on the hemolytic activity of patients' pre-drug serum. Eculizumab up to 2000μg/mL did not block hemolytic activity in the sera of either non-responder. However, hemolytic activity both in the two non-responders and in control patient was blocked completely using a different anti-C5 antibody (ALXN-Ab) at 50μg/mL and higher suggesting that hemolysis is C5 dependent in the sera of non-responders. Therefore, the DNA of C5 from UPN1 and 2 was sequenced, and a single missense C5 heterozygous mutation at exon 21, c.2684G 〉 A, which predicts p.Arg885His, was found in each case. Since this one base substitution generates a new ApaLI restriction site, the PCR products covering exon 21 of C5 from the other seven poor-responders (UPN3–9) and seven responders were easily screened to verify this mutation. All PCR products from poor-responders were partially cleaved by ApaLI and confirmed as having c.2684G 〉 A in one allele, while no mutation was found in all responders. To determine the prevalence of c.2684G 〉 A among the Japanese population, DNA samples from Japanese healthy volunteers have been analyzed. At this moment, we estimate the prevalence around a few %, because this mutation was found in 9 out of over 250 patients with PNH ( 〈 3.6%) who received eculizumab, and in 2 out of 96 healthy volunteers (2.1%). Thus, we have observed 9 poor-responders, whose sera exhibited hemolytic activity even in the presence of high concentrations of exogenously added eculizumab. However, their hemolytic activity was completely blocked by a different anti-C5 monoclonal antibody that binds to a site on C5 other than that which is bound by eculizumab. A single missense C5 heterozygous mutation, c.2684G 〉 A, which predicts p.Arg885His, was commonly identified in poor-responders, but not in responders. These data suggest that the poor-responders have normal levels of wild type C5 plus a functional variant that does not bind eculizumab, and that the variant is responsible for the component of hemolytic activity in the poor-responders that is refractory to eculizumab. In order to verify that the polymorphism in C5 is truly responsible for the phenomena, we have initiated studies to express recombinant C5 with the mutation and characterize its activity and function. We are also working to both determine a more reliable prevalence of this C5 polymorphism in the Japanese population and to evaluate whether it is specific to the Japanese population. Disclosures: Nishimura: Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau. Hase:Alexion Pharma G.K.: Employment, Equity Ownership. Lan:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Tamburini:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties. Kanakura:Alexion Pharmaceuticals: Consultancy, Research Funding.

Abstract: Eculizumab is a humanized monoclonal antibody targeting the terminal complement protein C5 and inhibiting terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). In the Japanese AEGIS PNH-eculizumab study, 2 poor-responders were identified out of 29 cases. Currently, more than 300 patients have been treated with eculizumab, and a total of 11 poor-responders were identified all of whom are Japanese. To clarify the mechanism of difference in the responsiveness of eculizumab, blood samples from poor and good responders were analyzed after obtaining informed consent. Approval for these studies was obtained from the institutional review boards at each study site taking care of patients as well as from Osaka University. The levels of lactate dehydrogenase in these two patients were markedly elevated before eculizumab treatment, and were not decreased during the 12 weeks AEGIS study. From the pharmacokinetic analysis, peak and trough levels of eculizumab during the study were well above the minimal level required to completely inhibit complement-mediated hemolysis in PNH patients. The pharmacodynamics of eculizumab were determined by measuring the capacity of the patients’ serum to lyse chicken erythrocytes in a standard hemolytic assay. Serum samples analysed from these two patients failed over the entire treatment period, to show a suppression of hemolysis, prompting further study of the effect of exogenous eculizumab on the hemolytic activity of patient pre-drug sera. Eculizumab up to 2000μg/mL did not block hemolytic activity in the sera of either poor-responder. However, hemolytic activity both in the two poor-responders and in control patient was blocked completely using a different anti-C5 antibody (N19/8) at 50μg/mL. Therefore, the DNA of C5 from Japanese PNH patients with a good or poor response to eculizumab was sequenced, and a single missense C5 heterozygous mutation at exon 21, c.2654G 〉 A, which predicts p.Arg885His, was found in all of the 11 poor responders identified to date, but not in any of the responders. Among about 300 Japanese patients treated with eculizumab, 11 patients (about 3.7%) have been identified as poor responders. A similar prevalence (3.5%) was seen in healthy volunteers, since we determined that 10 out of 288 Japanese healthy volunteers have the same mutation. This polymorphism was also identified in 1 out of 120 China Han healthy volunteers, but not in 100 persons of British ancestry living in England and Scotland, and not in 90 persons of Mexican ancestry in Los Angels. To close the genotype-phenotype loop, electrophoretically pure recombinant C5 (rC5) and rC5 mutant (rC5m) containing c.2654G 〉 A were generated and functionally compared in various in vitro experiments. As a preliminary experiment, we confirmed that natural C5, rC5, and rC5m restored classical pathway lysis equivalently when added to C5-depleted serum. Eculizumab did not block classical pathway lysis reconstituted with rC5m but did block rC5 and nC5-dependent lysis. By contrast, as observed with patient sera, N19/8 inhibited lysis reconstituted with nC5, rC5, and rC5m. Finally, while eculizumab bound nanomolar concentrations of rC5 using surface plasmon resonance, with clear association and dissociation phases, there was no detectable binding with rC5m in the same assay up to the highest concentration (1 µM) of eculizumab examined. A single missense C5 heterozygous mutation, c.2654G 〉 A, which predicts p.Arg885His, was commonly identified in poor-responders, but not in responders. This polymorphism had at least spread to other East Asian countries. After determining that the poor responders likely express both wild-type C5 and a structural variant C5, we then showed that the hemolytic activity supported by this structural variant in vitro, like the effects on patient sera, was not blocked by eculizumab but was fully blocked by N19/8, and that the variant was incapable of binding eculizumab. Collectively, these data are consistent with the hypothesis that the functional capacity of the mutant C5 together with its inability to bind to and undergo blockade by eculizumab fully account for the poor response in patients carrying this mutation. (JN and MY contributed equally to this work) Disclosures: Nishimura: Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Yamamoto:Alexion Pharm: Research Funding. Ohyashiki:Alexion: Research Funding. Noji:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Hase:Alexion Phama: Employment, Equity Ownership. Lan:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Johnson:Alexion Pharmaceuticals, Inc.: Employment. Tamburini:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership, Patent inventor but do not receive royalties, Patent inventor but do not receive royalties Patents & Royalties. Kinoshita:Alexion: Honoraria. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau.

Abstract: [Background] Myelodysplastic syndrome is an intractable disorder characterized by ineffective hematopoiesis. Although allogeneic hematopoietic stem cell transplantation is the only curative therapy for eligible patients, hematopoiesis-supportive pharmacotherapy is practically important for transplant-ineligible patients to overcome transfusion dependency and infections. Vitamin K2 (VK2, menatetrenone) is a drug used to aim at improvement of hematopoiesis in MDS patients (Leukemia 14: 1156, 2000). However, the exact mechanism how VK2 improves hematopoiesis remains largely unknown. It was reported that VK2 induces MDS cells to undergo apoptosis (Leukemia 13: 1399, 1999). Here, we investigated our hypothesis that VK2 exerts its hematopoiesis-supportive effects through acting on mesenchymal stem/stromal cells (BM-MSCs) in the bone marrow microenvironment. [Methods] Normal bone marrow (BM) samples from healthy adult volunteers were purchased from AllCells (Emeryville, CA). BM-CD34+ cells were isolated from BM-mononuclear cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Human BM-MSCs were isolated according to our previously published methods (Stem Cells 32:2245, 2014). In co-culture experiments, BM-MSCs with or without VK2 treatment were seeded on a 24-well culture plate. BM-CD34+ cells were applied on the MSC-grown plate and co-cultured in SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng/mL SCF, 100 ng/mL Flt-3 ligand, 50 ng/mL TPO and 20 ng/mL IL-3. After 10 days of co-culture, the number and surface marker expression of the expanded hematopoietic cells were examined by flow cytometric analysis. [Results] We first tested the direct effect of VK2 on BM-CD34+ cells. BM-CD34+ cells were treated with VK2 at various concentrations ranged from 0 µM to 10 µM for 24 hours and then cultured in SFEM in combinations with cytokines. Surprisingly, viable hematopoietic cells were hardly detected in the expansion culture of BM-CD34+ cells treated with 10 µM VK2. Even with 1 µM treatment, the number of CD45+ cells was decreased, as compared to that of expan sion culture of untreated BM-CD34+ cells. The apoptosis analysis showed that the percentage of AnnexinV+ PI+ cells in the expanded hematopoietic cells is increased by VK2 treatment. We next examined the effect of VK2 on the hematopoiesis-supportive capability of BM-MSCs. BM-MSCs were pretreated with VK2 at various concentrations and then co-cultured with BM-CD34+ cells. The numbers of CD34+ cells and CD45+ cells were increased in a VK2 dose-dependent manner. These results demonstrated that VK2 shows different effects on distinct stem/progenitor cells: the induction of apoptosis in BM-CD34+ cells and the enhancement of hematopoiesis-supportive capability of BM-MSCs. We then investigated whether apoptosis-related cell death of BM-CD34+ cells by VK2 treatment is ameliorated in the presence of BM-MSCs. Both BM-CD34+ cells and BM-MSCs were treated with VK2 for 24 hours, and then co-cultured. The number of CD34+ cells was not decreased significantly in contrast to its severe decrease in single culture of VK2-treated BM-CD34+ cells. We further analyzed the effect of VK2 on BM-MSCs. Subpopulation analysis in co-culture of CD34+ cells with VK2-treated BM-MSCs showed that the expansion efficacy of CD34+CD38+ cells is higher in comparison to that of CD34+CD38- cells. In addition, the percentages of CD34-CD33+ cells and CD34-CD13+ cells were higher than those in co-cultures with untreated BM-MSCs. Therefore, VK2-treated BM-MSCs supported the expanded CD34+ cells to skew their phenotype toward myeloid lineage. The presence of a transwell in the co-culture system was unrelated to the expansion pattern of CD34+ cells, which suggested the involvement of soluble factors with respect to the underlining mechanism. We therefore compared the levels of hematopoiesis-supporting cytokine mRNA expression in VK2-treated and untreated BM-MSCs: VK2-treated BM-MSCs showed lower expression of CXCL12/SDF-1 mRNA and a trend toward higher expression of GM-CSF mRNA. [Summary] VK2 acted on BM-MSCs to support their ability to enhance expansion and myeloid differentiation of BM-CD34+ cells probably via altered GM-CSF and CXCL12/SDF-1 expression in MSCs. These findings may help to identify the mechanisms of therapeutic effects of VK2 in patients with MDS (Figure). Disclosures No relevant conflicts of interest to declare.

Abstract: The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 μmol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.

Abstract: Background and objectives: Although intravenous (IV) FAMP in combination with Rituximab was reported to be effective against indolent B-NHL (JCO2005; 23:694), IV administration of FAMP for 3 to 5 consecutive days is inconvenient in an outpatient setting. We conducted a multicenter phase 2 study to evaluate the efficacy and safety of oral FAMP with Rituximab for relapsed or refractory indolent B-NHL. Patients and treatment: Eligible patients were: aged 20 to 74 years, with indolent B-NHL, with measurable lesions (the greatest transverse diameter 〉 1.5 cm by CT), performance status 0 or 1, with no major organ dysfunctions. Mantle cell lymphoma was excluded. Prior chemotherapies were limited to 〈 = 2 regimens, and prior rituximab treatments up to 16 times were allowed. Patients who received nucleoside analogs or stem cell transplant were excluded. Patients who had progressive disease within 6 months of receiving rituximab therapy were also excluded. Based on the results of the preceding phase 2 study (JCO2006; 24:174), oral FAMP 40 mg/m2 was administered on day 1 to 5, with rituximab 375 mg/m2 on day 1, repeated every 4 weeks, for up to 6 cycles. Results: Forty-one patients were enrolled and received oral FAMP with rituximab. Thirty-eight patients (93%) were follicular lymphoma, two with MALT lymphoma, and one with small lymphocytic lymphoma. Thirty-four patients (83%) received rituximab with or without chemotherapy prior to enrollment. Median treatment cycles were 6 cycles (range: 2 to 6, 66% of patients completed 6 cycles of treatment). Investigator-assessed overall response rate and complete response rate were 83% (34/41; CI: 68 to 93%) and 76% (31/41; CI: 60 to 83%), respectively. Central evaluation is pending. With median follow-up period of 9 months, median progression-free survival was not reached, and 28 patients (68%) were still alive without disease progression. Toxicity was primary hematologic, transient and manageable. Most common grade 4 hematologic toxicities included lymphopenia (98%), neutropenia (66%), and leukopenia (42%). Grade 3 or greater non-hematologic toxicities were observed in 29% of patients including one each of grade 4 stomatitis and hyperuricemia. Dose reduction of oral FAMP (from 40 to 30 mg/m2) was necessary in 44% of patients at some points of treatment courses. One patient died from Pneumocystis jiroveci pneumonia and other 2 from disease progression after the end of the study. Conclusion: Oral FAMP combined with rituximab is highly effective with acceptable toxicities for patients with relapsed or refractory indolent B-NHL who have been mostly pretreated with rituximab, and more convenient than the combination with IV FAMP. Prolonged prophylactic therapy against Pneumocystis jiroveci pneumonia would be recommended.

Abstract: Abstract 3042 Introduction: Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of diseases. However, severe graft versus host disease (GVHD) remains a life-threatening complication after allogeneic stem cell transplantation, and it is very difficult to treat steroid-resistant GVHD. Mesenchymal stem cells (MSCs) have a homing activity to inflammatory sites and modulate immune response via PGE2 and so on, suggesting that MSCs might be effective for refractory aGVHD. Recently, clinical studies have suggested that MSC infusion can also reduce the severity of GVHD. We report here the results of our multicenter phase I/II study of MSCs for treatment of steroid-resistant aGVHD. Methods: Patients with steroid-resistant, grade II-IV, aGVHD were treated with MSCs derived from bone marrow of healthy volunteers and expanded in vitro (JR-031). MSCs were administered twice a week for 4 weeks. All patients received 2 × 106 cells/kg of MSCs for each infusion. Patients with PR after 4 weeks were given continued weekly MSCs infusion for an additional 4 weeks. Results: From January 2009 to November 2010, 14 patients were treated. The median age was 52 years old (range: 4–62), Male (n=5) and Female (n=9). Nine patients were diagnosed as grade II aGVHD, 5 patients were diagnosed as grade III and no patients were diagnosed as grade IV aGVHD. Thirteen of 14 patients (92.9%) responded to MSCs, achieving CR (n=8) or PR (n=5) 4 weeks after treatment. Eleven patients (78.6%) survived 24 weeks after treatment. Seven patients showed severe adverse effects including thrombocytopenia, neutropenia and hepatic dysfunction, however, no patients had severe infections which was shown by existing salvage therapy for aGVHD. Conclusion: The prognosis of steroid-resistant aGVHD was poor, and the most effective salvage therapy remains to be clarified. In this study, we showed that in vitro expanded MSCs, JR-031, might be a safe and effective salvage therapy for patients with steroid-resistant aGVHD. Disclosures: Kato: Research Grant for Tissue Engineering (H17-014) and a Research Grant for Allergic Disease and Immunology (H20-015) from the Japnese Ministry of Health, Labor and Welfare.: Research Funding.

Abstract: Background: CHOP plus rituximab (R-CHOP) is the standard of care for previously untreated DLBCL. R-CHOP comprises CHOP and one-dose rituximab in each 21-day cycle; however, the schedule of rituximab administration has not been fully optimized. Dose-dense rituximab was expected to increase its peak concentration to enhance the synergistic effect with chemotherapy at early phase of treatment. To compare weekly administration of rituximab combined with CHOP (RW-CHOP) with standard R-CHOP in patients with previously untreated DLBCL, we conducted a multicenter, randomized phase II/III study (JCOG0601, UMIN000000929). Methods: Previously untreated patients with CD20+ DLBCL were eligible. Other major inclusion criteria were as follows: aged 20-79 years; ECOG performance status 0-2, at least 1 measurable lesion and preserved organ functions. At the beginning of the study, patients with advanced stage disease and the low or low-intermediate risk group by the International Prognostic Index (IPI) were eligible. These criteria were amended in September 2010 to allow enrollment of the patients with any IPI risk and any clinical stage because of slow accrual. Patients were randomly assigned to standard R-CHOP (rituximab 375 mg/m2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, vincristine 1.4 mg/m2 [max 2 mg], all IV on day 1, and prednisone 100 mg/day PO [40mg/m2 for aged 〉 65] on days 1-5, every 3 weeks) or RW-CHOP (standard CHOP with eight doses of weekly rituximab [375mg/m2 IV on days1, 8, 15, 22, 29, 36, 43 and 50] ). Six cycles of CHOP were given in stage I non-bulky patients, 8 cycles were given in stage I bulky and II-IV patients, and rituximab was given 8 times regardless of cycles of CHOP. Randomization was stratified by institution, presence or absence of bulky mass and patient age. The primary endpoint of phase III part was progression-free survival (PFS). Secondary endpoints included overall survival (OS) and adverse events (AE). Assuming 3-year PFS in the R-CHOP arm to be 77% and expecting a 7% increase in 3-year PFS of the RW-CHOP arm, required sample size was 211 per arm with a one-sided alpha of 5%, power of 80%, an accrual period of 7 years, and a follow-up period of 3 years. Results: Between December 2007 and December 2014, a total of 422 patients were randomized to study treatments but primary analysis was performed in 421 patients: 213 to the R-CHOP arm and 208 to the RW-CHOP arm, because of one consent withdrawal. Baseline characteristics of 421 eligible patients were as follows (R-CHOP vs. RW-CHOP): median age, 61 vs. 62 years; male sex, 54.5% vs. 55.8%; Ann Arbor stage I/II/III/IV, 14.6/32.9/26.8/25.8% vs. 16.3/42.8/20.2/20.7%; and IPI score ≤2, 77.0% vs. 87.5%. With a median follow-up of 63.4 months (range: 3.2-119.2) among all patients, there was no significant difference in PFS between the arms (hazard ratio [HR], 0.95; 90.6% confidence interval [CI] , 0.68 to 1.31; one-sided log-rank P = 0.39). The 3-year PFS and OS were 79.2% and 88.7% with the R-CHOP arm and 80.3% and 90.4% with the RW-CHOP arm, respectively. The complete response rate and overall response rate were 77.0% and 93.0% in the R-CHOP arm and 82.2% and 91.8% in the RW-CHOP arm, respectively. Major AEs were hematological toxicities and infections. Grade (G) 3/4 neutropenia and G 3/4 thrombocytopenia were observed in 97.7% and 8.0% in the R-CHOP arm and 97.1% and 5.3% in the RW-CHOP arm, respectively. G3 febrile neutropenia was occurred in 33.8% in the R-CHOP arm and in 22.1% in the RW-CHOP arm. The frequency of severe AE was 2.3% in the R-CHOP arm and 3.8% in the RW-CHOP arm. Safety profile was comparable. No unexpected AEs were experienced. Conclusion: In combination of standard CHOP and rituximab, dose-dense weekly rituximab at early phase of treatment did not improve the PFS in patients with untreated DLBCL. Figure. Figure. Disclosures Ohmachi: Celgene: Honoraria; Takeda Pharmaceutical Co., Ltd,: Honoraria; Pfizer: Honoraria; Chugai Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Meiji Pharma: Honoraria. Kinoshita:Takeda: Honoraria; Takeda: Research Funding; Ono: Research Funding; MSD: Research Funding; Solasia: Research Funding; Janssen: Honoraria; Ono: Honoraria; Zenyaku: Research Funding; Eisai: Research Funding; Gilead: Research Funding. Tobinai:Kyowa Hakko Kirin: Honoraria, Research Funding; Zenyaku Kogyo: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; HUYA Bioscience International: Consultancy, Honoraria; SERVIER: Research Funding; Abbvie: Research Funding. Fukuhara:Sumitomo Dainippon: Research Funding; Solasia: Research Funding; Symbio: Research Funding; Sanofi: Research Funding; Pfizer: Research Funding; Otsuka Pharmaceutical: Research Funding; Ono: Honoraria, Research Funding; Novartis pharma: Research Funding; Nippon-shinyaku: Research Funding; MSD: Research Funding; Mundipharma: Honoraria, Research Funding; Mitsubishi Tanabe: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Japan Blood Products Organization: Research Funding; Janssen: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Eisai: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; Daiichi-Sankyo: Research Funding; Chugai: Research Funding; Celgene: Research Funding; Baxalta: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bayer Yakuhin: Research Funding; Alexionpharma: Research Funding; AbbVie: Research Funding; Astellas: Research Funding; Nihon Ultmarc: Research Funding; Taiho: Research Funding; Teijin Pharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Takeda: Honoraria. Uchida:Takeda Pharmaceutical: Honoraria; Chugai Pharmaceutical: Honoraria; Kyowa Hakko Kirin: Honoraria; Meiji Seika Pharma: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Nippon Shinyaku: Honoraria; Novartis: Honoraria; Teijin: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria; Janssen Pharma: Honoraria; Otsuka Pharmaceutical: Honoraria; Eisai: Honoraria. Yamamoto:Solasia Pharma: Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria, Research Funding; ARIAD Pharmaceuticals: Research Funding; Bayer: Research Funding; Celgene: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Ono: Consultancy, Honoraria, Research Funding; AbbVie: Research Funding; Boehringer Ingelheim: Consultancy; Chugai: Consultancy, Honoraria, Research Funding; Meiji Seika Pharma: Consultancy; MSD: Research Funding; Takeda: Honoraria, Research Funding; Zenyaku: Research Funding; Kyowa Hakko Kirin: Honoraria; Otsuka: Honoraria; Pfizer: Honoraria; Sumitomo Dainippon: Honoraria; Mundipharma: Consultancy, Honoraria; HUYA: Honoraria; SymBio: Research Funding; Gilead Sciences: Research Funding. Miyazaki:Kyowa Hakko Kirin,: Honoraria, Research Funding; Celgene: Honoraria; Chugai Pharma,: Honoraria, Research Funding; Sumitomo Group: Research Funding; Nippon Shinyaku: Research Funding; Takeda: Research Funding; Astellas Pharma: Research Funding; Shionogi Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Teijin Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Toyama Chemical Co: Research Funding; Mochida Pharmaceutical Co. Ltd.: Research Funding; Novo Nordisk: Research Funding. Tsukamoto:Kyowa-Kirin: Research Funding; Pfizer: Research Funding; Chugai: Research Funding; Eisai: Research Funding. Iida:Teijin Pharma: Research Funding; Toyama Chemical: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Research Funding; Chugai: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astellas: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Gilead: Research Funding; MSD: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Sanofi: Consultancy. Yoshida:Taiho Pharma: Honoraria; Takeda Pharma: Honoraria; Celegene: Honoraria; Chugai Pharma: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Masaki:Ono: Research Funding; Kyowa Hakko Kirin: Research Funding; Phizer: Research Funding; Astellas: Research Funding; Eisai: Research Funding. Yakushijin:Mundipharma Co.,: Research Funding; Chugai Co.,: Research Funding; Kyowa-kirin Co.,: Research Funding; Merch Sharp & Dohme Corp.,,: Research Funding; Daiichi-Sankyo Inc.,: Research Funding; Eisai Co.: Research Funding. Suehiro:Kyowa Hakko Kirin: Research Funding; Ono Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding. Nosaka:Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical Co.LTD.: Honoraria; Eisai Co. Ltd.,: Honoraria; Kyowa Kirin Pharmaceutical Development, Inc.,: Honoraria; Chugai Pharmaceutical Co.LTD.,: Honoraria; Celgene Co. LTD.,: Honoraria. Dobashi:Celgene Co.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Sysmex Co.: Research Funding. Kuroda:Chugai Pharma: Honoraria, Research Funding. Takamatsu:Taisho Toyama Pharmaceutical: Research Funding; TAIHO Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Research Funding; Chugai Pharma: Research Funding; Takeda Pharmaceutical: Research Funding; Celgene: Honoraria. Maruyama:Ono Pharmaceutical: Honoraria, Research Funding; Fujifilm: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Asahi Kasei Pharma: Honoraria; AstraZeneca: Research Funding; Solasia Pharma: Research Funding; Pfizer: Research Funding; Nippon Boehringer Ingelheim: Research Funding; Dai-Nippon-Sumitomo: Honoraria; Dai-ichi-Sankyo: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Biomedis International: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Novartis: Research Funding; Otsuka: Research Funding; Amgen Astellas BioPharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Abbvie: Research Funding; Astellas Pharma: Research Funding; Mundipharma International: Honoraria, Research Funding. Ando:Eisai: Research Funding; Meiji Seika Pharma: Research Funding; Takeda Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding; Japan Blood Products Organization: Research Funding. Ishizawa:Eisai: Honoraria; Janssen: Honoraria; Chugai: Honoraria; Celgene: Honoraria; Otsuka: Research Funding; Sanofi: Research Funding; Phizer: Research Funding. Ogura:Celltrion: Consultancy, Research Funding; Mundi Pharma: Consultancy; SymBio: Research Funding; Takeda: Honoraria; Cellgene: Honoraria; MeijiSeika Pharma: Consultancy. Hotta:SymBio: Consultancy; CellSeed Inc.: Membership on an entity's Board of Directors or advisory committees. Tsukasaki:Celgene: Honoraria; Eisai: Research Funding; Chugai Pharma: Honoraria, Research Funding; HUYA: Consultancy, Research Funding; Ono Pharma: Consultancy; Daiich-Sankyo: Consultancy; Mundy Pharma: Honoraria; Kyowa-hakko/Kirin: Honoraria; Seattle Genetics: Research Funding. Nagai:HUYA Bioscience International: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Gilead Sciences Inc.: Honoraria, Research Funding; Bayer Yakuhin Ltd.: Research Funding; Sanofi K. K.: Honoraria; Zenyaku Kogyo Co., Ltd.: Honoraria, Research Funding; Solasia Pharma K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Roche Ltd.: Honoraria; Esai Co., Ltd.: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; SymBio Pharmaceuticals Limited: Research Funding; Janssen Pharmaceutical K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; AstraZeneca plc.: Research Funding; Abbvie G. K.: Research Funding.

Abstract: Rituximab plus cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) is the standard of care for untreated diffuse large B-cell lymphoma (DLBCL). However, the schedule for rituximab administration has not been optimized. To compare standard R-CHOP with CHOP plus dose-dense weekly rituximab (RW-CHOP) in patients with untreated DLBCL, we conducted a phase 2/3 study (JCOG0601, jRCTs031180139). Patients were randomly assigned to R-CHOP (CHOP-21 with 8 doses of rituximab once every 3 weeks [375 mg/m2]) or RW-CHOP (CHOP-21 with 8 doses of weekly rituximab [375 mg/m2] ) groups. The primary end point of the phase 2 component was percent complete response (%CR) of the RW-CHOP arm, whereas that of the phase 3 component was progression-free survival (PFS). Between December 2007 and December 2014, 421 untreated patients were randomly assigned to R-CHOP (213 patients) or RW-CHOP (208 patients). The %CR in the RW-CHOP arm was 85.3% and therefore met the prespecified decision criteria for the phase 2 component. With a median follow-up of 63.4 months, the 3-year PFS and overall survival were 79.2% and 88.7% in the R-CHOP arm and 80.3% and 90.4% in the RW-CHOP arm, respectively. There was no significant difference in PFS (hazard ratio, 0.95; 90.6% confidence interval, 0.68-1.31). Although the safety profile and efficacy of RW-CHOP was comparable with R-CHOP and its tolerability was acceptable, weekly rituximab in combination with CHOP during the early treatment period did not improve PFS in untreated patients with DLBCL. This trial was registered at jrct.niph.go.jp as #jRCTs031180139.

Abstract: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells. In some cases with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS), it is known that glycosylphosphatidylinositol-anchored protein deficient (PNH-type) cells can be often detected at low frequencies (about 0.01%) through the high-resolution flow cytometry-based methFod. Because these patient groups are reported to have a good reactivity towards immunosuppressive therapies as opposed to the other patient groups lacking PNH-type cells, detection of these cells is potentially useful in determining a treatment plan for the patients with bone marrow failure syndromes. To confirm this preliminary information, a large cohort study is needed. Method A nationwide multi-center prospective observational study (OPTIMA) was started in July 2011 to determine the prevalence of patients with bone marrow failure syndromes who carried PNH-type cells and to clarify the significance of the presence and quantitative changes of these cells with regard to the clinical features. Each of the six laboratories in different universities was assigned as a regional analyzing center. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Quality of the assay was managed in all the laboratories by periodic blind validation tests using standard blood samples containing 0.01% PNH-type cells. Until July 2013, 1214 cases were examined; 461 (38%) were positive for PNH-type cells and 141 (11.6%) had ≥1% PNH-type cells. Out of 1214, 783 patients were diagnosed to have AA (n=386), MDS (n=341), and PNH (n=56) based on the case report forms. PNH-type cells were detected in 56.2%, 19.1% and 100% of patients with AA, MDS and PNH, respectively. In a half of patients having ≥1% PNH-type cells, lactate dehydrogenase levels exceeded the ≥1.5×upper limits of normal. Conclusion Our study has successfully established the high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells (below 0.01%). Also, by implementing a uniform protocol to six individual laboratories across the country, a system has been established for the patients to undergo the detection test with equal accuracy in all of these laboratories. Further accumulation of case studies and prolonged observations are required to determine the clinical significance of the minimal PNH-type cells, especially in terms of its relation to response to immunosuppressive therapy. Disclosures: Obara: Alexion Pharmaceuticals: Honoraria. Chiba:Alexion Pharmaceuticals, In: Research Funding. Sugimori:Alexion Pharma: Honoraria. Noji:Alexion Pharmaceuticals: Honoraria. Yonemura:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals, In: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Ninomiya:Alexion Pharma: Honoraria. Nishimura:Alexion Pharma: Research Funding, Speakers Bureau. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau. Nakao:Alexion Pharmaceuticals, In: Research Funding.

Abstract: The prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is poor. Many cases in complete remission experience relapse of leukemia despite intensive chemotherapy or hematopoietic stem cell transplantation. This clinical observation suggests that minimal residual disease (MRD) still exists after these intensive therapies. Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms to support MRD in the bone marrow microenvironment (Clin Cancer Res 14:9, 2008). Mesenchymal stromal/stem cells (MSCs) are a cellular component of bone marrow (BM) and maintain physiological precursor B lymphopoiesis (Nature Rev Immunol 6:107, 2006). We investigated our hypothesis that survival of BCP-ALL cells is supported by their direct adhesion to BM-MSCs in BM microenvironment. First, we confirmed that BCP-ALL cells exhibited their drug resistant phenotype through adhesion to human BM-MSCs using human BCP-ALL cell line, Nalm6. We isolated human BM-MSCs from normal bone marrow samples (AllCells, Emeryville, CA). When Nalm6 cells were co-cultured with human BM-MSCs, they were prone to adhere to BM-MSCs. Nalm6 adhered to BM-MSCs (Nalm6/ad) were more resistant to the treatment with various anti-cancer drugs including doxorubicin than Nalm6 in suspension (Nalm6/su). Immunoblot analysis showed that expression level of anti-apoptotic protein Bcl-2 and phosphorylation level of pro-survival kinase Akt are higher in Nalm6/ad compared with those in Nalm6/su. In cell cycle analysis using Ki67/PI fluorescence-activated cell sorter (FACS) assay, the percentage of populations in G0 phase and S/G2/M phase was higher in Nalm6/ad compared with that in Nalm6/su, which indicated the increase of MRD and the high proliferation of leukemic cells in adhesive population, respectively. Therefore, we considered that detachment of Nalm6 from BM-MSCs is important to restore chemosensitivity of BCP-ALL cells and to eliminate these cells. Accordingly, we screened drugs that are capable of disrupting the adhesion of Nalm6 to BM-MSCs, and found that several proteasome inhibitors had a such activity. BM-MSCs were treated with bortezomib (10nM for 24h or 100nM for 1h), carfilzomib (3 nM for 24 h or 100 nM for 1 h) or oprozomib (10 nM for 24 h or 300 nM for 4 h), and then washed with PBS and co-cultured with Nalm6 cells. We confirmed that the number of BM-MSCs is not affected with these drugs irrespective of their concentration and incubation time by MTT assay. Co-cultured cells were divided into suspension cells and adherent cells. Each population was stained with monoclonal antibodies against CD19 and CD90 to detect Nalm6 and BM-MSCs, respectively, and analyzed by FACS. When BM-MSCs were pre-treated with bortezomib, carfilzomib or oprozomib, the number of Nalm6/ad was significantly decreased, and inversely, the number of Nalm6/su was increased, as compared to that of co-cultures with untreated BM-MSCs. Intriguingly, BM-MSCs treated with bortezomib showed aberrant expression of secreted protein acidic and rich in cysteine (SPARC). Immunoblot analysis showed that SPARC expression of BM-MSCs was transiently increased after bortezomib treatment. In co-cultures with BM-MSCs transfected with siRNA targeting SPARC, the number of Nalm6/ad was increased. In addition, the number of Nalm6 adhered to BM-MSCs pre-treated with recombinant human SPARC was lower than that adhered to control non-treated BM-MSCs. Therefore, SPARC showed anti-adhesive property and was one of the molecules associated with adhesion of BM-MSCs to Nalm6. Finally, we tested whether bortezomib shows therapeutic effects in vivo. Bortezomib administration supported survival of BCP-ALL xenograft model mice utilizing Nalm6, which was concomitant with the decrease in leukemia cells in the femur and in the size of spleen, as compared to those in non-treated control mice. In summary, Nalm6 cells that adhered to BM-MSCs contributed to construction of chemoresistant population similar to MRD. Here, we found that this population could be detached by treatment of BM-MSCs with bortezomib through transient increase in their SPARC expression. Our findings suggest that bortezomib or other proteasome inhibitors might be promising drugs to treat BCP-ALL through attenuating the adhesion of leukemic cells to BM-MSCs, and shed new insight into a therapeutic strategy in BCP-ALL by targeting BM-MSCs. Disclosures No relevant conflicts of interest to declare.