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Complement C5 inhibition is the standard of care (SoC) for patients with paroxysmal nocturnal hemoglobinuria (PNH) with significant clinical symptoms. Constant and complete suppression of the terminal complement pathway and the high serum concentration of C5 pose challenges to drug development that result in IV-only treatment options. Crovalimab, a sequential monoclonal antibody recycling technology antibody was engineered for extended self-administered subcutaneous dosing of small volumes in diseases amenable for C5 inhibition. A 3-part open-label adaptive phase 1/2 trial was conducted to assess safety, pharmacokinetics, pharmacodynamics, and exploratory efficacy in healthy volunteers (part 1), as well as in complement blockade–naive (part 2) and C5 inhibitor–treated (part 3) PNH patients. Twenty-nine patients were included in part 2 (n = 10) and part 3 (n = 19). Crovalimab concentrations exceeded the prespecified 100-µg/mL level and resulted in complete and sustained terminal complement pathway inhibition in treatment-naive and C5 inhibitor–pretreated PNH patients. Hemolytic activity and free C5 levels were suppressed below clinically relevant thresholds (liposome assay

Introduction Crovalimab is a novel anti-complement component 5 (C5) monoclonal antibody engineered with the Sequential Monoclonal Antibody Recycling Technology (SMART-Ig; Fukuzawa et al, Sci Rep. 2017) to extend half-life and enable infrequent, subcutaneous (SC) self-administration in C5-mediated diseases. Crovalimab is being investigated as a therapy for paroxysmal nocturnal hemoglobinuria (PNH), a disease for which C5 inhibition is the standard of care. The Phase I/II COMPOSER trial (NCT03157635; Röth, et al. Blood. 2020) is a global, open-label, multicenter study of crovalimab consisting of 4 sequential parts. Parts 1, 2, and 3 assessed the pharmacokinetics (PK) and safety of crovalimab in healthy volunteers, C5 inhibitor-naive patients, and patients switched from eculizumab, respectively. Part 4 assessed an optimized crovalimab dose and regimen in naive and switched patients with PNH. Because eculizumab and crovalimab bind to different C5 epitopes, drug-target-drug complexes (DTDCs) consisting of eculizumab, C5, and crovalimab motifs can temporarily form in the circulation of patients who switch treatments. DTDCs can form in a range of sizes, from single crovalimab-C5-eculizumab motif to larger complexes with multiple motifs. Larger DTDCs are a concern because they take longer to clear and may be more likely to induce type III hypersensitivity reactions. Objectives Describe the impact of DTDC formation on the safety, PK, and pharmacodynamics of crovalimab in patients with PNH who switched from eculizumab to crovalimab and to describe the effect of crovalimab dose on DTDC size distribution and kinetics. Study Design and Methods Using data from COMPOSER Parts 1-3, a biochemical mathematical model was developed to investigate the kinetics of the formation and dissociation of DTDCs under the assumption that larger complexes are formed by the reversible binding of smaller complexes. The model was calibrated using concentration-time profiles of total C5, total crovalimab, and the concentration of eculizumab at the time of crovalimab initiation. DTDC size distributions were measured using size-exclusion chromatography coupled to enzyme-linked immunosorbent assay. Using model-based simulations, an optimized crovalimab dosing strategy was identified to reduce the formation of large DTDCs while maintaining serum concentration of crovalimab above the target level of ≈ 100 μg/mL. The optimized dose and regimen were a loading series of 1000 mg intravenously on day 1 and 340 mg SC on days 2, 8, 15, and 22, followed by maintenance dosing of 680 mg SC every 4 weeks starting on day 29. The loading dose series increased the total crovalimab dose received during the first month of treatment to reduce the formation of larger DTDCs, in line with the lattice theory of complex formation. This optimized dosing strategy was investigated in Part 4 patients who switched from eculizumab. Results In COMPOSER, 19 patients with PNH were enrolled in Part 3 and switched from eculizumab to crovalimab. DTDCs were observed in all patients from Part 3 (Figure; larger DTDCs are found in fractions 1-4 and smaller crovalimab-containing complexes, such as single motifs and single crovalimab molecules, are found in fractions 5 and 6). Two Part 3 patients experienced clinical manifestations compatible with type III hypersensitivity reactions that were ascribed to DTDCs. The DTDC size distribution in Part 4 patients, who received the optimized dosing strategy, evolved differently than in Part 3 patients, consistent with model predictions. In the switched patients from Part 4, large DTDC levels started to decrease on day 8 and continued to decrease, in contrast to Part 3, in which they started to decrease on day 15. On day 22, the mean percentage of the largest DTDCs was reduced by 56% in patients in Part 4 relative to patients in Part 3. Part 4 patients achieved and maintained serum crovalimab concentrations above ≈ 100 µg/mL throughout follow-up. Despite DTDCs being observed in all Part 4 patients who switched from eculizumab, no adverse events suggestive of a type III hypersensitivity reaction occurred. Conclusions The optimized crovalimab regimen resulted in lower concentrations of large DTDCs than in patients who received the Part 3 regimen and reduced the persistence of DTDCs in patients who switched treatment. This regimen is now being evaluated in the Phase III COMMODORE 1 (NCT04432584) and COMMODORE 2 (NCT04434092) studies. Figure Disclosures Nishimura: F. Hoffmann-La Roche Ltd: Consultancy, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; Alexion: Honoraria, Research Funding; Chugai: Consultancy. Soubret:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Buatois:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Charoin:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Sreckovic:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Bucher:F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company, Ended employment in the past 24 months, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; ANAVEON AG: Current Employment. Hernández-Sánchez:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Jordan:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Ramos:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; Genentech, Inc: Current Employment, Other: Received fellowship support from Genentech, Inc.. Arase:Osaka University: Current Employment; Chugai: Consultancy; Alexion: Research Funding; F. Hoffmann-La Roche. Ltd.: Consultancy, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Hotta:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Isaka:Osaka University: Current Employment; Chugai: Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Ito:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Kanakura:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; Chugai Pharmaceutical: Consultancy. Kim:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Kinoshita:Alexion: Honoraria; F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Morii:F. Hoffmann-La Roche Ltd: Honoraria, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding; Chugai: Honoraria, Research Funding. Panse:Apellis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai: Speakers Bureau; Grunenthal: Consultancy, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Peffault De Latour:Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Apellis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding. Röth:Apellis: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding; Biocryst: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Sica:F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding. Takamori:Alexion: Research Funding; F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Ueda:Chugai: Research Funding; Novartis: Honoraria; Alexion: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.; Sanofi: Consultancy, Honoraria. Yoon:Kyowahako Kirin: Research Funding; Novartis: Consultancy, Honoraria; Janssen: Consultancy; F. Hoffmann-La Roche: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding; YuhanPharma: Research Funding; Amgen: Consultancy, Honoraria. Paz-Priel:Genentech, Inc: Current Employment; F. Hoffmann-La Roche Ltd: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Sostelly:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland..

Background Azacitidine (AZA) is one of the hypomethylating agents (HMAs) proven to significantly prolong overall survival (OS) for patients with higher-risk myelodysplastic syndromes (h-MDS) via a prospective phase 3 trial. However, the optimal treatment schedule of AZA for h-MDS has not yet been prospectively determined. As a 7-day treatment schedule includes weekends, a 5-day administration has also been applied with h-MDS, though solid evidence has not been established. Based on this background, we planned to compare AZA treatment on 7-day and 5-day schedules. Patients Patients diagnosed with de novo or treatment-related MDS (FAB-defined RAEB and RAEB-t), aged 16 years or older were eligible for this study, if they showed adequate performance status (ECOG PS 0-2) and no history of HMA treatment or chemotherapy. Candidates for allogeneic stem-cell transplantation were excluded. Study design This was a multicenter, randomized, open-label, phase 3 trial to compare the efficacy of AZA treatment for 7-days (AZA-7) to 5-days (AZA-5). The primary endpoint was 2-year OS rate (2y OS). AZA was given subcutaneously at 75 mg/m² every 28 days. To test the non-inferiority of AZA-5 to AZA-7, 2y OS of AZA-7 was estimated at 30%, and the delta was defined as 11%, for which 410 patients were needed to complete the trial. However, because of the poor recruitment, this study closed prematurely, and the protocol-planned interim analysis (when 200 patients were registered) was performed. This protocol was approved by the IRB at each trial center. All patients provided written informed consent, and the trial was conducted in accordance with the Declaration of Helsinki. The efficacy of AZA was evaluated using IWG2006 criteria, and treatment was continued until study completion, or relapse, unacceptable toxicity, or disease progression was observed. Efficacy was analyzed by intention-to-treat. Secondary endpoints were the hematological response rate, 2-year leukemia-free survival (2y LFS) rate, the cytogenetic response, and occurrence of adverse events. Results Between January 2013 and Jun 2018, 201 patients were randomly assigned to AZA-7 (n=99) or AZA-5 group (n=102). The median age of all patients at the enrollment was 73.5 years old (range, 48 to 91). Between the two groups, there was no significant difference in the baseline characteristics, such as sex, type of MDS (de novo or treatment-related), percent of BM blasts, FAB and WHO classification, and IPSS and IPSS-R risk. After excluding 5 and 6 patients from AZA-7 and AZA-5, respectively (poor data entry, change in diagnosis), at the time of last follow-up, 59 and 67 patients died in AZA-7 and AZA-5, respectively. The Kaplan-Meier estimates of the 2y OS and 2y LFS were 35.1% (95% CI, 23.8 to 46.7) and 27.3% (95% CI, 17.3 to 38.3) for AZA-7, and 22.4% (95% CI, 13.1 to 33.3) and 20.5% (95% CI, 11.6 to 31.2) for AZA-5, respectively. There was no significant difference in the frequency of erythroid, platelet or neutrophil improvement, or hematological response (complete or partial response, or any hematological improvement) between the two groups. The most common non-hematological adverse event of grade 3 to 4 (in ≥20% of the patients in either group) was febrile neutropenia (33.7% for AZA-7, and 30.5% for AZA-5). Conclusion and Discussion Because of the premature termination of this trial, statistical analysis for the primary endpoint (2y OS of 35.1% for AZA-7, and 22.4% for AZA-5) could not provide any solid evidence. However, considering the difference of more than 10% in 2y OS, and no major differences in the safety profiles, we favor AZA-7 to AZA-5 for MDS patients with RAEB and RAEB-t. Figure Disclosures Kiguchi: Tejin Co., Ltd.: Research Funding; Taiho Pharmaceutical Co., Ltd.: Research Funding; SymBio Pharmaceutical Co., Ltd.: Research Funding; Celgene Co., Ltd.: Research Funding; Janssen Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharmaceutical Co., Ltd.: Research Funding; MSD CO., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myeres Squibb Co., Ltd.: Research Funding; Sanofi K.K., Ltd.: Research Funding; Celltrion, Inc.: Research Funding. Usuki:Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc: Research Funding, Speakers Bureau. Suzuki:kyowa Hakko Kirin: Consultancy, Honoraria; MSD k.k.: Research Funding; Nippon Shinyaku: Honoraria; Novartis: Honoraria; takeda: Research Funding; Incyte and Pfizer: Research Funding; astellas: Research Funding; Celgene: Honoraria; Chugai: Honoraria, Research Funding; daiichi sankyo: Research Funding; eisai: Research Funding. Tomita:Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Taiho Pharma: Research Funding. Handa:Ono: Research Funding. Maeda:Janssen Pharmaceutical K.K..: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Iyama:Otsuka Pharmaceutical Co., Ltd.: Honoraria; Otsuka Pharmaceutical Factory: Honoraria; Astellas Pharma: Honoraria; Daiichi Sankyo: Honoraria; Allexion Pharma: Honoraria; CSL Behring: Honoraria. Yamauchi:Astellas, AbbVie: Consultancy; Pfizer, Chugai, Teijin, Solasia: Research Funding. Kiyoi:Daiichi Sankyo Co., Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Japan Inc.: Honoraria; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding; FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria.

Background: Therapy-related myeloid neoplasm (tMN) is a late complication of cytotoxic chemotherapy and radiotherapy. Because patients with malignant lymphoma (ML) tend to receive various types of chemotherapy, the developmental mechanism of tMN remains unclear.In myelodysplastic syndrome (MDS), clonal hematopoiesis of indeterminate potential (CHIP) has recently been consideredas a disease background,and preleukemic mutations have been detected as CHIP in peripheral blood samples of some patients with tMN before chemotherapy. Nevertheless, the clonal evolution from CHIP-related genetic modifications to tMN remains to be validated by the sequential analysis of bone marrow (BM) samples. Materials and methods: We performed targeted deepsequencing (TDS) using BM samples and ML tissues of five patients with tMN after treatment for ML between 2005 and 2017 to examine the sequentialclonal evolution of tMN. For DNA sequencing with MiSeq, we used the Myeloid Neoplasms Panel with 50 known mutation target genes of hematopoietic diseases. This study was approvedby the Institutional Review Board of Tokyo Medical University (no. 2151). Results : TDS revealed variedclonal evolution patterns in three patients. In patient 1, a small clone having NRAS, SETBP1, and SMC1A mutations was detectedat the diagnosis of diffuse large B-cell lymphoma [DLBCL; variant allele frequency (VAF): 0.07, 0.05, and 0.07, respectively], whereas other clones having TP53 and TET2 mutations emerged as a dominant clone at tMN by clonal sweeping.In patient 2, a clone having RUNX1 and TET2 mutations (VAF: 0.41 and 0.40, respectively) was detected 6 years before the diagnosis of nodular sclerosis classical Hodgkin lymphoma (NSCHL). The clone seemed to acquired NRAS and NF1 mutations at the NSCHL and tMN diagnoses (VAF: 0.31 and 0.38, respectively).This pattern is similar to the clonal evolution of MDS to secondary acute myeloid leukemia. In patient 3, we detected a clone having a DNMT3A mutation as the primary clone at DLBCL diagnosis; however, this clone was not detectedat the tMN diagnosis. Moreover, a clone having a SF3B1 mutation that was detected as a minor clone (VAF, 0.07) at the DLBCL diagnosis seemingly acquired the TP53 mutation and evolved as a dominant clone at tMN.This pattern demonstrates a clonalevolution from CHIP to tMN.We detected no common nonsynonymous mutations with BM mononuclear cells (BMMCs) in ML tissues of patients with tMN, excluding patient 2. The same RUNX1, TET2, and NRAS mutations observed in BMMCs were detectedat the NSCHL diagnosis in patient 2, however we speculated that these mutations were derived from nonmalignant T cells. Conclusion: This study suggests that the primaryclone as CHIP at the ML diagnosis was not always responsible for tMN, and clones that resisted chemotherapy or radiotherapy may have evolved into tMN by several clonal evolution patterns, including clonal sweeping and linear evolution. Disclosures Ohyashiki: Asteras KK,: Research Funding; Celegene KK,: Honoraria, Research Funding; Nihon-Seiyaku,: Research Funding; Novartis KK,: Honoraria, Research Funding; MSD,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Asahikase: Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Chugai KK,: Honoraria, Research Funding; Jansen Pharma KK,: Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Eizai,: Research Funding.

Introduction: Multiple myeloma (MM) is one of the hematological malignancy that affects the plasma cells in bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs. However, most patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Insulin-like growth factor (IGF) cause intracellular signaling that ultimately results in cellular growth and proliferation. Because IGF signaling pathways have crucial functions in hematological malignancies and solid tumors, IGF pathways might regulate myeloma cell survival. Materials and Methods: In this study, we investigated whether IGF1R inhibitor could suppress myeloma cells. We also investigated whether IGF1 was involved in proteasome sensitivity against myeloma cells and cytokines were induced by IGF1 in human umbilical vein endothelial cells (HUVEC). Results: We first investigated the IGF signaling pathways by microarray gene expression data from the online Gene Expression Omnibus (GEO). IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Gene expression of IGF1, IGF1R, IGFBP2, IGFBP5, IGFBP7 and IGF2 are involved in myeloma and normal control samples from the public microarray datasets of GSE39754. We next investigated the proteasome inhibitor, bortezomib or carfilzomib activity. Proteasome inhibitors exhibit cell growth inhibition against myeloma cells. However, in the presence of IGF1, proteasome inhibitors activity was reduced. Linsitinib is an orally bioavailable small molecule inhibitor of the IGF1R. We investigated whether linsitinib could inhibit the proliferation of myeloma cells. We found linsitinib treatment inhibited the growth of myeloma cells in a dose dependent manner. Activity of Caspase 3/7 and cytotoxicity were also increased. Combined treatment of myeloma cells with carfilzomib and linsitinib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We examined the intracellular signaling. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after carfilzomib and linsitinib treatment. We next blocked IGF1R function by small interfering RNA (siRNA). We found drug sensitivity of the cells to the carfilzomib was increased compared to mock-transfected cells. Caspase 3/7 activity and cytotoxicity were increased after carfilzomib treatment in siRNA transfected cells. We next investigated the in vitro effects of IGF1 on cultured HUVEC. Because the entire bone marrow cavity is hypoxic condition, we investigated the IGF signaling pathways in the hypoxic condition by microarray gene expression data from GEO. Gene expression of IGF1R and IGF2 are increased in the hypoxia from the public microarray datasets of GSE36837. Gene amplification was confirmed by RT-PCR analysis. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IGF1 stimulation in the time dependent manner. We also found that gene expression of monocyte chemotactic protein-1 (MCP-1) was enhanced by IGF1 stimulation. Conclusion: The IGF1 signaling pathway is involved in proteasome inhibitor sensitivity and plays a key role in chemokine production of the HUVEC. Our data also suggested that administration of IGF1R inhibitor, linsitinib, might be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. Disclosures Ohyashiki: MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Jansen Pharma KK,: Research Funding; Chugai KK,: Honoraria, Research Funding; Nihon-Seiyaku,: Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Eizai,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Asahikase: Research Funding; Asteras KK,: Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding.

Background The percentage of blasts in the bone marrow in the early stage of remission induction therapy for acute myeloid leukemia (AML) has been reported to affect not only remission rate, but also long-term prognosis. If early post-treatment response to therapy was determined to affect prognosis, this finding could be a basis for selection of a proactive treatment such as hematopoietic stem cell transplantation; however, the proper assessment period and the percentage of blast cell survival vary among reports. Therefore, we retrospectively analyzed the relationship between prognosis and the percentage of blasts in the bone marrow in the early post-treatment stage in cases registered for the AML201 study conducted by the Japan Adult Leukemia Study Group (JALSG). Patients and Methods The JALSG AML201 study was a multicenter randomized study in which 1064 patients with newly diagnosed AML patients were registered between December 2001 and December 2005. For remission induction therapy, we compared standard-dose idarubicin or high-dose daunorubicin in combination with cytarabine. For consolidation therapy, we compared standard-dose multiagent chemotherapy or high-dose cytarabine alone, and no apparent differences were observed in overall survival (OS) between both treatment groups. The percentage of blasts in the bone marrow on day 15 was strongly associated with remission; using the receiver operating characteristic curve as a reference, we divided patients into a ≥5% and a Results Among the 1064 patients registered for the JALSG AML201 study, 600 patients for whom bone marrow examination was performed on Day 15 were selected as subjects for the present study. The patients consisted of 375 men and 225 women with a median age of 47 years (15-64 years). A total of 377 patients (62.8%) had Conclusion As an early assessment of bone marrow following remission induction therapy, having Disclosures Fujita: Chugai Pharmaceutical CO.,LTD.: Honoraria. Kiyoi:Novartis Pharma K.K.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Celgene Corporation: Consultancy; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding. Naoe:Amgen Astellas BioPharma K.K.: Honoraria; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Pfizer Inc.: Research Funding; CMIC Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Bristol-Myers Squibb: Honoraria; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding.

Introduction: Acute myeloid leukemia (AML) with hyperleukocytosis is considered to have a poor prognosis due to a high early death rate secondary to complications such as disseminated intravascular coagulation (DIC), tumor lysis syndrome (TLS), and leukostasis. The usefulness of pre-treatment reduction of leukemic cells is controversial. We reviewed the clinical characteristics and outcome of adult patients with newly diagnosed AML with or without hyperleukocytosis registered to the Japan Adult Leukemia Study Group (JALSG) AML201study and examined whether early death in induction therapy for this AML subtype influenced the outcome. Methods: We retrospectively reviewed the records of 1,022 patients enrolled in the JALSG AML201 study, which was a phase 3 clinical trial for AML newly diagnosed from December 2001 to December 2005. The JALSG AML201 study administered cytarabine from day 1 to 7 as the induction therapy combined with idarubicin or daunorubicin (Ohtake S, et al. Blood. 2011, 24;117:2358-65.). None of the patients received pre-treatment with oral hydroxyurea or leukapheresis. Patients with performance status (PS) of 4 or severe comorbidities such as septic shock were excluded. Patients' characteristics and outcomes such as rate of early death (defined as death occurring within the first thirty days of treatment), achievement of complete remission (CR), overall survival (OS) rate and cumulative incidence of recurrence (CIR) were compared between patients with hyperleukocytosis and those without hyperleukocytosis. Result: We analyzed retrospectively the records of 1,057 patients with AML registered with the JALSG AML201 study and excluded 35 patients whose leukocyte count or outcome was not recorded. We found 113 AML patients with an initial leukocyte count greater than 100 × 109L−1 (HiLC group). There were 66 males and 47 females, and the median age was 45 years (range: 16-64). Median of leukocyte count before treatment was 160 × 109L−1 (range: 102-382 × 109L−1). According to the French-American-British (FAB) classification, there were four as M0, thirty-four as M1, fifty-six as M2, thirty as M4, and nineteen as M5. Cytogenetic analysis was performed in 107 patients. Eight patients had favorable karyotype, 90 had intermediate karyotype, and nine had unfavorable karyotype. Fifteen percent of patients with hyperleukocytosis had PS of two or greater. We compared the outcome of this AML subset with 909 patients with a leukocyte count lower than 100 × 109L−1 (median count: 10,900, range: 0.77-99.5× 109L−1) who received the same induction chemotherapy as the JALSG AML201 protocol (LoLC group). There was no significant difference between the two groups for age and sex; median age of the patients in LoLC group was 47 years (range: 15-64) with 542 males and 367 females. The frequency of the patients with a favorable karyotype or PS of 0 or 1 was lower in HiLC group than in LoLC group, whereas the rate of FAB M4 or M5 was higher in HiLC as compared to LoLC group. Eighty-two patients (72.5%) in HiLC group and 714 patients (78.5%) in LoLC group achieved CR. Sixty (53%) and twenty-two (19.5%) achieved CR after one and two cycles of treatment, respectively. In contrast, 577 (63%) and 137 (15%) in LoLC group achieved CR after one and two cycles of chemotherapy, respectively. The rate of CR was not significantly different between two groups (p=0.118). However, 5 year OS was significantly lower in HiLC as compared to LoLC group (26.9% vs. 49.4%, p < 0.001). Interestingly, the rate of early death was not different between the two groups (1.65% vs. 1.77%, p = 1.0), although the rate of severe complications, such as DIC, was higher in HiLC compared to LoLC group (32% vs. 10%, p < 0.001). The frequency of brain hemorrhage (1% vs. 1%, p = 1.0) and gastrointestinal hemorrhage (4% VS. 3%, p = 0.294) was not different between two groups. One patient died of brain hemorrhage and one of DIC in HiLC group, whereas five patients died of brain hemorrhage, four of sepsis, and three of pneumonia in LoLC within the first thirty days of treatment. CIR was higher in HiLC (64.7%) as compared to LoLC group (51.6%) (p = 0.0048). Conclusion: We conclude that hyperleukocytosis on presentation did not significantly affect the rate of early mortality during the induction phase of the treatment. It is considered that intensive chemotherapy without pre-treatment would be possible if appropriate supportive cares were given to manage fatal complications such as DIC. Disclosures Kiyoi: MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; AlexionpharmaLLC.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Phizer Japan Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Celgene Corporation: Consultancy; Chugai Pharmaceutical Co. LTD.: Research Funding. Naoe:Amgen Astellas BioPharma K.K.: Honoraria; CMIC Co., Ltd.: Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties.

It has been demonstrated that the chromosomal translocation t(7;11)(p15;p15) in patients with human acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) invariably involves fusion of the nucleoporin gene, NUP98, on chromosome 11 and the class 1 HOX gene, HOXA9, on chromosome 7, and that the fusion gene NUP98-HOXA9 is an important gene in myeloid leukemogenesis. Here are reported 2 novel chromosome 7p15 targets of the t(7;11)(p15;p15) chromosomal translocation in 2 patients with CML and myelodysplastic syndrome (MDS). Southern blot and polymerase chain reaction (PCR) analyses of leukemia cell DNA failed to show rearrangement of HOXA9,whereas NUP98 was found to be rearranged in both cases. Reverse transcription-PCR analysis using a NUP98 primer and a degenerate primer corresponding to the third helix of the homeodomain of HOXA demonstrated that NUP98 was fused in-frame to HOXA11 in the patient with CML and toHOXA13 in the patient with MDS. The chromosomal breakpoints on 7p15 were located within introns of HOXA11 orHOXA13 genes. In both patients chimericNUP98-HOXA9 transcripts were also observed. These findings suggest that AbdB-type HOXA genes are common targets of t(7;11)(p15;p15) chromosomal translocations and that a single translocation can produce more than oneNUP98-HOXA fusion gene, presumably because of altered splicing.

Multiple myeloma (MM) is one of the common hematological malignancies and is a uniformly fatal disorder of B cells characterized by accumulation of abnormal plasma cells in the bone marrow. Proteasome inhibitor, bortezomib, and immunomodulatory drugs such as thalidomide and lenalidomide play important roles in the treatment of MM patients. Although novel agents including, e.g. bortezomib, have significantly improved the response and survival of patients with MM, a large number of patients eventually have relapsed. For the patients who relapse after treatment with novel agents, the prognosis is still poor. Thus circumstanced, alternative strategies are required for continued disease control. Phosphoinositide 3-kinases (PI3Ks) are a family of proteins involved in the regulator of cell growth, metabolism and proliferation. PI3K signaling pathway also plays a critical regulatory role in MM pathology, including survival, cellular proliferation, migration and angiogenesis. Therefore, PI3K signaling pathway may present attractive targets for MM treatment. Copanlisib also known as BAY80-6946 is a potent and highly selective reversible PI3K inhibitor. Copnalisib is currently investigated in a pivotal phase 2 clinical trial against hematological malignancy such as malignant lymphoma. We hypothesized that treatment with PI3K inhibitor and proteasome inhibitors together would result in enhanced therapeutic activity in MM cells. In this study, we investigated the efficacy of copanlisib by using the MM cell lines, RPMI8226, MM1.S and MM1.R and primary sample. 72 h treatment of copanlisib exhibits cell growth inhibition of MM cell lines in a dose dependent manner. The treatment of proteasome inhibitors, bortezomib and carfilzomib exhibits cell growth inhibition partially against RPMI8226 cells in the presence of feeder cell line, HS-5. We examined the intracellular signaling in the presence of HS-5. Phosphorylation of Akt and activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) was partially reduced by carfilzomib or bortezomib in the presence of HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in RPMI8226 cells. We examined the intracellular signaling after treatment of copanlisib. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after copnlisib treatment in a dose dependent manner. Because PI3K signaling pathway regulates MM cell migration, we next evaluated the chemotactic response of MM cells to stromal cell-derived factor 1α (SDF-1α). We found that 4 h treatment of SDF-1α significantly induced the migration of MM cells compared to control medium. Treatment of copanlisib inhibited SDF-1α-stimulated chemotaxis in a dose dependent manner. We found that phosphorylation of Akt was reduced after copanlisib treatment suggesting that intracellular PI3K signaling pathway may play the important role in SDF-1α induced chemotaxis of MM cells. We investigated the copanlisib activity against MM cells. Combined treatment of MM cells with proteasome inhibitor, carfilzomib or bortezomib, and copanlisib caused significantly more cytotoxicity than each drugs alone. Phosphorylation of Akt was reduced and cleaved PARP was increased after copanlisib with or without proteasome inhibitor. We also found that copanlisib which was combinaed with carfilzomib or borteomib exhibited cell growth inhibition against MM primary sample. Data from this study suggested that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against stroma-associated drug resistance of MM cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. Disclosures No relevant conflicts of interest to declare.

Background: The inhibition of BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has markedly improved the prognosis of chronic myeloid leukemia (CML). Recently, it has been recognized that some CML patients with a complete molecular response (CMR) are able to maintain treatment-free remission (TFR) after discontinuation of TKIs. However, no predictive prognostic factors for successful discontinuation of the treatment have yet been identified. We set out to further clarify the role of predictive biomarkers in molecular relapse and non-relapse after ABL TKI discontinuation. Materials and methods: Patients in sustained CMR (MR 4.5) undergoing TKI therapy were eligible for inclusion in the study. Molecular relapse was defined as loss of major molecular response (MMR) of at least one point. Genomic DNA was obtained from whole blood using a DNA Extractor WB Kit (Wako, Osaka, Japan), and was subjected to polymerase chain reaction (PCR) amplification using primers designed to detect a deletion site (2903 bp) in intron two of the BCL2L11 gene (forward: 5′-AATACCACAGAGGCCCACAG-3′; reverse: 5′-GCCTGAAGGTGCTGAGAAAG-3′) and JumpStart RedAccuTaq LA DNA polymerase (Sigma Aldrich, St. Louis, MO, USA). Results: 32 CML patients (17 men, 15 women, median age 58.4 years) were included in this study (Sokal category; low 24, intermediate 7, high 1). Six patients were treated with IFNα before TKI treatment, and 3 were treated with IFNα after stopping TKI. Median duration from TKI initiation to discontinuation was 79.3 months (range; 22 to 138 months); median duration of CMR before TKI discontinuation was 47.3 months (range; 5 to 97 months). Seven patients showed loss of MMR; 6 relapsed within 6 months and one showed late relapse at 25 months after discontinuation. The cumulative incidence of MMR loss was estimated as 18.8% at 12 months and at 24 months. Fluctuation of BCR-ABL transcript levels below the MMR threshold (> two consecutive positive values) was observed in 6.25% of patients at 24 months after ABL TKI discontinuation. Treatment-free remission was estimated as 81.2% at 12 months and at 24 months. The median period of restoration of second CMR was 6.0 months in re-treated patients. No patient died during the follow-up period. TKI-free remission was estimated as 78.1% at 30 months. There was only a significant difference in BCL2L11 (BIM) deletion polymorphism between the patients who maintained and those who lost MMR (p = 0.0253). No significant difference was observed in prior IFNα therapy, time to complete cytogenetic response (CCyR), time to MMR, and time to CMR between relapsing and non-relapsing patients. Conclusion: Our study shows a specific association between BCL2L11 (BIM) deletion polymorphism and clinical outcome after ABL TKI discontinuation in patients with long-lasting molecular undetectable residual disease. BCL2L11 (BIM) deletion polymorphism may predict relapse after ABL TKI discontinuation, which may have an impact on future ABL TKI discontinuation trials. These results further illustrate the importance of single nucleotide polymorphisms in successful long-term treatment of CML. Disclosures Ohyashiki: Bristol-Myers Squibb KK : Research Funding, Speakers Bureau; Novartis KK: Research Funding, Speakers Bureau.

Abstract 410 Background: ATRA with anthracycline-based chemotherapy markedly improved the prognosis of APL, leading to disease free survival rates 70–80%. But continuous relapse in post-remission period has been a major problem. The role of maintenance therapy for patients in molecular remission after consolidation therapy is uncertain and previous study showed ATRA with or without 6-MP/MTX for 1 to 2 years would be the choice. JALSG APL204 study was intended to decrease the relapse rate in post remission period and applied new synthetic retinoid Tamibarotene (Am80) for maintenance therapy. Am80 is an oral synthetic retinoid with benzoic acid and RARα specific agonist invented in Japan in 1984. High differentiation potential and low affinity to cytoplasmic retinoic acid binding protein may contribute to a superior anti-APL cell effect than ATRA. Early phase II study showed CR rate was 58% for relapsed APL previously treated with ATRA (Blood 90:967–973, 1997). Furthermore Am80 has lower incidence of muco-cutaneous side effects because of no binding affinity to RARγ. Methods: APL204 study was designed as prospective randomized phase III trial comparing Am80 with ATRA in maintenance therapy. Patients (pts) with untreated PML/RARA positive APL between the age of 15 to 69 are eligible. Induction therapy was stratified according to the initial WBC counts: WBC Results: Between April 2004 to December 2010, 347 pts were enrolled and 345 pts were evaluable. Median age was 48 (range 16–68) years old, male : female = 183 : 162 and M3 : M3v = 322 : 23. Median WBC count at diagnosis was 700 (70–127,000)/μl and number of pts in each induction group were A=133, B=56, C=70, D=86 pts. In induction therapy, overall CR rate was 94.5% (326/345) and 95.5%(A), 92.9%(B), 88.6%(C), 98.8%(D) in each group. During consolidation therapy 11 pts (3.4%) were dead, including 9 pts (2.8%) for therapy related toxicity and 2 pts (0.6%) for resistant disease. Two hundred and seventy pts (85.7%) were evaluable for maintenance randomization, 134 pts in Am80 and 136 pts in ATRA. With median follow up of 51 (15–97) months, 5-year RFS was 90.9% (Am80) and 83.2% (ATRA) (P=0.1284), OS 92.4% and 98.1% (P=0.3543) in each group. In group C (WBC ≧10,000/μl), 5-year RFS was 87.7% (Am80) and 59.9% (ATRA) (P=0.0297), it was statistically significant, but in group A and B there was no significant differences. Overall relapse rate was 13.7% (37 pts) after randomization. The most frequent adverse effect of Am80 was a transient hyperglycemia. For all patients in APL204 study, 5-year EFS were 76.6%, RFS 82.9%, OS 87.5%. Conclusions: Maintenance therapy with Am80 has a moderate effect to decrease the relapse rate compared with ATRA but the difference was not statistically significant at 5 years. Both ATRA and Am80 maintenance seem to be effective compared to our previous APL97 study. Notably Am80 is significantly effective in high risk group as WBC ≧10,000/μl (Group C). These results may lead to new strategy for high risk APL. In the future study applying ATO in consolidation therapy and Am80 in maintenance therapy may be a promising strategy to improve the curability and decrease the toxicity of therapy for newly diagnosed APL. This trial was registered at University Hospital Medical Information Network (UMIN)-CTR ID C000000154 in Japan. Disclosures: No relevant conflicts of interest to declare.

We analyzed 23 patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) showing der(1;7)(q10;p10) (hereafter der(1;7)), to identify the exact predictive factor of this cytogenetic change. Eight (34.8%) patients, including 6 MDS and 2 AML patients, had a prior history of genotoxic exposure, especially radiation and/or antimetabolites. Patients with der(1;7) consisted of three groups; one-third of patients had a prior history of genotoxic agents, one-third had additional cytogenetic changes at the time of MDS/AML diagnosis without prior exposure history, and the remaining one-third had neither prior exposure history nor additional cytogenetic changes. The current study demonstrated that the poor outcome of MDS/AML with der(1;7) is due to the high frequency of associated risk factors, i.e., prior history of genotoxic exposure, the presence of additional cytogenetic changes, or both. Identification of prognostic disadvantage might be required for appropriate strategy in managing MDS/AML patients with rare der(1;7) abnormality.