Chugai Pharmaceutical Research Articles

Phase 2 Study of E7777, a Diphtheria Toxin Fragment-Interleukin-2 Fusion Protein, in Japanese Patients with Relapsed or Refractory Peripheral and Cutaneous T-Cell Lymphoma

Phase 2 Study of E7777, a Diphtheria Toxin Fragment-Interleukin-2 Fusion Protein, in Japanese Patients with Relapsed or Refractory Peripheral and Cutaneous T-Cell Lymphoma

Molecular Characterization and Novel Therapeutic Strategy for X-Linked Sideroblastic Anemia Associated with ALAS2 Missense Variants

Background: Congenital sideroblastic anemia (CSA) is an inherited anemia characterized by the presence of ring sideroblasts (RSs) in bone marrow. X-linked sideroblastic anemia (XLSA) is the most common form of CSA and is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Each ALAS2 mutation has a variable effect on the protein's enzymatic function, and only half of XLSA patients are responsive to pyridoxal 5′-phosphate (PLP) treatment. Thus, novel therapeutic strategies should be explored. We have established an in vitro XLSA model in human induced pluripotent stem cell-derived erythroid progenitors (HiDEP-1) by disrupting GATA-1-binding intronic enhancer region of ALAS2, which accounts for a minor XLSA subtype with PLP refractoriness (Saito and Ono et al. MCB 2019; ASH 2018). However, evidence regarding molecular characterization of ALAS2 missense variants is lacking, presumably due to difficulties in introducing the desired missense mutations in primary erythroblasts. In this study, we optimized a protocol for introducing point mutations in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells and subsequently characterized XLSA models harboring ALAS2 missense variants. Methods: Based on homology-directed CRISPR/Cas9 system, we introduced an ALAS2 mutation from arginine at amino acid residue 170, one of the XLSA hot spots, into leucine (R170L) or histidine (R170H). A twenty-nucleotide targeted genomic sequence, located upstream of protospacer adjacent motif (PAM) near ALAS2 R170, was cloned into plasmid vectors expressing Cas9 nuclease and green fluorescent protein (GFP). Each homologous recombinant template was designed as a 127 single-stranded oligodeoxynucleotide (ssODN) asymmetrically distributed from the Cas9-mediated cleavage site. PAM silent mutations were added to avoid continuous cleavage after successful mutation. HUDEP-2 cells were co-transduced with gRNA/Cas9/GFP-expressing vectors and ssODNs using Amaxa Nucleofector 2b (Lonza). After 48 hours, cells were isolated by GFP-positive expression; subsequently, single-cell dilution was performed, followed by clonal analysis. Results: Both XLSA clones (ALAS2 R170L and R170H) appeared pink/pale colored, reflecting impaired hemoglobin biosynthesis. Consistently, quantitative RT-PCR analysis demonstrated decreased globin gene (HBB) expression. On the other hand, XLSA clones did not show RSs and were morphologically similar to wild-type controls. When XLSA clones were induced to undergo erythroid differentiation by co-culturing with OP9 cells in a 100 mM sodium ferrous citrate (SFC)-supplemented medium, increased numbers of RSs were observed in mutant clones, mimicking findings in XLSA patients. Electron microscopy confirmed an aberrant mitochondrial iron deposit in XLSA clones. Also, HBB expression and intracellular heme concentration were significantly lower than those in wild-type controls. Intriguingly, while OP9 co-culture and SFC addition promoted erythroid differentiation in both wild-type and XLSA clones, XLSA clones exhibited a more immature morphological phenotype than wild-type controls. Expression profiling revealed that 317 and 86 genes were commonly up- and downregulated >2-fold in XLSA clones compared with those in wild-type controls. Gene ontology analysis showed significant (p < 0.01) enrichment of genes associated with mitochondrial gene expression and organization, suggesting that mitochondrial alteration is involved in XLSA pathogenesis. On the other hand, 188 genes in total were differentially expressed between ALAS2 R170L and R170H. Genes associated with ferroptosis, such as GPX4 and GCLC, showed a significantly lower expression in ALAS2 R170H clones, possibly accounting for phenotypic differences among patients with ALAS2 R170L or R170H mutation. Finally, supplementation with 5-aminolevulinic acid (ALA) significantly improved the compromised heme biosynthesis in XLSA clones, suggesting ALA treatment to be a promising therapeutic option for XLSA. Conclusion: The established models may act as useful tools for exploring the precise molecular mechanisms of XLSA harboring missense mutations and for drug testing. Also, our homology-directed CRISPR/Cas9-based protocol can be applied to establish a wide variety of CSA as well as congenital anemia models. Disclosures Fukuhara: Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Gilead: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Celgene Corporation: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; AbbVie: Research Funding; Bayer: Research Funding; Solasia Pharma: Research Funding. Onishi:Celgene: Honoraria; Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria.

Phase 2 Study of Tirabrutinib (ONO/GS-4059), a Second-Generation Bruton's Tyrosine Kinase Inhibitor, Monotherapy in Patients with Treatment-Naïve or Relapsed/Refractory Waldenström Macroglobulinemia

BACKGROUND Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma in which bone marrow is infiltrated by immunoglobulin M (IgM)-producing clonal lymphoplasmacytic cells. Tirabrutinib (ONO/GS-4059) is a second-generation Bruton's tyrosine kinase inhibitor with greater selectivity than ibrutinib. We performed a prospective, multicenter phase 2 study of tirabrutinib in patients with treatment-naïve (TN) or relapsed/refractory (R/R) WM. METHODS Patients with TN or R/R WM, serum IgM ≥500 mg/dL, ECOG performance status ≥1, and normal end-organ function were treated with tirabrutinib 480 mg once daily. The primary endpoint was major response rate (MRR, ≥ partial response [PR]) assessed by an independent review committee (IRC) according to the criteria of the VIth International Workshop on Waldenström Macroglobulinemia (IWWM) (Owen RG et al. Br J Haematol. 2013). Secondary endpoints included overall response rate (ORR, ≥ minor response [MR]), time to major response (TTMR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS Twenty seven patients (18 TN and 9 R/R) were treated as of July 3, 2019, and the median follow-up duration was 6 months. The median age of patients was 71 years (range 50-83), and 20 patients (74.1%) had ECOG performance status 0. Median serum IgM was 3600 mg/dL (range 730-6930). Median number of prior therapies among R/R patients was 2 (range 1-7), and 8 patients had received prior rituximab monotherapy or rituximab-containing chemotherapy. IRC-assessed MRR was 77.8% (95%CI: 52.4-93.6) in TN and 88.9% (95%CI: 51.8-99.7) in R/R. IRC-assessed ORR was 94.4% (95%CI: 72.7-99.9) in TN and 100% (95%CI: 66.4-100.0) in R/R. Median TTMR was 1.9 months (range 1.0-5.7) in TN and 2.1 months (range 1.0-3.7) in R/R. Median DOR, PFS, and OS were not reached. The most common adverse events (AEs) at any grade were rash (41%), neutropenia (22%), and leukopenia (15%), of which most were grade 1 or 2. Grade ≥3 AEs were neutropenia (7.4%), leukopenia, lymphopenia, atypical mycobacterial infection, rash erythematous, and erythema multiforme (3.7% each); there was no grade 4 or 5 AE. There were 4 bleeding events and all events were grade 1: mouth hemorrhage, petechiae, anal hemorrhage, and hematoma (3.7% each). Rash-related events occurred in 56% of patients and 2 events were grade 3: erythema multiforme and rash erythematous (3.7% each) which were manageable. CONCLUSION Although the follow-up time was relatively short, the results of this phase 2 study showed that tirabrutinib monotherapy is a highly effective treatment option for patients with TN and R/R WM, with a manageable safety profile. Disclosures Munakata: Ono: Research Funding. Sekiguchi:Ono, A2 Healthcare, Astellas, Janssen, Merck Sharp & Dohme. Otsuka, Pfizer, PPD SNBL, Sumitomo Dainippon Pharma, Daiichi Sankyo Company, Bristol-Myers Squibb: Research Funding. Shinya:Chugai Pharmaceutical Co., Ltd: Membership on an entity's Board of Directors or advisory committees. Suzuki:Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Handa:Ono: Research Funding. Shibayama:Astellas, Teijin, MSD, Shionogi, Eisai, Sumitomo Dainippon, Taiho, Nippon Shinyaku: Research Funding; Takeda, Novartis, Janssen, Chugai, Eisai, Mundi Pharma, Ono, Otsuka, Kyowa Kirin, Sumitomo Dainippon, AstraZeneca, Avvie, DaiichiSankyo, Fujimoto, Nippon Shinyaku, Sanofi, Bristol-Myers Squibb, Pfizer: Honoraria; Celgene, Chugai, Eisai, AstraZeneca: Membership on an entity's Board of Directors or advisory committees. Endo:Ono: Research Funding. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria. Iwaki:Ono: Research Funding. Fukuhara:AbbVie: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Mundi: Honoraria; Celgene Corporation: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Zenyaku: Honoraria; Gilead: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Bayer: Research Funding; Solasia Pharma: Research Funding. Tatetsu:Ono: Research Funding. Iida:Astellas: Research Funding; Abbvie: Research Funding; Gilead: Research Funding; Daichi Sankyo: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Teijin Pharma: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Sanofi: Research Funding; Celgene: Honoraria, Research Funding. Shiibashi:Ono: Employment. Izutsu:Chugai, Celgene, Daiichi Sankyo, Astra Zeneca, Eisai, Symbio, Ono, Bayer, Solasia, Zenyaku, Incyte, Novartis, Sanofi, HUYA Bioscience, MSD, Astellas Amgen, Abbvie, ARIAD, Takeda, Pfizer: Research Funding; Eisai, Symbio, Chugai, Zenyaku: Research Funding; Eisai, Chugai, Zenyaku: Honoraria; Celgene: Consultancy; Kyowa Kirin, Eisai, Takeda, MSD, Chugai, Nihon Medi-physics, Janssen, Ono, Abbvie, Dainihon Sumitomo, Bayer, Astra Zeneca, HUYA Japan, Novartis, Bristol-Byers Squibb, Mundi, Otsuka, Daiichi Sankyo, Astellas, Asahi Kasei: Honoraria. OffLabel Disclosure: Tirabrutinib. Clinical trial for WM/LPL.

Exposure-Response Relationship of the SMART-Ig Anti-hC5 Antibody Crovalimab (SKY59): Results from the Umbrella Phase 1/2 Composer Trial in Healthy Volunteers and PNH Patients

Exposure-Response Relationship of the SMART-Ig Anti-hC5 Antibody Crovalimab (SKY59): Results from the Umbrella Phase 1/2 Composer Trial in Healthy Volunteers and PNH Patients

Prognostic Effects of Pretreatment Statuses at Diagnosis in Patients with Chronic Myeloid Leukemia: Results of the New Target Observational Study 1

Prognostic Effects of Pretreatment Statuses at Diagnosis in Patients with Chronic Myeloid Leukemia: Results of the New Target Observational Study 1

HTLV-1 Dysregulates IL-6 and IL-10-JAK/STAT Signaling and Induces Leukemia/Lymphoma of Mature CD4+ T Cells with Regulatory T-Cell-like Signatures

Human T-cell leukemia virus, type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and chronic inflammatory diseases (e.g., HTLV-1 associated myelopathy, uveitis). Among viral genes encoded in the HTLV-1 provirus, HTLV-1 bZIP factor (HBZ) is the most important gene for its pathogenesis, since HBZ is constantly expressed in all ATL cases, and has a potential to enhance T-cell proliferation. We generated HBZ transgenic (Tg) mice and found that almost all mice developed systemic inflammation, such as dermatitis, and about 40% of mice suffered from T-cell lymphoma. In addition, this mouse demonstrates the similar immunophenotypes to HTLV-1-infected subjects, such as increase of effector/memory and Foxp3-expressing regulatory T cells (Tregs), indicating that it is a good animal model to analyze the molecular mechanisms of pathogenesis by HTLV-1. There is a clear correlation between the severity of inflammation and the incidence of T-cell lymphoma, suggesting that some immune factors are closely involved in oncogenesis by HBZ. Recent studies showed that several pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6), participate in not only inflammation but also tumor development. Especially, IL-6 has been reported to play important roles in oncogenesis, tumor progression, angiogenesis and migration. To clarify the roles of IL-6 in HBZ-mediated lymphomagenesis, we crossed HBZ-Tg mice with IL-6 knockout (KO) mice. Contrary to our hypothesis, inflammation was exacerbated, and incidence of T-cell lymphomas was markedly increased in HBZ-Tg/IL-6KO mice. In the pathological analysis of the lymphoma tissues, the percentage of Foxp3+ cells were much higher in HBZ-Tg/IL-6KO mice than in HBZ-Tg mice. Moreover, flow cytometric analysis also showed that CD4+Foxp3+ T cells were increased in spleens from HBZ-Tg/IL-6KO mice compared with HBZ-Tg mice or WT mice. Since IL-6 inhibits TGF-beta-induced Foxp3 expression and subsequently suppresses differentiation to Tregs, our results suggest that depletion of IL-6 induces aberrant differentiation of CD4+ T cells toward Treg-like cells in HBZ-Tg mice and accelerates lymphomagenesis of this subset. RNA-seq and flow cytometric analyses revealed that expression of IL-10 was significantly higher in CD4+ T cells of HBZ-Tg/IL-6KO than HBZ-Tg. Importantly, IL-10 accelerated the proliferation of CD4+ T cells of HBZ-Tg whereas it did not influence control T cells. We also found that HBZ interacted with both STAT1 and STAT3, which are critical transcription factors in IL-10-JAK/STAT signaling pathways, and enhanced their transcriptional activities. Foxp3-expressing T cells and IL-10 are known to be immune-suppressive, but these results indicate that increased Foxp3-expressing T cells and activation of IL-10-JAK/STAT signaling are associated with inflammation and lymphomagenesis by HBZ in vivo. Our results suggest that IL-6 and IL-10 have suppressive and promoting effects on HBZ-induced pathogenesis, respectively. It has been reported that progression of HTLV-1-associated diseases was observed in several HTLV-1 carriers after administration of anti-IL-6R antibody, Tocilizumab. Inhibition of IL-6/IL-6R signaling and consequent activation of Foxp3/IL-10/JAK/STAT axis seem to be important for HTLV-1 pathogenesis. Disclosures Ohshima: Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Matsuoka:Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Proteomic Analysis of Mitochondrial Membrane Protein FAM210B in Erythroid Cells

(Background) In the context of erythroid differentiation, the importance of transcription factor GATA-1 has been unequivocally demonstrated through cell-based ex vivoassays, knockout mouse models, and rare patients with anemia. GATA-1regulates the expression of erythroid-related genes such as globins, and those involved in heme biosynthesis and an unrecognized function. A novel FAM210B (C20orf108) gene was recently identified as a novel GATA-1 target gene (Kondo et al. Int J Hematol. 2016). FAM210B gene is abundantly expressed in the later stage of erythroid differentiation and encodes a protein containing an N-terminal mitochondrial-targeting sequence, which was considered as a mitochondrial membrane protein. Although the FAM210B protein has been suggested to regulate mitochondrial metabolism (Sun et al. Cell Death Dis. 2017), its detailed function remains to be elucidated. (Method) Endogenous FAM210B protein was deleted by CRISPR/Cas9 based on human iPS-derivederythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2103). To induce erythroid differentiation, HiDEP cells were co-cultured with OP9 stromal cells (ATCC) with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Suzuki et al. MCB2019). Affinity purification of the FAM210B complex was conducted in K562 erythroid cell line (ATCC) stably expressing His/Biotin-tagged FAM210B. MitoXpress Xtra (Agilent) and Human Oligo chip 25K (Toray) were used to evaluate oxygen consumption rate and transcription profiling, respectively. (Results) Clonal lines with HiDEP cells were established, which harbored deletion within coding exon 3 of FAM210B. Quantitative real-time polymerase chain reaction (RT-PCR) analysis confirmed a strong decrease in FAM210B expression. Microarray analysis revealed >1.5-fold up- and down-regulation of 104 and 53 genes caused by the FAM210B knockout, respectively. The down-regulated gene ensemble included TTC19, which is involved in mitochondrial respiratory chain complex III assembly, and genes encoding mitochondrial proteins (AARS2, YWHAE, CLIC1), whereas, the expressions for ALAS2, HBG, HBA, and HMOX1 were not significantly affected by FAM210B depletion. Intriguingly, when wild-type and FAM210B-depleted HiDEP cells were further induced to undergo erythroid differentiation, erythroid differentiation was more pronounced by FAM210B depletion. This was reflected by enhanced nuclear condensation and hemoglobinization, as well as upregulations for ALAS2, HBG, HBA, and HMOX1 in the FAM210B-depleted erythroblasts. Recent report suggests that FAM210B functions as an adaptor protein in the mitochondria (Yien et al. JBC 2018). Thus, FAM210B-interacting protein was purified from K562 cells stably expressing His/Biotin-tagged FAM210B.Mass spectrometry analysis based on the His/Biotin-purified material showed that FAM210B may interact with multiple mitochondrial proteins, including multiple subunits of mitochondrial adenosine triphosphate synthases and mitochondrial heat shock proteins. Further functional analysis showed that oxygen consumption rates might be decreased in FAM210B-depleted HiDEP cells, indicating that FAM210B was possibly involved in mitochondrial energy metabolism in erythroblasts. At present, we are conducting detailed biological analyses to characterize the role of FAM210B during erythroid differentiation. (Conclusion) Further characterization of FAM210B provides new insights in the study of erythroid differentiation, and this may lead to better understanding of the pathophysiology of certain anemias. Disclosures Fukuhara: Mundi: Honoraria; Janssen Pharma: Honoraria; Mochida: Honoraria; Gilead: Research Funding; Bayer: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; AbbVie: Research Funding; Solasia Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria. Onishi:MSD: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Celgene: Honoraria; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Efficacy and Safety of Blinatumomab in Asian Adults with Relapsed/Refractory B-Precursor ALL

Introduction: Blinatumomab is a bispecific T-cell engager (BiTE®) targeted immuno-oncology therapy with dual specificity for cluster of differentiation (CD) 19 and CD3 that redirects the patient's CD3-positive cytotoxic T cells to lyse CD19-positive malignant cells. Global studies have evaluated blinatumomab in patients with advanced Philadelphia chromosome-negative relapsed/refractory acute lymphoblastic leukemia (Ph- R/R ALL). In the global phase 3 TOWER study, blinatumomab monotherapy vs standard-of-care chemotherapy resulted in a significantly higher rate of complete remission (CR)/CR with partial hematologic recovery of peripheral blood counts (CRh)/CR with incomplete hematologic recovery of peripheral blood counts (44% vs 25%; P < 0.001) and longer median overall survival (7.7 vs 4.0 months; P = 0.01) (Kantarjian H, et al. NEJM. 2017;376:836-47). There are limited data on the efficacy and safety of blinatumomab in Asian patients, whose immunologic genetic background may differ from other patient populations. Therefore, we conducted a patient-level pooled analysis of the efficacy and safety of blinatumomab in 45 Asian adult patients with Ph- R/R ALL-19 from the blinatumomab arm of TOWER (NCT02013167) and 26 from a phase 1b/2 study in Japanese adults (NCT02412306). Methods: Patients in both studies were ≥ 18 years old and had Ph- R/R ALL, > 5% blasts, Eastern Cooperative Oncology Group performance status 0-2, and no central nervous system pathology. Patients received a maximum of 2 cycles of induction blinatumomab for 4 weeks by continuous intravenous infusion (cycle 1/week 1: 9 μg/day; cycle 1/weeks 2-4: 28 μg/day; subsequent cycles: 28 μg/day) followed by 2 weeks of no blinatumomab (each 6-week cycle). Responders (≤ 5% bone marrow blasts within 2 induction cycles) received blinatumomab 28 μg/day up to a maximum of 5 induction/consolidation cycles. In TOWER, patients who continued morphologic remission received up to 12 months of maintenance therapy. Patients could undergo stem cell transplantation at any time following the first treatment cycle. Results: Of the 45 Asian patients enrolled (26 female; median [range] age, 43 [18-75] years; prior hematopoietic stem cell transplantation, 20 [44.4%]; ≥ 1 prior salvage therapy, 30 [66.7%]), 44 received at least 1 cycle of blinatumomab 9-28 μg/day. Responses in the first 12 weeks of treatment (CR/CRh and minimal residual disease response) are shown in the Table. The Kaplan-Meier (KM) median overall survival time was 11.9 (95% CI: 9.9-17.1) months, and the KM median relapse-free survival time was 8.9 (95% CI: 3.8-10.7) months; median overall survival in the blinatumomab arm of TOWER was 7.7 months. Forty-one (93.2%) patients had grade ≥ 3 treatment-emergent adverse events (TEAEs), and 5 (11.4%) had fatal AEs. Grade ≥ 3 events TEAEs of interest included neurologic events (4.5%), cytokine release syndrome (2.3%), cytopenias (6.8%), and infections (20.5%). Conclusions: The safety and efficacy of blinatumomab in Asian patients were comparable with previous global studies with similar disease response rates and a favorable safety profile with no new safety signals. Disclosures Kobayashi: Astellas Amgen BioPharma: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; SymBio: Consultancy. Iida:Chugai Pharmaceutical Co., Ltd.: Research Funding. Minami:Astellas: Research Funding; Bayer: Honoraria, Other: Clinical trial, Research Funding; Taiho: Honoraria, Other: Clinical trial, Research Funding; Taisho-Toyama: Research Funding; Takeda: Honoraria, Research Funding; CSL Behring: Research Funding; Genomic Health: Honoraria; Daiichi Sankyo: Other: Clinical trial, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Eizai: Honoraria, Research Funding; Janssen: Honoraria; Kowa: Honoraria; Kyowa-Kirin: Honoraria, Research Funding; Nihon Shinyaku: Research Funding; Eli Lilly: Honoraria, Research Funding; Merck Serono: Honoraria, Research Funding; MSD: Honoraria, Other: Clinical trial, Research Funding; Nippon Chemiphar: Honoraria, Research Funding; Ono Yakuhin: Honoraria, Other: Clinical trial, Research Funding; BMS: Honoraria, Other: Clinical trial, Research Funding; Celgene: Honoraria; AstraZeneca: Other: Clinical trial; Boehringer: Honoraria, Research Funding; Otsuka: Honoraria; Pfizer: Honoraria, Other: Clinical trial, Research Funding; Sanofi: Honoraria, Research Funding; Shire Japan: Honoraria; Abbvie: Honoraria; Nihon Medi-Physics: Honoraria; Asahi-Kaseo Pharma: Research Funding; Amgen Inc: Other: Clinical trial; Nihon Kayaku: Research Funding; Shionogi: Research Funding; Novartis: Honoraria, Other: Clinical trial; Chugai: Honoraria, Other: Clinical trial, Research Funding; Yakult: Research Funding; Teijin Pharma: Research Funding. Maeda:Mundipharma Co Ltd.: Honoraria; Kyowa Kirin Co. Ltd.: Honoraria; Astellas Pharma Inc.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Yoon:Novartis: Consultancy, Honoraria; Yuhan Pharma: Research Funding; MSD: Consultancy; Kyowa Hako Kirin: Research Funding; Genentech, Inc.: Research Funding; Janssen: Consultancy; Amgen: Consultancy, Honoraria. Tran:Amgen: Employment, Equity Ownership. Morris:Amgen: Employment, Equity Ownership. Franklin:Amgen: Employment, Equity Ownership. Chong:Amgen Asia Holding Limited: Employment, Equity Ownership. Kiyoi:Astellas Pharma Inc.: Honoraria, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; FUJIFILM Corporation: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Perseus Proteomics Inc.: Research Funding.

Phase 2 Study of Bosutinib in Japanese Patients with Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

Phase 2 Study of Bosutinib in Japanese Patients with Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

Safety of Mogamulizumab in Mycosis Fungoides and Sézary Syndrome: Final Results from the Phase 3 Mavoric Study

Safety of Mogamulizumab in Mycosis Fungoides and Sézary Syndrome: Final Results from the Phase 3 Mavoric Study

Integrated Analysis of Copy-Number Alterations and Gene Mutations in 2,000 Patients with Myeloid Neoplasms

Integrated Analysis of Copy-Number Alterations and Gene Mutations in 2,000 Patients with Myeloid Neoplasms

High Natural Killer Cell Count at Diagnosis Predicts a Good Response to Immunosuppressive Therapy in Aplastic Anemia

Introduction: Aplastic anemia (AA) is an autoimmune-mediated disease with a complex mechanism. Innate as well as acquired immune system have been considered to participate in its pathogenesis. Although several studies have demonstrated the role of T and B cells in AA, there is limited research focusing on natural killer (NK) cells, which are important in innate and adaptive immune system. NK cells interact with T cells and alter their immune responses in a variety of diseases. Recent investigation suggests that NK cells serve immunoregulatory roles by attenuating autologous CD8+ T-cell response in AA (Chen et al. Cell Immunol 335:6-14, 2019). Here we assessed the kinetics of NK cells before and after immunosuppressive therapy (IST) in patients with AA to determine the association between NK cells and clinical courses. Methods: Patients with severe AA, in Japan, who required IST as first-line therapy were prospectively enrolled between May 2012 and October 2017. The IST regimen comprised rabbit anti-thymocyte globulin (ATG, thymoglobulin®, 2.5 or 3.5 mg/kg/day for 5 days), cyclosporine A (6 mg/kg/day for minimum 6 months), and methylprednisolone (2 mg/kg/day for 5 days) with subsequent tapering of the dose for 28 days. Flow cytometry was used to assess lymphocyte subsets, consisting of CD3+, CD4+, and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD4+CD25+CD127+ regulatory T cells (Tregs), before and after ATG administration on days 0, 14, 28, 60, 90, 120, and 180. Plasma rabbit ATG levels were measured using a rabbit IgG ELISA kit on days 14 and 28 (Bethyl Laboratories, Montgomery, TX, USA). Receiver-operator characteristic (ROC) curves were generated to differentiate between response and no response to IST. Results: A total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-ATG, respectively. Median follow-up duration was 445 days (range; 183-2165 days) from first ATG infusion. After 6 months, 50 patients (58%) responded to the therapy, including 4 (5%) who achieved a complete response and 45 (56%) a partial response. The response rates did not differ significantly between the two dosages in ATG groups at 6 months (2.5 mg/kg, 63% vs. 3.5 mg/kg, 58%, P = 0.820). The median absolute number of NK cells before IST was 92/µL (0-1,085/µL). To judge patients' responses to IST, we defined 138/µL as the absolute number of NK cells count before IST as the cut-off value using ROC curves. The response rate of patients with higher NK cells (≥ 138 /mL) to IST at 6 months was significantly higher than those with lower NK cells (Figure 1, < 138 /mL, 78% vs. 52%, P = 0.031). Multivariate logistic regression analysis identified the absolute number of NK cells before IST and reticulocyte at diagnosis (odds ratio [OR] = 0.16; 95% confidence interval [CI], 0.04-0.62; P = 0.008 and OR = 0.20; 95% CI, 0.06-0.67; P = 0.009, respectively) and the r-ATG plasma concentration on day 28 (OR = 0.11; 95% CI, 0.03-0.39; P = 0.001) as independent predictors of response to IST. The median absolute number of NK cells on days 90, 120, and 180 after IST was higher in the responders (113/mL, 118/mL, and 159/mL, respectively) than in the non-responders (57/mL, 53/mL, and 64/mL, P = 0.001, P<0.001, P<0.001, respectively). On the other hand, the recoveries in the number of CD3+, CD4+, CD8+, and Tregs cells were similar in both groups (Figure 2). Three relapses (among 21 responders) in higher NK cells group, and four relapses (among 28 responders) in lower NK cells group were observed. No patients developed clonal evolution in both groups. Two patients in the lower NK cell group died after IST. The etiologies of death included infection and unknown causes. Transplantation-free survival (TFS), failure-free survival (FFS), and overall survival (OS) at two years were similar between the higher and lower NK cell groups [TFS, 90% (95% CI, 64%−97%) vs. 78% (95% CI, 63%−87%); P = 0.147; FFS, 66% (95% CI, 45%−81%) vs. 50% (95% CI, 35%−62%); P = 0.147; OS, 100% (95% CI, 100%−100%) vs. 94% (95% CI, 76%−98%); P = 0.277]. Conclusions: The current study indicates that NK cells play a role in the pathogenesis of AA and may be the predictors of response to IST. Recovery of NK cell count correlated with hematopoietic recovery following IST, indicating that they may be used as markers to assess disease activity, treatment response and relapse. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

One and Half Million Hematopoietic Stem Cell Transplants (HSCT). Dissemination, Trends and Potential to Improve Activity By Telemedicine from the Worldwide Network for Blood and Marrow Transplantation (WBMT)

HSCT is the only curative option for many malignant and non-malignant diseases. The WBMT was founded as an umbrella organization of societies involved in cellular therapies with the mission of promoting excellence in HSCT. As a non-governmental organization in official relation with the World Health Organization (WHO), the WBMT assisted in the founding of regional societies (Latin America Blood and Marrow Transplantation Group and African Blood and Marrow Transplantation Group), performs global activity surveys, conducts workshops and provides expert support for programs in evolving countries. In this retrospective evaluation we analyzed worldwide activity trends in HSCT up to the year 2016 and evaluated possibilities of improving availability of HSCT by the use of telemedicine. Methods: HSCT activity was collected annually from member societies, national registries and individual centers including donor type (allogeneic/autologous), stem cell source (bone marrow/peripheral blood stem cells/cord blood) and indications for transplant. Transplant rates (TR) were calculated as HSCT/10 million inhabitants without adjustment for patients transplanted in a country other than that of primary residence. Country team density (TD) was defined as teams/10 million inhabitants. Workshops were organized in a number of locations where there was little or no HSCT activity or where improvement in one or more aspect of local or regional HSCT activity was requested, including Vietnam, Brazil, China, South Africa and Morocco. Other countries were paired with established centers using WBMT affiliated partners. In two countries, a pilot program was established involving a 6 month physician training in a JACIE/FACT accredited center followed by daily telemedicine-guided supervision of clinical activities. Results: From 1957-2016 a total of 1,298,897 HSCT (57.1% autologous) procedures were collected. By the end of 2016, HSCT activity was reported from 87 of the 195 WHO member states. A total of 89,070 HSCT from 1662 centers was reported in 2016. Assuming a frequency of 84,000/year, 1.5 million HSCT will be reached in 2019, only 7 years after the 1 million report in 2012 (Figure 1). The global activity/year increased continuously from 10,000/year in 1991 to 82,718 first HSCT/year in 2016 with a global increase of >7% (7.0% in autologous and 7.8% in allogeneic HSCT). As in previous years, slightly more autologous (53.5%) than allogeneic and more related (53,6%) than unrelated HSCT were reported. The further increase in related HSCT was caused mainly by an increase of non-identical family donors (39.5% of related HSCT). Increase in activities according to regions is given in Figure 2. TR and TD varied according to region and are highest in Nord America with 511.2 TR, followed by Europe with 390.9 TR, Latin America with 63.9 TR, APBMT with 46.2 TR and Africa/EMRO with 32.8 TR. In contrast, TD was highest in Europe (7.5 TD) followed by Nord America (6.0 TD), APBMT (1.9 TD), Latin America (1.9 TD) and Africa/EMRO (0.4 TD). Commonest indications were lymphoproliferative diseases for autologous and leukemia for allogeneic HSCT and continue to rise (Figure 3 autologous and Figure 4 allogeneic HSCT). Graft source were predominantly peripheral blood in autologous (99.7%) and 65% in unrelated HSCT, while umbilical cord blood as a stem cell source (13.8% of all unrelated) declined. More than 150 HSCT were performed in one country and one center without activities using daily telemedicine-guided supervision. In conclusion, the global distribution and activities are increasing continuously by more than 7,0% per year with numbers currently running at app. 90,000/year. Of note is the increase of haploidentical HSCT activity, while the use of umbilical cord blood HSCT continues to decrease. TR data show significant gaps between regions. Supervisory telemedicine is a powerful tool to overcome lack of experience and establish JACIE/FACT compatible new programs with collateral benefits for conventional hematology, blood banking, microbiology and virology. Abbreviations: EUR, Europe; AMR/PHA, America; SEAR/WPR, South-East Asia/Western Pacific; AFR/EMRO, African/Eastern Mediterranean. Disclosures Niederwieser: Daichii: Speakers Bureau; Cellectis: Consultancy. Atsuta:Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Janssen Paharmaceutical K.K.: Honoraria. Worel:Sanofi Genzyme, Malinckrodt Therakos: Research Funding; Jazz, Sanofi, Celgene, Novartis, Malinckrodt Therakos: Honoraria; Sanofi Genzyme, Malinckrodt Therakos: Speakers Bureau. Galeano:Szabo SA: Other: (Equity interest). Novitzky:Astellas, Roche: Consultancy. Szer:Prevail Therapeutics: Honoraria, Other: Travel, Research Funding; Novartis: Honoraria, Other: Travel, Research Funding; MSD: Honoraria, Other: Travel, Research Funding; Celgene: Honoraria, Other: Travel, Research Funding; Amgen: Honoraria, Other: Travel, Research Funding; Alexion: Honoraria, Other: Travel, Research Funding; Sanofi: Honoraria, Other: Travel, Research Funding; Takeda: Honoraria, Other: Travel, Research Funding; Pfizer: Honoraria, Other: Travel, Research Funding. Kröger:Celgene: Honoraria, Research Funding; DKMS: Research Funding; JAZZ: Honoraria; Medac: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Riemser: Research Funding; Sanofi-Aventis: Research Funding. Weisdorf:Incyte: Research Funding; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy. Pasquini:Amgen: Consultancy; BMS: Research Funding; Medigene: Consultancy; Pfizer: Other: Advisory Board; Novartis: Research Funding; Kit Pharma: Research Funding.

The Global State of Hematopoietic Stem Cell Transplantation for Multiple Myeloma: An Analysis of the Worldwide Network of Blood and Marrow Transplantation (WBMT) Database and the Global Burden of Disease Study

Background: Multiple myeloma (MM), is a clonal plasma cell neoplasm characterized by destructive bony lesions, anemia, and renal impairment. MM is a global disease - worldwide in 2016, there were 138509 incident cases with an age standardized incidence rate (ASIR) of 2.1 per 100 000 persons, with a 126% global increase in incident cases from 1990 to 2016 (Cowan AJ et al JAMA Oncology 2018). Access to effective care, including proteasome inhibitors, immunomodulatory agents, and autologous hematopoietic stem cell transplantation (HSCT) is largely limited to high-income sociodemographic index (SDI) countries. SCT remains the standard of care for eligible patients, and in general is more affordable and accessible worldwide than novel therapies. We sought to evaluate the rates and utilization of ASCT globally from 2006-2015 to better characterize access to SCT for patients with MM. Methods: This was a new analysis of a retrospective survey of WBMT sites, conducted annually between 2006-2015, as described previously (Niederwieser et al BMT 2016). Incidence data estimates were reported from the Global Burden of Disease study (Institute for Health Metrics and Evaluation. 2019 'GBD Results Tool.' Global Health Data Exchange. Seattle WA: University of Washington. Accessed 1 June 2019). South Asia and East Asia regions were combined for this analysis. Outcome measures included total number of autologous and allogeneic stem cell transplants by World Bank (WB) regions, and percentage of newly diagnosed MM patients who underwent ASCT, calculated by the number of transplants per region in calendar year / gross annual incidence of MM per region. Results: From 2006 to 2015, the number of autologous HSCT performed worldwide for MM increased by 107% (Figure 1). Activity increased in each region from 2006 to 2015 from 56% in USA and Canada to 335% in Latin America. Utilization of autologous HSCT was highest amongst the Northern America and European WB regions, with an increase from 13% to 24% in Northern America, and an increase from 15% to 22% in Europe. The activity increased considerably in the Latin American countries (335,46% increase) and the utilization reached >10%. In contrast, the utilization of autologous HSCT was much lower in the Africa/Mediterranean and Asian/Pacific region, with autologous HSCT utilization only changing marginally from 1.8% in 2006 to 4% in 2015 despite increase in activity. The number of first allogeneic HSCT performed globally for MM declined after a peak in 2012 by -3% since 2006 mostly in North America. Allogeneic HSCT remains highest amongst the European WB region (increase 8%). The increase in activity was accompanied by an increase in team numbers from 1327 in 2006 to 1581 in 2015 but also by an increase of activity in the teams. Discussion: Autologous HSCT utilization has increased worldwide in high-income SDI WB region countries for MM yet has not increased proportionally amongst low-middle income WB regions. There is a disparity in autologous HSCT utilization amongst high-income regions, exceeding 20% in North America and Europe, while remaining poorly utilized in Africa and the East Mediterranean. Latin America has increased their utilization and is for the first time above 10%. However, we are limited with respect to use of incidence data in LMIC countries from the GBD, likely due to under reporting. Conflicting clinical trial data likely contributed to the decline in some regions for first allogeneic HSCT in MM. More work is needed to improve access to transplantation services for MM patients, especially in low to middle income countries. Conclusion: Although autologous HSCT numbers and rates have increased globally, there are marked disparities in usage amongst high versus low to middle income countries. More work is needed to improve access to HSCT for MM globally. Figure 1 Disclosures Cowan: Celgene: Consultancy, Research Funding; Cellectar: Consultancy; Juno: Research Funding; Sanofi: Consultancy; Abbvie: Research Funding; Janssen: Consultancy, Research Funding. Atsuta:Janssen Paharmaceutical K.K.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria. Worel:Sanofi Genzyme, Malinckrodt Therakos: Research Funding; Jazz, Sanofi, Celgene, Novartis, Malinckrodt Therakos: Honoraria; Sanofi Genzyme, Malinckrodt Therakos: Speakers Bureau. Libby:Alnylam: Consultancy; Abbvie: Consultancy; Pharmacyclics and Janssen: Consultancy; Akcea: Consultancy. Pasquini:Novartis: Research Funding; Kite Pharmaceuticals: Research Funding; BMS: Research Funding; Medigene: Consultancy; Amgen: Consultancy; Pfizer: Consultancy. Galeano:Szabo SA: Other: (Equity interest). Szer:Amgen: Honoraria, Other: Travel, Research Funding; Alexion: Honoraria, Other: Travel, Research Funding; Pfizer: Honoraria, Other: Travel, Research Funding; Sanofi: Honoraria, Other: Travel, Research Funding; Takeda: Honoraria, Other: Travel, Research Funding; Prevail Therapeutics: Honoraria, Other: Travel, Research Funding; Novartis: Honoraria, Other: Travel, Research Funding; MSD: Honoraria, Other: Travel, Research Funding; Celgene: Honoraria, Other: Travel, Research Funding. Kroeger:Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Riemser: Research Funding; JAZZ: Honoraria; Sanofi-Aventis: Honoraria; Novartis: Honoraria, Research Funding; Medac: Honoraria; DKMS: Research Funding. Weisdorf:Fate Therapeutics: Consultancy; Incyte: Research Funding; Pharmacyclics: Consultancy. Niederwieser:Cellectis: Consultancy; Daichii: Speakers Bureau.

Establishment of a Predictive Model of GvHD-Free, Relapse-Free Survival after Allogeneic Hematopoietic Stem Cell Transplantation Using a Machine Learning Algorithm

Background: GvHD-free, relapse-free survival (GRFS) is a useful composite endpoint that measures survival without relapse or significant morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aimed to establish a predictive model of GRFS after allo-HSCT, using a machine learning algorithm within the Kyoto Stem Cell Transplantation Group (KSCTG), a multi-institutional joint research group of 17 transplantation centers in Japan. Methods: Consecutive patients with hematologic diseases who had undergone their first allo-HSCT between 1986 and 2016, were included in the study (n = 2,565). The primary endpoint was GRFS, and the secondary endpoints were: overall survival (OS), relapse, non-relapse mortality, and grades II-IV acute GvHD. Data were randomly split into the training set (70% of the dataset) and the validation set (30% of the dataset). The model was trained and tested using a 5-fold cross validation on the training set. A stacking ensemble of multiple machine learning algorithms was applied to develop a model. Predictions were first generated using different machine learning algorithms such as AdaBoost, Random Survival Forest, and Dynamic-DeepHit. A metamodel was subsequently trained using these predictions as inputs, to generate the final prediction (Figure 1). All models were trained using 28 input variables containing information on the patient and transplant characteristics. We used Cox Proportional-Hazards (Cox-PH) model as the benchmark case and evaluated the models' performance using the Harrell's Concordance Index (C-index). The model's final prediction can be directly used as a risk score for each patient. We validated this risk score in the validation set using the competing risk hazard model. Due to the small size of the validation set (N=770), we only classified the final risk scores into 2 groups as follows: high risk (above median) and low risk (below median) for each risk category. Results: The median age of the patients at transplantation was 48 years (range from 7 to 76). The sources of stem cells were related bone marrow (BM) in 650 patients, related peripheral blood (PB) in 382, unrelated BM in 978, unrelated PB in 24, and cord blood in 522. Myeloablative conditioning was administered in 1,293 patients and reduced-intensity conditioning in 949 patients. Refined disease risk indexes were low in 310 patients, intermediate in 1,197 patients, high in 545 patients, and very high in 368 patients. Median follow-up period of survivors was 4.5 years. For GRFS, our model achieved a higher C-index across all risk categories (C-index: 0.631) than other top-of-the-art competing risk models (Cox-PH: 0.620, Random Survival Forest: 0.627, Gradient Boosting: 0.609, Component-wise Gradient Boosting: 0.599, Dynamic DeepHit: 0.603). The model also showed the highest C-index for overall survival (C-index: 0.653), relapse (C-index: 0.703), non-relapse mortality (C-index: 0.636), and grades II-IV acute GvHD (C-index: 0.594). For GRFS, the probability after 1 year and 2 year was 24.9% and 15.2% for the high risk and 48.0% and 33.6% for the low risk, respectively (hazard ratio [HR] compared to the low risk: 1.88; 95% CI: 1.60-2.20) (Figure 2). The OS at 5 years was 43.4% for the high risk and 70.6% for the low risk (hazard ratio [HR] compared to the low risk: 2.41; 95% CI: 1.96-2.97). The cumulative incidence function (CIF) for non-relapse mortality at 5 years was 32.1% for the high risk and 21.4% for the low risk (HR 2.01; 95% CI: 1.38-2.91). The CIF for relapse at 5 years was 37.1% for the high risk and 16.4% for the low risk (HR 1.67; 95% CI: 1.25-2.23). Conclusions: A predictive model of GRFS after allo-HSCT was established, which could benefit physicians and patients during the clinical decision-making process. This also forms the basis for research on new prognostic biomarkers by evaluating their predictive power based on our model. Disclosures Kanda: MSD: Honoraria; Takeda: Honoraria; Bristol-Meyers Squib: Honoraria; Otsuka: Honoraria; Daiichi Sankyo Company: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Astellas: Honoraria; Chugai: Honoraria; Kyowa Hakko Kirin: Honoraria; JCR Pharmaceuticals: Honoraria; NextGeM Incorporation: Patents & Royalties: 2019-011392. Imada:Bristol-Meyer Squibb K.K.: Honoraria; Celgene K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinyaku Co.,Ltd.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria; Novartis Pharma K.K.: Honoraria; Astellas Pharma Inc.: Honoraria. Takaori-Kondo:Takeda: Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria.

Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

Optimum Imatinib Exposure Have Possibility of Leading to Appropriate Immune Response after Imatinib Discontinuation in CML Patients

Introduction: Imatinib, the first tyrosine kinase inhibitor (TKI), has dramatically improved the prognosis of chronic myeloid leukemia (CML) patients. Recently, many trials of TKI discontinuation revealed that approximately 40% to 60% of CML patients who treated long time TKI therapy reached the treatment free remission (TFR), thus now TFR is proposed as one of the goals in CML treatment. Achieving deep molecular response (DMR) by TKI therapy is a minimum requirement of challenge to TKI discontinuation in CML patient, actually CML patients with molecular residual disease (MRD) showed worse consequence than undetectable MRD (IJH 2017). On the other hand, it was known that some patients have continued TFR with detectable BCR-ABL fusion gene, these patients hadn't shown indubitable molecular relapse while BCR-ABL+ malignant cells continued to exist for prolonged time. We hypothesized that the malignant cells were eliminated by host immune systems in these fluctuated patients. Here, we focused on T-cell response, so we analyzed T-cell related markers to identify biomarkers that can predict patients which can continue TFR or not in Japanese CML patients. Furthermore, we confirmed the action of imatinib for T-cell response in vitro. Methods: Japanese CML patients treated with imatinib for at least three years and confirmed in DMR for at least two years were eligible. Patients who received other TKI or stem cell transplantations were excluded. Patients were re-confirmed in MR4.5 before discontinue imatinib and they were sampled peripheral blood at pre- and 1, 3 months after stopping imatinib (figure 1). Peripheral blood mononuclear cells (PBMCs) were subjected to staining with T-cell markers and analyzed by mass cytometry and flowcytometry. Plasma were subjected to detecting Imatinib trough concentrations. Purchased PBMCs of healthy individuals were cultured and analyzed by flowcytometry in vitro assay. Results: Samples of 68 CML patients were analyzed. We classified these CML patients into two groups (Non-retreatment and Retreatment groups) by clinical courses after stopping imatinib (figure 2). Frequency of CD4+ T cells and CD8+ T cells in CD3+ T cells were no difference between both groups. FoxP3+CD4+ regulatory T cells (Treg) were also no difference between both groups, but kinetics of Treg, especially Fraction II (Fr.II : FoxP3hiCD45RA-) of Treg from Pre-stopping imatinib to 1 month after stopping imatinib significantly increased in non-retreatment groups. Kinetics of Treg / CD8+ T cells ratio also significantly increased in non-retreatment groups, and predicted curve made by these kinetics of each groups were significant (figure 3). The expression of PD-1 or other suppressive co-stimulatory molecules in CD8+ T cells of non-retreatment groups at after stopping imatinib had tendency to decrease. Phosphorylated LCK in CD8+ T cells of non-retreatment groups at after stopping imatinib had tendency to increase. Next, we did in vitro assay to confirm the effect of pre-treatment of imatinib in imatinib free T cells. Pre-treatment of imatinib suppressed the proliferations of Treg Fr.II after TCR stimulation dose dependently, but not CD8+ T cells (figure 4). Frequency of phosphorylated LCK in Treg Fr.II increased after TCR stimulation even if pre-treated imatinib at reasonable dose, but didn't increased under the condition of high dose imatinib. Conclusion: Treg population and Treg / CD8+ T cells ratio in PBMCs elevated after stopping imatinib in non-retreatment groups of CML patients. Population of CD8+ T cells showed no differences in two groups but CD8+ T cells were tending to activate after stopping imatinib in non-retreatment groups. These data indicate that the kinetics of Treg after stopping imatinib connect with the immune response of imatinib discontinued CML patients. In vitro data indicate that Treg were more sensitive for imatinib treatment than CD8+ T cells, so kinetics of Treg may possibly become the biomarker of ability of immune responses. Our data suggested that optimum imatinib exposure induce appropriate immune responses leading good prognosis, and excess imatinib exposure induce exhaust immune responses leading poor prognosis. Disclosures Nishikawa: Taihou Pharmaceuticals: Research Funding; Kyowa Hakko Kirin: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Ono Pharmaceutical: Research Funding, Speakers Bureau; Chugai Pharmaceuticals: Research Funding, Speakers Bureau; Asahikasei Pharma: Research Funding; Sysmex: Research Funding; Daiichi Sankyo: Research Funding; Zennyaku: Research Funding. Takahashi:Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Chug Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Asahi Kasei Pharma: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Eisai Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding.

MRD Detection in Myeloma Cells: Comparison between Inexpensive 1-Tube 10 Color Multiparameter Flow Cytometry and Euroflow Multiparameter Flow Cytometry

Background: Achievement of MRD-negative status in MM patients has been demonstrated to be associated with better outcome. EuroFlow MM-MRD has been a global standard for MRD detection by MFC. In Japan, EuroFlow is limited to research use only, because the dynamics of the population have raised concerns about increasing health care expenditure in Japan. Therefore, it is desirable to develop an inexpensive FCM method with equivalent sensitivity to EuroFlow for detecting MRD. Aims: In this study, we analyzed the number and percentage of total plasma cells (PC), abnormal PC with EuroFlow MM-MRD and 10-color-MFC in 50 MM patients. Patients and Methods: Bone marrow samples collected from 50 MM patients were subjected to MRD detection by EuroFlow MM-MRD and 10-color-MFC simultaneously. EuroFlow MM-MRD was performed according to EuroFlow standard operating procedure. 10-color MFC was performed at BML corporation. Briefly, CD38 multiepitope FITC, CD138 V450, CD45 V500-C, CD56 PE, CD19 APC-H7, CD27 APC R700, CD81 BV605, CD117 PE-Cy7, cIgk APC and cIgl PerCP-Cy5.5 of monoclonal antibody were used according to the ICCS(International Clinical Cytometry Society) guidelines. Data were analyzed with FACSuite ver 1.2.1(Becton Dickinson) using a FACSLylic(Becton Dickinson). Wilcoxon signed-rank test was used for the comparison of paired variables. Results: The representative example of 10-color-MFC were shown in figure 1. We first compared the percentage of normal plasma cells in bone marrow. The percentage of normal plasma cells analyzed with 10-color-MFC and EuroFlow were 0.287% (range 0.001-1.0322%) and 0.257%(range0.0002-1.37%), respectively. The good correlation between them was observed (r=0.509). The percentage of myeloma cells determined with 10-color-MFC and EuroFlow were 0.0705%(range 0-0.9086%) and 0.0940% (range 0-1.3000%), respectively. Quantification of abnormal plasma cells were highly similar between 2 tests (r=0.977, figure 2). To compare the percentage of abnormal plasma cells analyzed with 10-color-MFC and EuroFlow, p-value determined with Wilcoxon signed-rank test was 0.0069 in 50 sample. However, when total bone marrow cells were 3 million cells, limit of detection (LOD) and limit of quantification (LOQ) in 10-color MFC were defined as 0.001% and 0.0017%, respectively. The correlation in samples with less than 3 million of bone marrow cells was not good (p=0.424). Only when 3 million or higher number of bone marrow cells were analyzed, better correlation is thought to be observed (p=0.0001). High concordant analytical results were observed with an overall qualitative concordance of 95%. Conclusion: Our 10-color-MFC demonstrated high sensitivity to detect MRD with good correlation and also inexpensive (about 100 dollars as of July 2019). However, 3 million or higher number of bone marrow cells is inevitable to demonstrate good correlation. In the country where high medical expenditure is not acceptable, our inexpensive MFC is considered as good alternative methods to detect MRD in MM patients. Disclosures Tsukada: Takeda Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Sanofi: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Fujimoto Pharmaceutical: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Asahi Kasei Pharma Corporation: Honoraria; MOCHIDA PHARMACEUTICAL CO., LTD.: Honoraria. Suzuki:Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

Genomic Landscape of Young ATLL Patients Identifies Frequent Targetable CD28 Fusions

Introduction Adult T-cell leukemia/lymphoma (ATLL) is a T-cell neoplasm induced by human T-cell leukemia virus type 1 (HTLV-1). In endemic areas, HTLV-1 infection typically occurs during breastfeeding but the median age of ATLL presentation in Japan is >70. Approximately 5% of ATLL patients in Japan present at age <50. Here, we hypothesized that ATLL in younger patients will have distinct genetic alterations. Methods DNA and RNA samples were extracted from frozen lymph nodes. Targeted capture sequencing for 98 genes and analysis of RNA sequencing (RNAseq) were performed in 8 samples of young patients with ATL (defined as ≤ 50 years in the current study) Results and discussion The most frequently mutated genes were CCR4 and CARD11, each in 3 of the analyzed cases (37.5%). Mutations of PLCG1, PRKCB and STAT3 were frequent in a previous genomic analysis (Kataoka et al. Nat Genet. 2015) that were not age-selected, but were not identified in our 8 cases. Three (37.5%) cases harbored concurrent CTLA4-CD28 and ICOS-CD28 fusions. This contrasts with previous reports, in which cases with both fusions were found in <1% of PTCL and ATLL patients. We confirmed the presence of both fusions in all 3 cases by RT-PCR. The structure of the CTLA4-CD28 fusion suggests that the extracellular portion of CTLA4 is expressed on the cell surface where it can interact with CD80 and CD86 to activate signaling through the intracellular CD28 portion. In contrast to CTLA4-CD28, the ICOS-CD28 fusion links the N-terminal signal peptide of ICOS with the extracellular and intracellular portions of CD28. This fusion should simply result in overexpression of CD28 and haploinsufficiency of ICOS. To evaluate the function of CTLA4-CD28, we used Ba/F3 cells, which are strictly dependent on exogenous IL-3 for proliferation. We transduced Ba/F3 cells with CTLA4-CD28 or a CTLA4-CD28 mutant (mut) with three amino acid substitutions that abrogate CD28 signaling. We then cultured these cells in the presence or absence of Raji cells, which express CD80 and CD86. In the absence of Raji cells, neither CTLA4-CD28 nor CTLA4-CD28mut expression conferred IL-3-independent growth. However, Ba/F3 cells expressing CTLA4-CD28 cultured in the presence of Raji cells achieved IL-3 independence. This was not the case for Ba/F3 cells expressing CTLA4-CD28mut, indicating a requirement on functional CTLA4-CD28 signaling. Western-blot analysis identified activation of CD28 signal in Ba/F3 cells with CTLA4-CD28 co-cultured with Raji cells. These data indicate that ligand-bound CTLA4-CD28 can promote downstream signaling in the absence of an endogenous TCR complex. Gene set enrichment analysis (GSEA) comparing the 3 ATLL cases with CD28 fusions to the 5 cases that lacked fusions demonstrated enrichment of previously defined gene signatures associated with AKT and RAF signaling, both of which are downstream of CD28 activation. GSEA also identified enrichments of T-cell function-related signatures, including pathways involved in interferon responses, in cases with CD28 fusions Cases with CTLA4-CD28 and ICOS-CD28 fusions become haploinsufficient for CTLA4 and ICOS. As expected, these cases had lower expression of both genes by RNAseq. Interestingly, these cases also had higher expression of CD80. By immunohistochemistry, ATLL cells with CTLA4-CD28 and ICOS-CD28 fusions expressed CD80 and macrophages in the tumor microenvironment expressed CD86. Thus, both intra- and intercellular interactions could drive CTLA4-CD28 and ICOS-CD28 signaling in these cases. Treatment of Ba/F3 cells expressing CTLA4-CD28 and cultured in the presence of Raji cells with a CTLA4 blocking antibody suppressed proliferation in a dose-dependent fashion. A previous case report (Mol Genet Genomic Med, 2015) described a patient with Sezary Syndrome and CTLA4-CD28 fusion who had a deep but transient response to the anti-CTLA4 antibody ipilimumab. This study did not clarify the extent to which response resulted from blocking of cell-autonomous CTLA4-CD28 signaling versus activation of a cell non-autonomous immune response. Nonetheless, it strongly supports the testing of CTLA4 blockade in additional cases of T-cell malignancies, including ATLL, that harbor CTLA4-CD28 fusions. Disclosures Stevenson: Celgene: Research Funding. Ohshima:Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Weinstock:Verastem Oncology: Research Funding; Celgene: Research Funding.

Graft-Versus-Host Disease-Free, Relapse-Free Survival in Allogeneic Stem Cell Transplantation for Adult T-Cell Leukemia/Lymphoma Provides a Novel Donor Selection Paradigm

Background: Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide sustained remission for adult T-cell leukemia/lymphoma (ATL), its prognosis is still unsatisfactory. Alternative donor sources are often utilized in allo-HSCT for ATL, but advantages and disadvantages of each donor source have not been fully elucidated. Graft-versus-host disease (GVHD) -free, relapse-free survival (GRFS) has been proposed as a novel and clinically meaningful endpoint of allo-HSCT. Whereas GVHD has been relatively accepted in ATL to induce substantial graft-versus-tumor (GVT) effects, severe GVHD is associated with non-relapse mortality and disturbs quality of patients' life. Analyses of GRFS may illustrate important aspects to optimize the balance between regulating GVHD and enhancing GVT effects in ATL patients. Patients and methods: We retrospectively analyzed the data of 1363 patients with ATL who received first allo-HSCT from 2006 through 2015 in Japan. Data were collected from the nationwide database maintained by the Japan Society of Hematopoietic Cell Transplantation. The probability of OS and GRFS was estimated according to the Kaplan-Meier method, and univariate comparisons among the groups were performed using the log-rank test. The Cox proportional hazard model was used for the multivariate analysis of OS and GRFS. Results: Median age was 57 years-old (range: 20-78) and median observation period of survivors was 3.1 years (range: 0.0-10.5). One-year OS and GRFS were 45% and 24%, respectively. OS of cord blood transplantation (CB) (37% at 1 year, n = 359) was worse than those of related bone marrow transplantation (R-BM) (48%, n = 121), related peripheral blood stem cell transplantation (R-PB) (50%, n = 264) and unrelated bone marrow transplantation (UR-BM) (47%, n = 619) (p < 0.001). However, in the cases of complete remission (CR) at allo-HSCT, OS of CB (n = 132) was 52% at 1 year and not inferior to those of R-BM (54%, n = 52), R-PB (57%, n = 88) and UR-BM (58%, n = 280) (p = 0.14). The cumulative incidence of relapse in CB was 47% at 1 year and higher than those of R-BM (40%), R-PB (43%) and UR-BM (35%) (p = 0.002). However, in CR cases, the cumulative incidences of relapse were lower in UR-BM (21% at 1 year) and CB (26%) than those in R-BM (31%) and R-PB (31%), though not significant (p = 0.21). Notably, there were no significant differences in GRFS of CB, R-BM, R-PB and UR-BM (21%, 25%, 21% and 26% at 1 year, p = 0.08, Figure 1A). It was attributed to low incidence of chronic GVHD which needed systemic therapy in CB (9% at 1 year, R-BM: 22%, R-PB: 28% and UR-BM: 18%, p < 0.001, Figure 1B) and low incidence of non-GVHD/relapse mortality in R-PB (9% at 1 year, CB: 20%, R-BM: 13% and UR-BM: 16%, p < 0.001, Figure 1C). In R-BM and UR-BM, there were no significant differences as for OS and GRFS between HLA-matched and HLA-mismatched transplantations. In R-PB, OS of HLA haploidentical transplantation (Haplo, n = 36) was 39% at 1 year and tended to be inferior to those of HLA 6/6-matched (53%, n = 183) and HLA 5/6-matched transplantation (49%, n = 45) (p = 0.11). There was no significant difference as for GRFS among these 3 groups (Haplo: 22%, 6/6-matched: 20%, 5/6-matched: 27% at 1 year, p = 0.69). Finally, multivariate analysis revealed that male sex, higher age (> 50 years), worse PS (> 1) and non-CR at allo-HSCT were associated with lower OS and GRFS. Importantly, donor sources and HLA mismatch were not associated with OS and GRFS. Conclusions: Our results imply that all donor sources are feasible for CR cases and can be selected depending on their clinical characteristics such as frailty and history of infection. While the main issue of CB is high non-GVHD/relapse mortality, it is warranted to suppress severe GVHD in R-PB. Thus, GRFS provides important clues to improve the outcome of allo-HSCT for ATL. Figure Disclosures Shindo: Novartis: Research Funding. Nakano:Novartis: Honoraria. Ichinohe:Astellas Pharma: Research Funding; Chugai Pharmaceutical Co.: Research Funding; CSL Behring: Research Funding; Eisai Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Ono Pharmaceutical Co.: Research Funding; Pfizer: Research Funding; Nippon Shinyaku Co.: Research Funding; MSD: Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Repertoire Genesis Inc.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Takeda Pharmaceutical Co.: Research Funding; Zenyaku Kogyo Co.: Research Funding; Alexion Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; JCR Pharmaceuticals: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Mundipharma: Honoraria; Novartis: Honoraria. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria.