Intrapatient Complex Transcriptional Responses to Conventional Chemotherapy in Relapsed Acute Myeloid Leukemia Revealed By Single Cell RNA-Seq Analysis

Resistance to anthracycline and cytarabine based conventional chemotherapy often occurs and results in extremely poor prognosis in patients with acute myeloid leukemia (AML). Although chemotherapy resistance is the most critical clinical problem, the mechanisms by which AML confers resistance to conventional chemotherapy are not yet fully understood. In this study, we investigated the key mechanisms of chemotherapy resistance through single cell RNA-sequencing analysis using paired bone marrow AML cells longitudinally collected from two AML-MRC patients at diagnosis and relapse after anthracycline-based chemotherapy. AML blasts were sorted by CD45/SSC gating and subjected to single cell RNA-seq analysis. Single cell RNA-seq was performed using 10x Genomics' Chromium System. Mean estimated number of cells per sample was 3.403 (2,731-4,200) and median detected genes per cell ranged 3,030 to 3,918 among four samples. Data collected from paired samples were combined in following analysis. Transcriptome based clustering following UMAP dimensionality reduction distinguished 5 and 9 cluster groups in each paired sample. Chemotherapy sensitive cluster groups dominant at diagnosis and chemotherapy resistant cluster groups dominant at relapse were clearly divided. In each paired sample, a few AML cells at diagnosis were allocated to chemotherapy resistant cluster groups. This suggested that transcriptionally identifiable less frequent cells resistant to chemotherapy existed at diagnosis and may expand during and/or after chemotherapy maintaining its transcriptional features. Next, to determine whether these transcriptional features are correlated with DNA mutation profiles, we labeled DNA mutation status to each cell and compared frequencies of mutation. As far as we detected, AML recurrent mutations such as DNMT3A R882C and TP53 missense mutation were not related to chemotherapy resistant cluster groups, although this method was relatively limited by the nature of RNA-seq-based mutation detection. Then we sought to determine transcriptional features of resistant clones. Gene set enrichment analysis identified some gene groups such as E2F signaling pathway, MYC signaling pathway, hedgehog signaling pathway and TNFA signaling pathway as transcriptional signatures related to emergence after chemotherapy. Analysis of known hematopoietic differentiation gene signatures showed distinct differentiation profiles in each cluster groups, whereas resistant cluster groups were not necessarily related to hematopoietic stem cell signatures. Intrapatient variations of transcriptional signatures among the resistant cluster groups were detected, which indicated that accurate detection of transcriptional features related to chemotherapy resistance may be difficult by using bulk RNA-seq method. As for other cluster groups which were not dominant both at diagnosis and relapse, these cluster groups hardly changed its frequencies between at diagnosis and relapse, which suggested less proliferative leukemia cells persisted during chemotherapy and have various transcriptional features although whether these persisting cells contribute to relapse was unclear. Since enriched transcriptional signatures in resistant cluster groups were not consistent between the two patients, further analysis using samples collected from more patients would be needed to determine common critical chemotherapy resistant transcriptional signature. In conclusion, our analysis suggested that a transcriptionally identifiable small fraction of cells showing gene signatures related to chemotherapy resistance at diagnosis may expand during chemotherapy and revealed intrapatient transcriptional complexity of response to chemotherapy, which cannot be uncovered by bulk RNA-sequencing. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

EVI1-Positive AML Cells Show Distinct Features and High Dependency on ETS Transcription Factor ERG

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1 is indispensable for normal hematopoiesis. We have previously reported that EVI1 expression is restricted to hematopoietic stem cell fraction and EVI1-expressing cells show robust long-term reconstitution capacity using Evi1-IRES-GFP knock-in (EVI1-GFP) mice, which enable us to track Evi1 expression on a single cell basis. In this study, we tried to elucidate the functional implication of EVI1 expression in AML using these mice. We generated murine EVI1-GFP AML model by retrovirally transducing MLL-AF9 or -ENL fusion gene into Lineage- Sca-1+ c-kit+ (LSK) cells from EVI1-GFP mice followed by transplantation into lethally irradiated syngeneic mice. Clonogenic and leukemogenic potentials of AML cells, especially leukemic cells with a granulocyte-macrophage progenitor phenotype (L-GMPs) from these mice, were compared according to GFP expression. Remarkably, GFP-positive L-GMPs tended to show lower colony-forming activity in semi-solid media and lower leukemia-initiating potential than GFP-negative L-GMPs. GFP-positive L-GMPs, however, induced a more aggressive form of AML, characterized by shorter survival in the secondary transplantation model. When EVI1-GFP AML mice underwent cytotoxic chemotherapy with cytarabine, the GFP-positive fraction was enriched during myelosuppression, indicating the survival advantage of EVI1-positive cells. To investigate the downstream target of EVI1, we employed murine EVI1-AML model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody. To identify leukemia-specific targets of EVI1, the result was compared with the result of ChIP-seq obtained from 32D-cl3 murine hematopoietic progenitor cells with 3×FLAG tag inserted into 3'-end of the coding region of the EVI1 gene. Gene ontology analysis revealed that genes involved in immune processes are explicitly enriched in the leukemia samples. Among the list of EVI1-bound genes, we tried to refine functional downstream targets of EVI1, which are upregulated in murine EVI1-AML cells and of which expressions are positively correlated with EVI1. By combining the ChIP-seq data with murine transcriptome data that compare hematopoietic progenitor cells expressing empty-vector and EVI1+ AML cells, and public gene expression datasets of human AML (Valk et al. NEJM 2004), we picked out 18 genes as candidate EVI1 downstream genes. Functional screening using EVI1-AML cells and shRNAs against these genes revealed that silencing of ETS transcription factor ERG (ETS-related gene) markedly suppressed proliferation and colony-forming activity of EVI1-AML cells, as well as rendered them vulnerable to cytotoxic agents. Normal c-kit+ hematopoietic progenitor cells were less affected by shRNAs against ERG. By comparing MLL-ENL immortalized murine hematopoietic cells with high and low EVI1 expression, EVI1-high MLL-ENL cells showed higher ERG dependency than EVI1-low MLL-ENL cells. Pharmacological inhibition of ERG also led to marked inhibition of EVI1-AML cells and EVI1-high MLL-ENL cells. Finally, knockdown of ERG remarkably delayed AML development in bone marrow transplantation model of EVI1-AML and EVI1-expressing MLL-ENL AML. Our data suggest that EVI1-positive AML cells are characterized by an aggressive nature and resistance to cytotoxic agents, as well as low leukemia stem cell capacity. ERG would be a novel downstream target of EVI1, on which survival of EVI1-expressing AML cells depends. Disclosures Masamoto: Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; MSD K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau. Kurokawa: Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau.

The Outcomes of Systemic Chronic Active EBV Infection Treatment By Allogeneic Hematopoietic Stem Cell Transplantation: An Analysis of Japanese Registry Data

Background and aims Systemic chronic active Epstein-Barr virus infection (sCAEBV) is classified as T- or NK-cell neoplasms in the WHO classification revised in 2017. Although allogeneic stem cell transplantation (allo-HSCT) is efficacious for sCAEBV, the effects are yet to be analyzed in a large number of cases due to the disease rarity. To investigate the outcomes and the prognostic factors of allo-HSCT in sCAEBV under the definition of the WHO 2017 classification, we analyzed retrospectively using the database of Japanese Society for Transplantation and Cellular Therapy (JSTCT). Methods Data collection We used the clinical data of hematopoietic stem cell transplantation (HSCT) recipients of the Transplant Registry Unified Management Program (TRUMP) sponsored by JSTCT and Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT). Patients who underwent HSCT to cure EBV-associated diseases, secondary hemophagocytic lymphohistiocytosis (HLH), and virus-associated hemophagocytic syndrome between January 1993 and December 2016 were selected in TRUMP database, and on our behalf, JDCHCT sent out survey questions to the institutions of these patients to collect additional data to check if the diagnosis of sCAEBV matches our criteria, to analyze disease status at the time of HSCT, and to evaluate the efficacy of different treatment methods. The diagnosis of sCAEBV sCAEBV was diagnosed according to criteria suggested in 2016 by the Research group of Measures against Intractable Diseases by Ministry of Health, Labour and Welfare of Japan: (1) elevated EBV DNA load in peripheral blood (PB) (> 10 2.5 copies/μg DNA), (2) detection of EBV infection in T or NK cells from the affected tissues or PB, (3) systemic inflammatory symptoms such as fever, lymphadenopathy, liver dysfunction, progressive skin lesions, vasculitis, and uveitis persisting for > 3 months, and (4) exclusion of other possible diseases, such as primary EBV infection, autoimmune disease, immunodeficiencies, and lymphomas. Patients who fulfilled all (1) to (4) were diagnosed as sCAEBV. These criteria are compatible with the definition of sCAEBV described in the WHO definition of 2017. The definitions of disease activities and responses The disease activities are defined in previous reports (Blood. 2012;119, p673 and BMT. 2016;51, p879) as follows: positive of fever, ALT level elevation, vasculitis, progressive skin lesions, or uveitis. We defined the complete resolution of disease activity as complete response (CR) and CR with a significant decrease in PB EBV-DNA load (< 10 2.5 copies/μg DNA) as virological CR (vCR). Results 81 patients who met the diagnostic criteria of sCAEBV were analyzed. The median age at HSCT was 24 years old, and the three-year overall survival rate (3-year OS) was 74.0%. Of 74 patients whose viral load after HSCT evaluated, 49 (66.2%) achieved vCR. The multivariate cox proportional hazard model revealed that advanced age, adolescent and young adult (AYA) (age, 15-39; n = 48) and adult (age, > 40; n = 13), was a risk factor of poor OS. The hazard ratios (HR) of AYA and adult groups were 10.14 and 4.63 respectively. It also showed that the presence of HLH at HSCT (HR 4.55), high sIL-2R (≥ median, 691 U/mL) at HSCT (HR 5.27), and conditioning without total body irradiation (HR 3.23) were independently associated with poor survival. Moreover, the median survival time of patients with active disease and extremely high sIL-2R level (≥ 3 × median, 2073 U/mL) was 0.9 months, whereas the other groups did not reach the median. Conclusion Although HSCT is the only curative treatment for sCAEBV, the strategies need improvements in high-risk cases, especially of high sIL-2R. Disclosures Arai: ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Kyowa Kirin CO., LTD.: Honoraria, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Elsai Co Ltd: Research Funding; Abbott Japan LLC: Honoraria; Nippon Shinyaku Co. Ltd: Honoraria, Research Funding; Otsuka Pharmaceuticals Co. Ltd: Research Funding; Novartis Pharma KK: Honoraria; Takeda Pharmaceuticals Co Ltd: Honoraria, Research Funding; Shionogi & Co Ltd: Research Funding; Asahi Kasri Pharma Corporation: Research Funding; Sanofi: Honoraria; Pfizer japan: Honoraria; Astellas Pharma Inc.: Honoraria. Yamamoto: Bristol-Myers Squibb Company: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; NIPPON SINYAKU CO., LTD: Honoraria; Novartis Pharma: Honoraria; ONO PHARMACEUTICAL CO.: Honoraria; Otsuka Pharmaceutical: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria. Nakamae: Astellas Pharma Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Simon-Kucher & Partners: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; Novartis: Honoraria, Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb Company: Honoraria, Research Funding; Alexion: Research Funding; PPD-SNBL K.K: Research Funding; CMIC HOLDINGS Co., Ltd: Research Funding. Ichinohe: Repertoire Genesis Inc.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria; Celgene: Honoraria; Zenyaku Kogyo Co.: Research Funding; Takara Bio Inc.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Nippon Shinyaku Co: Research Funding; Ono Pharmaceutical Co.: Honoraria, Research Funding; Kyowa Kirin Co.: Honoraria, Research Funding; FUJIFILM Wako Chemicals.: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Eisai Co.: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co.: Honoraria; AbbVie Pharma: Research Funding; Astellas Pharma: Honoraria, Research Funding. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria; Meiji Seika Pharma Co, Ltd.: Honoraria.

Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Background Clonal hematopoiesis (CH) in apparently healthy individuals has been drawing an increasing attention of recent years due to its implication in the risk of hematopoietic malignancies and also in cardiovascular complications. However, our knowledge on CH has been largely based on genetic studies, while few functional analyses have been performed using human materials with most studies being confined to artificial models using mice. The major challenge here is the difficulty of isolating CH clones from wild-type (WT) cells in human bone marrow (BM) to elucidate the effect of CH-mutations. Methods To investigate cellular phenotypes of mutated and WT cells separately in CH, we developed a Fluidigm C1-based single cell-sequencing platform for simultaneous genotyping and gene expression analysis. We analyzed a total of 10,178 hematopoietic stem/progenitors (HSPCs) derived from BM of patients with (n=11) and without (n=17) CH. Results In the analysis of HSPCs from CH(−) elderly individuals, we found a significant positive correlation between age and expression of gene sets implicated in inflammatory responses including TGFβ signalling and IL-6. We next analysed CH(+) samples, including those with mutations in TET2, DNMT3A, SF3B1 and IDH1 in which we investigated the pathways enriched in differentially expressed genes between mutated and unmutated cells. Regardless of mutation type, mutant cells showed an upregulation of genes implicated in an enhanced cell proliferation, while genes related to inflammatory responses were significantly downregulated. These results suggest that mutant HSPCs show an enhanced cell proliferation and an attenuated response to an inflammatory microenvironment in aged BM, compared with endogenous WT counterparts. Based on these observations, we further investigated the role of the BM microenvironment in CH, in which we compared the phenotype of WT cells in age-matched CH(+) and CH(−) cases. Compared with those from CH(−) cases, WT cells from these CH(+) cases showed an enhanced response to proinflammatory cytokines, including IL-6, interferons and TNF, suggesting a possibility that CH(+) BM might be characterized by a more enhanced inflammatory microenvironment, compared with CH(−) BM. To understand the different phenotype of WT cells between CH(+) and CH(−) cases, we next investigated a possible effect of mutant cells on endogenous WT cells. Such an effect was first documented in an IDH1-mutated CH case. IDH1-mutated cells showed a gene expression profile suggestive of an enhanced cell proliferation compared with endogenous IDH1-WT cells (WT IDH1). However, on the basis of comparison of gene expression between WT cells from CH(−) cases, it was better explained by suppressed proliferation of endogenous WT cells, rather than enhanced proliferation of mutant cells. In agreement with this, mouse BM cells treated with 2-hydroxyglutalate (2HG), an oncometabolite produced by IDH1-mutant cells, mimicked the endogenous WT cells in IDH1-mutated cases, including downregulated E2F target genes and upregulated inflammation-related genes, compared with control BM cells. A non-cell autonomous effect of mutations was also seen in cases with TET2-mutated CH, in which not only TET2-mutated cells but also endogenous WT cells exhibited a significantly different gene expression profile, compared with those from CH(−) cases. Of interest, when BM cells from WT Ly5.1/5.2 mice were co-transplanted with those from heterozygous conditional Tet2 knock-out (Ly5.2) or WT (Ly5.2) mice into lethally irradiated mice (Ly5.1) and flow-sorted Lin − WT donor competitor cells were analysed using single cell sequencing, the WT HSC-like cells co-transplanted with Tet2-mutant competitors exhibited enhanced cell proliferation and IFNα and IFNγ pathway genes, compared with those co-transplanted with WT competitors. Conclusions Taken together, these results suggest that mutant cells in CH(+) BM have non-cell autonomous effects on endogenous WT cells, which might be responsible for an accelerated inflammatory microenvironment of aged BM, favor positive selection of CH-clones, and also affect the phenotype of endogenous WT cells, contributing to the pathogenesis of CH. Disclosures Nakagawa: Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Inagaki: Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Yoda: Chordia Therapeutics Inc.: Research Funding. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.

Allogeneic Stem Cell Transplantation Conditioned with Myeloablative Regimens Containing Total Body Irradiation in Adolescent and Young Adult Patients with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia Who Were Treated with Pediatric-Type Chemotherapy

Background: Although introduction of pediatric-type Berlin-Frankfurt-Munster (BFM) chemotherapy has markedly improved the prognosis of adolescent and young adult (AYA) patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph-negative ALL), their higher toxicity has become an emerging issue with an increased post-remission mortality in AYA patients than pediatric patients. Allogeneic stem cell transplantation (allo-SCT) with myeloablative conditioning (MAC) regimens containing total body irradiation (TBI) is recognized as an important treatment option for patients with higher risk diseases. However, safety and efficacy of allo-SCT with TBI-MAC have not been fully investigated among AYA patients who received pediatric-type chemotherapy. Patients and methods: AYA was defined as 16 to 39 years old. Of the 143 AYA patients with Ph-negative ALL who underwent the first allo-SCT in the first remission between 2007 and 2016 at the 20 institutions, 106 patients who were treated with one BFM chemotherapy regimen before transplant and were conditioned with MAC regimens containing more than or equal to 8 Gy of TBI dose. The reasons for the transplant were surveyed with a multi-answer questionnaire. Overall survival (OS) was defined as the interval from the date of transplant to the date of death. Fisher's exact test was used to compare binary variables. The cumulative incidence (CI) of non-relapse mortality (NRM) and relapse were evaluated with Gray's test, considering relapse and NRM as a competing risk, respectively. OS was estimated with the Kaplan-Meier method and compared using the log-rank test. Factors associated with at least borderline significance (p < 0.20) in univariate analyses were subjected to multivariate analysis. The Fine-Gray and Cox proportional hazard model were used for multivariate analysis of risk factors and prognostic factors, respectively. Values of p < 0.05 were considered to indicate statistical significance. Results: Of the 106 patients included in this study, 61 were male, and 45 were female. The median age at transplant was 29 years (range, 16-39 years). Donor types were related, unrelated, and cord blood in 47 (44%), 47 (44%), and 12 (12%) patients, respectively. The difficulty of continuing chemotherapy due to side effects as the reason for transplant was included in 13 patients (12%). As chemotherapy before transplant, pediatric-type and adult-type regimens were used in 56 (53%) and 50 patients (47%), respectively. There was no significant difference in baseline characteristics and transplant procedures between the pediatric-type group and the adult-type group, except for proportion of patients over 30 years (14% vs. 64%, respectively; p < 0.01), those with more than or equal to 2 of hematopoietic cell transplant comorbidity index (13% vs. 36%, respectively; p < 0.01), and transplant between 2007 and 2011 (36% vs. 64%, respectively; p < 0.01). The CI of NRM, the OS rates, and the CI of relapse were not significantly different between two groups (NRM: 4% vs. 12% at three years after transplant, respectively; p = 0.26), (OS: 86% vs. 70%, respectively; p = 0.14), and (relapse: 16% vs. 24%, respectively; p = 0.32), respectively. Multivariate analysis for NRM revealed that more than or equal to 1 of performance status at transplant (hazard ratio [HR] = 4.8; p < 0.01) and transplant due to side effects of chemotherapy (HR = 3.5; p = 0.04) were identified as independent risk factors, but not pediatric-type chemotherapy (HR = 0.48; p = 0.23). The proportions of transplant due to side effects of chemotherapy were similar between two groups (13% vs. 12%, respectively; p = 1). No independent prognostic factor for OS was found, while transplant between 2007 and 2011 (HR = 2.5; p = 0.04) was extracted as an independent risk factor of relapse. Regarding transplant complications, significant differences were not shown in CI of grade II to IV acute graft-versus-host disease (GVHD), chronic GVHD, bacteremia, cytomegalovirus reactivation, hemorrhagic cystitis, and avascular necrosis between two groups. No characteristic cause of NRM was found in the pediatric-type group. Conclusion: These findings suggested that conventional allo-SCT with TBI-MAC can be performed without increasing NRM in AYA patients with Ph-negative ALL even after pediatric-type chemotherapy. Disclosures Kimura: MSD: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Astellas: Honoraria; Pfizer: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical: Honoraria; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Eisai: Honoraria; Nippon Kayaku: Honoraria; Takeda Pharmaceutical: Honoraria; SymBio Pharmaceutical: Honoraria. Usuki: MSD: Speakers Bureau; Alexion: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Research Funding, Speakers Bureau; Eisai: Speakers Bureau; Nippon shinyaku: Research Funding, Speakers Bureau; Astellas-Amgen-Biopharma: Research Funding; Nippon Boehringer Ingelheim: Research Funding; Takeda: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Research Funding; Ono: Research Funding, Speakers Bureau; Brisol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Sumitomo Dainippon: Research Funding; Daiichi Sankyo: Research Funding, Speakers Bureau; Symbio: Research Funding, Speakers Bureau; Gilead: Research Funding; Abbvie: Research Funding; Astellas: Research Funding, Speakers Bureau; Mundipharma: Research Funding; Yakult: Speakers Bureau; PharmaEssentia: Speakers Bureau. Sakaida: Bristol Myers Squibb: Research Funding; Chugai: Research Funding; Ono: Research Funding; Kyowa Kirin: Research Funding. Fujisawa: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding. Handa: Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding; Sanofi: Honoraria, Research Funding; Ono: Honoraria; BMS: Honoraria; Janssen: Honoraria; Daiichi Sankyo: Research Funding; Celgene: Honoraria, Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding. Hatta: Bristol-Myers Squibb: Honoraria; Novartis KK: Honoraria; Pfizer Japan Inc.: Honoraria; Otsuka Pharmaceutical.: Honoraria. Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding; Sanofi: Research Funding; MSD: Honoraria.

HLA-B Leader Dimorphism Impacts on Outcomes of HLA-Matched Related/Unrelated Transplantation: Analysis of the Japanese Society for Transplantation and Cellular Therapy

Background: HLA-B leader encodes methionine (M) or threonine (T) at position 2 and gives rise to TT, MT, or MM genotype. HLA-B M leaders promote higher HLA-E expression than T leaders, enhancing T cell and natural killer (NK) cell recognition on HLA-E via NKG2A and NKG2C related to cytomegalovirus (CMV) recognition. The dimorphic HLA-B leader informs acute graft-versus-host disease (GVHD) risk in HLA 1 allele mismatched unrelated hematopoietic stem cell transplantation (HCT) (Petersdorf EW et al. Lancet Haematol. 2020 and Blood. 2020) and haploidentical HCT (Fuchs EJ et al. 62nd ASH Annual Meeting 2020). However, the impact of the HLA-B leader genotype in HLA-matched related/unrelated donor HCT from the viewpoints of CMV reactivation has not been elucidated fully yet. We performed this retrospective study to explore the significance of HLA-B leaders in HLA-matched related/unrelated HCT in the Japanese population. Methods: All clinical data of 10,110 patients who underwent 8/8 HLA matched related/unrelated donor bone marrow/peripheral blood HCT for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS) between 1996 - 2019 were provided by the Japanese Data Center for Hematopoietic Cell Transplantation. All 8 alleles at HLA-A, -B, -C, and DRB1 were matched by genotyping, and sibling pairs whose genotype were unknown but matched 8/8 for HLA antigen at a serologic level were included. Study outcomes were overall survival (OS), relapse, non-relapse mortality (NRM), grade II-IV acute GVHD, grade III-IV acute GVHD, and chronic GVHD. Multivariable models using Cox regression analysis assessed transplant outcomes associated patient age, patient sex, patient performance status, donor sex, donor age, diagnosis, disease risk index (DRI), donor source (bone marrow or peripheral blood), related/unrelated donor, myeloablative (MAC)/reduced-intensity conditioning, patient CMV serostatus and patient/donor HLA B-leader (TT, MT, MM). In subgroup analysis, we adopted results of CMV antigenemia instead of CMV serostatus to evaluate the impact of CMV reactivation for HCT outcomes. All statistical analyses were performed with EZR. Results: This study included 5,212 AML patients (51.9%), 2,995 ALL patients (29.6%) and 1,864 MDS patients (18.4%). In DRI, low risk was 501 (5.4%), intermediate risk was 5,750 (61.6%), high risk was 2,711 (29.0%) and very high risk was 378 (4.0%). Median patients age was 44 (range 0-77) years. Bone marrow was the graft source in 7,183 recipients (71.0%). Related donors were 5,378 (53.2%). MAC was used in 5,891 (70.3%) patients. The number of TT patients/donor was 7,419 (73.4%), MT patients was 2,496 (24.7%) and MM patients was 195 (1.9%). MM patients was associated with significant lower OS (hazard ratio [HR] 1.329 [95% CI, 1.053 - 1.677]); p = 0.017 and higher NRM (HR 1.391, [95% CI, 1.018 - 1.902]); p = 0.039) compared to TT patients (Table). There was no significant correlation between MM patients and grade II-IV/III-IV acute GVHD. In subset analysis for each diagnosis, MM genotype didn't affect outcomes in AML patients, whereas MDS and ALL patients with MM genotype showed lower OS (MDS: HR 1.829, [95% CI, 1.070 - 3.128]; p = 0.023), (ALL: HR 1.638, [95% CI, 1.032 - 2.599]; p = 0.037) compared to TT genotype (Table). In subgroup analysis for HLA-B leader genotype, CMV reactivated patients were significant better for OS (HR 0.467, [95% CI, 0.266 - 0.819]; p < 0.001) and lower NRM (HR 0.342, [95% CI, 0.153 - 0.763], p = 0.009) only in MM patients. Conclusions: MM HLA-B leader genotype is a risk factor for worse OS and higher NRM compared to TT genotype in HLA matched related and unrelated HCT, particularly MDS and ALL patients in the study. On the other hand, CMV reactivation could be favorable for OS and NRM in MM leader patients suggesting that promoting NK cell reconstitution and education due to CMV reactivation might benefit MM leader patients. Figure 1 Figure 1. Disclosures Kanda: CHUGAI PHARMACEUTICAL Co., Ltd.: Honoraria; DAIICHI SANKYO Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co., Ltd.: Honoraria; Megakaryon Co: Honoraria, Membership on an entity's Board of Directors or advisory committees; NextGeM Inc: Patents & Royalties; Novartis Pharma K.K.: Honoraria; Ono Pharma Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Sanofi K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; SymBio Pharmaceuticals, Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA LIMITED.: Honoraria; Bristol-Myers Squibb Co: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Amgen Astellas BioPharma: Honoraria. Ichinohe: Takeda Pharmaceutical Co.: Honoraria; Kyowa Kirin Co.: Honoraria, Research Funding; FUJIFILM Wako Chemicals.: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; CSL Behring: Honoraria, Research Funding; Taiho Pharmaceutical Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Honoraria, Research Funding; Ono Pharmaceutical Co.: Honoraria, Research Funding; Nippon Shinyaku Co: Research Funding; Takara Bio Inc.: Research Funding; Zenyaku Kogyo Co.: Research Funding; Repertoire Genesis Inc.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical Co.: Research Funding; Eisai Co.: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Bristol-Myers Squibb: Honoraria; AbbVie Pharma: Research Funding; Astellas Pharma: Honoraria, Research Funding. Atsuta: Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; Meiji Seika Pharma Co, Ltd.: Honoraria; Astellas Pharma Inc.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria.

Clonal Evolution Pattern and Prognostic Significance of Clonal Architecture in KMT2A-Rearranged Acute Myeloid Leukemia

MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities that occur in acute myeloid leukemia (AML). Mutational landscapes in KMT2A-rearranged AML have been reported; however, most studies are missing data at relapse. Therefore, matched diagnostic and relapse samples were analyzed in this study, and the clonal evolution pattern in KMT2A-rearranged AML was examined. Further, the prognostic significance of the clonal architecture was investigated. Sixty-two diagnostic and 16 relapse samples obtained from pediatric patients with KMT2A-rearranged AML enrolled in the Japan Children's Cancer Group (JCCG) AML-05/AML-99 study were analyzed for 338 genes using targeted sequencing. The data were analyzed with the published data of 105 diagnostic and 9 relapse samples with KMT2A-rearranged AML. Additionally, as a control, the mutation data of matched diagnostic and relapse samples of 107 patients with non-KMT2A-rearranged AML were collected. Among 25 patients with KMT2A-rearranged AML with matched data at diagnosis and relapse, mutations of signaling pathway genes (FLT3, KRAS, NRAS, PTPN11, CBL, and BRAF) were frequently detected in diagnostic samples (25 mutations/25 patients). However, 21 of 25 (84.0%) mutations were lost at relapse. In contrast, 7 of 19 (36.8%) mutations of other pathway genes were lost at relapse, and the percentage was significantly lower than that of mutations in the signaling pathway genes (P = 0.002). Six mutations in the signaling pathway genes and 11 mutations in other pathway genes were acquired at relapse. Particularly, mutations of transcription factor genes (WT1, SPI1, GATA2, and RUNX1) were acquired at relapse: 7 of 8 (87.5%) mutations were detected only at relapse. These results suggest that mutations of signaling pathway genes are unstable in the clonal evolution of KMT2A-rearranged AML. Mutations of other pathway genes, especially those of transcription factor genes, may contribute to relapse in patients with KMT2A-rearranged AML. Next, attention was turned to the KRAS mutations (KRAS-MT) because we have previously shown that KRAS-MT are independent adverse prognostic factors in KMT2A-rearranged AML (Blood Adv. 2020). Among 25 patients with KMT2A-rearranged AML with matched data at diagnosis and relapse, 10 (40.0%) patients harbored KRAS-MT at diagnosis. Interestingly, KRAS-MT were lost at relapse in 9 of 10 (90.0%) patients. Among 107 patients with non-KMT2A-rearranged AML with matched data at diagnosis and relapse, 10 (9.3%) patients harbored KRAS-MT at diagnosis. The frequency of KRAS-MT was significantly higher in KMT2A-rearranged AML (40.0% vs. 9.3%, P = 0.0006). This may be explained on the basis of the fact that KRAS-MT is associated with a high relapse rate in KMT2A-rearranged AML, but not in non-KMT2A-rearranged AML. KRAS-MT was lost at relapse in 5 of 10 (50.0%) patients with non-KMT2A-rearranged AML. The percentage of KRAS-MT loss at relapse was higher in KMT2A-rearranged AML. However, it was not statistically significant (90.0% vs. 50.0%, P = 0.14). Therefore, KRAS-MT may be unstable in clonal evolution regardless of disease subtypes in AML. The underlying mechanisms of the paradox between the high relapse rate in patients with KRAS-MT and frequent loss of KRAS-MT at relapse in patients with KMT2A-rearranged AML should be examined in future studies. The loss of KRAS-MT at relapse suggests that the mutations were in subclones at diagnosis. Therefore, we finally examined the prognosis of 167 patients according to the clonality of KRAS-MT at diagnosis. In patients with KMT2A-MLLT3 (n = 67), those with subclonal KRAS-MT (n = 6) had adverse 5-y event-free survival compared with both patients with wild-type KRAS (KRAS-WT) (n = 56) (KRAS-WT vs. subclonal KRAS-MT: 58.7% vs. 16.7%, P = 0.04) and patients with clonal KRAS-MT (n = 5) (clonal KRAS-MT vs. subclonal KRAS-MT: 80.0% vs. 16.7%, P = 0.07). However, 5-y overall survival (OS) was similar among the three groups. In contrast, among patients with KMT2A-MLLT10 (n = 37), those with clonal KRAS-MT (n = 5) had adverse 5-y OS compared with both patients with KRAS-WT (n = 20) (KRAS-WT vs. clonal KRAS-MT: 59.7% vs. 0.0%, P = 0.006) and patients with subclonal KRAS-MT (n = 12) (subclonal KRAS-MT vs. clonal KRAS-MT: 58.3% vs. 0.0%, P = 0.04). According to these results, the effects of the clonality of KRAS-MT on prognosis may depend on which KMT2A fusion is present. Disclosures Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Saito: Toshiba corporation: Research Funding. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; ChordiaTherapeutics, Inc.: Consultancy, Research Funding.

NGS Evaluation of the Eqol-MDS Trial: Preliminary Analysis of Eltrombopag for Thrombocytopenia of Low-Risk MDS

Background: In myelodysplastic syndromes (MDS), thrombocytopenia is an adverse risk factor. Treatments in this setting are scarce. In a randomized international phase 2 trial (EQoL-MDS, EudraCT number 2010-022890-33), we reported effecacy and safety of eltrombopag for the treatment of thrombocytopenia in the first 90 patients with lower-risk MDS with a platelet (PLT) count < 30 Gi/L (Oliva et al. Lancet Hem 2017). However, there are concerns of regulatory agencies regarding the use of thrombopoetin rececptor agonists in MDS due to previous reports signalling disease progression in clinical trials with the use of romiplostim and of eltrombopag, the latter in high risk MDS. Objective: We are further evaluating safety by conducting a comprehensive analysis of mutations in a panel of major driver or candidate driver genes in cases enrolled in the EQoL-MDS trial using targeted-capture sequencing. Methods: Serial (every 3 months) sequencing was performed using the SureSelect custom kit (Agilent Technologies), for which 350 genes were selected from known oncogenes or tumour suppressor genes in haematological malignancies. Relevant somatic mutation data with (i) VAF > 0.05; (ii) depth > 100; (iii) P value for EBCall < 0.0001, were filtered by exclusion based on (i) synonymous SNVs; (ii) variants present only in unidirectional reads; (iii) variants occurring in repetitive genomic regions; (iv) missense SNVs with VAF of 0.4-0.6 or <0.04; and (v) known variants listed in SNP databases. The present analysis has been conducted at baseline, at 12 and 24 weeks. Results: We present preliminary results of the first 21 cases (13 eltrombopag, 9 placebo) enrolled in the trial and with biological samples. Mean age was 62 (± 15) and 13 patients were male. According to the WHO 2016 classification, 11 patients had MDS with single lineage dysplasia (SLD), 7 had multi lineage dysplasia (MLD), 1 placebo case had excess blasts-1, and 1 placebo case had unclassifiable. IPSS-R risk was very low, low and intermediate in 4, 8 and 1 eltrombopag cases, respectively, and 5, 2 and 1 placebo cases, respectively. Karyotype was normal in 16 cases, del(20q) was detected in 4 cases and +14 in 1 case. At study entry, in total 49 genes were mutated (Figure), where one or more of the 49 driver genes were mutated in all but 1 placebo patient (Table). The table shows characteristics and events of patients according to treatment arm. Noteworthy, in the eltrombopag arm, two cases experienced a loss of gene mutations, one obtaining International Working Group defined complete remission of MDS, while 1 MDS EB-1 case had a gain in ZRSR2. Two placebo cases experienced a gain in mutations Conclusions: Treatment with eltrombopag in lower risk MDS is effective and safe. Preliminary analyses do not suggest that eltombopag induces disease progression neither at a clinical, nor a molecular level. Loss of mutations may occur during eltrombopag treatment with complete remission. Figure 1 Figure 1. Disclosures Oliva: Novartis: Other: Advisory Board; Celgene BMS: Consultancy, Other: Advisory Board, Patents & Royalties; Alexion: Other: Advisory Board; Argenx: Other: Advisory Board; Daiichi: Other: Advisory Board; Amgen: Other: Advisory Board. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Kulasekararaj: Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Ra Pharma: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria, Speakers Bureau; Achilleon: Consultancy, Honoraria, Speakers Bureau; Biocryst: Consultancy, Honoraria, Speakers Bureau; Akari: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau. Latagliata: Novartis: Honoraria; Pfizer: Honoraria; BMS Cellgene: Honoraria. Santini: Astex: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Ogawa: Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company. OffLabel Disclosure: Eltrombopag (Revolade) is indicated for chronic immune (idiopathic) thrombocytopenic purpura (ITP) patients aged 1 year and above who are refractory to other treatments (e.g. corticosteroids, immunoglobulins).Revolade is indicated in adult patients with chronic hepatitis C virus (HCV) infection for the treatment of thrombocytopenia, where the degree of thrombocytopenia is the main factor preventing the initiation or limiting the ability to maintain optimal interferon-based therapy.Revolade is indicated in adult patients with acquired severe aplastic anaemia (SAA) who were either refractory to prior immunosuppressive therapy or heavily pretreated and are unsuitable for haematopoietic stem cell transplantation.

Prognostic Factors of Idiopathic Hypereosinophilic Syndrome: A Nationwide Survey in Japan

Background Idiopathic hypereosinophilic syndrome (iHES) is a rare disease characterized by prolonged hypereosinophilia and organ damage without any known cause of eosinophilia. Since iHES was first proposed, the concept of the disease has continued to change, and its clinical features, optimal treatment, and prognosis have not yet been elucidated. Therefore, to clarify the clinical features and prognostic factors of iHES, we conducted a nationwide survey and collected detailed clinical information of iHES in Japan. Methods We conducted a nationwide postal questionnaire survey of iHES. A first simple questionnaire was sent to departments of hematology across the country to determine presence or absence of iHES in their department. Subsequently, a detailed questionnaire was sent to the department that responded that they had experience with iHES. After collecting the questionnaires, the validity of the diagnosis was determined. Only those that corresponded to iHES were used for subsequent analysis. In this research, iHES was defined as follows: absolute eosinophil counts more than 1,500/µL, presence of organ damage due to eosinophilia, with no known cause of eosinophilia. Allergy, collagen diseases, infection, asthma, drugs, vasculitis, and other diseases that can cause eosinophilia were ruled out. The absence of hematopoietic malignancies was confirmed by bone marrow examination, fluorescence in situ hybridization for FIP1L1-PDGFRA fusion gene, and chromosomal analysis by G-banding. Results The 1st questionnaire was sent to 492 departments of hematology, and we identified 152 patients with iHES in Japan. Among those patients, a detailed clinical information was collected from 68 patients. Of the 68 patients, 23 did not meet the criteria for iHES, and the remaining were subjected to subsequent analysis. Of the 45 patients with iHES, 27 (60%) were male, and 18 (40%) were female. The median age of diagnosis was 54 (range: 16-95) years, and the median number of involved organs per patient was 2 (range: 1-7). Symptoms caused by hypereosinophilia were consisted of systemic symptom (22, 49%), hematopoietic disorder (17, 38%), skin (16, 36%), digestive (15, 33%), respiratory (14, 31%), cardiovascular (14, 31%), and kidney (4, 9%). The median white blood cell and absolute eosinophil count were 18,800/µL (5,300-73,000/µL) and 9587/µL (2,067-63,370/µL), respectively. The median hemoglobin level was 13.3 g/dL (6.6-16 g/dL), and the median platelet count was 255 × 10 9/L (4.7-54 × 10 9/L). The median levels of lactate dehydrogenase and C-reactive protein were 299 U/L (123-972 U/L) and 0.96 mg/dL (0.02-13.9 mg/dL), respectively. Of the 45 cases, 37 (82%) required treatment, and 35 (78%) received corticosteroid as 1st line treatment. Although 28 (80%) patients responded to corticosteroid, 12 (34%) patients required subsequent 2nd line treatment, and 2 (6%) patients died. In addition, 6 patients required 3rd line treatment. Six of 45 patients died from any cause during the follow-up, and the median follow-up period for censored cases was 3.1 years (0.2-23 years). The median survival from diagnosis for all cases was 2.5 years (0.1- 23 years). In univariate analysis, hemoglobin less than 10 g/dL (P=0.02), the presence of renal symptoms (P<0.001), and the presence of respiratory symptoms (P<0.01) were statistically significant factors for overall survival. In multivariate analysis, hemoglobin less than 10 g/dL was a statistically significant factor for overall survival (HR, 17.2; 95% CI, 1.51-197; P=0.02). Conclusion In this nationwide survey, we clarified the clinical characteristics of iHES in Japan. In addition to clinical features at the time of diagnosis, the response rate to corticosteroid and long-term prognosis were also clarified. Furthermore, the presence of anemia was found to be a poor prognostic factor for iHES. Further accumulation of cases is necessary to establish the optimal treatment strategy for iHES. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee. Toyama: Celgene Corporation: Other: Lecture fee; Otsuka Pharmaceutical Co., Ltd.: Other: Lecture fee; NIHON PHARMACEUTICAL CO., LTD.: Other: Lecture fee; ONO PHARMACEUTICAL CO., LTD.: Other: Lecture fee; DAIICHI SANKYO COMPANY, LIMITED: Other: Lecture fee; CHUGAI PHARMACEUTICAL CO., LTD.: Other: Lecture fee; Takeda Pharmaceutical Company Limited: Other: Lecture fee. Matsuda: Ono Pharmaceutical: Other: Lecture fee; Kyowa Kirin: Other: Lecture fee. Komatsu: Fujifilm Wako Pure Chemical Corporation: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Japan Tobacco Inc.: Consultancy; Otsuka Pharmaceutical Co. Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharma KK: Consultancy, Research Funding, Speakers Bureau; Shire Japan KK: Consultancy, Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Consultancy, Current Employment, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding. Kurokawa: Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

Combination Therapy Strategy for Lymphoma Based on Cancer Energy Metabolism of OPB-111077, a Novel Antitumor Agent Inhibiting Mitochondrial Oxidative Phosphorylation

Development of antitumor agents targeting energy metabolism pathways of cancer cells has attracted much attention in recent days. In general, most cancer cells are known to display Warburg effect. Recently, it has been reported that cancer cells exist as heterogeneous groups in the microenvironment surrounding cancer cells and that not only glycolysis but also mitochondrial oxidative phosphorylation (OXPHOS) is important for cancer growth. In addition, many research groups have reported that the acquisition of anticancer drug resistance is caused by metabolic reprogramming from glycolysis to OXPHOS. Therefore, OXPHOS inhibitors could be useful antitumor agents. OPB-111077 is an orally active novel antitumor agent and has the inhibitory effect on mitochondrial OXPHOS. OPB-111077 had the inhibitory effect on mitochondrial respiratory chain complex I which led to the inhibition of energy production and activation of AMPK-mTOR energy stress sensor pathway. In in vitro growth inhibition studies, OPB-111077 showed potent inhibitory effects on the growth of various human blood tumor cell lines derived from leukemia, multiple myeloma and lymphoma with IC 50 values between 18.6 and 525.3 nM, and on the growth of human solid tumor cell lines derived from liver cancer, lung cancer, gastric cancer and breast cancer with IC 50 values ranging from 92.6 to 1727.7 nM. In in vivo studies using SCID mice bearing tumors from human tumor cell lines, daily oral administration of OPB-111077 demonstrated significant antitumor effects against leukemia, lymphoma, liver, gastric and breast cancer cell line-derived tumor, dose-dependently. Here, we report the combination therapy strategy based on the cancer energy metabolism of OPB-111077. We found that OPB-111077 combined with alkylating agent, cyclophosphamide or bendamustine, showed the synergistic effect on xenograft mice model using human diffuse large B-cell lymphoma (DLBCL) cell line. To clarify this mechanism, we examined the effect of alkylating agents on mitochondrial respiration in vitro using flux analyzer and found that alkylating agents induced OXPHOS nature in the DLBCL cell line. Furthermore, the administration of alkylating agents to DLBCL cell line xenograft model mice followed by mRNA expression analysis of their tumor tissue confirmed that expression of OXPHOS-related markers was induced. These results suggest the administration of alkylating agents induced reprogramming to OXPHOS predominant nature in tumor. The tumor environment is actively reprogrammed to OXPHOS by alkylating agents, and it is changed to a state in which the antitumor effect of OPB-111077 can be more exerted. Thus, alkylating agents enhance the effect of OPB-111077 by tilting glycolytically dominant tumors to OXPHOS dominant. The alkylating agents, cyclophosphamide and bendamustine, are used as standard therapies for lymphoma. OPB-111077 could be treated in a wide range of applications as a combination therapy with the alkylating agents for lymphoma. In clinical, we have confirmed that OPB-111077 has tolerable and controllable toxicity profile of single agent in some advanced cancer patients including DLBCL. Bendamustine and rituximab (BR) therapy is indicated for the treatment of patients who are ineligible for transplantation of relapsed / refractory (R/R) DLBCL. However, there is still a high unmet need for patients with R/R DLBCL due to no standard treatment for transplant-ineligible patients. We are conducting combination therapy of OPB-111077 with bendamustine and rituximab in Phase 1 clinical trial for R/R DLBCL in Japan. Disclosures Ohi: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. Okuno: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. Tanaka: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. Ohtani: Otsuka Pharmaceutical Co.,Ltd.: Current Employment. OffLabel Disclosure: Compound: OPB-111077 Purpose: Development of novel antitumor agent