Abstract: Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. So, we analyzed 50 paired tumor-normal samples of myeloid neoplasms using whole exome sequencing, among which we identified recurrent mutations involving STAG2, a core cohesin component, and two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex which is composed of four core subunits (SMC1, SMC3, RAD21 and STAG proteins), and is engaged in cohesion of sister chromatids, DNA repair and transcriptional regulation. To extend the findings in the whole-exome analysis, an additional 534 primary samples of various myeloid neoplasms was examined for mutations and deletions in a total of 9 components of the cohesin complexes, using high-throughput sequencing and SNP arrays. In total, mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205), in a mutually exclusive manner. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles revealed that majority of the cohesin mutations existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we examined several myeloid leukemia cell lines with or without cohesin mutations for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of several components of cohesin was significantly reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we introduced the wild-type RAD21 allele into RAD21-mutated cell lines (Kasumi-1), which effectively suppressed the proliferation of Kasumi-1, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, about half of cohesin-mutated cases in our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Of note, the number of mutations determined by our whole exome analysis was significantly higher in cohesin-mutated cases compared to non-mutated cases. Since cohesin participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Citation Format: Ayana Kon, Lee-Yung Shih, Masashi Minamino, Masashi Sanada, Yuichi Shiraishi, Yasunobu Nagata, Kenichi Yoshida, Yusuke Okuno, Masashige Bando, Shunpei Ishikawa, Aiko Sato-Otsubo, Genta Nagae, Aiko Nishimoto, Claudia Haferlach, Daniel Nowak, Yusuke Sato, Tamara Alpermann, Teppei Shimamura, Hiroko Tanaka, Kenichi Chiba, Ryo Yamamoto, Tomoyuki Yamaguchi, Makoto Otsu, Naoshi Obara, Mamiko Sakata-Yanagimoto, Tsuyoshi Nakamaki, Ken Ishiyama, Florian Nolte, Wolf-Karsten Hofmann, Shuichi Miyawaki, Shigeru Chiba, Hiraku Mori, Hiromitsu Nakauchi, H. Phillip Koeffler, Hiroyuki Aburatani, Torsten Haferlach, Katsuhiko Shirahige, Satoru Miyano, Seishi Ogawa. Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2013-4602

Abstract: The impacts of graft‐versus‐host disease (GVHD) and graft‐versus‐leukemia (GVL) effect might differ depending on minimal residual disease (MRD). Therefore, we examined 1,022 recipients who underwent their first allogeneic hematopoietic stem cell transplantation (HSCT) for Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph‐positive ALL) in first complete remission. MRD status at HSCT was negative in 791 (77·4%) and positive in 231 (22·6%). The impact of GVHD as a time‐dependent covariate on transplant outcomes were analyzed while adjusting for other possible variables. Mild acute GVHD [hazard ratio (HR), 0·90; 95% confidence interval (CI), 0·70–1·16; P  = 0·901] and chronic GVHD (HR, 0·82, 95% CI, 0·58–1·14; P  = 0·238) were not significantly associated with overall mortality, whereas severe acute GVHD (HR, 2·26, 95% CI, 1·64–3·11; P   〈  0·001) resulted in inferior overall survival due to high non‐relapse mortality. Moreover, even in the subgroup analyses stratified according to MRD status, acute and chronic GVHD were not significantly associated with better overall survival. Therefore, less intensive GVHD prophylaxis to achieve a GVL effect is not recommended for Ph‐positive ALL.

Abstract: [Background] Previous studies have shown that a graft-versus-leukemia (GVL) effect was augmented in patients who developed graft-versus-host disease (GVHD). The benefit of the GVL effect is counter-balanced by treatment-related mortality (TRM) due to GVHD. In addition, the development of the ability to detect minimal residual disease (MRD) has changed the landscape of risk stratification. Therefore, patients with positive-MRD require a more intensive GVL effect to reduce relapse, whereas a "mild" GVL effect may be sufficient in patients with negative-MRD. However, it is uncertain whether the influence of a GVL effect would differ depending on the MRD status at HSCT. Here, we conducted a nationwide retrospective study to evaluate the impact of GVHD and a GVL effect according to the MRD status for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL) in the TKI era. [Patients & Methods] Clinical data were obtained from the Transplant Registry Unified Management Program (TRUMP), which is the registry database of the Japan Society for Hematopoietic Cell Transplantation (JSHCT). We examined 1022 recipients who underwent their first allogeneic hematopoietic stem cell transplantation (HSCT) for Ph-positive ALL in first complete remission between 2005 and 2017. We excluded patients who lacked MRD status at HSCT or who received in vivo T-cell depletion or high-dose post-transplantation cyclophosphamide. The impacts of acute GVHD (aGVHD) and chronic GVHD (cGVHD) as time-dependent covariates on transplant outcomes were analyzed while adjusting for other significant variables in multivariate analyses. In the analysis of cGVHD, only patients who survived at least 100 days without hematological relapse were included. This retrospective study was approved by the data management committee of TRUMP and by the Institutional Review Board of Jichi Medical University Saitama Medical Center. [Results] The median age at HSCT was 45 years (range, 16 to 71 years). MRD status at HSCT was negative in 791 (77.4%) and positive in 231 (22.6%). The median observation period of the survivors was 1505 days (range, 18 to 4944 days). To graphically illustrate the impacts of aGVHD and cGVHD, a Simon-Makuch plot were drawn in the whole cohort and in the groups limited to negative-MRD and positive-MRD at HSCT (Figure 1 and 2). The impacts of acute GVHD on overall survival, hematological relapse, and non-relapse mortality (NRM) were summarized in Table. In multivariate analyses, the HRs for hematological relapse with positive-MRD at HSCT (0.80 for grade 1-2 aGVHD and 0.31 for grade 3-4 aGVHD) were smaller than those with negative-MRD at HSCT (1.07 for grade 1-2 aGVHD and 0.45 for grade 3-4 aGVHD), respectively. In addition, the risk of hematological relapse gradually decreased proportionally to the severity of aGVHD. Because the risks of NRM for grade 1-2 aGVHD were not significant regardless of MRD-positivity, grade 1-2 aGVHD was not significantly associated with superior overall mortality. Grade 3-4 aGVHD was significantly associated with inferior overall survival in the whole cohort and in the group limited to negative-MRD at HSCT because of high NRM. Meanwhile, grade 3-4 aGVHD was not significantly associated with overall mortality due to the potent GVL effect in the analysis limited to positive-MRD at HSCT. In the analysis of cGVHD, although cGVHD reduced the risk of hematological relapse, the survival advantage of cGVHD was not significant regardless of MRD-positivity. Because the NIH severity score was not available in our database, further evaluations of cGVHD considering the severity score are needed. [Conclusion] Our study showed that both MRD status at HSCT and the severity of aGVHD might be associated with the intensity of a GVHD-associated GVL effect for Ph-positive ALL. However, because GVHD had no apparent survival benefit regardless of MRD-positivity at HSCT or the severity of GVHD, less intensive GVHD prophylaxis to obtain the GVL effect is not recommended for Ph-positive ALL. Disclosures Kanda: Nippon-Shinyaku: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria; Pfizer: Research Funding; Novartis: Research Funding; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; CSL Behring: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Research Funding; Tanabe Mitsubishi: Research Funding; Sanofi: Research Funding; Celgene: Consultancy, Research Funding; Taiho: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Eisai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; CSL Behring: Research Funding; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Taisho-Toyama: Research Funding; Asahi-Kasei: Research Funding; Taiho: Research Funding; Tanabe Mitsubishi: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Mochida: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria; Eisai: Consultancy, Honoraria, Research Funding; Takara-bio: Consultancy, Honoraria; Otsuka: Research Funding; Asahi-Kasei: Research Funding; Mochida: Consultancy, Honoraria; Taisho-Toyama: Research Funding; Ono: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Takara-bio: Consultancy, 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. Tanaka:Bristol-Myers Squibb: Research Funding. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Kako:Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria.

Abstract: Background: The prognosis of relapsed/refractory (R/R) myeloid malignancies remains poor, and the development of novel treatment strategies is crucial. Although chimeric antigen receptor (CAR)-modified T cell therapy for B cell malignancies has shown excellent clinical efficacy, the use of CAR-T cells for myeloid malignancies has been more challenging, partly due to the heterogeneous expression of candidate target antigens in leukemia cells and shared expression of those antigens in normal myeloid cells or progenitor cells. We previously developed the piggyBac-modified chimeric antigen receptor (CAR)-T cells targeting CD116, also known as GM-CSF receptor alpha chain (GMR) (Nakazawa Y, et al. J Hematol Oncol. 2016 and Hasegawa A, et al. Clin Transl Immunology. 2021). GMR CAR-T cells showed substantial antitumor effects against both acute myeloid leukemia and juvenile myelomonocytic leukemia. Moreover, modulation of the spacer and antigen recognition site of the CAR vector further enhanced the anti-tumor effects of GMR CAR-T cells. GMR CAR-T cells showed an acceptable safety profile with limited cytotoxicity on normal hematopoietic cells except monocytes. Based on these results, we have initiated a first-in-human clinical trial of GMR CAR-T cell therapy in March 2021 in Japan. Study Design and Methods: The study is a phase I/II, single-center, dose-escalation study with a traditional 3+3 dose-escalation design (Table 1). Maximum of 18 patients will be recruited. Primary objectives of this study are to determine the safety and severe adverse events of piggyBac-modified GMR CAR-T cells for CD116 + relapsed/refractory myeloid malignancies by assessing the dose-limiting toxicity within 28 days from the single infusion of GMR CAR-T cells. The patients with CD116 + myeloid malignancies aged more than 1 year with myeloid malignancies who experienced an induction failure or a relapse after hematopoietic stem cell transplantation (HSCT) will be recruited. CD116 is defined as positive when a CD116 relative mean fluorescence (RFI) in leukemia cells ≥ 2. RFI was calculated by dividing the MFI of samples with that of the isotype control. Major exclusion criteria are as follows, acute promyelocytic leukemia, acute graft-versus-host disease (GVHD) (Grade ≥ 2), extensive chronic GVHD, and concurrent treatment with corticosteroid (≥ 6 mg/m2). Statistical analysis will be performed when the data will be fixed. Peripheral blood mononuclear cells will be harvested from the patient by leukapheresis and then will be transduced with GMR CAR vector by piggyBac transposon system. All manufacturing process of GMR CAR-T cells is performed in Cell Processing Center in Shinshu University Hospital under good manufacturing practice conditions. The patient will be treated with lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide, followed by CAR-T cell infusion. The dose of CAR-T cells will be 3 x 10 5 and 1 x 10 6 in cohorts 1 & 2 and cohort 3, respectively (Table 1). Kinetics of GMR CAR-T cells will be determined by quantifying the GMR CAR gene in the peripheral blood using real-time PCR after the CAR-T cell infusion. All the patients will be required to receive HSCT by day 56 following CAR-T cell infusion. The primary endpoint of the study is the safety, pharmacokinetics, and efficacy of GMR CAR-T therapy (Trial registration: jRCT2033210029). Conclusion: We herein described the protocol of first-in-human GMR CAR-T cells for relapsed/refractory myeloid malignancies. By employing the optimized CAR vector and production protocol, the safety and efficacy of GMR CAR-T cells will be evaluated in this study. Figure 1 Figure 1. Disclosures Saito: Toshiba corporation: Research Funding. Yagyu: AGC Inc.: Research Funding. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Abstract: While in the past germline (GL) events have been investigated in myeloid disorders, these studies focused on targeted genotyping of empirically selected candidate genes, and a comprehensive characterization of GL-encoded susceptibility has not been undertaken in these diseases. Whole exome sequencing (WES) data sets obtained in the molecular discovery projects, allow for a targeted or unbiased analysis of inherited alterations to identify those with a pathogenic importance. To that end, initial WES studies, including published analyses from TCGA leukemia cohort, have focused mainly on somatic mutations. MDS is a disease of the elderly, typically presenting with complex phenotypes and clinical histories. Inherited predisposition, if present, would be expected to follow complex, non-mendelian genetic traits, have low penetrance and interact with various extrinsic factors such as exposures or coexisting conditions, and family histories may not be informative. Therefore, in this disorder predisposing GL alterations may be difficult to discern. Here, we have applied WES in patients with MDS and other myeloid neoplasms (N=117). We also investigated AML data set available through TCGA (N=201). In addition to defining somatic mutations in paired (tumor, GL DNA), we also performed an unbiased search for GL SNPs and mutations. Our strategy was comprehensive; firstly it focused on selection of non-synonymous and, possibly deleterious SNPs. Subsequently, putative candidates were further prioritized based on their genotypic frequency in the general population, and finally, according to perceived importance for the leukemogenesis. For the purpose of this study, we used a maximum minor allele frequency threshold of .05 in matched general population for alterations to be prioritized. As a result, we identified a large variety of new and, as expected, previously reported GL polymorphisms and mutations. Known pathogenic as well as novel GL alterations (42 non-synonymous sites, reported (N=33) and novel (N=9)) were found in telomerase genes (TERT, DKC1, GAR1, POT1, SMG6, NOP10, TINF2, NHP2, WRAP53) and other bone marrow failure genes (ELANE, GFI1, HAX1, GATA2, CSF3R, WAS) or many others. Subsequently, we hypothesized that there may also be GL variants of pathogenic significance in genes that are also frequently affected by somatic events. Known examples of such genes include SETBP1, NF1, CBL and many others. Such GL events may be directly disease-prone or indirectly predispose to somatic lesions within the same gene (as recently shown for JAK2 V617F mutation). Since the complexity of the entire analysis exceeds the boundaries of this abstract we focus here on exemplary results of genes most frequently affected by somatic mutations, including e.g., TET2 (8%), ASXL1 (15%), DNMT3A (26%), CEBPA (6%), TP53 (8%) and others. In this illustrative subset alone, 647 non-synonymous variants across 74 sites, in 14/16 genes of interest were found (FLT3, TET2 and TP53 were most frequently affected). Following, bio-analytic filtering we identified 30 rare variants, of which 11/30 were present at a significantly higher frequency compared to control population. Of these, 5/11 were located within such gene as ASXL1: S737N (p=.002), P1213R (p=.005), G543S (p=.023), L1286V (p=.026), L1216F (p=.036), 3/11 in FLT3: D324N (p 〈 .001), A912V (p=.002) ,F298L (p=.003), the remaining were in DNMT3A: R693H (p=.042), KIT: N293S (p=.019), and KRAS: C180X (p=.002). Of note is the FLT3 polymorphism, D324N, which has been previously linked to AML susceptibility. Excluding reported polymorphic sites, 33% of non-synonymous sites were not reported in population databases. Our data identified several GL variants that appear to associate with specific phenotypic features. For instance, a significantly decreased overall survival was seen in patients who had a RUNX1/RUNX1-IT (p=.03) GL mutation. Furthermore, patients diagnosed with MDS/MPN were found to have a greater odds of presenting with concurrent GL and somatic variants in TET2 gene compared to non-MDS/MPN patients [OR 7.4; 95% CI 2.5-21.8). In conclusion, our data suggests that both GL and somatic events in myeloid disorders are important for the pathogenesis of myeloid malignancies. They may interact with each other and with cytogenetic abnormalities, which can lead to deletion of protective alleles or amplification of disease-prone alleles. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia & MDS International Foundation: Research Funding.