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Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the sole curative therapy for myelodysplastic syndromes (MDS). However, whether bridging therapy (BRT) including azacitidine (AZA) and combination chemotherapy (CCT) prior to allo-SCT should be performed is unclear. We analyzed BRT and the outcomes of patients with myelodysplastic syndrome with excess blasts (MDS-EB) who were registered in a prospective observational study in order to clarify the optimal allo-SCT strategy for high-risk MDS. A total of 371 patients were included in this study. Among 188 patients (50.7%) who were considered for allo-SCT, 141 actually underwent allo-SCT. Among the patients who underwent allo-SCT, 64 received AZA, 29 received CCT and 26 underwent allo-SCT without BRT as an initial treatment. The multivariate analysis identified BRT as independent factors influencing overall survival (AZA vs. without BRT, hazard ratio [HR] 3.33, 95% confidence interval [95%CI] 1.44–7.70, P = 0.005; CCT vs. without BRT, HR 3.82, 95%CI 1.60–9.14, P = 0.003). In a multivariate analysis, BRT showed an independent association with progression-free survival (AZA vs. without BRT, HR 2.23, 95%CI 1.03–4.83, P = 0.041; CCT vs. without BRT, HR 2.94, 95%CI 1.29–6.69, P = 0.010). Transplant-eligible patients with MDS-EB should undergo upfront allo-SCT without BRT.

Intravascular large B-cell lymphoma (IVLBCL) is a very rare subtype of malignant lymphoma that is difficult to diagnose. Cases of myocardial infarction caused by IVLBCL are even rarer. Herein, we report a case presenting with heart failure and delayed enhancement in the hypokinetic cardiac septum on contrast-enhanced cardiac magnetic resonance imaging. Myocardial biopsy showed large B-cell lymphoma cells in the microvessels within the myocardium. To the best of our knowledge, this is the first report of imaging findings of cardiac involvement in IVLBCL.

Although some studies have suggested the effectiveness of hyperbaric oxygen (HBO) therapy for hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the role of HBO has not been established. We compared the treatment outcomes of 8 patients with viral HC (adenovirus [ADV], n = 2; BK virus [BKV], n = 6) treated with HBO (HBO[+]) and 8 patients (ADV, n = 2; BKV, n = 6) treated with conventional therapy (HBO[−]), such as urinary catheterization and intravenous cidofovir. HBO therapy was performed at 2.1 atmospheres for 90 min/day until clinical improvement was achieved. The median number of HBO treatments was 10 (range 8–12). The median duration of HBO treatment was 19.5 days (range 10–23 days). All 8 HBO(+) patients achieved complete remission (CR) at a median of 14.5 days (range 5–25 days). Of the 8 HBO(−) patients, 5 (62.5%) obtained CR and 3 remained symptomatic for 2–6 months. The cumulative incidence of transplant-related mortality at day 100 after allogeneic HSCT was significantly higher in the HBO(−) patients than in the HBO(+) patients (14.2 vs. 0%, P

The outcome of immunosuppressive therapy (IST) and prognosis in patients with aplastic anaemia (AA) secondary to chemotherapy or radiotherapy for cancers remains unknown. A total of 43 of 2559 patients with AA referred to our hospital had previously received chemoradiotherapy for various types of solid tumours (n = 25) or haematological malignancies (n = 18). Their cancer status was complete remission (CR) in 27, non-CR in 13, and unknown in three. Small populations of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] granulocytes were detected in 16 patients (37·2%). Of 18 patients who were treated with IST, 50% improved regardless of the presence of GPI(-) cells. The overall survival (OS) rate was significantly higher in patients with a history of solid tumours patients than in those of haematological malignancies (median OS, 87 vs. 11 months, P = 0·0003), and in patients treated with IST than in those of untreated patients (median OS, 115 vs. 20 months, P = 0·028). Cancer aggravation occurred in two of four patients who were treated with IST while in non-CR of their original cancers. Progression to myelodysplastic syndromes was observed in two patients not possessing GPI(-) cells. IST should thus be considered for patients with AA secondary to chemoradiotherapy for cancers, particularly when their original solid tumours are in CR.

The loss of killer cell Ig-like receptor ligands (KIR-Ls) due to the copy number–neutral loss of heterozygosity of chromosome 6p (6pLOH) in leukocytes of patients with acquired aplastic anemia (AA) may alter the susceptibility of the affected leukocytes to NK cell killing in vivo. We studied 408 AA patients, including 261 who were heterozygous for KIR-Ls, namely C1/C2 or Bw6/Bw4, for the presence of KIR-L–missing [KIR-L(−)] leukocytes. KIR-L(−) leukocytes were found in 14 (5.4%, C1 [n = 4], C2 [n = 3], and Bw4 [n = 7]) of the 261 patients, in whom corresponding KIR(+) licensed NK cells were detected. The incidence of 6pLOH in the 261 patients (18.0%) was comparable to that in 147 patients (13.6%) who were homozygous for KIR-L genes. The percentages of HLA-lacking granulocytes (0.8–50.3%, median 15.2%) in the total granulocytes of the patients with KIR-L(−) cells were significantly lower than those (1.2–99.4%, median 55.4%) in patients without KIR-L(−) cells. KIR2DS1 and KIR3DS1 were only possessed by three of the 14 patients, two of whom had C2/C2 leukocytes after losing C1 alleles. The expression of the KIR3DS1 ligand HLA-F was selectively lost on KIR-L(−) primitive hematopoietic stem cells derived from 6pLOH(+) induced pluripotent stem cells in one of the KIR3DS1(+) patients. These findings suggest that human NK cells are able to suppress the expansion of KIR-L(−) leukocytes but are unable to eliminate them partly due to the lack of activating KIRs on NK cells and the low HLA-F expression level on hematopoietic stem cells in AA patients.

Clonal hematopoiesis by hematopoietic stem progenitor cells (HSPCs) that lack an HLA class I allele (HLA − HSPCs) is common in patients with acquired aplastic anemia (AA); however, it remains unknown whether the cytotoxic T lymphocyte (CTL) attack that allows for survival of HLA − HSPCs is directed at nonmutated HSPCs or HSPCs with somatic mutations or how escaped HLA − HSPC clones support sustained hematopoiesis. We investigated the presence of somatic mutations in HLA − granulocytes obtained from 15 AA patients in long-term remission (median, 13 years; range, 2-30 years). Targeted sequencing of HLA − granulocytes revealed somatic mutations ( DNMT3A , n = 2; TET2 , ZRSR2 , and CBL , n = 1) in 3 elderly patients between 79 and 92 years of age, but not in 12 other patients aged 27 to 74 years (median, 51.5 years). The chronological and clonogenic analyses of the 3 cases revealed that ZRSR2 mutation in 1 case, which occurred in an HLA − HSPC with a DNMT3A mutation, was the only mutation associated with expansion of the HSPC clone. Whole-exome sequencing of the sorted HLA − granulocytes confirmed the absence of any driver mutations in 5 patients who had a particularly large loss of heterozygosity in chromosome 6p (6pLOH) clone size. Flow–fluorescence in situ hybridization analyses of sorted HLA + and HLA − granulocytes showed no telomere attrition in HLA − granulocytes. The findings suggest that HLA − HSPC clones that escape CTL attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations.

Hematopoietic stem cells (HSCs) that lack HLA-class I alleles as a result of copy-number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations often constitute hematopoiesis in patients with acquired aplastic anemia (AA), but the precise mechanisms underlying clonal hematopoiesis induced by these HLA-lacking (HLA-) HSCs remain unknown. To address this issue, we generated induced pluripotent stem cells (iPSCs) from an AA patient who possessed HLA-B4002-lacking (B4002-) leukocytes. Three different iPSC clones (wild-type [WT], 6pLOH+, and B*40:02-mutant) were established from the patient's monocytes. Three-week cultures of the iPSCs in the presence of various growth factors produced hematopoietic cells that make up 50% to 70% of the CD34+ cells of each phenotype. When 106 iPSC-derived CD34+ (iCD34+) cells with the 3 different genotypes were injected into the femoral bone of C57BL/6.Rag2 mice, 2.1% to 7.3% human multilineage CD45+ cells of each HLA phenotype were detected in the bone marrow, spleen, and peripheral blood of the mice at 9 to 12 weeks after the injection, with no significant difference in the human:mouse chimerism ratio among the 3 groups. Stimulation of the patient's CD8+ T cells with the WT iCD34+ cells generated a cytotoxic T lymphocyte (CTL) line capable of killing WT iCD34+ cells but not B4002- iCD34+ cells. These data suggest that B4002- iCD34+ cells show a repopulating ability similar to that of WT iCD34+ cells when autologous T cells are absent and CTL precursors capable of selectively killing WT HSCs are present in the patient's peripheral blood.

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT) and often requires stem cell infusion from the same donor when the patient fails to respond to conventional therapies. We screened the peripheral blood (PB) of 14 patients who developed donor-type LGF at 2 to 132 months after allo-SCT for the presence of the markers for immune-mediated bone marrow (BM) failure. Increased glycosylphosphatidyl inositol-anchored protein-deficient (GPI-AP-) leukocytes, which accounted for .009% to 0.147% of the total granulocytes, were detected in 5 patients (severe aplastic anemia, n = 2; follicular lymphoma, n = 1; acute lymphoblastic leukemia, n = 1; myelodysplastic syndromes; n = 1) and 4.7% to 81.2% HLA-allele–lacking leukocytes (HLA-LLs) were detected in 2 patients (acute myelogenous leukemia, n = 1; and myelodysplastic syndromes, n = 1). Three of the 5 patients with increased GPI-AP- leukocytes were treated with antithymocyte globulin (ATG), and 2 patients achieved transfusion independence. These results suggest that immune mechanisms that are similar to acquired aplastic anemia underlie condition of approximately one-half of the patients with donor-type LGF, and that in patients with increased GPI-AP- cells, donor-derived hematopoiesis may be restored by ATG therapy alone without donor stem cell infusion.

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the sole curative therapy for myelodysplastic syndromes (MDS). Several studies have addressed the efficacy of bridging therapy before allo-SCT including hypomethylating agents, combination chemotherapy (e.g. induction chemotherapy regimen for acute myeloid leukemia [AML]), and low-dose chemotherapy. However, these studies were retrospective, so selection bias was unavoidable, and the optimal bridging therapy prior to allo-SCT remains unclear. We analyzed the bridging therapy and outcomes of patients with MDS registered in a prospective observational study, the JALSG-AML/MDS/CMML Clinical Observational Study-11 (CS-11). Methods: We studied 393 patients with MDS-refractory anemia with excess blasts (MDS-EB) ≤70 years old at registration for CS-11 between 2011 and 2016. We collected new data using an online survey software program (Survey Monkey ®) and analyzed these data following their integration into the CS-11 data. This study was approved by the ethical committee of Kanazawa University (No. 2018-227) and all participating institutions. Results: A total of 371 patients (94.4%) had additional data available and who were included in this study. The median age of the patients at registration of CS-11 was 64 (range: 16-70) years, and 279 (75.2%) were male. The patients' cytogenetic subgroups were divided into very good (n=3 [0.8%]), good (n=117 [31.5%]), intermediate (n=60 [16.2%]), poor (n=35 [9.4%]), very poor (n=148 [39.9%]), and missing data (n=8 [2.1%]), according to Revised International Prognostic Scoring System (IPSS-R). A total of 188 patients (50.7%) were considered for allo-SCT at the diagnosis of MDS-EB, and 141 patients (75.0%) underwent allo-SCT, whereas 181 (48.9%) patients were not considered for allo-SCT at their diagnosis. The overall survival (OS) of the patients considered for allo-SCT at their diagnosis was significantly higher than that of the patients who were not considered for allo-SCT (3-year OS, 33.7% vs. 13.9%, P Disclosures Usuki: MSD K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Kyowa-Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon-Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Amgen-Astellas Biopharma K.K.: Research Funding; Mundipharma K.K.: Research Funding; Nippon-Boehringer-Ingelheim Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Celgene K.K.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Research Funding; Ono Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Research Funding; AbbVie GK: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Research Funding, Speakers Bureau; Yakult Honsha Co., Ltd.: Research Funding, Speakers Bureau; Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Apellis Pharmaceuticals, Inc.: Research Funding; Incyte Biosciences Japan G.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sanofi K.K.: Speakers Bureau; Amgen K.K.: Research Funding. Handa: 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; Shionogi: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding. Fujisawa: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding. Suehiro: Amgen BioPharma: Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Incyte: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Nippon Shinyaku: Honoraria; Novartis: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding. Maeda: Bayer Yakuhin, Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Novartis Pharmaceuticals: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding. 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. Kiyoi: Astellas: Honoraria; celgene: Honoraria; Daiichi Sankyo: Honoraria; Dainippon Sumitomo: Honoraria; Eisai: Honoraria; Fijifilm: Honoraria; Kyowa Kirin: Honoraria; Otsuka: Honoraria; Perseus Proteomics: Honoraria; Pfizer: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Zenyaku Kogyo: Honoraria. Matsumura: Ono: Research Funding; Otsuka: Consultancy, Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Shionogi: Research Funding; Taiho: Research Funding; Takeda: Research Funding; Sumitomo Dainippon: Research Funding; Nihon Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding, Speakers Bureau; Japan Blood Products Organization: Research Funding; Mundipharma: Research Funding; AYUMI Pharmaceutical: Research Funding; Eli Lilly Japan: Research Funding; Novartis: Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding; MSD: Research Funding; Mitsubishi Tanabe: Research Funding; Amgen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Kyowa Kirin: Research Funding; Eisai: Research Funding; Chugai: Research Funding; Astellas: Speakers Bureau; Asahi Kasei: Research Funding; Addvie: Research Funding. Miyazaki: Pfizer: Honoraria; Kyowa-Kirin: Honoraria; Abbvie: Honoraria; Bristol-Myers Squibb: Honoraria; Nippon-Shinyaku: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Sumitomo-Dainippon: Honoraria, Research Funding; Janssen: Honoraria; Eisai: Honoraria; Daiichi-Sankyo: Honoraria; Takeda: Honoraria; Chugai: Honoraria; Sanofi: Honoraria.

In immune-mediated acquired aplastic anemia (AA), the presence of an HLA allele, which is highly overrepresented or lost due to somatic mutations, may represent a specific immune pathophysiology and a clinical manifestation. HLA-B*14:02 is one of the most overrepresented class I alleles in AA and is also frequently affected by a somatic loss of expression; the inherited B*14:02 genotype was correlated with high-risk clonal evolution in two independent cohorts in the U.S. (Babushok DV et al. Blood Adv 2017; Zaimoku Y et al. manuscript in preparation). In contrast, HLA-B*14:02 is virtually absent in Japanese, in whom somatic mutations of AA have frequently been detected in HLA-B*40:02, B*54:01, and A*02:06, and occasionally in A*02:01, A*02:07, A*31:01, B*13:01, B*40:01, B*40:03, B*44:03, B*55:02, and B*56:01 (Mizumaki H et al. Haematologica 2021). A class II allele HLA-DRB1*15 is highly overrepresented in AA across various ethnic groups, including those in the U.S. and Japanese. This retrospective study in the Japanese population aimed to explore the clinical significance of disease-associated non-B*14:02 HLA class I and II alleles in AA. A total of 423 enrolled patients with AA (very severe [n = 81], severe [n = 266], transfusion dependent non-severe [n = 76]; median age 60 [range, 1-86] years) had undergone genotyping for HLA-A, HLA-B, HLA-C, and HLA-DRB1 at 2-field resolution. The HLA allele frequencies in these patients were compared to those in a Japanese HLA haplotype dataset (n = 19183; Ikeda N et al. Tissue Antigens 2016). The most overrepresented allele in AA was HLA-DRB1*15:02, followed by DRB1*15:01, B*40:02, and A*02:06 (Table); DRB1*13:02 and B*44:03, which are in linkage disequilibrium, were markedly underrepresented, consistent with a well-known protective role of DRB1*13 against autoimmune diseases. HLA-DRB1*15:02 was also significantly correlated with age and its frequency among patients aged The overall response rate to anti-thymocyte globulin-based immunosuppressive therapy at 6 months was 63% (139 of 220 treated and evaluable patients). A trend for a higher response was observed in patients harboring mutation-related HLA-B alleles (except for minor alleles B*13:01, B*40:03, and B*55:02) and the highly overrepresented or protective HLA-DRB1 alleles, but not in the HLA-A alleles (Figure D). A multivariate logistic regression revealed that the combination of the presence of any favorable alleles in HLA-B (odds ratio 3.6, P < 0.0001) or in HLA-DRB1 (odds ratio 2.3, P = 0.00085) was significantly and independently associated with a hematologic response; the tendencies for a lower or higher response in very severe disease and the presence of paroxysmal nocturnal hemoglobinuria clone did not reach statistical significance. Further, there was likely an additive effect when two favorable alleles coexisted in HLA-B or HLA-DRB1 (Figure E); the copy number of the favorable HLA-B and HLA-DRB1 alleles stratified the response rate to four groups: three or four copies, 95% (19 of 20); two copies, 72% (61 of 85); one copy, 59% (50 of 85); and zero copy, 30% (9 of 30). Only eight patients displayed clonal evolution to monosomy 7, myelodysplastic syndrome, or acute myeloid leukemia after immunosuppression without significant overrepresentation or underrepresentation of the pathogenic HLA alleles. Using a large dataset of homogeneous Japanese population with high-resolution HLA typing, we revealed, for the first time, a strong relationship between disease-associated (overrepresented, inactivated, or protecting) HLA alleles and the responsiveness to immunosuppressive therapy. Figure 1 Figure 1. Disclosures Takamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; SRL: Consultancy; Adaptive Biotechnologies, Eisai: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Honoraria; Kyowa Kirin: Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

A 48-year-old male underwent an osteosynthesis surgery for right patellar fracture without bleeding episodes around the surgery. After 7 months, he presented with a bleeding episode after a nail extraction surgery from his knee joint. He was diagnosed with mild hemophilia A after his second surgery. The patient's clinical course suggested that he had mild hemophilia A, although he had a past surgical history without any bleeding episodes. Early diagnosis is important in patients with mild hemophilia A because bleeding episodes complicated with surgery can be prevented by the administration of prophylactic replacement therapy.

Supplemental Digital Content is available in the text., Background. IVIG is occasionally used for preventing and treating severe infections of patients who are to undergo transplantation. Administration of IVIG, which includes high-titer antibodies (Abs) against HLA class I and II, might have a substantial influence on the HLA Ab test results of these patients. However, this issue has remained unreported. Methods. Anti-HLA Ab titers were determined in 4 types of IVIG preparations, fresh frozen plasma, and the sera of 11 patients with hematological diseases before and after IVIG administration. Results. Although anti-HLA Abs were not detected in any of the fresh frozen plasma products, various anti-HLA class I and II Abs were detected in all 4 IVIG preparations. Six out of 11 patients who had received IVIG showed a low titer of anti-HLA class II Abs, which were not detected before IVIG administration. Conversely, no anti-HLA class I Abs were detected in any of the 11 patients. Furthermore, all 4 (100%) patients who were positive for anti-HLA class II Abs initially and were assessable became negative for anti-HLA Abs after the discontinuation of IVIG treatment (median, d 79; range, d 22–192). Conclusions. IVIG preparations consist of high-titer anti-HLA class I and II Abs, but the latter can be transiently detected in the sera of patients who had received IVIG. When these patients are screened for the presence of donor-specific Abs, some may be incorrectly deemed positive for HLA class II Abs. Thus, caution is necessary when only donor-specific Abs specific to class II HLAs are detected in patients.

Hematopoietic stem cells (HSCs) that lack HLA-class I alleles as a result of copy-number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations often constitute hematopoiesis in patients with acquired aplastic anemia (AA), but the precise mechanisms underlying clonal hematopoiesis induced by these HLA-lacking (HLA

Acute myelogenous leukemia (AML) is a clinically heterogeneous disease that is frequently associated with relapse and a poor prognosis. Among the various subtypes, AML with the monosomal karyotype (AML-MK) has an extremely unfavorable prognosis. We performed screening to identify antitumor compounds that are capable of inducing apoptosis in primary leukemia cells harboring the AML-MK karyotype and identified a naturally occurring stilbene, Gnetin-C, with potent anti-tumor activities against AML cells from patients with various cytogenetic abnormalities, including patients with the AML-MK karyotype. Gnetin-C simultaneously inhibits the ERK1/2 and the AKT/mTOR pathways, two signals that are essential for the survival of leukemia cells. A combination of Gnetin-C with low doses of chemotherapeutic drugs led to synergistic anti-tumor effects against AML cells. In an immunodeficient mouse model of human leukemia, Gnetin-C attenuated the formation of leukemia, depleted leukemia cells and improved survival. These findings suggest that Gnetin-C has antitumor activities in AML and supports the therapeutic potential of blocking two different pathways in AML.

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria.

To identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[-]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002-) using a new monoclonal antibody specific to this allele, B4002- granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(-) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002- granulocytes that retained the HLA-A allele on the same haplotype (B4002-A+), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002-A-) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002-A+ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002+ granulocytes of 3 patients possessing B4002- granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.

Among the various manifestations of colonic involvement in malignant lymphomas, an ulcerative colitis-like appearance is rare. Herein, we describe a case in which extranodal colonic involvement resembled ulcerative colitis in a patient with advanced follicular lymphoma. A 59-year-old Japanese man with diarrhea and body weight loss was referred to our hospital. Computed tomography (CT) revealed systemic lymphadenopathy, splenomegaly, and thickening of the sigmoid colon and rectum walls. 18F-fluorodeoxy-glucose positron emission tomography (18F-FDG-PET/CT) revealed intense tubular 18F-FDG accumulation extending from the rectum to the colon at the hepatic flexure and much weaker accumulation in the systemic lymph nodes, bone marrow, and spleen. The isotope-enriched areas had an ulcerative colitis-like appearance as shown via colonoscopy. The patient was ultimately diagnosed with follicular lymphoma (stage IV A, grade 1) based on a pathological examination of the neck lymph nodes, iliac bone marrow, and colon. After six courses of chemotherapy (R-CHOP), 18F-FDG-PET/CT confirmed complete remission of the lymphoma including the colonic lesion. This is presumably the first case of ulcerative-like colitis caused by a follicular lymphoma. As a novel approach, the lymphoma-related colitis was detected by comparing the pathology results and the 18F-FDG-PET/CT results.

Langerhans cell sarcoma (LCS) is an extremely rare neoplasm of Langerhans cell origin characterized by systemic involvement and a poor prognosis. There are, however, few reports of LCS with central nervous system involvement. We experienced a patient with LCS recurrence in the brain that appeared during systemic chemotherapy. The brains lesions eventually responded to total cranial irradiation. A 60-year-old female presented with systemic lymphadenopathy. LCS was diagnosed based on neck lymph node biopsy findings. Two cycles of ESHAP induced marked regression of her lymphadenopathy, but FDG-PET/CT scan revealed new lesions in the central nervous system and her disorientation gradually worsened. We administered 37.5 Gy of total cranial irradiation which improved her consciousness and shrank the brain tumors as demonstrated by MRI. The patient's clinical course indicates that radiation therapy may be effective for central nervous system involvement of LCS even if the lesion is resistant to systemic chemotherapy.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that CTLs are involved in the development of AA, but the precise mechanisms underlying HLA missing and clonal hematopoiesis by such HLA(-) HSPCs are unknown. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in Japanese AA patients. The study also showed that B*54:01 was one of three HLA-alleles that were most likely to be possessed by 6pLOH(+) patients (29% [5/17]) when only patients not carrying HLA-B*40:02 were analyzed. These results prompted us to study the role of HLA-B*54:01 in the pathogenesis of AA in a larger number of patients. [Method] To identify HLA class I alleles other than HLA-B*40:02 that are closely involved in the auto-antigen presentation in AA, we studied leukocytes of 549 AA patients for the presence of 6pLOH as well as HLA alleles that are lost due to 6pLOH. To gain insight into the mechanism underlying clonal hematopoiesis by HLA-B*54:01-lacking HSPCs, we studied HSPCs derived from induced pluripotent stem cells (iPSCs) that were generated from an AA patient possessing B5401-lacking monocytes. We also investigated the association between male AA patients possessing B*54:01 and CAG microsatellites of androgen receptor (AR) gene which are related to transactivation of the AR gene. [Results] 6pLOH was detected in 91 (16.6%) of the total patients and in 48 (10.4%) of the 462 patients not possessing B*40:02. Among the HLA alleles possessed by the 48 patients, B*54:01 was the most frequent (23%). 6pLOH was detected in 17 (34%) of 50 patients possessing B*54:01, and the incidence was markedly higher in males (15/24, 62.5%) than in female patients (2/26, 7.7%, P Disclosures Yoshida: Nihon-shinyaku: Research Funding. Akashi:Novartis pharma: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MSD: Research Funding; Eisai: Research Funding; Asahi-kasei: Research Funding; Ono Pharmaceutical: Research Funding; Pfizer: Research Funding; Celgene: Research Funding, Speakers Bureau; sanofi: Research Funding; Chugai Pharma: Research Funding; Otsuka Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Taiho Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may play an important role in the regulation of the HSPC commitment, given the fact that a lack of GPI-APs due to PIGA mutations allows HSPCs to preferentially undergo commitment into mature blood cells under immune pressure against HSPCs in patients with acquired aplastic anemia. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs has not been clarified. TF-1 is one of a few myeloid leukemia cell lines that express CD109, the proliferation of which is dependent on GM-CSF. Since TF-1 undergoes erythroid differentiation in response to δ-5-aminolevulinic acid (δ-ALA), and its differentiation is reportedly inhibited by TGF-β, a lack of GPI-APs due to PIGA mutation and/or the knockout (KO) of CD109 may affect the differentiation of TF-1 cells. [Objectives/Methods] To gain insights into the role of GPI-APs on HSPCs, we established a PIGA-mutated TF-1 cell line by culturing TF-1 in the presence of α-toxin for several months, and a CD109 KO TF-1 cell line using a CRISPR-Cas 9 system. The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) on TF-1 cells using flow cytometry (FCM) and iron staining. We also determined the CD109 expression by HSPCs from healthy individuals and C57BL/6 mice using FCM and a quantitative PCR. [Results] Both GPI-AP-deficient TF-1 cells that had a PIGA mutation (7 nucleotide deletion at position 291-297 [TTGTCAC] in exon 2) and CD109 KO TF-1 cells showed slower proliferation than wild-type (WT) TF-1 cells. Similarly to TF-1 cells treated with δ-ALA, both mutant cells expressed GPA, exhibited erythroid morphology, and were positive for iron granules, suggesting that GPI-APs inhibited the erythroid differentiation of WT TF-1 cells that were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), and that the GPI-AP that plays a key role in the inhibition of erythroid differentiation is CD109. Since low levels (1-2 ng/ml) of TGF-β in the serum-containing culture medium were suspected to inhibit the erythroid differentiation of WT TF-1 through its binding to CD109, WT TF-1 cells were cultured in a serum-free medium Expi293 Expression Medium for 10 days. While control TF-1 cells cultured in the serum-containing RPMI1640 were negative for the expression of GPA, 77.0-84.5% of the cultured TF-1 cells expressed GPA and exhibited erythroid morphology. CD109 was expressed by 12.1-18.3% of CD34+CD38- cells, 4.5-7.4% of common myeloid progenitor cells (CMPs), 20.8-42.4% of megakaryocyte-erythrocyte progenitor cells (MEPs), and 14.2-22.0% of granulocyte macrophage progenitor cells (GMPs) in the bone marrow of healthy individuals, while murine CD48-CD150+CD34- LSK cells were negative for either CD109 protein or mRNA. [Conclusions] CD109 protects TF-1 cells from differentiating into erythroid cells in serum-containing culture. In contrast to keratinocytes, the CD109 on TF-1 cells, and possibly on HSPCs, may enhance TGF-β signaling, and the lack of the GPI-AP might make PIGA-mutated HSPCs insensitive to TGF-β, leading to the preferential commitment of mutant HSPCs to mature blood cells in immune-mediated bone marrow failure. Disclosures Nakao: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

[Background] The frequent loss of heterozygosity of the HLA haplotype in the short arm of chromosome 6 (6pLOH) in leukocytes is thought to offer compelling evidence of cytotoxic T cell (CTL) involvement in the development of acquired aplastic anemia (AA) because it represents the escape of hematopoietic stem/progenitor cells (HSPCs) with 6pLOH from the attack of CTLs that are specific to autoantigens presented by the lacked HLA class I allele. Although our previous study suggested that HLA-B*40:02 is the major allele involved in this phenomenon, the exact role of B*40:02 remained unclear because 6pLOH involving this allele is always associated with a lack of HLA-A and C alleles in the haplotype, and the presence of B*40:02-missing leukocytes were unable to be shown due to the lack of monoclonal antibodies (mAbs) specific to B61, the HLA-B antigen that corresponds to B*40:02. We recently succeeded in generating a mAb specific for HLA-B61 that enabled us to explore the role of B*40:02 in the development of AA. [Methods] Using the new mAb, we examined peripheral blood samples of 28 AA (12 with 6pLOH and 16 without 6pLOH) patients carrying this allele for the presence of B61(-) leukocytes using flow cytometry. HLA genes were enriched by sequence capture, a hybridization-based gene enrichment method, from genomic DNA of sorted B61(-) granulocytes, and were subjected to deep sequencing using an NGS (MiSeq). B61(+) granulocytes or T cells were used as controls. Potential mutations responsible for the B61-missing were identified when 10 or more variant reads were found only in B61(-) granulocytes. Thereafter, HLA-B alleles carrying those mutations were determined taking advantage of the nearest allele-specific SNPs. [Results] Among the 12 6pLOH(+) patients, 10 (83%) possessed 0.5%-60% B61-missing granulocytes that were not lacking HLA-A, in addition to 12% to 99% 6pLOH(+) granulocytes that lacked both B61 and an HLA-A allele on the same haplotype (Figure 1). B61(-) granulocytes that accounted for 0.5%-99% of the total granulocytes were detected in 9 (56%) of the 16 6pLOH(-) patients. The prevalence of missing B61 in the 28 AA patients was 21/28 (75%), much more frequent than those of the 3 other major alleles (A*02:01, 32%; A*02:06, 30%; A*24:02, 6%). B61(-) granulocytes were available for mutation analyses of HLA-B alleles in 15 of the 19 patients who possessed B61(-) granulocytes. The mean coverage of HLA-B gene was 426x. In total, 43 somatic mutations of HLA-B were identified in B61(-) granulocytes, all of which were present in B*40:02 but not in any of the other HLA-B alleles. Median variant allele frequency was 4.8% (range, 1.0% - 43%) and the number of mutations in each patient was 1 to 6 (Figure 2). Thirty-nine mutations were exonic while 4 were intronic. Exonic mutations included frameshift insertions (n=12), frameshift deletions (n=16), non-frameshift deletions (n=2), nonsense mutations (n=7), a missense mutation (n=1) and a start codon mutation (n=1). All four intronic mutations were considered to be a splice site mutation; two mutations deactivated 5' and 3' splice sites, whereas the other two were single base substitutions within intron 3, making alternative 5' splicing site with strong consensus sequence: GGC [A>G] TGAGT and TTC [C>G] TGAGT. Surprisingly, missense mutations in the alpha-2 and alpha-3 chain-coding region of HLA-B*40:02 were detected exclusively in the B61(+) granulocytes of two patients possessing B61(-) granulocytes, suggesting the inability of the mutant HSPCs to interact with CTLs. Variant allele frequencies of the two missense mutation were 40% and 45%, respectively. As a result of the mutation, virtually all granulocytes of the two patients were affected by B*40:02 mutations that allowed the HSPCs to escape the T cell attack. [Conclusions] The markedly high prevalence of leukocytes lacking HLA-B*40:02 as a result of either or both 6pLOH or structural gene mutations clearly indicates that antigen presentation by HSPCs to CTLs via the HLA-B allele plays a critical role in the pathogenesis of AA. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

[Background] Clonal hematopoiesis is currently known to be common in patients with acquired aplastic anemia (AA). One of the most common abnormalities underlying clonal hematopoiesis in AA patients is copy-number neutral loss of heterozygosity (LOH) in the short of 6 chromosome (6pLOH) caused by acquired uniparental disomy. Hematopoietic stem/progenitor cells (HSPCs) having undergone 6pLOH are thought to evade attack by cytotoxic T lymphocytes (CTLs) specific to auto-antigens by lacking particular HLA-A alleles. These HSPCs then produce HLA class I allele-lacking [HLA(-)] leukocytes to support hematopoiesis in patients with AA patients in remission. Our recent study showed that HLA(-) granulocytes are detected in about 24% of newly-diagnosed AA patients, and the aberrant granulocytes often account for more than 95% of the total granulocytes and persist for many years. The sustainability of 6pLOH(+) HSPC clones suggests that these HSPCs may suffer from secondary somatic mutations that confer a proliferative advantage on them over normal HSPCs. Alternatively, 6pLOH(+) HSPCs may persist and continue to support hematopoiesis according to their inherent sustainability, just like the PIGA mutant HSPCs we previously described (Katagiri et al. Stem Cells, 2013). To test these hypotheses, we determined the sequences of genes associated with the clonal expansion of HSPCs in HLA(-) granulocytes. [Patients and Methods] Eleven AA patients whose percentages of HLA(-) granulocytes ranged 6.4%-99.8% (median 94.2%) of the total granulocyte population were chosen for this study. The patients (male/female, 5/6 and age 27-79 [median 53] years) had been diagnosed with severe (n=5) or non-severe (n=6) AA 2-25 [median 12.5] years earlier, and 7 and 4 patients achieved complete response and partial response, respectively after treatments with cyclosporine (CsA) alone (n=4), CsA+antithymocyte globulin (ATG, n=3), CsA+anabolic steroids (AS, n=2), AS+romiplostim (n=1), and AS alone (n=1). The lineage combinations of HLA(-) cells were granulocyte, monocytes, B cells and T cells (GMBT) in 6, GMB in 4 and GM in 1. HLA(-) and normal [HLA(+)] granulocytes were sorted from the blood leukocytes of the 11 patients and the DNA of each cell population as well as that of buccal mucosa cells was subjected to target sequencing of 61 myelodysplastic syndrome (MDS)-related genes with MiSeq. DNA samples from 5 patients including 4 patients whose HLA(-) cell percentages were greater than 95% were further analyzed by whole-exome sequencing (WES) using HiSeq. The percentage of 6pLOH(+) cells in the total granulocytes or sorted HLA(-) granulocytes were estimated using digital droplet PCR or deep sequencing of HLA alleles. [Results] Target sequencing of 8 of the 11 patients revealed somatic mutations in the HLA(-) granulocytes of 3 patients. HLA(-) granulocytes-specific mutations were found in DNMT3A, PRR5L, SMC3A, and LRCH1 (Table). The variant allele frequencies (VAF) of these mutations were far lower (5.1%-20%) than those of HLA(-) granulocytes that accounted for 95% of sorted cells. WES revealed 22 non-synonymous and 9 synonymous mutations in the HLA(-) granulocytes from 4 of the 5 patientsthat included 3 new patients and 2 patients whose samples were negative for mutations revealed by the target sequencing. The VAF of these mutations ranged from 20.7-52.5% (median 44.1%, Table). Very-high VAFs of several mutant genes suggested that these mutations occurred simultaneously with or soon after the occurrence of 6pLOH. A patient who achieved remission after romiplostim therapy without ATG showed various gene mutations that were thought to have occurred after 6pLOH. Despite of their highly biased hematopoiesis supported by single or few clones, recurrent or MDS-related oncogenic mutations were not detected in any of the 11 patients. Of note, the percentages of 6pLOH(+) cells in the sorted HLA(-) granulocytes were ≤75% (36.7%, 46%, 74%, and 75%) in 4 patients, indicating the presence of granulocytes lacking HLA-A alleles through mechanisms other than 6pLOH. [Conclusions] HLA(-) HSPCs caused by 6pLOH or other unknown mechanisms support long-term hematopoiesis without the development of oncogenic driver mutations that are associated with clonal hematopoiesis of MDS; as such, clonal hematopoiesis by 6pLOH(+) HSPCs may not portend a poor prognosis. Disclosures Nakao: Alexion Pharmaceuticals: Honoraria, Research Funding.

[Background] An increase in the numbers of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] blood cells is often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS), and is associated with good response of their bone marrow (BM) failure to immunosuppressive therapy. Although some immune mechanisms are thought to play a role in the preferential commitment of hematopoietic stem/progenitor cells (HSPCs) with PIGA mutations in such BM failure patients, the exact mechanisms are unknown. Our previous studies suggested that GPI(-)T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) were less susceptible to TGF-ƒÀ-mediated inhibition of proliferation triggered by anti-CD3 and anti-CD28 antibodies than GPI(+)T cells of the same patient. The lower sensitivity of PIGA mutant HSPCs to TGF-ƒÀ, a cytokine capable of inhibiting the cell cycling of dormant HSPCs, than GPI(+) HSPCs may also explain the preferential commitment of GPI(-) HSPCs in immune-mediated BM failure. However, little is known about the GPI-APs that affect the sensitivity of HSPCs to TGF-ƒÀ. [Objectives/Methods] We assessed the roles of GPI-APs in the signal transduction of CD34(+) cells of a PNH patient and the myeloid leukemia cell line TF-1 in response to TGF-ƒÀ. We also assessed the TGF-ƒÀ-mediated inhibition of cell proliferation in TF-1 cells with or without a PIGA mutation. CD109, a GPI-AP that serves as a TGF-ƒÀ co-receptor in human keratinocytes, of TF-1 cells, was knocked out from TF-1 cells using a CRISPR-Cas 9 system, and the sensitivity to TGF-ƒÀ was compared between CD109(+) and CD109(-) TF-1 cells. [Results] The treatment of BM mononuclear cells from a florid PNH patient with TGF-ƒÀ induced SMAD2 phosphorylation in GPI(-) CD34(+) cells to a lesser degree than in GPI(+) CD34(+) cells (fold increase in pSMAD2 MFI, 1.0 vs. 2.6, Figure 1). TGF inhibited PIGA-mutant TF-1 (PNH-TF-1) proliferation to a lesser degree (percentage of inhibition, 19%}13%) than wild-type TF-1 cells (67%}3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-ƒÀ (53%}10% vs. 19%}13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-ƒÀ in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly CD34+CD38+CD123-CD45RA- megakaryocyte-erythroid progenitor cells, as well as on TF-1 cells. The pSMAD2 induction in CD109(-) TF-1 cells by TGF-ƒÀ was less pronounced (relative increase in pSMAD2 MFI, 7.65}2.15 vs. 10.74}2.28) than that in CD109(+) TF-1 cells (Figure 2). [Conclusions] CD109 deficiency is involved in the lower sensitivity of GPI(-) HSPCs to TGF-ƒÀ than GPI(+) HSPCs. This deficiency may account for the preferential activation of PIGA mutant HSPCs in immune-mediated BM failure, in which TGF-ƒÀ suppresses activation of wild-type HSPCs. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

A T-cell attack against hematopoietic stem cells (HSCs) is believed to be the principal mechanism underlying the development of acquired aplastic anemia (AA). The presence of leukocytes that lack HLA class I alleles as a result of the copy number-neutral loss of heterozygosity of the HLA haplotype due to uniparental disomy in the short arm of chromosome 6 (6pLOH) is compelling evidence for the involvement of cytotoxic T lymphocytes (CTLs) in the HSC destruction; this is based on the high response rate to immunosuppressive therapy (IST) in 6pLOH(+) patients (Katagiri, et al. Blood, 2011). However, target cells of the putative CTLs have not yet been characterized because of the small number of patients analyzed via flow cytometry (FCM) in our previous study. FCM can be substituted for SNP arrays by detecting HLA-A allele-lacking leukocytes (HLA-LLs) caused by 6pLOH. To gain insight into the CTL target responsible for the development of AA, we examined a total of 223 patients (213 idiopathic and 10 hepatitis-associated AA; 93 severe and 130 non-severe AA) for the presence of HLA-LLs and determined the lineage combinations of the aberrant leukocyte population. Of the 223 patients, 145 (65.0%) were heterozygous for the HLA-A allele and could be assessed for the presence of HLA-LLs by FCM. Eighteen (25.4%; 10 with severe AA and 8 with non-severe AA) of the 71 pre-treatment patients, and 26 (35.1%; 13 with severe AA and 13 with non-severe AA) of the 74 post-treatment patients were found to be positive for HLA-LLs. The lineage combinations of HLA-LLs in the 44 HLA-LL(+) patients were granulocytes (Gs), monocytes (Ms), B cells (Bs), and T cells (Ts, GMBT) in 13, GMB in 16 and GM in 11 patients. Surprisingly, HLA-LLs were found in Bs alone in three patients, and in one patient, the lineage combination pattern was TB (Figure). The presence of 6pLOH was confirmed via deep sequencing of isolated Bs from one of the three Bs alone patients. These lineage combination patterns were not observed to change for 1-40 months in 22 of 23 patients whose blood samples were available for follow-up analyses. In one patient with the GMB pattern, HLA-LLs decreased from 11.7% before treatment to 0% after 12 months of ATG therapy. The response rate to IST in GMBT patients (3/4, 75%) and in patients with GMB or GM (9/10, 90%) were similarly higher than in patients without HLA-LLs (22/39, 56%) or in patients who were homozygous for the HLA-A allele (23/36, 64%). Two of the three Bs alone patients showed complete responses at the time of sampling three years after ATG therapy and 20 years after CsA therapy, and another patient had a secondary myelodysplastic syndrome two years after response to ATG. The TB alone patient developed AA 20 years earlier, but had not been treated until recently because there was no need for blood transfusions, and is now improving in response to eltrombapag. This study revealed that the targets of putative CTLs in more than half of AA patients are hematopoietic progenitor cells with limited differentiation but long-lasting capacity, and in some patients, they are lymphoid progenitor cells that do not contribute to hematopoiesis. This suggests that CTL attack against non-HSCs including lymphoid precursors could trigger BM failure. Consistent with our previous report, the bystander effects caused by the immune response to non-HSCs such as myelosuppressive cytokines may play a major role in the development of AA. Disclosures No relevant conflicts of interest to declare.

Background: Acquired aplastic anemia (AA) is occasionally preceded or complicated by the other autoimmune diseases such as rheumatoid arthritis and ulcerative colitis (UC), but the pathogenetic links between these diseases remain unknown. A number of case reports attributed the development of AA in UC patients to mesalazine that had been administered before the onset of AA. However, some UC patients who were referred to our clinical because of "mesalazine-induced AA" had increased percentages of glycosylphosphatidylinositol-anchored protein-deficient (PNH-type) cells that serve as a marker for immune-mediated bone marrow (BM) failure. This suggested an immune pathophysiology of UC-associated AA similar to that of idiopathic AA. Methods: To address this issue, we analyzed clinical and laboratory data including HLA-DRB1 alleles and PNH-type cells of 14 AA patients (six males and eight females) associated with UC who were referred to our clinic from 2001 through 2015. Results: The median age of the patients was 55 (range, 22-76). Diagnoses of the 14 patients were severe AA in eight, non-severe AA in five, and hypomegakaryocytic thrombocytopenia in one. UC preceded AA in 11 patients and nine of them received mesalazine at the time of AA diagnosis, while three AA patients developed UC 12, 72, and 36 months after diagnosis of AA. Increased percentages of PNH-type granulocytes (0.35% to 3.2%) were detected in 11 (79%) patients. Of 9 PNH(+) patients with AA preceded by UC, seven had received mesalazine while two had not been treated with mesalazine. The prevalence of increased PNH-type cells in AA patients who developed UC was 67% (2/3). Six patients were positive for DRB1*15:02, a well-known allele associated with susceptibility both to UC and AA in Japanese patients (Table). Another major DR15 allele, DRB1*15:01, was possessed by four patients. Seven patients received immunosuppressive therapy with either cyclosporine (CsA) alone (two patient) or CsA+antithymocyte globulin (five patients) and six of them achieved a remission of AA. Conclusions: The high prevalence of increased PNH-type cells indicates that AA complicated by UC is a legitimate immune-mediated BM failure and that mesalazine may be irrelevant to the development of AA. The high frequency of DRB1*15:02 suggests the two diseases share an immune pathophysiology. Genome wide association studies on UC patients may help to identify genes associated with a susceptibility to AA. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.