ALBERT

Description: [Backgrounds] The controlling nutritional status (CONUT), one of the useful parameter of nutritional assessment tools, is a significant prognostic factor for various solid tumors. The CONUT score is an index calculated from the following factors; the serum albumin concentration (Alb), the total peripheral lymphocyte counts (Lymph) and total cholesterol concentration (Chol) (Table 1). Some predictive models specified the relationship between nutritional status and the prognostic value of malignant disease have been proposed. However, the role of the CONUT score in predicting clinical outcomes of diffuse large B cell lymphoma (DLBCL) patients has not been investigated. The aim of this study is to elucidate the impact of the pretreatment CONUT score on survival in patients with DLBCL who received rituximab (R) plus chemotherapy. [Patients and Methods] We retrospectively investigated 240 patients who were histologically diagnosed with DLBCL between June 2004 and November 2015. All patients received R-CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or R-THP-COP (cyclophosphamide, tetrahydropyranyl-adriamycin, vincristine, prednisone) regimen. We defined the best cutoff value of the CONUT score as 3 using a receiver operating characteristic curve. [Result] The mean and median of the CONUT score of all patients (median age 68, range 19 - 93, 140 male and 100 female) were 2.85 and 2 (range 0 - 12). The data of each parameter's median and range constituting CONUT (Alb, Lymph, Chol) was as follows: 4 (1.9 - 5.3), 1170 (105 - 13192), and 173 (49 - 287), respectively. Patients with High-CONUT score (≥3, n = 109) had significantly lower overall survival (OS) and progression-free survival (PFS) than those with Low-CONUT score (≤2, n = 131) (5-year OS, 63.0 vs. 83.6%, P = 0.006; 5-year PFS, 56.5 vs. 78.0%, P = 0.003). The conventional predictive factors such as International Prognostic Index (IPI; age, performance status, Ann Arbor stage, extra-nodal involvement sites and lactate dehydrogenase) were all significantly associated with a worse OS and PFS. A subsequent subgroup analysis based on age indicated that 70 years or elder patients (n = 108) with High-CONUT had a significantly worse 5-year OS and PFS as compared to Low-CONUT patients (OS, 50.0 vs. 77.2%, P = 0.008; PFS, 41.6 vs. 77.6%, P = 0.0004). In contrast, no significant differences were observed in the OS and PFS when High- and Low-CONUT patients less than 70-year-old were compared. The multivariate analysis of all of the significant parameters in patients older than 70 years indicated that High-CONUT was an independent prognostic factor for PFS (HR = 2.20, 95 % CI = 1.08-4.66, p = 0.03). [Discussion] The serum Alb concentration is a reliable indicator of nutritional status and systemic inflammation. Total peripheral Lymph, which play an important role in the immune response to the tumor, are known to indicate the immunological and nutritional status. It is also reported that Chol, an indicator of a patient's caloric reserves, increased the antigen-presenting function of monocytes. Organ function decreases with aging, and many elderly patients have comorbidities that compromise their capacity to tolerate standard dose chemotherapy. In addition, intensive chemotherapy is often complicated by deterioration of nutritional status as the elderly. Hence, elderly patients are an extremely heterogeneous population and optimal treatment strategy should be adapted in consideration of comorbidities. On the other hand, DLBCL is a curable disease even in the elderly population. Therefor prognostic stratification in older population should be focused on the real biological age of patients and on primary variables that reflect tumor aggressiveness, immune interaction and nutritional status. In this respect, the pretreatment CONUT score is considered suitable for prognostic model of elderly patients. Previously, we have reported that sarcopenia is an independent poor prognostic factor for PFS in male patients with DLBCL (Ann Hematol, 2015). In this cohort, sarcopenia has no effect on PFS in elderly patients. [Conclusion] The pretreatment CONUT score is easily able to predict the prognosis of elderly patients with DLBCL. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Bowel perforation after chemotherapy is a serious life-threatening complication of diffuse large B cell lymphoma (DLBCL) involving gastrointestinal tract. R Vaidya et al reported that 9 % of patients with gastrointestinal lymphoma developed a perforation, of which 55% occurred after chemotherapy, and DLBCL was the most common lymphoma associated with perforation (R Vaidya et al Ann Oncol 2013). We assume that the dose reduction of chemotherapy may have advantages to reduce the risk of gastrointestinal perforation and shorten the duration of neutropenia. This study aimed to investigate the incidence of gastrointestinal perforation after chemotherapy, whether reduced dose intensity chemotherapy could reduce the risk of the bowel perforation and how therapeutic modalities influence on the prognosis. PATIENTS AND METHODS: We retrospectively reviewed the medical records of 348 consecutive patients who were diagnosed as having DLBCL at the Gifu University Hospital between August 2004 and April 2015. Eighty-six patients (86/348, 24.7%) were identified to have gastrointestinal involvement of DLBCL by biopsy. The prognosis of DLBCL patients with GI involvement was retrospectively investigated regarding treatments. RESULTS: The involved sites were gastric (51 patients, 48.1%), duodenal (7 patients, 6.6%), colorectal (6 patients, 5.7%), small intestine (36 patients, 34.0%) with duplicate counts permitted. Fourteen patients (14/86, 16.3%) were provided surgical treatment prior to chemotherapy since many of them had already suffered from serious complications such as ileus, perforation and bleeding. Sixty-five patients (65/86, 75.5%) received R-CHOP or CHOP like regimen ± radiation therapy as a first-line chemotherapy. As for remaining patients, two patients received R-hyperCVAD regimen because their histology were Burkitt like, and another patients received Rituximab ± other low dose chemotherapy for their frailness. The 45 patients (45/65, 69.2%) of them received reduced dose intensity (RDI) chemotherapy at the first course because increased risk of gastrointestinal perforation with chemotherapy was concerned about by endoscopic findings. The reduction rates of chemotherapy determined by the attending physician were approximately from 10% to 50%. Six patients (6/65, 9.23%) developed gastrointestinal perforation after chemotherapy (stomach 1, duodenum 0, small intestine 5, colon 0). Three of them received full dose chemotherapy at the time of small intestinal perforation including 2 patients who developed grade 4 neutropenia after the perforation. One of them died of infection after the perforation. The perforation rate was 15.0% in full dose chemotherapy group and 6.67% in the RDI chemotherapy group (p=0.361). The median day of perforation after initiation of chemotherapy was 21 days (range; 5-63 days). The small intestine was the most common site of perforation and 4 patients (4/5, 80%) had the ulcerative type lesions in their perforation site. CONCLUSIONS: Prior to chemotherapy, risk of GI perforation should be evaluated. We consider that small intestinal ulcerative lesion is high risk of GI perforation. The RDI chemotherapy may be an optimal therapy for patients with primary GI DLBCL with high risk. Further and larger prospective study should be required to confirm this. Disclosures No relevant conflicts of interest to declare.

Description: Background: Burkitt lymphoma (BL) is a B-cell lymphoma with high proliferating ability, derived from germinal center B-cells (GCB). The MUM-1/IRF4 gene has been identified as a myeloma-associated oncogene which is activated at the transcription level as a result of t(6;14)(p25;q32) chromosomal translocation. In normal lymphohematopoietic tissue, MUM1 protein has an important role in lymphocyte activation and in terminal B cell differentiation, and is expressed in plasma cells and in a small percentage of GCB. It is known that some of BL express MUM1, suggesting late GCB origin, but its characteristics still remain controversial. Patients and methods: A total of 91 patients previously diagnosed as BL were retrospectively analyzed. Two patients were excluded from the study, because translocation or amplificationof BCL-2 was detected by FISH in addition to translocation of MYC. The other 89 cases were confirmed as BL based on morphology, immunophenotype and the result of FISH analysis for translocation of MYC and BCL-2. The expression of MUM-1 was examined by immunohistochemistry. Furthermore, to characterize MUM1-positive (MUM1+) BL, we have compared the clinicopathologic characteristics of MUM1+ BL and MUM1-negative (MUM1-) BL. Results: Forty out of the 89 cases showed positivity for MUM1. The clinical characteristics of 40 MUM1+ BL were as follows. There were 20 men and 20 women, with a median age of 28 years, ranging from 3 to 83 years old. Thirty three cases (83%) showed extranodal involvement at presentation. The most frequent sites of extranodal involvement were bone marrow (n=14), lower gastrointestinal tract (n=14), central nervous system (n=5) and peripheral blood (n=5). Eight (20%) of MUM1+ BL patients had a bulky mass, 27 (69%) were categorized as stage III/IV, and 14 (36%) had B symptoms. With regard to laboratory data at presentation, 35 patients (90%) had elevated level of LDH. According to international prognostic index, 27 cases (69%) were identified into high/high-intermediate. Compared with MUM1- BL, patients with MUM1+ BL showed significantly younger onset (p=0.0053) and a higher ratio of females (p=0.007). The MUM1+ BL was also featured by higher percentage of elevated level of LDH, and tended to have anemia (hemoglobin level 20 years old), MUM1+ cases showed more significantly worse prognosis (p=0.0096). (Figure 1) Conclusion: MUM1+ BL showed significantly worse prognosis, particularly in adult cases, compared with MUM1- BL. In addition, the difference of the onset age, sex ratio, and involved sites between the two groups was highlighted. Our results demonstrate that MUM1 expression might predict worse prognosis of BL, and MUM1+ BL should be distinguished from MUM1- BL. Disclosures No relevant conflicts of interest to declare.

Description: Background: We recently reported that the International Prognostic Scoring System for Waldenström macroglobulinemia (ISSWM), which is widely used to predict the prognosis of WM patients, might not be applicable to Japanese patients, and evidence of pleural effusion might be a novel adverse prognostic factor for symptomatic WM in the rituximab era. Further studies with a large number of patients are deemed to be conducted. Methods: We retrospectively analyzed the clinical data of 498 patients with WM diagnosed between January 2001 and December 2015 from 44 institutes involved with the Japanese Society of Myeloma. The overall survival (OS) was analyzed using Kaplan-Meier methods and compared using log-rank test. Several clinical characteristics at the diagnosis were assessed by Cox regression for univariate and multivariate analyses of the OS. Results: We included 420 cases diagnosed with symptomatic (n=314) and asymptomatic WM (n=106) in accordance with the classification of the Second International Workshop on WM. The median age at the diagnosis was 69 (range, 32-91) years, with 75.5% male, and 16.0% had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 2-4. Oral alkylating agents, purine analogs, cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) or CHOP-like regimens ± rituximab, rituximab monotherapy, or dexamethasone, rituximab and cyclophosphamide (DRC) were mainly administered as initial treatment. Rituximab-containing therapy was administered in 76.8% of all patients. The median follow-up was 45 months. The 5-year OS rate for all patients was 77.9%, while the rates for those with symptomatic and asymptomatic WM were 72.9% and 92.2%, respectively. Significant differences in the survival were seen between risk groups of ISSWM in symptomatic WM patients (5-year OS: high, 55.4%; intermediate, 81.2%; low, 90.2%; p65 years, platelet count ≤10×104/µL, serum β2-microglobulin (β2-MG) 〉3 mg/L, ECOG PS 2-4, abnormal karyotype, pleural involvement, WBC 1.5 mg/dL, CRP 〉2.0 mg/dL and sIL-2R 〉4000 U/mL were significant adverse prognostic factors for the OS. A multivariate analysis revealed that a platelet count ≤10×104/µL (hazard ratio [HR] 5.942; 95% confidence interval [CI] 2.265-14.761), serum β2-MG 〉3 mg/L (HR 2.748; 95% CI 1.091-7.655), ECOG PS 2-4 (HR 2.899; 95% CI 1.219-6.290), and pleural involvement (HR 11.066; 95% CI 3.672-29.829) were adverse independent risk factors for symptomatic WM. We constructed a prognostic model by combining these prognostic variables as follows: patients with good risk (n=219), no adverse factors or only serum β2-MG 〉3 mg/L or ECOG PS 2-4; patients with poor risk (n=81), ≥1 adverse factors with a platelet count ≤10×104/µL, pleural involvement, or both serum β2-MG 〉3 mg/L and ECOG PS 2-4. The 5-year OS rates were 82.3% for good risk and 44.4% for poor risk, and this prognostic model significantly stratified symptomatic WM patients separately by the survival (p

Description: Introduction: The intracellular enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step in the metabolism of the essential amino acid tryptophan along the L-kynurenine (KYN) pathway. Some metabolites derived from tryptophan via IDO catalysis such as KYN block antigen-driven specific T-cell proliferation and induce T-cell death. IDO activity might play an important role in regulating the immune response exerted by antigen presenting cells. Indeed, we have recently reported the utility of either serum KYN or the expression of IDO mRNA as prognostic factors for acute myeloid leukemia (AML) [Leuk Lymphoma: In press, Leuk Lymphoma 56:1398-405]. Here, we investigated the value of combination of serum KYN level and expression of IDO mRNA as a prognostic factor in AML patients. Patients and Methods: AML was diagnosed according to the WHO 2008 criteria based on standard cytological and histochemical assessments of smears of cryopreserved bone marrow cells from 29 consecutive adult patients between December 2005 and March 2014. All patients in this study had been enrolled in both our serum KYN study and expression of IDO mRNA study. All follow-up data were fixed on August 1, 2014. KYN concentrations in serum samples were measured by high-performance liquid chromatography. Bone marrow-derived mononuclear fractions were separated and IDO expression was analyzed by using extracted mRNA amplified by PCR. Results: We examined expression of IDO mRNA and serum L-kynurenine in a total of 29 patients (median age, 55 years; range, 18-74 years). Among them, 11, 14 and 4 patients were classified as having favorable, intermediate and unfavorable cytogenetic risk, respectively. Twenty seven patients underwent standard intensive chemotherapy, mainly consisting of cytosine arabinoside (Ara-C) and anthracycline. All patients with acute promyelocytic leukemia (n = 5) received induction therapy containing all-trans retinoic acid. Two patients received less intensive chemotherapy [J Cancer Res Clin Oncol. 133:547-53.], because of poor performance status. The median serum KYN level was 1.63 µM (range, 0.66-5.27 µM). We set the L-kynurenine cut-off value at 2.4 µM according to our previous report. The RT-PCR results showed that bone marrow from 12 (41%) patients were IDO-positive. No significant correlation was identified between serum KYN level and IDO expression. Complete remission (CR) rates were 57% and 86% for patients with KYN levels ≥2.4 and

Description: Background: The response to treatment and overall survival of patients with acute myeloid leukemia (AML) are heterogeneous. A number of prognostic factors related to patient and tumor characteristics have been described for AML, including age, performance status, and karyotype. The depletion of skeletal muscle (sarcopenia) and adipose tissue (adipopenia) are known to be associated with unfavorable prognosis in patients with some malignant diseases including lymphoma. Here, we studied the impact of sarcopenia and adipopenia on clinical outcomes of adult AML. Patients and Methods: We retrospectively analyzed 70 patients with adult AML (age ≥ 18 years) who received chemotherapy at Gifu University Hospital between December 2004 and September 2014. Skeletal muscle and adipose tissue were measured by the analysis of CT images at the L3 level before treatment. CT images were analyzed using SliceOMatic version 4.3 software (TomoVision, Montreal, QB, Canada), which enables specific tissue demarcation using previously reported Hounsfield unit (HU) thresholds. The CT HU thresholds were -29 to 150 for skeletal muscles and -190 to -30 and -50 to -150 for subcutaneous and visceral adipose tissue, respectively. These values were normalized for stature in order to calculate skeletal muscle index (SMI, cm2/m2) and adipose tissue index (ATI, cm2/m2). Results: Median age at diagnosis was 57 years (18-84 years), with 37 males and 33 females. SMI was significantly higher in male than female patients (P 〈 0.001). ATI was significantly higher in patients aged 60 years and over than in those under 60 years (P 〈 0.05). The sex-specific cut-offs for the SMI and ATI were determined by ROC curve analysis. Thirty-five (50%) and 31 (44%) patients were defined as sarcopenia and adipopenia, respectively. Sarcopenia and adipopenia did not significantly differ among various FAB subtypes or cytogenetic risk profiles. The rate of patients with poor performance status (ECOG ≥ 2) was significantly higher in the sarcopenic group (80% vs 45%, P 〈 0.05), whereas not in the adipopenic group (40% vs 47%). With a median follow-up of 33.5 months, the 3-year overall survival (OS) in the sarcopeniac group was 34% compared with 74% in the non-sarcopenic group (P 〈 0.001, Figure 1A) and 32% in the adipopenic group compared with 72% in the non-adipopenic group (P 〈 0.005, Figure 1B). In a multivariate analysis, sarcopenia (HR = 2.84, CI = 1.08-8.08, P 〈 0.05) and adipopenia (HR = 2.85, CI = 1.19-7.24, P 〈 0.05) remained predictive of OS. Conclusion: Sarcopenia and adipopenia are independent prognostic factors in patients with AML. Evaluation of skeletal muscle and adipose tissue depletion by CT imaging is a useful objective tool to predict patient outcomes, but a larger prospective study is needed to confirm this effect. Figure 1. Overall survival according to sarcopenic (A) and adipopenic (B) status. Figure 1. Overall survival according to sarcopenic (A) and adipopenic (B) status. Disclosures No relevant conflicts of interest to declare.

Description: Each transfusion requires 200-300 billion platelets in patients with thrombocytopenia. To continuously supply such a huge number of platelets by ex vivo generation, two distinct steps, megakaryopoiesis and platelet shedding, must be both considered. For the former, one approach is to increase the number of source cell, megakaryocytes. For example, the immortalized megakaryocyte cell line (imMKCL) system uses self-renewing megakaryocyte (MK) cell lines derived from induced pluripotent stem cells (iPSCs) (Nakamura et al., Cell Stem Cell, 2014). For the latter, there have been an idea of bioreactors whereby shedding of platelets from proplatelets could be promoted by flow-dependent shear force within the bone marrow in vivo (Junt et al., Science, 2007; Zhang et al., J Exp Med, 2012). Based upon this idea, we constructed a flow chamber type bioreactor recapitulating in vivo blood flow shear rate. However, this bioreactor failed to efficiently yield platelets, and moreover, the produced platelets had poor quality as indicated by high Annexin V levels (Exp Hematol, 2011 and unpublished result). Recently, we demonstrated two different kinetics of platelet biogenesis from bone marrow MKs, whereby either thrombopoietin (TPO) mostly regulates steady-state shedding of platelets from proplatelets, or interleukin-a (IL-1a) triggers inflammation-dependent rupture of MK cytoplasm contributing to a quick increase of platelet count at higher rate (Nishimura et al., J Cell Biol, 2015). However, the rupture type platelets revealed shorter half-life with relatively higher Annexin V levels. Therefore, to gain insights from platelet biogenesis in vivo, we focused on biophysical analysis of steady-state platelet biogenesis via proplatelets in bone marrow. Our observations strongly indicated that the presence of 'vorticity' defined by vortex turbulence in addition to shear-dependent 'stress' and 'strain' correlates with the efficient shedding of competent platelets. From this new finding, we developed an alternative bioreactor system, which enabled generation of 100 billion platelets from imMKCL in a 16L-scale liquid culture condition without any adherent machinery using two 10L-bioreactors. Furthermore, platelets generated via new bioreactors showed low Annexin V levels (

Description: (Background) Sideroblastic anemias are heterogeneous congenital and acquired refractory anemias characterized by bone marrow ring sideroblasts, reflecting excess mitochondrial iron deposition. While the disease is commonly associated with myelodysplastic syndrome, the congenital forms of sideroblastic anemias comprise a diverse class of syndromic and non-syndromic disorders, which are caused by the germline mutation of genes involved in iron-heme metabolism in erythroid cells. Although the only consistent feature of sideroblastic anemia is the bone marrow ring sideroblasts, evidence on the detailed molecular characteristics of ring sideroblasts is scarce owing to a lack of the biological models. We have recently established ring sideroblasts by inducing ALAS2 gene mutation based on human-induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells (ASH 2017) and have further extended the molecular characterization of human ring sideroblasts to gain new biological insights. (Method) We targeted the GATA-1-binding region of intron 1 of the human ALAS2 gene in HiDEP cells and established two independent clones [X-linked sideroblastic anemia (XLSA) clones]. A co-culture with OP9 stromal cells (ATCC) was conducted with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, and ascorbic acid. To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in a liquid suspension culture (Fujiwara et al. JBC 2014). Bone marrow glycophorin A (GPA)-positive erythroblasts of patients with XLSA and normal individuals were separated using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) after obtaining written informed consent. For transcription profiling, Human Oligo chip 25K (Toray) was used. (Results) We previously demonstrated that co-culture with OP9 cells in the medium supplemented with 100 uM sodium ferrous citrate (SFC) promoted erythroid differentiation of XLSA clones, which enabled the establishment of ring sideroblasts (ASH 2017). To confirm the importance of SFC in terminal erythroid differentiation, we further demonstrated that the addition of SFC, and not transferrin-loaded iron, induced the frequency of GPA+ cells and TfR1-GPA+ mature erythroid population, based on primary erythroblasts derived from human CD34-positive cells. Subsequently, to reveal the molecular mechanism by which abnormal iron mitochondrial iron accumulation occurs by co-culture with SFC, we evaluated the expressions of various metal transporters, demonstrating that the addition of SFC significantly increased the expressions of mitoferrin 1 (MFRN1; a ferrous iron transporter in mitochondria), divalent metal transporter 1 (DMT1), and Zrt- and Irt-like protein 8 (ZIP8; a transmembrane zinc transporter, recently known as a ferrous iron transporter) in the XLSA clone than the wild-type cells, which would have contributed to the formation of ring sideroblasts. Moreover, we performed expression analyses to elucidate the biochemical characteristics of ring sideroblasts. After co-culture with OP9 in the presence of SFC, ring sideroblasts exhibited more than two-fold upregulation and downregulation of 287 and 143 genes, respectively, than the wild-type cells. Interestingly, when compared with the expression profiling results before co-culture (ASH 2017), we noticed prominent upregulation of gene involved in anti-apoptotic process (p = 0.000772), including HSPA1A, superoxide dismutase (SOD) 1, and SOD2. In addition, we conducted a microarray analysis based on GPA-positive erythroblasts from an XLSA patient and a normal individual. The analysis revealed significant upregulation of genes involved in the apoptosis process, as represented by apoptosis enhancing nuclease, DEAD-box helicase 47, and growth arrest and DNA-damage-inducible 45 alpha, and anti-apoptotic genes, such as HSPA1A and SOD2. Concomitantly, when the XLSA clone was co-cultured with OP9 in the presence of SFC, the apoptotic cell frequency as well as DNA fragmentation were significantly reduced compared with the XLSA clone co-cultured without SFC, indicating that ring sideroblasts avoid cell death by inducing anti-apoptotic properties. (Conclusion) Further characterization of the XLSA model would help clarify its molecular etiology as well as establish novel therapeutic strategies. Disclosures Fukuhara: Celgene: Research Funding; Chugai: Research Funding; Daiichi-Sankyo: Research Funding; Boehringer Ingelheim: Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Japan Blood Products Organization: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Mitsubishi Tanabe: Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Research Funding; Nippon-shinyaku: Research Funding; Novartis pharma: Research Funding; Ono: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Symbio: Research Funding; Solasia: Research Funding; Sumitomo Dainippon: Research Funding; Taiho: Research Funding; Teijin Pharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Takeda: Honoraria; Baxalta: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bayer Yakuhin: Research Funding; Alexionpharma: Research Funding; AbbVie: Research Funding; Astellas: Research Funding; Nihon Ultmarc: Research Funding.

Description: [Introduction] Anthracycline-induced cardiotoxicity (ACT) has been a major problem in leukemia therapy, and reported to be associated with several candidates of single nucleotide polymorphisms (SNPs). C242T polymorphism (rs4673) in the cytochrome b-245 alpha chain (CYBA) gene, which encodes superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase light chain subunit, is focused on as one of ACT related SNPs. Several clinical studies indicated that rs4673 T allele increases the risk of ACT, however, it is unclear how rs4673 affects ACT. Here, we established a series of induced pluripotent stem cells (iPSCs) with a genotype of C/C, T/T or C/T of rs4673 in a uniform genetic background by using CRISPR/ Cas9, and evaluated the effect of each SNP genotype on ACT in vitro. [Methods and Results] To edit the SNP site using CRISPR/Cas9, three guide RNAs (gRNAs) were designed to induce double strand break (DSB) near the target site. Cas9 nuclease and gRNA expression vectors were transfected into HEK293T cells using jetPEI (Polyplus-transfection SAS), and the genome editing activity was assessed by T7 endonucleaseⅠ(T7E1) assay to select more efficient gRNA for cleavage near the target site. The indel frequencies of sgRNAs 1,2 and 3 were 29.4%, 25.6%, and 18.6%, respectively. Furthermore, oligo-DNA (100 nt) to edit single-base in the SNP site was prepared. To generate Cas9/gRNA ribonucleoprotein (RNP) complexes, 1 μg of Cas9 protein and 0.2 μg of gRNA molecules were mixed. RNP complexes and 50 pmol of oligo-DNA were electroporated into human iPSC 201B7cells (Riken BioResource Center) using Neon electroporation device (Invitrogen),single-base-edited iPSCs (C/C and T/T from C/T genotype in rs4673) were established by homology-directed repair (HDR). Successful HDR events were verified by Sanger sequencing. These established iPSCs were differentiated into cardiomyocytes using PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) in vitro. To facilitate nascent mesoderm induction, TBX6 was expressed transiently by a tetracycline-inducible lentiviral vector. Differentiated cardiomyocytes were revealed to be contracting and be positive for NK2 transcription factor related locus 5 (NKX2-5) and troponin T2 (TNNT2) by immunofluorescence staining.In order to evaluate the ACT depending on the difference of single base in rs4673, doxorubicin (DOX)was administered to each edited iPSC-derived cardiomyocyte. NADP+/NADPH before and after administration of DOX (1μM) in C/C cardiomyocytes was 1±0.18 and 0.93±0.14, respectively. On the other hand, it was 1±0.07 and 1.29±0.03 in T/T cardiomyocytes (p