Abstract: The immaturity of T cells in cord blood is well known in functional assays and phenotypic analyses. During the first several months after cord blood transplantation (CBT), the T cell compartment is recovered by peripheral expansion from those mature and naïve T cells in cord blood grafts and plays an important role in acute graft-versus-host disease (GVHD) and graft-versus-leukemia reaction. Recently, we have reported that adult patients with hematological malignancies receiving CBT from HLA-partially-mismatched unrelated donors (n=68) had a lower risk of severe acute GVHD ( 〉 grade II, 7% versus 26%) and transplant-related mortality (9% versus 29% at 1 year) and a higher probability of disease-free survival (74% versus 44% at 2 years) than HLA-matched unrelated bone marrow transplant (BMT) recipients (n=45) in our multivariate analysis (Takahashi et al., Blood, in press). We speculated that the immune reconstitution process over a period of several months after CBT might have contributed to these promising clinical results. Using four-color analysis with CD4, CD8, CD45RA, and CD62L, more than 90% of cord blood CD4+ and CD8+ T cells in the grafts belonged to the naïve fraction. Cytokine expression in cord blood T cells was also suppressed to 0.1% in CD4+ and to 0.9% in CD8+ with positive interferon-γ by intracellular staining, which were significantly lower than those in adult T cells (16.2% in CD4+ and 37.8% in CD8+). Circulating T cell counts normalized after 3 months for CD8+ and 4 months for CD4+ in our CBT recipients, both of which were significantly faster than in previously published studies, which were 9 months for CD8+ and 12 months for CD4+. After T cell recovery, peripheral blood T cells moved from the naïve to the central memory fraction immediately, and then moved to the effector memory fraction. A naïve subset of CD4+ T cells remained (median: 38 cells/μl on day 90, n=12) during the first 3 months, which was significantly higher than in the BMT control (median: 9 cells/μl on day 90, n=5, p=0.015), but showed a low level of CD8+ T cells (median: 14 cells/μl on day 90, n=12), almost the same as in BMT recipients (median: 13 cells/μl on day 90, n=5). Intracellular interferon-γ-producing T cells were detected at 3.4% (0.1–34.2%) in CD4+ and 32.3% (1.1–86.9%) in CD8+ at 1 month post-CBT (n=16), both of which were comparable to post-BMT. To investigate whether these T cells with memory phenotype are functional, we analyzed antigen-specific T-cell recovery using cytomegalovirus (CMV) as a specific antigen. CMV-responsive CD4+ T cells were detected within the first 4 months in all recipients with positive CMV antigenemia (n=13), but CD8+ T cells were detected only in 5 out of 13 cases, probably because of pre-emptive Gancyclovir administration in most antigenemia-positive patients. To conclude, naïve cord blood T cells rapidly increased in number and adopted a memory phenotype showing cytokine-production and antigen-recognition capacity in the early phase after CBT. These data suggest that mature T lymphocytes in cord blood have unique properties and contribute to the favorable clinical outcome of CBT.

Abstract: Background: Isocitrate dehydrogenase (IDH)-1 and -2 are TCA cycle-involved enzymes which convert isocitrate to alpha-ketoglutarate. Mutations that alter the enzymatic activity causes accumulation of a mal-metabolite D-2-hydroxyglutarate, which results in inhibition of DNA methylation and tumorigenesis. IDH-1 and IDH-2 mutation are present in approximately 7-10% and 10% of patients with acute myeloid leukemia (AML), respectively. Recently, whole exome sequencing has been used for the next-generation sequencing of AML, and certain gene mutations have been identified in patients with AML. The treatment strategies for leukemia have undergone drastic changes with the rapid development of new drugs. However, the proper use of newly developed agents poses a major challenge in AML treatment. Genome profiling analysis can be used to select the optimal treatment for patients with newly diagnosed AML. IDH mutant-specific inhibitors such as ivosidenib and enasidenib were already approved in the US, and combination treatment with venetoclax and Azacitidine was recently approved in Japan. Methods and Results: We lunched an actionable mutation profiling multicenter study named Hematologic Malignancies (HM)-SCREEN-Japan 01 (UMIN000035233), in which a comprehensive genomic assay was performed by Foundation One Heme (F1H) panel. The primary outcome was the frequency of each genomic alteration, as determined using F1H, which is a comprehensive genome profiling test based on next-generation sequencing, in the AML specimens. The secondary outcome was the association between each genomic alteration and the clinicopathological characteristics, prognosis, and quality of specimens used in the genetic analysis. The eligibility criteria were as follows: 1) histological diagnosis of AML through bone marrow aspiration; 2) fulfillment of either of the following conditions: i) newly diagnosed AML unfit for standard treatment (ND-unfit AML) or ii) R/R-AML; 3) sufficient sample collection via bone marrow aspiration; 4) Age of participants 20 years or above during registration; 5) provision of written informed consent by participants. Paraffin-embedded bone marrow samples were gathered from 17 Japanese faculties and the F1H reports were returned to the patients. The median turnaround time was 13 days (minimum 8 days). We found 13 patients (7.3%) with IDH1 mutation and 17 patients (9.6%) with IDH2 mutation out of 177 patients who joined this study and the F1H report was successfully returned. Only one patient had both mutations, and each mutation was mutually exclusive in all the other patients (Figure 1). The major amino acid alteration of IDH1 and IDH2 were R132C/G/H/L and R140Q/W, respectively. Frequently co-occurring mutations include FLT3 (44.8%), NPM1 (34.5%), DNMT3A (31.0%) and RUNX1 mutation (20.7%). Mutations of RAS pathway-related genes (e.g., KRAS, NRAS and PTPN11) were seen in 6 patients (20.7%). Any gene alterations didn't show statistically significant co-occurrence with IDH1 and IDH2 mutation. Serial genome profiling analyses were performed to evaluate the time-dependent changes in the genome profiles of patients administered FLT3 inhibitors, gilteritinib, and quizartinib for treating FLT3-mutated AML. Also in this cohort, we are examining the properties and distribution of IDH1/2 mutations during treatment with FLT3 inhibitors. In the several patients, expansion and persistence of IDH mutated clones seemed to be cause of resistance (Figure 2 as the representative result). The detailed clinical outcomes of AML patients with IDH1/2 mutations are under investigation. Conclusions: In our evaluation of the suitability of F1H for HM-SCREEN-Japan 01, we successfully identified IDH-1/2 mutation that can be used as therapeutic targets in AML, which have rarely been identified thus far. Figure 1 Figure 1. Disclosures Shibayama: Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Ono: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Nippon Shinyaku: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Fujimoto: Honoraria; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Mundi Pharma: Honoraria; Essentia Pharma Japan: Research Funding. Yamauchi: Otsuka: Research Funding; Ono Pharmaceutical: Honoraria; Pfizer: Honoraria, Research Funding; Chugai: Honoraria; Abbie: Research Funding; Astellas: Research Funding; Daiichi Sankyo: Research Funding; Solasia Pharma: Research Funding. Kondo: Otsuka Pharmaceutical: Consultancy, Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Bristol-Myers Squibb Company: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy; Pfizer: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Abbvie: Honoraria. Yamamoto: AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Bristol-Myers Squibb/Celgene: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Eisai: Honoraria, Research Funding; IQIVA/Incyte: Research Funding; IQIVA/HUYA: Honoraria; HUYA: Consultancy; Janssen: Honoraria; Kyowa Kirin: Honoraria; Meiji Seika Pharma: Consultancy, Honoraria, Research Funding; MSD: Honoraria; Mundipharma: Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ono: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Sanofi: Honoraria; Solasia Pharma: Research Funding; SymBio: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Yakult: Honoraria, Research Funding; Zenyaku: Honoraria, Research Funding; Micron: Honoraria; IQIVA/Genmab: Research Funding; ADC Therapeutics: Honoraria. Kuroda: Taiho Pharmaceutical: Research Funding; Fujimoto Pharmaceutical: Current Employment, Honoraria, Research Funding; Asahi Kasei: Research Funding; Shionogi: Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Sysmex: Research Funding; Eisai: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; MSD: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Dainippon Sumitomo Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Bristol-MyersSquibb: Consultancy, Honoraria, Research Funding; Janssen Pharmaceutical K.K: Consultancy. Usuki: Astellas: Research Funding, Speakers Bureau; Abbvie: Research Funding; Gilead: Research Funding; Symbio: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding, Speakers Bureau; Sumitomo Dainippon: Research Funding; Otsuka: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Brisol-Myers Squibb: Research Funding, Speakers Bureau; Ono: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Nippon Boehringer Ingelheim: Research Funding; Astellas-Amgen-Biopharma: Research Funding; Nippon shinyaku: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau; Pfizer: Research Funding; Alexion: Speakers Bureau; Eisai: Speakers Bureau; MSD: Speakers Bureau; PharmaEssentia: Speakers Bureau; Yakult: Speakers Bureau; Mundipharma: Research Funding. Yoshimitsu: Novartis: Honoraria; Takeda: Honoraria; Sanofi: Honoraria. Ishitsuka: Asahi kasei: Research Funding; Eli Lilly: Research Funding; MSD: Research Funding; Daiichi Sankyo: Consultancy, Other: Personal fees; Kyowa Kirin: Other: Personal fees, Research Funding; Ono Pharmaceutical: Other: Personal fees, Research Funding; Celgene: Honoraria, Other: Personal fees; Chugai Pharmaceutical: Honoraria, Other: Personal fees, Research Funding; BMS: Other; Takeda: Other: Personal fees, Research Funding; Mundipharma: Other: Personal fees; Taiho Pharmaceuticals: Other: Personal fees, Research Funding; Janssen Pharmaceuticals: Other: Personal fees; Huya Japan: Other: Personal fees; Novartis: Other: Personal fees; Pfizer: Other: Personal fees; Astellas Pharma: Other: Personal fees, Research Funding; Genzyme: Other: Personal fees; Sumitomo Dainippon Pharma: Other: Personal fees, Research Funding; Eisai: Other: Personal fees, Research Funding; Mochida: Other: Personal fees, Research Funding; Shire: Other; Otsuka Pharmaceutical: Other: Personal fees; Teijin Pharma: Research Funding. Ono: Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb Company: Honoraria; Celgene: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K: Honoraria; Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; DAIICHI SANKYO COMPANY, LIMITED.: Honoraria; Novartis Pharma KK: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; Merck Sharp & Dohme: Honoraria, Research Funding. Takahashi: Toyamakagaku: Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Research Funding; Kyowahakko-Kirin: Research Funding; Ono: Research Funding; Asahikasei: Research Funding; Eizai: Research Funding. Iyama: Alexion Pharmaceuticals: Honoraria, Research Funding; Astellas: Honoraria; CSL Behring: Honoraria; Daiichi Sankyo: Honoraria; Otsuka Pharmaceuticals Factory: Honoraria; Otsuka Pharmaceuticals Factory: Honoraria; MSD: Research Funding; Nippon Shinyaku: Honoraria; Novartis: Honoraria; Otsuka: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; SymBio Pharmaceuticals: Research Funding. Izutsu: Genmab: Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Fuji Film Toyama Chemical: Honoraria; Eisai: Honoraria, Research Funding; Incyte: Research Funding; Huya Biosciences: Research Funding; Chugai: Honoraria, Research Funding; Symbio: Honoraria; Solasia: Research Funding; Pfizer: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; MSD: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Beigene: Research Funding; Bayer: Research Funding; AstraZeneca: Honoraria, Research Funding; Allergan Japan: Honoraria; AbbVie: Honoraria; Takeda: Honoraria, Research Funding; Yakult: Research Funding. Minami: Novartis Pharma KK: Honoraria; Ono: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb Company: Honoraria; CMIC: Research Funding.

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor is a promising alternative option for patients who can not find an HLA-matched donor. However, HLA-mismatch allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of non-relapse mortality mediated by alloreactive T cells. Infusion of anti-thymocyte globulin (ATG) is used both as a treatment for and as a prophylactic against GVHD but ATG, like other immunosuppressive regimens, reacts against cells without distinguishing between donor and recipient cells. ATG is polyclonal and causes many side effects such as allergic reaction and increasing susceptibility to infection due to repression of T cells. Our ultimate goal is to develop a novel therapeutic approach to GVHD with anti-HLA monoclonal antibody specifically targeting the mismatched HLA molecule of donor origin to transiently ablate alloreactive T cells. To generate allele-specific anti-HLA antibody (ASHmAb), we established a rapid and efficient strategy using two different HLA-transgenic mouse strains, HLA-A24 and HLA-A2 and immunized them with HLA-A* 02:01 and HLA-A* 24:02 tetramers loaded with cytomegalovirus PP65 peptide, respectively. Antibodies were screened for HLA allele-specific complement-dependent cytotoxicity in vitro. We successfully established three killing ASHmAbs (kASHmAbs) against HLA-A* 02:01, -A* 02:01/–A* 03:01, and -A* 23:01/-A* 24:02. In vivo cytotoxicity and specificity of a kASHmAb against HLA-A* 02:01 (A* 02:01-kASHmAb) was examined in detail using a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγnull (NOG) mice were injected with either HLA-A2 or HLA-A26 positive human peripheral blood mononuclear cells (PBMCs) from healthy donors (i.e. HLA-A2 mice or HLA-A26 mice, respectively). Administration of A* 02:01-kASHmAb promoted the survival of HLA-A2 mice to 100%, with a mean survival of more than 6 months. In contrast, administration of A* 02:01-kASHmAb to nonreactive HLA was not effective; all HLA-A26 mice died within 2 months (p 〈 0.0001), demonstrating kASHmAb can successfully treat a GVHD mouse model. Next, we examined the cytotoxicity of the kASHmAb on the human graft in bone marrow using humanized NOG mice. Sublethally irradiated NOG mice were reconstituted with CD34 positive hematopoietic stem/progenitor cells from HLA-A2 positive cord blood. These humanized mice showed about 70-90% human T, B and myeloid cells in the peripheral blood. When these mice were administrated with A* 02:01-kASHmAb for five consecutive days (60 mg/day), mature human blood cells disappeared immediately from the mouse peripheral blood. Interestingly, human PBMCs were detectable again in the mouse blood in 2-4 weeks after the last kASHmAb administration, suggesting kASHmAb may have preferential cytotoxic effect on mature PBMCs sparing hematopoietic stem progenitor cells. The mechanism of this preferential killing is not clear, but an optimal dose of kASHmAb may be safely administered to GVHD patients without permanently ablating the graft and necessitating repeated transplantation. This study is the first reported instance of using the cytotoxic effect of HLA antibodies against GVHD. Unlike ATG, kASHmAb specifically killed donor cells and contributed to the survival of the recipients. While this novel therapeutic approach requires a panel of kASHmAbs to different HLA alleles, this may open a new door for treatment of severe GVHD. Furthermore, optimization of the kASHmAb administration protocol may contribute to tolerance induction. Disclosures: No relevant conflicts of interest to declare.

Abstract: Subsequent solid cancers after HCT occurred at younger ages than primary cancers and accounted for most common causes of deaths. Subsequent solid cancers after HCT showed similar or worse survival compared with primary cancers in the general population.

Abstract: Pretransplant imatinib improved both relapse and nonrelapse mortality in patients with BCR-ABL–positive acute lymphoblastic leukemia.

Abstract: Allogeneic hematopoietic cell transplantation (allo-HCT) is a promising treatment for adult acute lymphoblastic leukemia (ALL), an intractable hematological malignancy. The trends in allo-HCT outcomes over the past 30 years were examined to verify the efficacy of evolving treatment methods and to identify further challenges. We analyzed data from a registry database that included 8467 adult ALL patients who underwent their first allo-HCT between 1990 and 2019. The period was divided into three 10-year intervals for analysis. Five-year overall survival improved from 48.2% to 70.2% in the first complete remission (CR1), from 25.6% to 44.1% in subsequent CR, and from 10.0% to 22.7% in non-CR. Nonrelapse mortality improved over the 3 decades in each disease stage. However, the relapse rate only improved in CR1 every decade (26.3% to 15.9% in CR1, 33.4% to 32.8% in subsequent CR, and 53.6% to 54.8% in non-CR). Although there were continual improvements in adjusted survival for Philadelphia chromosome (Ph)-positive patients, the improvement was inadequate for Ph− patients with t(4;11), t(8;14), t(14;18), or hypodiploidy. Allo-HCT outcomes for adults with ALL have improved over the past 30 years. Improved outcomes in the future will require more effective prevention of relapse in patients with ALL not in CR1 and in those with high-risk chromosomal abnormalities.

Abstract: Unrelated cord blood transplantation (CBT) has now become more common, but as yet there have been only a few reports on its outcome compared with bone marrow transplantation (BMT), especially for adults. We studied the clinical outcomes of 113 adult patients with hematologic malignancies who received unrelated BM transplants (n = 45) or unrelated CB transplants (n = 68). We analyzed the hematopoietic recovery, rates of graft-versus-host disease (GVHD), risks of transplantation-related mortality (TRM) and relapse, and disease-free survival (DFS) using Cox proportional hazards models. The time from donor search to transplantation was significantly shorter among CB transplant recipients (median, 2 months) than BM transplant recipients (median, 11 months; P & lt; .01). Multivariate analysis demonstrated slow neutrophil (P & lt; .01) and platelet (P & lt; .01) recoveries in CBT patients compared with BMT patients. Despite rapid tapering of immunosuppressants after transplantation and infrequent use of steroids to treat severe acute GVHD, there were no GVHD-related deaths among CB transplant recipients compared with 10 deaths of 24 among BM transplant recipients. Unrelated CBT showed better TRM and DFS results compared with BMT (P = .02 and P & lt; .01, respectively), despite the higher human leukocyte antigen mismatching rate and lower number of infused cells. These data strongly suggest that CBT could be safely and effectively used for adult patients with hematologic malignancies.

Abstract: Background AL(acute leukemias) with 11q23 abnormalities involving the MLL (mixed lineage leukemia) gene comprise one category of recurring genetic abnormalities in the WHO classification. However, the prognosis of adult AL with 11q23/MLL between fusion partners are controversial especially in Asia. Methods We analyzed data on 61 Japanese adult patients diagnosed as AL with 11q23/MLL abnormalities, including the translocations t(4;11), t(9;11), t(6;11), t(11;19) between 1990 and 2006. Results Median age of the 61 patients was 48 years (range, 17–79 years). 54 patients were AML (acute myeloid leukemia) and 7 patients were ALL (acute lymphoblastic leukemia).The incidence by fusion partners in AML were as follows; t(9;11) in 29.6%, t(11;19) in 24.0%, t(6;11) in 18.5%. The incidence of patients with t(11;19) was higher than that in Caucasian and t(4;11) was lower than that in childhood. As for the FAB (French-American-British) subtype, 68.5% of the 11q23/MLL patients were M4/M5. Organ involvement was observed commonly (46.2%) in AML with 11q23/MLL patients except for t(11;19) (7.6%).The CR (complete remission) rate of AML with 11q23/MLL was not low (78.7%), but the CR duration was very short (10 months).The DFS (disease free survival) at 1year and the OS (overall survival) at 2 years were 20% and 30% in AML with t(9;11), 10% and 19% in AML with t(11;19), 10% and 30% in AML with t(6;11). In total, the DFS at 1year and the OS at 2 years were 26% and 25% in AML patients, respectively, while 50% and 50% in ALL patients, respectively. The DFS and the OS at 2 years were 44% and 53% in AML patients with 11q23/MLL who were treated with HSCT(hematopoietic stem cell transplantations), while 14% and 21% in those without HSCT respectively. Conclusions Although there were reports that the prognosis of AML with t(9;11) was not bad in Caucasian, the present study showed poor prognosis of AML with 11q23/MLL in Japanese adult patients regardless of the fusion partners. Our study revealed the short CR duration of the AML patients with 11q23/MLL in the face of the high CR rate. AML patients with 11q23/MLL who were treated with HSCT had superior outcomes to those without HSCT (P=.04 for DFS and P=.04 for OS) .This findings suggest that treatment strategies including HSCT should be considered for AML with 11q23/MLL during the first CR.

Abstract: BACKGROUND: Immune reconstitution following unrelated cord blood transplantation (UCBT) in adult patients is of great concern because of immaturity of cord blood immunological cells. STUDY DESIGN AND METHODS: Twenty-six adult patients (15 to 58 year-old) with hematological malignancies, who underwent UCBT and sustained engraftment were enrolled in this study. Infused number of immunological cells in thawed CB units including T cells (CD3+), B cells (CD19+), NK cells (CD3-CD56+), monocytes (CD14+) and also CD34 + cells was analysed using bead-contained TRUCOUNT tube (BD, CA). Dead cells after thawing were excluded by gating out with 7AAD dye. Immune reconstitution was analysed every 30 days by 120 days after CBT. Four-colour FACS Caliber and TRUCOUNT tube were utilized to calculate the absolute number of immune cells concentration in blood after UCBT. We put strict volume of 50μl fresh unmanipulated blood in each TRUCOUNT tube. RESULTS: Thawed-transplanted NC 2.3±x107/kg, CD34 was 0.72±0.3x105/kg (4.1x106 total), T cells; 3.1±1.6x106/kg with CD4/8 ratio of 3.2±2.0, B cells; 1.2±0.5x106/kg, NK cells; 1.0±0.5x106/kg and monocytes; 1.6±0.6x106/kg. There were no correlations between infused CD34+ cells number and T, B, NK and monocytes numbers. Monocytes increased in blood rapidly after CBT at 30 days, then, declined to the normal value. NK cells was recovered in the early after CBT and then did not so change in number from 30 to 120 days after CBT, while T cells increased time dependent manner, and B cells appeared late but influenced by acute GVHD grade. Within 120 days after CBT, T cells showed also CD4+dominant in most cases with relatively high CD25+CD4+ regulatory T (rT) cells compared to normal control. The patients with grade II to IV aGVHD showed significantly higher number of rT cells on 30 days (P 〈 0.05) compared to those with grade 0–I aGVHD. On day 30, the number of rT cells showed 7.7±5.9/μl in grade 0–I aGVHD and 19.4±13.3/ μl in grade II–IV. The patients with grade II to IV aGVHD showed significant delayed recovery of B cells on 90 days after CBT compared to those with 0–I aGVHD (P 〈 0.001). CONCLUSION: aGVHD in adult patients may influence on the number of regulatory T cells in the early period after UCBT and delayed recovery of B cells.

Abstract: Abstract 1360 Introduction: The tyrosine kinase inhibitor (TKI) imatinib is used as the first-line therapy for newly diagnosed chronic myeloid leukemia (CML). However, some patients fail to respond or become intolerant to imatinib. Nilotinib is a second-generation TKI with higher selectivity and more potent inhibitory effects on BCR-ABL than imatinib. Several studies have shown hematologic and cytogenetic responses to nilotinib in patients with imatinib-resistant or intolerant CML. Purpose: To investigate the safety and efficacy of nilotinib for patients with imatinib-resistant or intolerant, chronic (CP)- or accelerated (AP)-phase CML from the East Japan CML Study Group (EJCML) trial by evaluating molecular responses in terms of the BCR-ABL1 mutational status and plasma trough concentration of nilotinib. Methods: In this multicenter phase II clinical trial, nilotinib (400 mg bid) was administered orally for one year and the molecular responses were monitored by means of the international scale of quantitative PCR (IS-PCR). BCR-ABL1 mutations were analyzed by direct sequencing at the baseline and 12 months or at the time of the event for discontinuation of the treatment (i.e., progressive disease, insufficient effects, or severe adverse events). The plasma trough concentration of nilotinib was measured by high-performance liquid chromatography 3 months after nilotinib administration. Results: From March 2009 through February 2011, 51 patients were registered in this study, and data of 49 patients whose molecular responses were evaluated by the IS-PCR were analyzed (imatinib-resistant CML = 33, imatinib-intolerant CML = 16; CP CML = 46, AP CML = 3). The median follow-up period was 12.0 months (range = 0.1–13.3 months). At 6 and 12 months, the major molecular response (MMR; ≤0.1% IS) rates were 52.5% and 67.6%, respectively, and the complete cytogenetic response (CCyR)-equivalent (≤1.0% IS) rates were 75.0% and 85.3%, respectively. Five types of BCR-ABL1 mutations (M244V, F317L, N358D, F359V, and E459K) were detected in 6 patients (12.2%) at the baseline, but the M244V, N358D, and E459K mutations disappeared after the nilotinib treatment. Acquired BCR-ABL1 mutations (Y253H, I418V, and exon 8/9 35bp insertion) were detected in 3 patients (8.6%) at 12 months or at the time of the event; these patients did not achieve a CCyR or an MMR. No patients showed an acquired mutation of T315I. Most patients except 11 subjects (22.4%) still received the treatment. The reasons for discontinuation were progressive disease in one patient with an F317L mutation, insufficient effects in one patient without any mutation, and adverse events in 9 patients (thrombocytopenia in 5 patients, hyperbilirubinemia in 2 patients, headache in one patient, and heart disease in one patient). Among 30 patients without BCR-ABL1 mutations, the plasma trough concentration of nilotinib was significantly higher in 21 patients with an MMR than in those without an MMR by 12 months (median = 1255.1 ng/mL vs. 372.8 ng/mL, P = 0.0012 by Mann–Whitney U-test; see the figure). The concentration of 761 ng/mL was significantly associated with an MMR by 12 months in a receiver-operating characteristic (ROC) curve analysis of the best sensitivity (76.2%) and specificity (77.8%). Conclusion: The patients with imatinib-resistant or intolerant, CP or AP CML, even those having BCR-ABL1 mutations M244V, N358D, and E459K, achieved an MMR by 12 months of nilotinib treatment. The plasma trough concentration of the drug was related to the MMR by 12 months, and the plasma threshold of nilotinib should be set above 761 ng/mL. These findings suggest that nilotinib shows good efficacy and tolerability in Japanese patients with imatinib-resistant or intolerant, CP or AP CML. (ClicalTrials.gov, UMIN ID 000002201) Disclosures: No relevant conflicts of interest to declare.