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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.

Cytomegalovirus reactivation (CMVR) after allogeneic hematopoietic cell transplantation (HCT) is a frequent complication related to survival outcomes, but the impact of CMVR on relapse is still unclear, especially in acute lymphoblastic leukemia (ALL). In this nationwide retrospective study, we included patients with acute myeloid leukemia (AML) and ALL in first or second complete remission who underwent their first HCT using pre-emptive strategy for CMVR. Because ninety percent of cases with CMVR had occurred by day 64 and ninety percent of cases with grades II to IV acute GVHD had occurred by day 58, a landmark point was set at day 65. In the landmark analyses, 3793 patients with AML and 2213 patients with ALL who survived without relapse for at least 65 days were analyzed. In the multivariate analyses, CMVR was associated with a lower incidence of relapse in both AML (hazard ratio [HR], 0.81; 95% confidence interval [CI], 0.69-0.95; P = 0.009) and ALL (HR, 0.81; 95% CI, 0.66-0.99; P = 0.045). These findings were confirmed when we treated CMVR as a time-dependent covariate. Moreover, our study suggested that the protective effect of CMVR on relapse was independent of acute GVHD. A post-hoc subgroup analysis that combined AML and ALL showed that CMVR had a mild anti-leukemia effect without effect modification in contrast to the impact of CMVR on NRM. Our findings may provide important implications for the strategy used for CMV prophylaxis after HCT.

We aimed to evaluate the impact of pretransplant imatinib administration on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in adults with Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL). We retrospectively analyzed 738 patients with Ph(+) ALL that underwent allo-HSCT between 1990 and 2010 using data from the Transplant Registry Unified Management Program of the Japan Society of Hematopoietic Cell Transplantation. We compared the allo-HSCT outcomes between 542 patients who received imatinib before allo-HSCT during the initial complete remission period (imatinib cohort) and 196 patients who did not receive imatinib (non-imatinib cohort). The 5-year overall survival after allo-HSCT was significantly higher in the imatinib cohort than in the non-imatinib cohort (59% vs 38%; 95% confidence interval [CI], 31-45%; P < .001). Multivariate analysis indicated that pretransplant imatinib administration had beneficial effects on overall survival (hazard ratio [HR], 0.57; 95% CI, 0.42-0.77; P < .001), relapse (HR, 0.66; 95% CI, 0.43-0.99; P = .048), and nonrelapse mortality (HR, 0.55; 95% CI, 0.37-0.83; P = .005). In conclusion, our study showed that imatinib administration before allo-HSCT had advantageous effects on the clinical outcomes of allo-HSCT in patients with Ph(+) ALL.

Background: The number of solid organ transplantation (SOT) is increasing. While the remarkable improvement in SOT procedures provides recipients with a good chance of long-term survival, the prolonged immunosuppression has led toa variety of malignancies in solid organ transplant (SOT) recipients; post-transplant lymphoproliferative disorder (PTLD) is a life-threating complication among them. However, there remain some controversies regarding its clinical features. Method: We retrospectively surveyed the patients who had visited both departments: hematology and urology, or renal surgery in kidney center at Tokyo Women's Medical University Hospital from 2003 to 2017. We identified 61 patients diagnosed with hematological disorder in kidney-transplant recipients. Among them, PTLD were analyzed. Continuous variables were compared using Student's t-tests. Overall survival rate (OS) was calculated using the Kaplan-Meier method. P values < 0.05 were considered significant. Statistical analyses were performed using JMP Pro 14 (SAS Institute Inc., Cary, NC, USA) and GraphPadPrism version 7.0 (GraphPad Software, San Diego, USA). This study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review boards. Results: During the survey period, 3,133 patients had visited both departments: hematology and urology, or surgery in Kidney Center. Among them, 61 kidney-transplant recipients were diagnosed with hematological disorder; they comprised 27 PTLD, 10 plasmacytic disorder, 6 idiopathic thrombocytopenic purpura, 5 myelodysplastic syndromes, 4 acute leukemia, 3 polycythemia, 6 other diseases. Among the patients with PTLD (n = 27), 18 patients (66.7%) were male.Median patient ages at the diagnosis of PTLD and kidney transplant were 54 (25-79) and 43 (16-75) years, respectively. Eighteen and nine patients had received kidney-transplant from a living donor (15 related and three unrelated donors) and a cadaveric donor, respectively. The median time from kidney transplant to diagnosis of PTLD was 7.8 years; there was a significant difference in the time between the kidney-transplant recipients from a cadaveric donor and those from a living donor (Figure 1A, 11.3 and 5.6 years, P = 0.02). With regard to the histological subtypes of PTLD, they comprised 17 diffuse large B-cell lymphoma (63.0%), 2 Burkitt lymphoma (7.4%), 4 unclassified B-cell lymphoma (14.8%), 2 T-cell lymphoma (7.4%), and 2 unknown cases (7.4%). 24 of 27 patients had extranodal involvement: 9 central nervous system (33.3%), 7 gastrointestinal tract (25.9%) and other sites. First-line treatment procedure which all patients received was the modification of immunosupressants; complete response (CR) rate was 37.0%. After the failure of first-line therapy, 15 of 27 patients received chemotherapy: 4 rituximab monotherapy (30.8%), 6 CHOP-like regimens (46.2%), and other regimens (33.3%). Among them, five patients experienced relapse. They received salvage chemotherapy, and 2 and one patients achieved CR and partial response, respectively. Rest of them died of lymphoma and infection. The estimated five-year OS from the diagnosis of PTLD and the initiation of chemotherapy was 88.7% and 86.7%, respectively. During the survey period, 5 patients died; their causes of death were 3 lymphomas, 1 infection, and 1 heart failure. There were no deaths in kidney-transplant recipients from a cadaveric donor (Figure 1B). Discussion: While our cohort had more extranodal PTLD recipients than those reported previously, the modification of immunosupressants improved them in more than one-third of cases, which showed the usefulness of it as first-line treatment procedure for PTLD. Nearly half of cases were identified in recipients from a cadaveric donor; the results suggest that the incidence o PTLD among them is higher than those from a living donor because the number of kidney-transplant from a living donor was 10 times higher than those from a cadaveric donor. This study has several limitations due to its retrospective nature and small sample size. In addition, the estimated five-year OS might be better than actual because some patients became lost to follow-up. Further large-scale studies are warranted to validate the findings of this study. Disclosures Hagiwara: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Tanaka:Bristol-Myers Squibb: Research Funding.

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

Introduction: We previously reported that CD86 expressed on tumor cells from multiple myeloma (MM) patients is associated with a proliferative advantage of tumor cells and suppresses the antitumor immune response by inducing the immunosuppressive cytokine IL-10 from CD4+ T cells via CD86-CD28 interaction [Yamashita T, Clin Cancer Res 2009]. Furthermore, CD28 expressed on MM cells can mediate pro-survival signaling through CD86-CD28 interaction, resulting in chemotherapeutic resistance [Murray ME, Blood 2014]. Hock et al. reported that serum soluble CD86 levels (sCD86) were a significant independent prognostic marker in MM patients treated with conventional chemotherapy [Br J Haematol 2006]. However, it is unknown whether serum soluble CD86 is still a prognostic marker in the novel agent era and how sCD86 is produced in serum. In this study, we investigated the association of clinical characteristics and prognosis with serum sCD86 and elucidated the mechanism by which sCD86 is produced in MM patients. Materials and Methods: 1) Peripheral blood and bone marrow (BM) samples were obtained from 294 newly diagnosed (42 asymptomatic and 252 symptomatic) MM patients, 16 patients with monoclonal gammopathy of undetermined significance (MGUS), and 16 healthy controls. 2) sCD86 levels were measured using ELISA. 3) The expression of cell-surface antigen on plasma cells identified as CD138-positive and CD38-strong positive cells were analyzed using flow cytometry. Expression of the CD86 full-length (variant 1) and alternatively spliced variant deleting exon 6 (variant 3), which encodes for the transmembrane domain, in MM cell lines and CD138+ plasma cells isolated from BM samples from MM patients were analyzed using real-time PCR. Results: 1) sCD86 levels were significantly increased in MM patients compared with MGUS patients and healthy controls. Among MM patients, the levels were significantly higher in symptomatic than in asymptomatic patients and markedly increased in advanced stages (International Staging System [ISS] stage II/III and revised ISS II/III). We next investigated the differences in clinical characteristics between two groups according to serum sCD86 levels: high (≥2.16 ng/mL); and low ( Conclusions: We demonstrated that sCD86 is produced via expression of CD86 variant 3 in primary MM cells and that sCD86 is a prognostic marker in the novel agent era. These results suggest that serum sCD86 levels may reflect disease progression through CD28 expressed on myeloma cells and be a useful prognostic factor. Disclosures Inokuchi: Novartis: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria. Handa:Ono: Research Funding. Komatsu:Fuso Pharmaceutical Industries, Ltd.: Research Funding; Novartis K.K: Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau; Wako Pure Chemical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau. Imai:Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Ito:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Ono: Honoraria. Tamura:Celegene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Takeda Pharmaceutical: Honoraria.

[Background] We previously demonstrated that allogeneic hematopoietic stem cell transplantation (HSCT) in the first complete remission (CR1) was recommended for adult patients wih Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) who have a human leukocyte antigen (HLA)-matched related (Leukemia 2011;25:259-265) or unrelated donor (Bone Marrow Transplant 2013;48:1077-1083). However, pediatric-inspired high-intensity chemotherapy dramatically improved the prognosis of adult patients with Ph-negative ALL. Therefore, the optimal treatment strategy for Ph-negative ALL in CR1 has not been established in the era of high-intensity chemotherapy. [Methods] Outcomes of patients with Ph-negative ALL who underwent HSCT from HLA-matched related or unrelated donor in CR1 performed between 2002 and 2011 (HSCT-MRD group and HSCT-MUD group, respectively) were obtained from the Transplant Registry Unified Management Program (TRUMP), which is the registry database of the Japanese Society for Hematopoietic Cell Transplantation (JSHCT). Patients aged between 16 and 24 and between 25 and 65 were analyzed separately, and their outcomes were compared with those of patients who did not receive HSCT in CR1 in clinical studies by Japan Adult Leukemia Study Group (JALSG), ALL-202U study in which patients received chemotherapy designed for pediatric patients (Blood Cancer J 2014;4:e252)(202U group) and ALL-202O study in which patients were randomly assigned to receive adult-type chemotherapy with high-dose or intermediate-dose methotrexate (MTX) therapy (Leukemia 2018;32:626-632)(202O group), respectively. Median durations between diagnosis to HSCT were obtained from the TRUMP data, and only patients who were disease-free more than each median duration were included from the JALSG studies. Risk stratifications were performed based on the white blood cell (WBC) count and cytogenetics. [Results] In patients aged less than 25 who underwent HSCT from related donor, the median duration between diagnosis to HSCT was 188 days. Overall survival (OS) in 202U group (N = 93) was significantly superior compared with OS in HSCT-MRD group (N = 102) (OS at 5 years: 81.8% vs 67.8%, P = 0.016) (Figure 1(A)), and which was attributed to the higher non-relapse mortality in HSCT-MRD group (NRM at 5 years: 1.4% vs 9.5%, P = 0.015). Disease-free survival (DFS) and relapse rate (RR) were similar between two groups (DFS at 5 years: 70.2% vs 62.5%, P = 0.115, and RR at 5 years: 28.4% vs 28.4%, P = 0.689). Higher OS was preserved in younger ( In patients aged more than 24 who underwent HSCT from related donor, the median duration between diagnosis to HSCT was 158 days. OS rate was not different between 202O group (N = 147) and HSCT-MRD group (N = 152) (OS at 5 years: 57.0% vs 60.2%, P = 0.114) (Figure 1(B)). The lower RR was counterbalanced by the higher NRM in HSCT-MRD group (RR at 5 years: 51.6% vs 30.0%, P < 0.001, and NRM at 5 years: 4.1% vs 15.2%, P = 0.015). No difference in OS was demonstrated among any subgroups. Superior DFS in HSCT-MRD group was observed only in high-risk patients (DFS at 5 years: 26.5% vs 57.2%, P = 0.003), but it diminished when only patients who actually received high-dose MTX were included in 202O group (DFS at 5 years: 36.4% vs 57.2%, P = 0.092). In patients aged more than 24 who underwent HSCT from unrelated donor, the median duration between diagnosis to HSCT was 214 days. OS rate was not different between 202O group (N = 138) and HSCT-MUD group (N = 104) (OS at 5 years: 60.0% vs 65.8%, P = 0.563). Superior DFS in HSCT-MUD group was observed even in all patients (DFS at 5 years: 47.2% vs 64.2%, P = 0.029). RR in HSCT-MUD group was lower (RR at 5 years: 14.3%) than that in HSCT-MRD group. [Discussions & Conclusions] High-intensity chemotherapy may change the role of HSCT for Ph-nagative ALL. Improved outcomes in HSCT-MUD group were observed, and it was partially because patients who had long CR before HSCT could underwent HSCT from MUD in CR1. Disclosures Kako: Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria. Tanaka:Bristol-Myers Squibb: Research Funding. Ichinohe:JCR Pharmaceuticals: Honoraria; Celgene: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Astellas Pharma: Research Funding; Chugai Pharmaceutical Co.: Research Funding; CSL Behring: Research Funding; Eisai Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Ono Pharmaceutical Co.: Research Funding; Pfizer: Research Funding; Nippon Shinyaku Co.: Research Funding; MSD: Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Repertoire Genesis Inc.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Takeda Pharmaceutical Co.: Research Funding; Zenyaku Kogyo Co.: Research Funding; Alexion Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Mundipharma: Honoraria; Novartis: Honoraria. Kurahashi:Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd: Honoraria; Bristol-Myers Squibb, Ltd.: Honoraria. Usui:Alexion Pharmaceuticals: Other: Personal Fees; Bristol-Myers Squibb: Other: Personal Fees, Research Funding; Kyowa-Hakko-Kirin Co.Ltd.: Other: Personal Fees; Astellas Pharma Inc.: Other: Personal Fees; Huya Bioscience International: Other: Personal Fees; Nippon Shinyaku Co.: Other: Personal Fees; SymBio Pharmaceuticals: Other: Personal Fees; Celgene Corporation: Other: Personal Fees; Pfizer: Other: Personal Fees, Research Funding; Takeda Pharmaceutical Co. Ltd: Other: Personal Fees; Cmic Co: Other: Personal Fees; Otsuka Pharmaceutical Co.Ltd.: Other: Personal Fees; AbbVie Inc: Other: Personal Fees. Kiyoi:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Perseus Proteomics Inc.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding. Matsumura:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Otsuka Pharmaceutical: Consultancy, Research Funding; Pfizer: Research Funding, Speakers Bureau. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria.

Recent advances of monoclonal antibodies provide a potent anti-myeloma effect based on antibody-dependent cytotoxicity (ADCC). Elotuzumab is a monoclonal antibody which binds SLAMF7 on the surface of myeloma cells and induces ADCC by NK cells (Cancer Immunol Immunother 64:61-73,2015). Lenalidomide may enhance the killing activity ad IFN-gamma production in NK cells (Blood 126:50-60,2015). In Eloquent-2 trial, the combination therapy of Elotuzumab, Lenalidomide, and Dexamethasone (ELD) showed a significant relative reduction in the risk of disease progression or death (N Engl J Med 373:621-631,2015). However, the number of NK cells in patients with multiple myeloma is generally suppressed, and the activity is often exhausted (Cytometry 26:121-124,1996, Med Oncol 24:312-317,2007). It is unclear if they fully exhibit the ADCC against myeloma cells. Also, enough number of NK cells as effector cells is required to obtain effective ADCC. To enhance the ADCC activity of NK cells against multiple myeloma, cell therapy using expanded NK cells might be effective. Therefore, we conduct the clinical trial of ex-vivo expanded NK cell infusion therapy in combination with ELD for minimal residual disease (MRD) after autologous stem cell transplantation (ASCT). Clinical trial registry number: UMIN000033128 Study design: Non-randomized single-arm phase I/ II clinical trial. Study population: 20; Five for phase I part and 15 for phase II. Primary endpoint: Any grade of adverse events, and the response defined by IMWG criteria. Secondary endpoint: Overall survival, progression-free survival, and MRD. Study protocol: 1x106 - 5x107/kg of ex-vivo expanded NK cells are infused on day 2 of ELD regimen in patients who failed to achieve sCR or with detective MRD after ASCT. The NK cell infusion with ELD therapy will be repeated 4 cycles monthly. Major inclusion criteria: (1)Multiple myeloma diagnosed by IMWG diagnostic criteria, (2)Age: 20==, (3)ECOG Performance status0-2, (4)PR or better after induction therapy, (5) Transplant eligible patients without severe organ damages, (6)Patients failed to achieve sCR after stem cell transplantation or positive for minimal residual disease by 6-8 color Flow cytometry. Major exclusion criteria: (1) Patients who were previously transplanted, (2) Failed to obtain more than 2x106/kg of CD34+ cells, (3) Patients who are not eligible for harvest or transplant due to cardiac or pulmonary disorder, AST/ALT/total bilirubin>2.5x lower limit, serum Cr>=2.0mg/dl, (4) Patients who have severe allergy or uncontrollable active infection. Currently, two participants completed the protocol and are under the observation. Figure Disclosures Hagiwara: Bristol-Myers Squibb: Research Funding. Kanno:Agios Pharmaceuticals, Inc.: Honoraria. Tanaka:Bristol-Myers Squibb: Research Funding.

Deletion of long arm of chromosome 20 (del(20q)) is commonly observed in myelodysplastic syndromes (MDS). Reduced expression of genes located within the common deleted region (CDR) of del(20q) due to haploinsufficiency may play a role in molecular pathogenesis of MDS. In the previous study, we examined expression of genes located within the CDR which we determined using array-CGH, in bone marrow mononuclear cells in MDS patients with or without del(20q), indicating that BCAS4 expression was significantly reduced in bone marrow cells in MDS patients with or without del(20q). The BCAS4 gene, which was identified as a fusion transcript expressed in MCF7 cells, encodes 23kD protein. Although function of BCAS4 protein remains unclear, it could be a member of "cappuccino" family, which belong to lysosome-related organelles. Abnormality of genes encoding lysosome-related organelles cause variety of congenital disorders, including the Hermansky-Pudlak syndromes, which is characterized by oculocutaneous albinism and bleeding tendency due to platelet dysfunction as a result of lysosome abnormalities. In the present study we investigated clinical implication of BCAS4 expression level in MDS patients. Mononuclear cells separated from bone marrow samples taken at the time of MDS diagnosis were used for analysis. Written informed consent was obtained from patients before study. To analyze BCAS4 expression, quantitative RT-PCR was performed using cDNA from mononuclear cells as template by the TaqMan probe method (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems). Samples from 103 MDS patients, 64 males and 39 females with median age of 67 years (range: 20-91 years), with (n=14) or without (n=89) del(20q), were examined in the present study. Patients were classified as RCUD (n=12), RCMD (n=55), RARS (n=9), RAEB-1 (n=10), and RAEB-2 (n=13), according to WHO 2008 classification, and in RAEB-T (n=4) according to FAB classification. They also were categorized in four IPSS risk groups, low risk (n=30), intermediate-1 risk (n=46), intermediate-2 risk (n=18), and high risk (n=9). There was no significant difference in relative BCAS4 expression level between patients with del(20q) and those without del(20q), and among WHO subtypes. Higher IPSS risk groups (INT-2 and High) showed trend in association with reduced BCAS4 expression compared with lower IPSS risk groups (Low and INT-1) (P=0.104). We analyzed impact of BCAS4 expression on overall survival (OS). Based on BCAS4 expression level, 103 patients were divided into four groups, highest (Q1), intermediate (Q2, Q3), and lowest (Q4) quartiles. The Kaplan-Meier analysis demonstrated that Q4 showed significantly worse OS compared with remaining quartiles (Q1-Q3) (log-rank test, P=0.0031). The estimated 2-year OS rates in Q1-3 group and Q4 group were 75.1% and 48.9%, respectively. According to the COX proportional hazards model, univariate analysis showed lower BCAS4 expression (Q4 vs Q1-Q3) was associated with worse OS (hazard ratio 3.43, 95%CI 1.89-6.11, P=0.0001) as well as older age (65 years or older vs less than 65 years), and higher IPSS risk groups (INT-2 and High vs Low and INT-1). Multivariate analysis indicated that lower BCAS4 expression showed trend for association with worse OS (hazard ratio 1.90, 95%CI 0.96-3.64, P=0.0651) by analyzing with two variables (older age and higher IPSS groups). Next, we investigated whether OS is predicted by combination of three variables, BCAS4 expression level, IPSS risk groups, and age at diagnosis. We defined lower BCAS4 expression (Q4), higher IPSS (INT-2 and High), and older age (65 years or older), as risk factors. The Kaplan-Meier analysis showed that survival curves were well separated according to number of risk factors (0, 1, and 2 or more) (P The present study demonstrated that reduced BCAS4 expression is associated with inferior clinical outcome, indicating that BCAS4 expression level could be a useful prognostic marker in MDS, especially by combination with IPSS risk and patients age at diagnosis. Disclosures Tanaka: Bristol-Myers Squibb: Research Funding.

Background: Methotrexate-associated lymphoproliferative disorders (MTX-LPD) develops in patients with rheumatoid arthritis (RA) or other autoimmune disorders during low-dose MTX treatment. MTX-LPD includes wide variety disease spectrum, ranging from polymorphic proliferation to aggressive lymphoma. Although etiology of MTX-LPD has not been fully understood, approximately half of the MTX-LPD cases showed association with EB virus (EBV), suggesting that MTX treatment causes reduced immune response to EBV-positive cells, and results in MTX-LPD development. Natural killer (NK) cells play important roles in eradicating tumor and virus-infected cells. NK cell function is modulated by multiple cell surface receptors, including Killer immunoglobulin-like receptor (KIR). There are multiple KIR genes (inhibitory or activated), which are various in number and/or composition among individuals, on chromosome 19q. Previous reports demonstrated that combination of KIR genes affects NK cell function, and is associated with the risk of development of certain types of cancers, viral infections and collagen disease. There is no report about the association of KIR genotype and MTX-LPD. We consider that NK cells play a significant role in suppression of MTX-LPD development. In this study, we focused on examining genotype KIR and KIR-ligand (HLA class I). Methods: We retrospectively analyzed 35 MTX-LPD cases diagnosed between 2009 and 2019. Genomic DNA was extracted from mononuclear cells that were isolated from the bone marrow or peripheral blood samples of patients with MTX-LPD. KIR genotypes were analyzed using the KIR genotyping sequence-specific primers kit. The variations of KIR content and haplotype and their relationship with progression to malignant lymphoma (ML) and response to chemotherapy were investigated. HLA was analyzed using PCR-Luminex assay. The frequency of each HLA allele and each combination was determined by referring to the data base of an HLA laboratory. Chi-squared (χ2) tests and Wilcoxon rank sum tests were used to test associations between the variables. Results: Among the 35 patients, 25 were diagnosed with ML and 10 with polymorphic LPD. Diffuse large B cell lymphoma (DLBCL) was most common type in ML (57.7%). Table 1 showed characteristics of patients and summary of the results. All patients underwent MTX treatment for RA. The median duration of MTX administration at the time of MTX-LPD diagnosis is 11.5 years (range=0.8-27.2), and median MTX dose was 10mg/week (range=4-17.6). The duration and dose of MTX had no effect between ML and polymorphic LPD. Twenty-three patients required chemotherapy, and 12 patients had tumor regression after stopping MTX treatment. Relative patient populations requiring chemotherapy in ML or polymorphic LPD were 85% or 11%, respectively (P=0.0001). EBV-positive patients tended to regress tumors with MTX discontinuation alone (P=0.16). In KIR genotype analysis, patterns of number and combination of the KIR genes are mainly classified as haplotype "A" containing multiple inhibitory KIR genes with a KIR 2DS4 (an activated KIR [aKIR]) and haplotype "B" (other than haplotype "A"). Patients were classified in haplotype A (13 cases, 37%) and haplotype B (22 cases, 63%), respectively. ML patients showed higher ratio in haplotype A (ML 46.2% vs LPD 11.1% P=0.045). There was no difference in number of aKIR or iKIR between ML and polymorphic LPD patients. In HLA Class I analyses, there was significant difference in frequencies of HLA-C haplotype between lymphoma and polymorphic LPD patients (P=0.026). Furthermore, HLA- C1 / C1 patients were more relapsed or refractory to chemotherapy than C1 / C2 patients (P = 0.17). Conclusion: This is the first report showing clinical significance of KIR genotypes in MTX-LPD. Patients with haplotype A, a suppressive haplotype, seems to be at high risk for developing lymphomas that require chemotherapy during MTX treatment. HLA- C1/C1 patients are more likely to develop lymphomas that respond poorly to treatment, suggesting that the activity of NK cells may be lower because ligands can match with KIRs that are more restricted than C1/C2. Considering the potential NK functions with KIR genotype would improve the understanding of the prognosis and lead to prevention for MTX-LPD. Disclosures Hagiwara: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Harigai:Bristol Myers Squibb Co: Other: personal fees, Research Funding; Eisai Co: Other: personal fees, Research Funding; Ayumi Pharmaceutical Co: Other: personal fees, Research Funding; AbbVie Japan GK,: Other: personal fees, Research Funding; Eli Lilly Japan K.K: Other: personal fees; Kissei Pharmaceutical Co.: Other: personal fees; Teijin Pharma Ltd: Other: personal fees, Research Funding; Mitsubishi Tanabe Pharma Co: Research Funding; Nippon Kayaku Co.: Research Funding; Pfizer Japan Inc.: Other: personal fees; Chugai Pharmaceutical Co., Ltd.: Other: personal fees; Japan College of Rheumatology: Other: personal fees; Boehringer Ingelheim Japan, Inc: Other: personal fees; GlaxoSmithKline K.K: Other: personal fees; Oxford Immuotec,: Other: personal fees. Tanaka:Bristol-Myers Squibb: Research Funding.

Background. The recent advance of antiretroviral therapy decreased the morbidity of opportunistic infections. However, the incidence of HIV-associated lymphoma remains high. Also, the outcome of the HIV-associated lymphoma is unclear in the era of rituximab. In order to address these clinical questions, we performed a nation-wide epidemiological study. Methods. Patients with HIV-associated lymphoma were extracted from the database of Japanese society of hematology blood disease registry from January 2012 to December 2015. We analyzed the patient's age, sex, subtypes of lymphoma, the international prognostic index (IPI) for diffuse large B cell lymphoma, and overall survival. Results. Eighty-one patients were extracted from the database. Eighty patients were available for the survival analysis. Seventy-six (93.8%) patients of them were male. The median age was 52.5(25-88) year-old. However, there were two peaks of age; the first peak was 38-40-year-old and the second was 59-62-year-old. Sub-types of lymphomas were diffuse large B cell lymphoma (DLBCL)(48.1%), Burkitt lymphoma(19.8%), primary CNS lymphoma(8.6%), plasmablastic lymphoma(7.4%), peripheral T cell lymphoma(3.7%), Hodgkin's lymphoma(3.7%), primary effusion lymphoma(2.5%), MALT lymphoma(1.2%), Follicular lymphoma(1.2%) and Adult T cell lymphoma/leukemia(1.2%). Extra-nodal involvement at the diagnosis was observed in 61.7%. The involved sites were the brain, stomach, small bowel, colon, thyroid and the others. In DLBCL, the patients with IPI high and high-intermediate risk was 51.3%. The median observation period was 26 months. Estimated 3 years overall survival (OS) in all cases was 68.8+/-0.63%. Although there was no statistical significance, however, the 3 years, OS of Burkitt lymphoma tended to be better than that of DLBCL (84.6%+/-10.0 versus 67.7+/-8.8%). Log-rank analysis showed the OS in DLBCL patients with IPI high-intermediate and high risk was significantly worse than the patients with low, and low-intermediate risk (p Conclusion. Our study showed diversity in the pathological subtype of HIV lymphoma. In the era of rituximab, the outcome seemed to be improved in patients with DLBCL and Burkitt lymphoma. However, the survival remains short in patients with poor prognostic factors and primary CNS lymphoma. Figure. Figure. Disclosures Hagiwara: Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Shiseki:NOVARTIS Pharma: Honoraria, Research Funding; Bristol-Myers Sqibb: Honoraria; Otsuka: Speakers Bureau. Tanaka:Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Otsuka: Honoraria; Pfizer: Honoraria.

Post-transplant lymphoproliferative disorder (PTLD) following hematopoietic stem cell transplantation (HSCT) is a rare, but life-threatening complication. PTLD typically develops within 6-12 months of HSCT, which is before the reconstitution of EBV-specific cytotoxic T-cell immunity. Several risk factors for developing PTLD have been reported in the literature, including the use of antithymocyte globulin (ATG) and ex vivo T-cell depletion (TCD). However, only a few large-scale retrospective studies have been conducted and risk scores have not yet been well defined. Therefore, to further evaluate the probability of and risk factors for PTLD and establish a risk scoring system, we conducted a retrospective multicenter analysis using data from a Japanese nationwide transplant outcome registry database. A total of 71,904 patients aged 16 years or older who received HSCT between January 1990 and December 2016 were included in this study. We excluded patients for whom information regarding the transplantation type or PTLD development was unavailable, resulting in 64,539 patients ultimately being analyzed. The probability of developing PTLD was estimated using the Kaplan-Meier method, and differences between groups were evaluated using the log-rank test. To identify risk factors for PTLD, univariate and multivariate analyses were performed using the Cox's proportional hazard regression model for patients who received allogeneic HSCT. Among the 64,539 patients analyzed, 40,195 received allogeneic HSCT, 24,215 received autologous HSCT, and 129 received syngeneic HSCT. A total of 299 patients developed PTLD, including 267 who had received allogeneic HSCT, 31 who had received autologous HSCT, and one who had received syngeneic HSCT. The median age of patients with PTLD after allogeneic HSCT was 44 years (range, 16-76), and males accounted for approximately 60%. The underlying disease was acute myeloid leukemia and myelodysplastic syndrome in 20,474 patients (51%), acute lymphoblastic leukemia in 6,808 (17%), chronic myeloid leukemia and myeloproliferative disorder in 2,827 (7%), malignant lymphoma in 7,824 (19%), aplastic anemia (AA) in 1,327 (3%) and others in 932 (2%). ATG was used in 4,026 patients (10%) who underwent allogeneic HSCT. In contrast, ex vivo TCD was used only in 296 (0.7%). The donor type was human leukocyte antigen (HLA)-matched-related donor (MRD) in 13,058 patients (33%), HLA-mismatched-related donor (MMRD) in 4,095 (10%), HLA-matched-unrelated donor (MURD) in 10,216 (25%), HLA-mismatched-unrelated donor (MMURD) in 1,993 (5%) and unrelated cord blood (CB) in 10,118 (25%), but was unknown for the remaining 715 patients (2%).The probability of PTLD at 2 years after HSCT was 0.79% with a median onset of 131 (range, 15-6,356) days in patients after allogeneic HSCT. In contrast, the probability of PTLD was 0.11% with a median onset of 198 (range, 21-4,759) days in those after autologous HSCT. Only one patient developed PTLD at 29 days in those after syngeneic HSCT (0.78%). In multivariate analyses performed for patients after allogeneic HSCT, the following variables were identified as risk factors for PTLD: the use of ATG, donor type (MMRD, MURD, MMURD and CB), disease (AA), the number of HSCT, year of HSCT, and acute graft-versus-host disease (grade II-IV). To predict the risk of developing PTLD before HSCT, we established a novel scoring system using three pre-transplant risk factors that correlated with the development of PTLD. We assigned point(s) for the three risk factors by referring to each hazard ratio of the multivariate analysis as shown in the Table. Risk scores were calculated by adding each point(s), and patients were then successfully classified into 4 risk groups: low (score 0-1), intermediate (score 2), high (score 3), and very high risk (score 4-5), with probabilities of PTLD at 2 years after HSCT of 0.3, 1.1, 3.0, and 8.6%, respectively (Figure). Although ex vivo TCD is described as a risk factor for PTLD as well as ATG, it was not identified as a significant risk factor for PTLD in our cohort, probably due to the small number of the affected patients. This scoring system enables us to predict higher risk patients before HSCT. The close monitoring of EBV-DNA may be beneficial for the early diagnosis of these patients and more rapid initiation of treatment interventions, such as rituximab and EBV-specific cytotoxic T cells. Disclosures Mori: Janssen: Honoraria; Novartis Pharma: Research Funding; Taisho Toyama Pharmaceutical Co: Honoraria; CHUGAI: Honoraria; Kyowa Hakko Kirin: Honoraria; MSD: Honoraria; Novartis Pharma: Honoraria; Asahi Kasei: Research Funding; SHIONOGI: Honoraria; Pfizer: Honoraria; Japan Blood Products Organization: Honoraria; Shire Japan: Honoraria; Astella Pharma: Honoraria; Celgene: Honoraria; MSD: Research Funding; Eisai: Honoraria; Ono: Honoraria. Kanda:Nippon-Shinyaku: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Pfizer: Research Funding; MSD: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria. Tanaka:Pfizer: Honoraria; Otsuka: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis Pharma: Honoraria. Ichinohe:Celgene: Honoraria; Takeda Pharmaceutical Co.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Zenyaku Kogyo Co.: Research Funding; Mundipharma: Honoraria; Bristol-Myers Squibb: Honoraria; Alexion Pharmaceuticals: Honoraria; MSD: Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; Repertoire Genesis Inc.: Research Funding; JCR Pharmaceuticals: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Novartis.: Honoraria; Nippon Shinyaku Co.: Research Funding; Pfizer: Research Funding; Ono Pharmaceutical Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Eisai Co.: Research Funding; CSL Behring: Research Funding; Chugai Pharmaceutical Co.: Research Funding; Astellas Pharma: Research Funding. Suzuki:Celgene: Honoraria; Sawai Pharmaceutical: Honoraria; Ohtsuka: Honoraria; MSD: Research Funding; Meiji Seika Pharma: Honoraria; Takeda Pharmaceuticals: Honoraria; Shionogi: Honoraria; Novartis: Honoraria; Mochida Pharmaceutical: Honoraria; Chugai Pharmaceutical: Honoraria; Kyowa-Hakko Kirin: Honoraria; Bristol-Myers Squibb: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Gilead Sciences: Consultancy; MundiPharma: Consultancy; Jazz Pharmaceuticals: Consultancy.

nts Introduction Myelodysplastic syndrome (MDS) is clinically and biologically heterogeneous. Although the pathobiology of MDS is complex and not fully understood, the development of MDS involves a series of genetic changes, which influence hematopoietic growth and differentiation, resulting in the accumulation of abnormal cells and the impairment of normal hematopoiesis. Recently, Lindsley et al. reported the difference in the mutation of MDS genes in adolescent and young adult (AYA) patients and elder patients. These results suggested that MDS in AYA patients was biologically different from MDS in elderly patients. In addition, AYA patients have some specific issues such as infertility, late complications, and psychological and social problems. For AYA patients with MDS, similar to MDS patients of other age, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curable treatment option. However, limited evidence was available indicating the effectiveness of allogeneic HSCT in the treatment of MDS in AYA patients, because about 90% of patients noted are usually in an older age group. Thus, we aimed to investigate the epidemiology of AYA patients with MDS who received allogeneic HSCT for the first time, and estimate prognostic factors after HSCT, using registry data of Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT). Methods We enrolled AYA patients aged 16-39 years from JDCHCT, who received allogeneic HSCT for the first time between January 2000 and December 2015. Active disease was defined as the presence of >5% blast in bone marrow and/or presence of blast in peripheral blood. We set the primary endpoint as the 5-year overall survival (5yOS). Five-year cumulative incidence of relapse (5yCIR) and 5-year cumulative incidence of non-relapse mortality (5yCINRM) were also evaluated as secondary endpoints. OS was evaluated using the log-rank test. Other endpoints were evaluated using Gray's method. To evaluate the prognostic factor for 5yOS, we employed the multivariable Cox proportional hazards model. Covariates were as follows: age (16-29 vs 30-39), sex (male vs female), Performance Status (PS) according to the Eastern Cooperative Oncology Group (0-1 vs 2-4), disease status (active vs non-active), karyotype risk according to the International Prognostic Scoring System (IPSS, good vs intermediate vs poor), conditioning regimen (Reduced intensity conditioning regimen [RIC] vs myeloablative conditioning regimen), donor type (related bone marrow transplantation [BMT] or peripheral blood stem cell transplantation [PBSCT] vs unrelated BMT or PBSCT vs cord blood transplantation [CBT]), prophylaxis of graft-versus-host disease (tacrolimus-based vs cyclosporine A-based), and years of HSCT (2000-2007 vs 2008-2015). Results We analyzed 628 AYA patients with MDS who received HSCT for the first time. Patient characteristics are described in Table 1. Median age was 30 years, 327 patients (52.7%) were of age ≥30 years, and 356 patients (56.7%) were male. At diagnosis, 140 patients (37.4%) were at IPSS low or intermediate-1 risk. At HSCT, 257 (40.9%) patients had active disease and 315 (52.1%), 166 (27.4%), and 124 (20.5%) patients had good, intermediate, and poor risk karyotype, respectively. RIC was administered for 121 (19.9%) patients. Regarding donor type, 230 (36.6%) received related BMT or PBSCT, 297 (47.3%) received unrelated BMT or PBSCT, and 101 (16.1%) received CBT. Regarding primary endpoint, 5yOS after allogeneic HSCT was 69.6% (65.6-73.2%) (Figure A). Five-year CIR and 5yCINRM were 11.9% (9.5-14.7%) and 20.7% (17.4-24.2%), respectively (Figure B, C). Regarding prognostic factors influencing 5yOS, active disease (adjusted hazard ratio [aHR]: 1.54, 95% confidence interval [95%CI]: 1.10-2.17, p-value = 0.013), poor karyotype (aHR: 2.06, 95%CI: 1.38-3.07, p-value < 0.001), PS 2-4 (aHR: 2.03, 95%CI: 1.19-3.48, p-value = 0.013), unrelated BMT or PBSCT (aHR: 2.10, 95%CI: 1.33-3.30, p-value = 0.001) and CBT (aHR: 2.40, 95%CI: 1.46-3.95, p-value < 0.001) were significantly associated with 5yOS, but other factors were not (Table 2). Conclusion In the present study, the epidemiology of AYA patients with MDS who received allogeneic HSCT for the first time, and the prognostic factors influencing 5yOS were evaluated. Our results provide physicians with additional information for the treatment of AYA patients with MDS. Disclosures Mori: Novartis Pharma: Research Funding; Astella Pharma: Honoraria; Shire Japan: Honoraria; Taisho Toyama Pharmaceutical Co: Honoraria; Japan Blood Products Organization: Honoraria; Eisai: Honoraria; Janssen: Honoraria; MSD: Research Funding; SHIONOGI: Honoraria; Pfizer: Honoraria; CHUGAI: Honoraria; Novartis Pharma: Honoraria; MSD: Honoraria; Ono: Honoraria; Celgene: Honoraria; Asahi Kasei: Research Funding; Kyowa Hakko Kirin: Honoraria. Tanaka:Bristol-Myers Squibb: Honoraria, Research Funding; Otsuka: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria. Ishiyama:Alexion Pharmaceuticals, Inc.: Honoraria.

Background: High incidence of graft failure is a major obstacle for successful unrelated cord blood transplantation (UCBT). To ensure engraftment, unrelated cord blood (UCB) unit with higher total nucleated cell (TNC) and CD34 cell doses and less HLA mismatches between UCB units and patients were usually selected. However, it remains unclear whether HLA haplotype between the UCB units and patients should be matched. Using data from Japanese registry, we analyzed the effect of haplotype matching on the outcomes after single UCBT. Methods: Patients with hematologic diseases, aged 16 years or more, and have undergone their first UCBT between 2005 and 2014 were included in the study. The effect of haplotype matching (0, 1, and 2 haplotype matches) on neutrophil engraftment, overall mortality, relapse, and non-relapse mortality was analyzed after adjusting for other significant variables. Haplotype was estimated based on HLA-A, -B, -C, and -DRB1 allele information and Japanese allele frequency data. The haplotypes of both the patient and UCB unit were determined in 1,347 cases. Cases with more than 4 allele mismatches (n = 96) were excluded from the study. Results: The median age of the patients included in the study was 55 years (range 16-79 years). Median TNC and CD34 doses were 2.7 x 107/kg (range, 1.4-8.1x107/kg) and 0.8 x 105/kg (range, 0.1-5.2x105/kg), respectively. The number of allele mismatches were 0 in 82, 1 in 154, 2 in 252, 3 in 565, and 4 in 294 patients. The graft-versus-host disease (GVHD) prophylaxes were calcineurin inhibitors in combination with methotrexate in 707, calcineurin inhibitors in combination with Mycophenolate mofetil in 430, and other prophylaxes in 208 patients. The cumulative incidence of neutrophil engraftment at day 42 among groups with 0, 1, and 2 HLA haplotype matches was 79%, 82%, and 88%, respectively (Gray test, P=0.008) (Figure 1). In the multivariate analysis, the group with no haplotype match was marginally associated with worse neutrophil engraftment compared to the group with 1 haplotype match group (0 match vs. 1 match, HR 0.88, P=0.087), whereas the group with 2 haplotype matches was significantly associated with better neutrophil engraftment (2 match vs. 1 match, HR 1.39, P=0.005). Other significant variables were recipient sex, CD34 cell dose, transplant year, performance status, ABO matching, GVHD prophylaxis, and disease status. Tendency of worse neutrophil engraftment in the group with no haplotype matches was more apparent in patients with double mismatches at the same locus (0 match + no double mismatch vs. 1 match, HR 0.93, P=0.429, 0 match + double mismatch vs. 1 match, HR 0.80, P=0.050). Haplotype matching did not have any effect on overall survival and non-relapse mortality. Group with 2 haplotype matches was significantly associated with higher risk of relapse than the group with 1 haplotype match, whereas, there was no difference in the risk of relapse between the groups with no match and 1 match. Conclusions: In addition to the CD34 cell dose and HLA matching, HLA haplotype matching might be considered to obtain better neutrophil engraftment. Two haplotype matches (8 of 8 allele matches) should be avoided if the relapse risk is high. Figure 1. Figure 1. Disclosures Tanaka: Pfizer: Honoraria; Otsuka: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis Pharma: Honoraria. Ichinohe:Zenyaku Kogyo Co.: Research Funding; Celgene: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Taiho Pharmaceutical Co.: Research Funding; Mundipharma: Honoraria; Otsuka Pharmaceutical Co.: Research Funding; Nippon Shinyaku Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; MSD: Research Funding; Novartis.: Honoraria; JCR Pharmaceuticals: Honoraria; Alexion Pharmaceuticals: Honoraria; Repertoire Genesis Inc.: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co.: Research Funding; Pfizer: Research Funding; Ono Pharmaceutical Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Eisai Co.: Research Funding; CSL Behring: Research Funding; Chugai Pharmaceutical Co.: Research Funding; Astellas Pharma: Research Funding.

Definitive risk factors for the development of adult T-cell leukemia (ATL) among asymptomatic human T-cell leukemia virus type I (HTLV-1) carriers remain unclear. Recently, HTLV-1 proviral loads have been evaluated as important predictors of ATL, but a few small prospective studies have been conducted. We prospectively evaluated 1218 asymptomatic HTLV-1 carriers (426 males and 792 females) who were enrolled during 2002 to 2008. The proviral load at enrollment was significantly higher in males than females (median, 2.10 vs 1.39 copies/100 peripheral blood mononuclear cells [PBMCs]; P < .001), in those 40 to 49 and 50 to 59 years of age than that of those 40 years of age and younger (P = .02 and .007, respectively), and in those with a family history of ATL than those without the history (median, 2.32 vs 1.33 copies/100 PBMCs; P = .005). During follow-up, 14 participants progressed to overt ATL. Their baseline proviral load was high (range, 4.17-28.58 copies/100 PBMCs). None developed ATL among those with a baseline proviral load lower than approximately 4 copies. Multivariate Cox analyses indicated that not only a higher proviral load, advanced age, family history of ATL, and first opportunity for HTLV-1 testing during treatment for other diseases were independent risk factors for progression of ATL.

BACKGROUND : There have been few reports on outcome of adolescent and young adults (AYAs) with acute myeloid leukemia (AML) who received allogeneic hematopoietic cell transplantation (allo-HCT). We performed a retrospective analysis of nationwide registration data of the Japan Society of Hematopoietic Cell Transplantation collected between 1990 and 2013 to assess the allo-HCT outcomes of AYA patients with AML in Japan. PATIENTS & METHODS : 2973 patients with de novo AML (excluding acute promyelocytic leukemia, myeloid leukemia associated with Down syndrome, and secondary AML) whose age was 0-29 years old at their first allo-HCT either in first or second remission (CR), primary induction failure, or first relapse were identified. Outcomes including overall survival rate (OS), disease-free survival rate (DFS), cumulative incidence of relapse (CIR), and treatment-related mortality (TRM) were compared between children (0-14 years old at HCT, N=1123) and AYAs (15-29 years old, N=1850). RESULTS : AYA patients had male predominance, higher incidence of intermediate risk cytogenetics but lower incidence of high-risk cytogenetics [-7/7q-, -5/5q-, complex karyotype, t(6;9), or t(16;21)], and higher prevalence of French-American-British M0, M1, and M2 morphology, while M5 and M7 were lower. Compared to children, AYA patients were transplanted more frequently by HLA-matched-related or unrelated donors, while less frequently by HLA-mismatched related or unrelated cord blood donors. As for conditioning regimen, proportion of myeloablative conditioning (MAC) using total-body-irradiation (TBI) was higher in the AYAs, while that of non-TBI MAC and reduced-intensity-conditioning (RIC) were lower. Cyclosporine was more frequently used than tacrolimus as graft-versus-host-disease (GVHD) prophylaxis in the AYAs. Five-year OS and DFS rates were significantly poorer in the AYAs: 58% vs 54% (p Finally, we analyzed the impact of transplant center types on HCT outcomes among the 317 adolescent patients (15-18 years old) who were transplanted at 1CR or 2CR, because they could be transplanted either by pediatricians or by adult hematologists according to their referral pattern. 92 cases were transplanted in pediatric centers, 203 in adult centers, and 22 in combined pediatric and adult centers. Basic characteristics and method of HCT did not differ significantly among the three groups. Interestingly, there was no difference in OS, DFS, CIR, or TRM. CONCLUSIONS : AYAs with AML showed inferior post-transplant survival, which was mainly due to higher TRM in the AYAs. Disclosures No relevant conflicts of interest to declare.

Backgrounds: Primary myelofibrosis (PMF) is associated with poor prognosis and a marked reduction in life expectancy, with median survival durations ranging from 3.5 to 6 years, according to previous studies with great variation. We conducted a 17-year nationwide survey (1999-2015) to elucidate the clinical outcomes of patients with PMF in Japan, and evaluated the change of survival duration over time. Patients and Methods: Questionnaires were sent annually to approximately 500 hematology departments of board-certified member institute of the Japanese Society of Hematology to collect data about clinical features and prognoses of patients with PMF. Newly diagnosed patients with PMF were enrolled in this study and were followed up annually to collect prognostic information. Approximately 50 patients were enrolled per year, yielding an eventual total of 780 patients with PMF that were included in a 17-year study period (1999-2015). Results: The median age at diagnosis was 66 years. At the time of the analysis, 374 enrolled patients (48%) had died after a median survival duration of 47 months (95% CI, 41-53 months). Of the patients for whom the final cause of death was known, infection (n = 89) and transformation into acute leukemia (n = 91) were the most frequent causes. The 3-year overall survival (OS) rate was 59% (95% CI, 55%-63%). Significant improvements in the 3-year OS rate were not observed during the 17-year period of analysis (p = 0.235)(Figure A). Among the proposed prognostic models for predicting the outcomes of PMF patients, the Dynamic International Prognostic Scoring System of PMF (DIPSS) plus model was the most feasible for our cohort. However, the calculated areas under ROC curves were 0.609, and only modest accuracy was observed in this regard. Forty-three patients (median age of 52 years; range, 24-66 years) received allogeneic hematopoietic stem cell transplantation (alloSCT) at a median of 343 days after diagnosis. The estimated median survival duration after first alloSCT was 134 months (range, 71 to not reached), with a 3-year OS rate of 84% (95% CI, 68%-93%). The median survival duration after diagnosis among patients who received alloSCT was 207 months (range, 105 to not reached), and this is significantly longer than that calculated for patients who did not receive alloSCT (median: 45 months; range, 38-49 months; p < 0.001)(Figure B). Ruxolitinib therapy was given to 47 patients, but did not have an impact on survival duration. Conclusions: The survival curves of patients diagnosed during different subperiods of our study period exhibited complete overlap, indicating a lack of improvement in survival duration over time. However, only alloSCT among current treatment modalities had an impact on the natural course of PMF. Regarding prognostic prediction, DIPSS plus was the optimal model for survival prediction even among Japanese patients with PMF. However, the ROC analysis indicated that the prognostic covariates in DIPSS plus were not sufficient, and additional information regarding gene mutation statuses (e.g., CALR and ASXL1) might be required to predict the precise outcomes of PMF patients. A long-term registration study is required for further evaluations of prognosis and the impact of treatments on survival. Figure Figure. Disclosures Shibayama: Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Ozawa:Sumitomo Dainippon Pharma Co. Ltd: Research Funding; Takara Bio Inc: Research Funding; Celgene Japan: Consultancy; JCR Pharmaceutical Inc: Consultancy.

The PLCG1 gene encodes phospholipase C g isoform, which is involved in diverse physiological and pathological cellular processes through catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerole. Until now, roles of phospholipase C g 1 in hematopoiesis have been shown in animal models. Plcg1 deficient mouse lacks erythropoiesis and die in early embryonic stage. In zebrafish model, it was demonstrated that phospholipase C g 1 is required in granulocyte maturation. Physiological and/or pathological role of phospholipase C g 1 in human hematopoiesis has not been elucidated. In human, the PLCG1 gene is located on long arm of chromosome 20. Deletion of long arm of chromosome 20 (del(20q)) is commonly observed in myelodysplastic syndromes (MDS). Previously, we determined the common deleted region (CDR) of del(20q) in MDS, and the PLCG1 gene is located within the CDR. Reduced expression of genes located in the CDR due to haploinsufficiency may play role in molecular pathogenesis of MDS. Therefore, we analyzed PLCG1 expression in bone marrow mononuclear cells in MDS patients with or without del(20q), and investigated its clinical significance in the present study. Mononuclear cells separated from bone marrow samples taken at the time of diagnosis with written informed consent from patients were used. To analyze PLCG1 expression, quantitative RT-PCR was performed. Total RNA was extracted from mononuclear cells and subjected to cDNA synthesis. Real-time RT-PCR was carried out using cDNA as template by the TaqMan probe method (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems) were performed. The human PLCG1 primer-probe set was from Applied Biosystems. A total of 109 MDS patients, 65 males and 44 females with median age of 69 years (range: 22-91 years), with (n=20) or without (n=89) del(20q), were included in the present study. They were classified as RCUD (n=11), RCMD (n=55), RARS (n=11), RAEB-1 (n=16), and RAEB-2 (n=16) according to WHO classification. They were categorized in four IPSS risk groups, low risk (n=21), intermediate-1 risk (n=52), intermediate-2 risk (n=20), and high risk (n=6). Relative PLCG1 expression level was significantly reduced in MDS patients with del(20q) compared to control subjects (n=20) (P=0.011). Median values of relative PLCG1 expression level in MDS patients with del(20q) and control subjects were 0.94 and 1.96. In addition, relative expression level of PLCG1 in whole MDS cohort significantly lower than that in control subjects (P=0.046). Expression patterns of PLCG1 among, were not different. Median values of relative PLCG1 expression level in five WHO-subtypes, RCUD, RCMD, RARS, RAEB-1, and RAEB-2 were 1.50, 1.55, 1.29, 1.13, and 1.12, respectively, but no statistically difference was observed. WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) showed trend in association with reduced PLCG1 expression compared with those with low blast counts (RCUD, RCMD, and RARS) (median value: 1.01 vs. 1.54, P =0.11). To investigate prognostic implication of PLCG1 expression in MDS, we analyzed impact of PLCG1 expression on overall survival (OS). Based on PLCG1 expression level, 109 patients were divided into four groups, high (Q1), intermediate (Q2, Q3), and low (Q4) quartiles. Kaplan-Meier analysis demonstrated that the lowest quartile (Q4) showed significantly worse survival compared with remaining quartiles (Q1-Q3) (P =0.0015). The estimated 5-year OS rates in Q1-3 group and Q4 group were 63.2% and 29.7%, respectively. Percentage of patients with WHO-subtypes with high blast count (RAEB-1 and RAEB-2) was significantly higher in Q4 than other quartiles (46.9% vs 22.1%, P =0.019 by Fisher's exact test). The present study demonstrated that reduced PLCG1 expression is associated with inferior clinical outcome, indicating that PLCG1 expression could be a useful prognostic marker in MDS. Association between reduced PLCG1 expression and WHO-subtypes with high blasts counts, suggesting that PLCG1 dysfunction play a role in disease progression. Disclosures No relevant conflicts of interest to declare.

Follicular lymphoma (FL) is a low-grade B-cell lymphoma, and the response of FL to treatment, as well as progression-free survival and overall survival (OS), are improved by the application of combined chemotherapy with rituximab (R). The cyclophosphamide, adriamycin, vincristine, and prednisolone with rituximab (R-CHOP) regimen is the standard first-line therapy for patients with FL. However, many patients have coexistent diseases (comorbidities), and it is believed that therapeutic decision-making is influenced by comorbidities. In this study, we retrospectively analyzed treatment decision-making and its outcome for patients with FL. Based on this analysis, we investigated how the presence of comorbidity affects the survival of patients with FL. We analyzed 113 patients who were diagnosed with FL at our institute between September 2001 and March 2014. The treatment strategy was classified as observation without treatment, chemotherapy, radiotherapy, and others. Chemotherapy was subclassified as R-CHOP, reduced R-CHOP (the chemotherapeutic dose was reduced), and other chemotherapy. The severity of the comorbidity present at the time of diagnosis of FL was estimated according to the Charlson Comorbidity Index (CCI) (Table 1). The clinical characteristics of the 113 patients are shown in Table 2. The median age of the patients was 60 years. Six patients were observed without treatment, and 8 patients underwent resection or localized radiation for lymphoma. R-CHOP and other chemotherapy regimens were utilized in 84, and 15 patients, respectively. The 5-year OS rates of patients treated with R-CHOP, reduced R-CHOP, and other chemotherapy regimens were 92.3%, 75.5%, and 74.1%, respectively. As shown in a previous study (Andre Wieringa, et al., Br J Haematol, 2014, 165, 489-496), the 5-year OS rate of patients with CCI score ≥2 was inferior to that of patients with CCI 0-1 (CCI ≥2 (n = 32); 76.5%, CCI 0-1(n=81); 92.4%, p = 0.0361, Figure 1a). When we analyze the patients treated with R-CHOP, the overall response rate was 92.4% (complete response [CR] = 77.2%, partial response [PR] = 15.1%) in patients with a CCI score of 0-1 and 94.4% (CR = 77.8%, PR = 16.7%) in those with CCI score ≥ 2. In addition, there was no significant difference in the 5-year OS rate between patients with a CCI score of 0-1 or ≥2 (91.0% vs. 85.0%, p = 0.779, Figure 1b). Although, the percentage of patients with CCI ≥2 is lower than that of the other therapies (CCI ≥2; R-CHOP 25%, other chemotherapy 67%), it is supposed that R-CHOP is effective even if the patients have severe comorbidities. In 6 patients among these, treatment was discontinued or the chemotherapeutic dose was reduced due to myelosuppression during the designated course of therapy .The 5-year OS rate of these patients was favorable, even among those with high CCI score(CCI 0,1; 94.1%, CCI ≥2; 100%). Thus, it appears that R-CHOP with appropriate dose modification during the designated course will improve patient survival. Our results indicate that patients with severe comorbidities can be treated effectively by adjusting R-CHOP. Table 1. The items used to estimate the Charlson comorbidity index in each patient Comorbidity Score No. of patients Cardiac disease 1 5 Peripheral vascular disease 1 1 Cerebrovascular disease 1 3 Chronic pulmonary disease 1 6 Connective tissue disease 1 8 Ulcers 1 3 Mild liver disease 1 4 Diabetes 1 16 Tumors 2 22 Moderate or severe liver disease 3 1 Metastatic solid tumors 6 2 Table 2. Clinical characteristics of the patients Characteristic No. of patients (n = 113) Percentage (%) Sex Male 53 47 Female 60 53 Age >60 years 56 50 ≤60 years 57 50 IPI Low 55 49 Low-intermediate 38 34 High-intermediate 17 15 High 3 2 FLIPI Low 41 36 Intermediate 36 32 High 36 32 Therapy None 6 4 Resection only 3 3 Radiotherapy only 3 3 Resection and radiotherapy 2 2 R-CHOP 71 63 Discontinued or dose was reduced 23 20 Reduced R-CHOP 13 12 Other chemotherapy 15 13 IPI, international prognostic index; FLIPI, follicular lymphoma international prognostic index; R-CHOP, cyclophosphamide, adriamycin, vincristine, and prednisolone with rituximab Figure 1. Overall survival of patients treated with the cyclophosphamide, adriamycin, vincristine, and prednisolone with rituximab (R-CHOP) regimen according to the Charlson Comorbidity Index (CCI) Figure 1. Overall survival of patients treated with the cyclophosphamide, adriamycin, vincristine, and prednisolone with rituximab (R-CHOP) regimen according to the Charlson Comorbidity Index (CCI) Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Introduction: Adult T-cell leukemia-lymphoma (ATL) is a distinct peripheral T-cell lymphomacaused by human T-lymphotropic virus type I (HTLV-1), which is endemic in Japan. Only a small percentage of HTLV-1 carriers infected through breast-feeding develop the disease. Previous nationwide surveys during 1980s-2000s in Japan provided a comprehensive view on the clinio-epidemiological profiles of ATL, including the establishment of the subtype-classification (i.e., the Shimoyama criteria: acute, lymphoma, chronic, and smoldering type) based on the diversity of the clinical features and prognosis. Treatment strategies have been developed by the subtype-classification; watchful waiting for indolent ATL, intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive ATL. The aim of this 11th survey is to characterize the current clinio-epidemiological features of ATL in Japan in the light of previous surveys. Methods: In 2013-14, we conducted an anonymous, cross-sectional, hospital-based survey of patients with ATL who were newly diagnosed during 2010-2011 across Japan. First, we asked 2500 hospitals with departments of hematology and dermatology to participate in this survey, and then we sent questionnaires to 128 participating hospitals to accumulate data of ATL. Skilled five hematologists reviewed the data of each questionnaire sheet, and the agreements of ATL diagnosis including the subtypes between the central review and participating hospitals were evaluated using Kappa statistics. Most of the collected data were analyzed by descriptive statistics. Results: A total of 996 ATL patients were registered; of these, 41 were eliminated because of the insufficient information for the diagnosis of ATL. Overall diagnostic agreement on subtype of ATL was substantial, with a kappa value 0.71 (standard error, 0.021); most of the disagreement was within aggressive/indolent category, acute vs. lymphoma types or acute vs. unfavorable chronic types. Among the 955 evaluable patients (M/F ratio, 1.12), 70% were diagnosed in the southwest region of Japan (Kyusyu/Okinawa) where HTLV-1 is endemic, which was consistent with the past surveys. The mean age at diagnosis was 68.3 years (median 68.8, range 24.7-100.3), which was significantly higher than 60.3 years in 1994-1995 and 57.7 years in 1986-1987. The proportion of ATL patients aged over 65 years was 79 % in this survey (whereas, 48% in the 1990-1991 survey), which indicates that the allo-HSCT is not applicable to the majority of current ATL patients in Japan. The proportion of acute, lymphoma, chronic, and smoldering types was 48, 28, 10 and 13%, respectively. The proportion of smoldering and chronic types tended to be greater than previous surveys (7% and 10% in the 1988-1997 surveys combined). The proportion of subtype by age group in this survey showed that the older in age at diagnosis are, the higher proportion of lymphoma subtype (10% in those aged younger than 40 years, 20% in those 50-59, and 31% in those 70 years or older. Discussion: This 11th survey revealed several clinio-epidemiological features of current ATL in Japan different from those in past two decades. First, a considerably increasing proportion of elderly ATL patients were diagnosed, which may be expected from a recent distribution of nationwide HTLV-1 carriers in Japan (Satake, et al, 2011). Second, there is an increasing tendency in the proportion of indolent types of ATL. This may be partly influenced by participation of the dermatology departments for the first time, because early phase of ATL localized in skin were frequently diagnosed in the dermatology department. Third, the proportion of lymphoma subtype was increased with age, which suggests that aging-related unknown mechanisms may affect the subtype distribution. More detailed analysis and subsequent surveys are planned to further evaluate clinical features and the efficacy of modern therapies on prognosis of ATL. Disclosures Ishizawa: Kyowa Kirin: Speakers Bureau; GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Pfizer: Speakers Bureau. Ishida:Bristol-Myers Squibb: Honoraria. Ishida:Kyowa Hakko Kirin Co., LTD: Honoraria, Other: Research Funding to my institution; Bayer Pharma AG: Other: Research Funding to my institution; Celgene K.K.: Other: Research Funding to my institution. Tobinai:Gilead Sciences: Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Background: Although many salvage regimens have been used in patients with relapse or refractory lymphoma, these have limitations in elderly or patients with complications due to toxicity and restricted tolerability. In particular, the treatment strategy for patients with severe comorbidity remains to be determined. The combination of ifosfamide, carboplatin, and etoposide (ICE) has been used for relapse and refractory non-Hodgkin lymphoma since the 1990s. Ifosfamide, an alkylating agent, is a key drug in the ICE regimen, which is non-cross resistant with cyclophosphamide. In practice, there are various regimens of ICE treatment. The outpatient-based fractionated regimen of ICE (fractionated ICE) described in 2003 by Herzberg et al involves a different method of ifosfamide-administration from that originally described. Although ifosfamide 5000 mg/m2 is continuously infused for 24 h according to the original ICE regimen, it is infused in three equally divided doses over 2 h on days 1-3 of each cycle in the fractionated ICE regimen. Unlike the original ICE regimen, the modified regimen can be performed in outpatient clinics. The efficacy and tolerability of fractionated ICE as both a salvage and stem cell mobilization regimen have been confirmed in relapse/refractory patients. Many studies have reported the efficacy of the addition of rituximab to chemotherapy for diffuse large B-cell lymphoma patients (DLBCL). However, no study has reported the efficacy of rituximab addition to the fractionated ICE regimen. In this study, we analyzed the efficacy and toxicity of fractionated ICE with rituximab (fractionated R-ICE) as a salvage regimen in relapse/refractory DLBCL patients (Table 1). Further, we compared the response and survival rate after fractionated R-ICE between patients with severe comorbidity and other patients. Method: We retrospectively analyzed the records of 66 patients with relapse or refractory DLBCL diagnosed between 2000 and 2014. Comorbidities were evaluated using Charlson Comorbidity Index (CCI), and National Cancer Institute Common Toxicity Criteria were used to define toxicities. Result: Among the 66 patients, 55 received salvage therapies, 30 received fractionated R-ICE, and 25 received it as a second-line salvage therapy. Efficacy and toxicity is demonstrated in Table 2. The overall response rate (ORR) to fractionated R-ICE was 46.7% (n=14) (complete response [CR], 26.7% [n=8], partial response, 20% [n=6]); 1 year survival rate after relapse was 56.7%, and the duration of 50% survival after relapse was 2.4 years. During the cycles, myelosuppression was the most serious toxicity, followed by grade 4 hematological adverse events were 18 (60%) patients, respectively. A previous study showed poor prognosis in patients with high CCIs before the first R-CHOP treatment. In our study, the ratio of patients with chemotherapy dose reduction less than 30% was comparable between low (0 or 1) and high (≥2) CCIs estimated before the fractionated R-ICE therapy. There was no significant difference of survival duration after relapse between low and high CCI (Figure 1). The items used for estimating CCI such as diabetes (n=5), heart disease (n=4), tumor (n=4), and collagen disease (n=3) did not affect survival. Conclusion: Although patients in our study were old (median: 71 y, range 50-85 y), the CR rate was similar to that of previous studies involving ICE therapy (CR rate 12.5-37%) or R-ICE therapy (CR rate 25-53%). Our results suggest that comorbidities do not have significant impact on the outcome of patients with relapse or refractory DLBCL treated with fractionated R-ICE. Although myelosuppression was severe in patients with high CCI scores, there was no increased incidence of infection or other adverse events. Fractionated R-ICE is supposed to be useful as a salvage therapy, which can be performed in outpatient clinics for relapse/refractory DLBCL patients including those who were older or having high CCI scores. Download : Download high-res image (57KB) Download : Download full-size image Download : Download high-res image (104KB) Download : Download full-size image Download : Download high-res image (47KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Introduction: The immunophenotypic analysis of plasma cells using flow cytometry (FCM) is useful for the diagnosis for multiple myeloma (MM) and the detection of MM cells after treatment. CD138 is a well-known surface marker identifying plasma cells, although decreased CD138 expression on plasma cells is frequently observed in MM patients with relapsed or progressive disease. Thus, more stable MM antigens are needed. The SLAM family molecule CD229 has recently been reported to be specifically overexpressed on MM cells including their clonogenic precursors, suggesting that it may be a good marker to identify MM cells. Furthermore, some surface antigens, i.e., costimulatory molecule CD86 (B7-2), its receptor CD28, IL-6 receptor (CD126), insulin-like growth factor-1 (CD221), and coinhibitory molecule B7-H1 (PD-L1, CD274), were reported to be associated with poor prognosis. This study aimed to validate the potential of CD229 as a new MM cell marker and the efficacy of immunophenotypes on MM cells as prognostic markers in a multicenter study. Patients & Methods: Two-hundred thirteen patients comprising 144 newly diagnosed (18 asymptomatic and 126 symptomatic) MM patients, 25 refractory/relapsed MM patients, and 44 monoclonal gammopathy of undetermined significance (MGUS) patients were enrolled. Immunophenotyping of plasma cells in bone marrow was performed with standard 3-color FCM, in which plasma cells were gated by CD38-highly positive cells and 9 parameters, i.e., expression of MM antigens (CD138, CD229) and prognosis-related antigens previously reported (CD28, CD45, CD56, CD86, CD126, CD221, CD274) on MM cells, were analyzed. Expression levels of prognosis-related antigens were compared between patients in high-risk categories, i.e., International Scoring System (ISS) stage II/III, high-risk chromosomal abnormalities [t(4:14), t(14:16), del17p] by FISH, high-risk group stratified by the International Myeloma Working Group (IMWG) [ISS stage II/II and t(4:14) and/or del17p], high serum lactase dehydrogenase (LDH) level, or revised ISS stage III, and other patients. The revised ISS is a powerful new tool to predict outcome in MM patients treated with novel agents based on 3 factors: serum LDH level, ISS stage, and high-risk chromosomal abnormalities [Oliva S, et al. EHA2014, Abstract #S1289]. Differences between continuous variables were evaluated using the Mann-Whitney U-test. Results: 1) CD229 was expressed on almost all MM cells, even on low CD138-expressing MM cells. MM cells from ISS stage II/III patients expressed significantly lower levels of CD138 than those from ISS stage I patients (P = 0.0004). Furthermore, although CD138 expression levels in symptomatic MM patients were lower than those in patients with asymptomatic MM and MGUS and the expression was decreased in refractory/relapsed MM patients, CD229 expression levels on plasma cells were similar in MGUS and MM patients. 2) Newly diagnosed MM patients with high-risk chromosomal abnormalities or in the IMWG high-risk group had higher levels of CD86 and CD126 compared with other patients (P = 0.0056 and 0.0248 in high-risk chromosomal abnormalities, P = 0.0221 and 0.0396 in IMWG high-risk group, respectively). Furthermore, patients in revised ISS stage III had higher levels of CD126 compared with others (P = 0.0646). 3) Although the serum IL-6 level was significantly higher in ISS stage II/III patients, in patients with high LDH levels, and in revised ISS stage III patients compared with other groups, there was no correlation between serum IL-6 levels and expression levels of the IL-6 receptor CD126 on MM cells. Conclusion: CD229 has the potential to become a new marker for the identification of MM cells and target for immunotherapy. High expression levels of CD86 and CD126 were associated with high-risk patients, and CD126 may be associated with survival in patients treated with novel agents. FCM analysis may be useful for predicting prognosis as well as detecting malignant clones. Further studies are in progress to clarify the significance of those molecules on MM cells. Disclosures No relevant conflicts of interest to declare.

Introduction: B7-H1 (also known as PD-L1 or CD274), a co-inhibitory molecule of the B7 family, is detected on various tumor cells and associated with tumor evasion from cytotoxic T lymphocyte (CTL)-mediated immune surveillance. Our previous study showed that B7-H1 expression levels on plasma cells from multiple myeloma (MM) patients were significantly upregulated compared with those cells from monoclonal gammopathy of undetermined significance patients and healthy volunteers. B7-H1-expressing MM cells had a proliferative advantage and were resistant to antimyeloma agents (Tamura et al. Leukemia 2013). However, it remains unknown whether cellular responses in B7-H1-expressing MM cells are affected by the interaction of B7-H1 molecules with their receptors, i.e., PD-1 and CD80. We thus investigated the reverse signal derived from B7-H1 binding to their receptors on MM cells and examined the clinical characteristics of B7-H1-highly positive MM patients in a multicenter study. Methods: 1) We established B7-H1-expressing MM cell lines called MOSTI expressing high levels of CD38 and CD138 from bone marrow mononuclear cells of myeloma patients and B7-H1-positive MM cells (B7-H1.KMS28PE) stably transfected with the B7-H1 gene. 2) B7-H1 knockdown MOSTI cell lines were obtained using B7-H1-specific short-hairpin RNA expressing a lentiviral vector. 3) The proliferative potential was examined by BrdU incorporation using flow cytometry (FCM) and the MTT assay. 4) Drug-induced apoptotic cells were stained with annexin V and propidium iodide (PI) and detected in FCM. 5) Magnetic Dynabeads were coupled with PD-1-Ig or CD80-Ig fusion proteins. B7-H1-expressing MM cells were co-cultured with the beads, and the binding capacity of the beads to B7-H1+ MM cells, drug sensitivity, and cell proliferation of B7-H1+ MM cells were analyzed. 6) We classified 105 cases with newly diagnosed MM into two groups according to B7-H1 expression levels on plasma cells and compared the clinical characteristics associated with prognosis between B7-H1-highly-expressing MM patients (n=43) and other patients (n=62). Results: 1) Knockdown of B7-H1 expression in MOSTI cells significantly suppressed cell proliferation and increased melphalan-induced apoptosis. These results demonstrated that B7-H1 expression is directly associated with aggressive myeloma behavior including cell growth and drug resistance. 2) B7-H1 molecules on MOSTI and B7-H1.KMS28PE cells bound more strongly to PD-1-Fc than to CD80-Fc. The binding of PD-1-Fc to MOSTI cells was inhibited by anti-B7-H1 antibody in a concentration-dependent manner. In MOSTI cells treated with PD-1-Fc beads, apoptosis induced by both melphalan and bortezomib was markedly inhibited in comparison with the cells treated with control Ig. PD-1-Fc bead-treated B7-H1.KMS28PE cells were also resistant to melphalan-induced apoptosis. However, CD80-Fc bead-treated cells did not show drug resistance. Resistance to antimyeloma agents via the reverse signal from PD-1 to MM cells was inhibited by the PI3K/AKT inhibitor LY294002. In Western blot analysis, phospho-AKT expression was significantly upregulated in PD-1-Fc-treated MM cells. However, the cell growth of PD-1-Fc-treated MOSTI cells was the same as that of control Ig-treated cells. These data indicate that the reverse signal delivered from B7-H1 expressed on MM cells bound to PD-1 induced the drug resistance of MM cells thorough the Akt signaling pathway. 3) Patients with B7-H1 highly-expressing MM cells tended to have the poor-risk cytogenetic abnormality t(4;14) (P=0.0703). Furthermore, fibroblast growth factor receptor 3 was significantly upregulated on MM cells in those B7-H1-highly positive patients (P=0.0141). Expression levels of CD56, CD45, and CD221, which were reported to be poor prognostic markers in MM, were siginificantly higher in B7-H1-highly positive patients compared with others. Conclusion: Our study revealed a new mechanism via which the interaction between B7-H1 on MM cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces the drug resistance of MM cells through the Akt signaling pathway. Furthermore, B7-H1 expression on MM cells may be associated with t(4;14) translocation and poor prognostic MM antigens. Thus, B7-H1 may be a reasonable target for immunotherapy. Disclosures No relevant conflicts of interest to declare.

Background: Steroid-refractory acute graft vs. host disease (SR-aGVHD) remains a significant complication of allogenic hematopoietic stem cell transplantation (allo-HSCT). In this phase II-III multicenter clinical study we evaluated the efficacy and safety of MSCs(JR-031; JCR Pharmaceuticals Co., Ltd., Japan) for SR-aGVHD. Method: 25 patients (age: 5-66 years, median 33years; male 15, female 10) who developed SR-aGVHD (grade III 22, grade IV 3) after allo-HSCT (Nov. 2011-Sept. 2012) were enrolled and given 8 biweekly infusions of 2x106 cells/kg of JR-031 for 4 weeks, with an additional 4 infusions weekly after 28 days in patients with partial response (PR). They were followed up to 24 weeks. Results: At 4 weeks after the first dose, 6 (24%) patients showed complete response (CR), 9 (36%) patients showed PR, 4 (16%) patients showed mixed response (MR) and 1(4%)patient showed no change (NC). During 24 weeks of observation period, 12 (48%) patients achieved durable CR (CR >=28days). As for response by organ, 80% (16/20) of GI, 66.7% (8/12) of skin and 66.7%(4/6) of liver GVHD completely responded (stage 0). 15 (60%) patients survived up to 24 weeks after the first dose.All patients experienced at least one adverse event(AE). Common AEs were leukopenia (12 patients), thrombopenia (9 patients), sepsis, anemia, TMA and hepatic dysfunction (6 patients). Among 10 death, causal relationship with JR-031 were not completely ruled out in 4 cases (TTP, pneumonia, sepsis and relapse of underlying malignancy), however there was no case in which the causality was strongly suggested. Conclusion: It was suggested that JR-031 is a safe and effective therapy in the treatment of patients with SR-aGVHD. Disclosures Miyamura: Novartis: Honoraria, Speakers Bureau.

Background: Multidrug resistance (MDR) expressed in leukemia cells is a major obstacle in the use of chemotherapy for leukemia treatment. The Wnt signaling pathway is essential for maintenance, activation, regulated self-renewal and proliferation in both normal hematopoietic and leukemia cells. Frizzled-1 (FZD1), part of the Wnt receptor family, has been reported to be associated with drug resistance in a number of solid cancers, but the involvement of FZD1 in leukemia is yet to be studied. Here, we investigated whether FZD1 is associated with MDR1/P-glycoprotein (P-gp) in multidrug resistance of leukemic cell lines and examined FZD1 and MDR1/P-gp expression levels in AML cells from patients. Methods and Patients: Drug resistant cell lines K562/ADM, K562/Vin and NOMO-1/ADM as well as their parental cell lines were transduced with a GFP-containing FZD1 lentiviral vector (shFZD1) or a scrambled control (shC). The effect of FZD1 knockdown and change in MDR1/P-gp were confirmed by quantitative RT-PCR, western blot analysis, and flow cytometry. Cell growth was evaluated by cell counts over time and colony formation assays. The P-gp-mediated MDR phenotype was verified by assessing the rhodamine123 (Rh123) efflux. Drug sensitivity was examined in both the presence and absence of adriamycin (ADM), daunomycin (DNR) or vincrine (VCR). We also analyzed target genes downstream of the Wnt/β-catenin signaling pathway including β-catenin. Clinical samples were obtained from 112 AML patients with a normal karyotype at initial diagnosis after acquiring written informed consent. Results: The expression level of FZD1 in drug resistant cells was higher than that of their parent cells. Effective knockdown of FZD1 was confirmed and FZD1 expression was reduced by 82% (K562/Vin) and 50%-61% (NOMO-1/ADM and K562/ADM) when compared to shC. Interestingly, knockdown of FZD1 also resulted in a significant reduction of MDR1 by 40%-50% in resistant cells. Western blotting showed a reduction in the P-gp level as well as the FZD1 protein in shFZD1 cells. Mean fluorescence intensities of P-gp were also reduced significantly in K562/Vin (50.6%±16.4%), K562/ADM (68.4%±16.3%) and NOMO-1/ADM (56.9%±0.6%) cells. Rh123 intracellular content in these shFZD1 cells significantly increased 2.5-3.4 folds, indicating a reduction in P-gp function. The cytotoxicity assay revealed restoration of drug sensitivity to a varying extent. In addition, cell growth and colony formation were significantly suppressed. To investigate whether these results were caused by the activation of the Wnt/β-catenin pathway, immunofluorescence of β-catenin was performed, and the results showed that accumulation of nucleic β-catenin was reduced after inhibition of FZD1. We then assessed β-catenin, TCF7L2, LEF1, CyclinD1, p21 and p27 target genes in shFZD1 cells. The expression levels of proteins revealed that β-catenin and TCF7L2 were significantly decreased in shFZD1 cells. Elevated p27 and decreased cyclin D1 protein levels were also observed. A total of 112 untreated AML patients with normal karyotypes who received anthracycline-based chemotherapy were analyzed. Eighty-five patients achieved complete remission (CR), and the remaining 27 patients had no response (NR). Thirty-four of these 85 patients relapsed (CR-Re) after remission, and the remaining 51 patients maintained CR. No significant difference in FZD1 expression was observed between the CR and NR groups. However, a statistical difference was observed between the NR group and the CR group when the CR-Re group was excluded from the analysis (median 5.989 vs. 1.856; p = 0.003). Expression of FZD1 was also significantly higher in the CR-Re group than in the CR group (10.738; p = 0.002). The NR group had significantly higher MDR1/P-gp levels than the CR group. Furthermore, a statistically significant relationship was observed in the NR group between FZD1 and MDR1 (p = 0.005), MDR1 and P-gp expressions (p Conclusions: These data indicate that FZD1 promotes the activation of β-catenin/TCF7L2 and increases the proliferative capacity of cells through the regulation of cyclin D1 expression. FZD1 seems to modulate multidrug resistance through the Wnt/β-catenin signaling pathway, suggesting that AML patients who have higher FZD1 expression at diagnosis tend to relapse and show chemotherapy resistance. Therefore, FZD1 likely participates in the development of MDR in leukemic cells. Disclosures No relevant conflicts of interest to declare.

Allogeneic hematopoietic cell transplantation (HCT) from a matched sibling donor (MSD) may provide a cure for acute myeloid leukemia in first complete remission (AML/CR1), although the procedure is associated with a higher rate of treatment-related mortality (TRM) than autologous HCT, and it remains uncertain which modality is preferable as post-remission treatment. We therefore conducted a retrospective registry-based analysis to compare the outcomes of patients with AML/CR1 receiving autologous peripheral blood (PB) grafts (n=398, median age: 47, range: 17-80) and those receiving allogeneic MSD bone marrow (BM) grafts (n=633, median age: 38, range: 16-73) or allogeneic MSD PB grafts (n=475, median age: 42, range: 16-74) between 1995 and 2011. Consequently, the 5-year overall survival (OS) rates for the autologous PB, allogeneic BM and allogeneic PB recipients were 62% (95% confidence interval [CI], 57-67%), 61% (95% CI, 57-65%; P=0.90) and 54% (95% CI, 49-59%; P=0.07), respectively (Fig. 1-A), and the 5-year leukemia-free survival (LFS) rates were 57% (95% CI, 52-62%), 58% (95% CI, 54-63%; P=0.49) and 51% (95% CI, 46-56%; P=0.12), respectively(Fig. 1-B). Meanwhile, the 5-year cumulative incidence of TRM was 8% (95% CI, 5-11%), 16% (95% CI, 13-19%; P=0.009) and 19% (95% CI, 15-23%; P=0.0001), respectively, while that of relapse was 35% (95% CI, 30-40%), 26% (95% CI, 22-29%; P=0.003) and 30% (95% CI, 26-35%; P=0.08), respectively. A multivariate analysis performed with autologous PB HCT as the reference showed a hazard ratio (HR) for OS of 0.93 (95% CI, 0.73-1.18; P=0.53) for allogeneic BM HCT and 1.08 (95% CI, 0.83-1.39; P=0.57) for allogeneic PB HCT and an HR for LFS of 0.86 (95% CI, 0.69-1.09; P=0.21) and 0.98 (95% CI, 0.77-1.24; P=0.85), respectively. Stratifying the patients according to cytogenetics (favorable, intermediate and poor) and age ( Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Bendamustine is an alkylator containing a benzimidazole ring; it only has partial cross-resistance with the other alkylating agents. Several studies have shown that bendamustine is effective as a single agent or as combined treatment with rituximab for low-grade B-cell lymphoma. Although bendamustine has a favorable safety profile, the occurrence of myelosuppression and lymphocytopenia is relatively frequent. In lymphocytopenia, CD4-positive (CD4+) T-cell recovery is specifically impaired. If this occurs over a prolonged period of time, patients are at risk of opportunistic infections, such as cytomegalovirus (CMV) reactivation. This is a risk for bendamustine-treated patients, although the actual frequency of CMV reactivation in these patients is not clear. In this study, we prospectively evaluated the occurrence of CMV reactivation in relapsed or refractory low-grade B-cell lymphoma patients who were treated with bendamustine alone or in combination with rituximab. We simultaneously investigated the immune state of the patients. We analyzed CMV pp65 antigen in leukocytes, immunoglobulin (Ig) G, IgM, and IgA levels, and the CD4+:CD8+ lymphocyte ratio prior to treatment and after the third and sixth courses of treatment. There were 29 patients enrolled in the study. The median age was 68 years old (range, 28–83 years). Patients were treated a median of 2 times prior to bendamustine treatment; these treatments included chemotherapy and radiotherapy. There were 15 patients (51%) who completed 6 courses of chemotherapy. The immune status of the patients is shown in Figure 1. The median CD4+ lymphocyte count prior to treatment was 218/µL, which decreased to 75/µL by the end of treatment. The median of IgG, IgM, and IgA concentrations before treatment were 909, 52, and 147 mg/dL, respectively; by the end of treatment, the IgG, IgM, IgA concentrations had decreased to 832, 30, and 110 mg/dL, respectively. The extent of CD4+ T-cell depression during treatment appeared to be more severe than the Ig depression. CMV pp65 antigen was detected in 3 patients after the third course of treatment, and in 2 patients after the sixth course of treatment. None of the patients had any symptoms and CMV pp65 antigen count returned to negative without any treatment. The median CD4+ lymphocytes count in patients with or without CMV reactivation is shown in Table 1. There is a trend that the depression of CD4+ cells after the 6th course of bendamustine increases the risk of CMV reactivation. This is the first study to prospectively analyze CMV reactivation in low-grade B-cell lymphoma patients treated with bendamustine. In this study, we found that CMV reactivation by bendamustine occurred in approximately 15% of patients. On the other hand, the risk of symptomatic CMV disease, which would require medical intervention, does not appear to be high in bendamustine-treated patients. Most patients with relapsed or refractory low-grade B-cell lymphoma who are administered bendamustine therapy are in an immunosuppressed condition at the beginning of treatment. This is due to heavy chemotherapy pretreatment, which is often performed using rituximab. In addition to this, the decrease in CD4+ lymphocytes observed during bendamustine treatment, is likely to increase the risk of opportunistic infections, such as CMV reactivation. In future studies, we aim to investigate the risk factors for symptomatic cytomegalovirus infection in patients treated with bendamustine, especially in combination with rituximab. We hope that our study, which evaluates the risk of opportunistic infections in bendamustine treated patients, will be beneficial in determining the optimal timing for a preemptive approach for CMV infection. This will be helpful in establishing safe bendamustine treatment regimens in relapsed or refractory low-grade B-cell lymphoma patients. Figure 1 Figure 1. Table 1. The median number of lymphocytes, CD4+ lymphocytes and CD4/8 ratio in patients with or without cytomegalovirus reactivation Parameter All patients CMV reactivation p value + - Before treatment (n=24) Lymph (/µl) 996 1518 (n=5) 987 (n=19) 0.24 CD4+ cells (/µl) 218 261 218 0.96 CD4/8 ratio 0.69 0.60 0.69 0.54 After the 3rd course (n=23) Lymph (/µl) 980 1330 (n=5) 944 (n=18) 0.39 CD4+ cells (/µl) 130 118 130 0.52 CD4/8 ratio 0.20 0.12 0.20 0.16 After the 6th course (n=15) Lymph (/µl) 628 579 (n=5) 902 (n=10) 0.27 CD4+ cells (/µl) 75 36 118 0.035 * CD4/8 ratio 0.20 0.17 0.21 0.35 *; significant Disclosures No relevant conflicts of interest to declare.

The preferentially expressed antigen of the melanoma (PRAME) gene was first identified in melanoma tissue as a tumour-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. While expression level of PRAME is quite low in most of normal tissues of human, including bone marrow normal CD34+ cells, except for testis, PRAME overexpression is observed in various human cancers, including hematological malignant disorders. Although clinical and biological significance of PRAME expression in human cancers has not been fully elucidated, it has been demonstrated that high PRAME expression is associated poor clinical outcomes in a number of solid cancers. Association between PRAME expression and clinical outcomes in hematological malignancies has not been clarified. In acute myeloid leukemia, high expression of PRAME was shown to be associated with worse progression-free survival or overall survival by some research groups but not by other groups. Our previous study demonstrated that inhibition of PRAME expression in leukemic cells results in cell cycle arrest and induction of apoptosis, suggesting oncogenic function of PRAME expression in leukemogenesis. Clinical significance of PRAME expression in myelodysplastic syndromes (MDS) also has not been fully elucidated. In the present study, we examined PRAME expression of bone marrow cells in MDS patients to clarify clinical significance of PRAME expression. Bone marrow samples of MDS patients were used for analysis. Samples were taken at the time of diagnosis with written informed consent from patients. Total RNA extraction, cDNA synthesis and quantitative real-time RT-PCR by the TaqMan probe method using an ABI 7500 real-time PCR system (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems), were performed. Expression levels were obtained using the standard curve method in each experiment, after normalization with the GAPDH gene for each sample in duplicate wells. The human PRAME primer-probe sets were from Applied Biosystems (assay ID: Hs00196132_m1). Data including patients’ demographic, disease status, medical history, clinical and laboratory findings, and outcome, were collected from medical records and laboratory data base. A total of 111 MDS patients, 68 males and 43 females with median age of 69 years (range: 20-91 years) were included in the present study. They were classified as RCUD (n=15), RCMD (n=61), RARS (n=8), RAEB-1 (n=15), and RAEB-2 (n=12) according to WHO classification. Based on the IPSS, they were categorized in four risk groups, low risk (n=29), intermediate-1 risk (n=55), intermediate-2 risk (n=23), and high risk(n=4). Expression level of PRAME was varied among the MDS patients analyzed. Median value of relative PRAME expression level of 111 MDS patients and that of 19 control subjects were 0.073 and 0.07, and there was no significant difference in distribution of expression level of PRAME. However, when we compared expression patterns of PRAME among five WHO-subtypes, statistical difference was observed (P=0.0116). Relative PRAME expression level in WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) was significantly higher than that in WHO-subtypes with less blast counts (RCUD, RCMD, RARS) (median value: 0.44 vs. 0.05, P=0.0012). To investigate prognostic implication of PRAME expression in MDS, we analyzed impact of PRAME expression on overall survival (OS). Based on PRAME expression level, 111 patients were divided into two categories, ‘high expression group’ (above median value) and ‘low expression group’ (below median value). Kaplan-Meier analysis demonstrated that high expression group showed significantly poorer overall survival than low expression group (P=0.0165). The estimated 5-year OS rates in high expression group and low expression group were 63.5% and 78.4%, respectively. The present study demonstrated that high PRAME expression is associated with poorer clinical outcome, indicating that PRAME expression could be a useful prognostic marker in MDS. Biological significance of PRAME expression in MDS is unclear. Expression level of PRAME was higher in WHO-subtypes with high blasts counts, suggesting that PRAME may play role in disease progression in MDS. Further studies should be necessary to clarify clinicopathological and biological significance of PRAME expression in MDS. Disclosures: No relevant conflicts of interest to declare.

Background Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for the patients with acute myeloid leukemia (AML), but a major obstacle to success is represented by the relapse of the disease. Donor T lymphocytes can induce graft-versus-leukemia (GVL) effect, and it contributes to eliminate leukemic cells. We retrospectively analyzed the risk of relapse after allo-HCT for the patients with AML. Purpose of this study is to determine the impact of donor sources for the risk of relapse and survival. Patients and Methods Clinical data were collected from the Transplant Registry Unified Management Program (TRUMP) in Japan. An individual patient data-based analysis was performed on patients with AML who underwent allo-HCT between 2006 and 2010. Patients with CR1 or CR2 were defined as having standard-risk disease, and patients with CR3≤ or non-CR were defined as having advanced-risk disease. Cox proportional hazards models were used to evaluate variables that may affect overall survival. Fine and Gray proportional-hazards model were used to evaluate variables that may affect morphologic relapse. Death in remission was treated as a competing risk when analyzing cumulative incidence of relapse. Death or relapse without GVHD was treated as a competing risk for acute and chronic GVHD. Results We analyzed a cohort of 3,829 patients with AML: 543 (14%) received bone marrow from related donors (RBM), 768 (20%) received peripheral blood from related donors (RPB), 1,576 (41%) received bone marrow from unrelated donors (URBM), and 942 (25%) received cord blood (CB). The median age at allo-HCT was 46 years (range, 16 to 65 years). The cumulative incidence of relapse at 5 years in all-risk patients, standard-risk disease, and advanced-risk disease were 36%, 22%, and 42%, respectively. The 5-year unadjusted OS rates in all-risk patients, standard-risk patients and advanced-risk patients were 44%, 61%, and 26%, respectively. In multivariate analysis, the uses of antigen matched URBM (HR 0.47, 95% CI 0.36-0.61, p Conclusion In this study, we found that the uses of URBM and CB for standard-risk patients could reduce risk of relapse, but they did not result in a favorable OS. Reduction of NRM could in part be attributed a better outcome in standard-risk patients who transplanted from URBM or CB. However, it is difficult to determine how much of the benefit is attributed to the incidence of GVHD after allo-HCT, the use of URBM and CB were associated with a higher incidence of grade II-IV acute GVHD compared with matched RBM. Although relapse is the major cause of treatment failure after allo-HCT, immune modulation mediated by donor T lymphocytes may play a role in disease control for the patient with AML. Disclosures: No relevant conflicts of interest to declare.

Background Allogeneic hematopoietic cell transplantation (HCT) is one of the most curative therapeutic modalities for hematological malignancies. Recent advances in availability of stem cell sources, conditioning regimens, managements of graft-versus-host disease (GVHD) and supportive care have improved the outcomes of HCT. But even now, relapse is still the leading cause of treatment failure and some long-term survivors after HCT suffer late relapse of the original disease. To better understand the risk and risk factors of late relapse in HCT recipients, we conducted a nationwide retrospective study of the patients who received first HCT for hematological malignancies in Japan. Patients and Methods The study population included HCT recipients reported to the Japan Society for Hematopoietic Cell Transplantation. From this database, we extracted the data of patients with hematological malignancies who received first HCT between 1974 and 2011. There were 29,479 recipients selected according to this criterion. Then, we excluded 1,192 (4.0%) cases from the study because of missing key variables. Results A total of 28,287 recipients were evaluated in this study. Median age at transplant was 37 years old (range, 0-88), and 41.0% (n=11,605) were female. Underlying diseases were acute lymphoblastic leukemia (ALL) (n=7,300, 25.8%), acute myeloid leukemia (AML) (n=11,526, 40.7%), chronic myelogenous leukemia (CML) (n=3,038, 10.7%), myelodysplastic syndrome (MDS) (n=3,235, 11.4%), malignant lymphoma (ML) (n=2,956, 10.5%) and plasma cell disorder (PCD) (n=232, 0.8%). Type of transplant included bone marrow transplantation (BMT) from HLA-matched or 1 locus serological mismatched (“matched”) related donor (n=8,002, 28.3%), BMT from more than 1 locus-mismatched (“mismatched”) related donor (n=261, 0.9%), BMT from HLA-matched unrelated donor (n=9,074, 32.1%), BMT from HLA-mismatched unrelated donor (n=1,112, 3.9%), peripheral blood stem cell transplantation (PBSCT) from “matched” related donor (n=4,385, 15.5%), PBSCT from “mismatched” related donor (n=467, 1.7%), cord blood transplantation (CBT) from “matched” unrelated donor (n=2,326, 8.2%) and CBT from “mismatched” unrelated donor (n=2,556, 9.0%). Myeloablative conditioning regimens were applied to 77.0% (n=21,773) of recipients and reduced-intensity conditioning regimens to 22.1% (n=6,247). Median follow-up of survivors was 1,934 days (range, 18-11,123). Event-free survival of all cases was 41.8% and cumulative incidence of relapse (RI) of them was 33.1% at 5 years. Disease-free survival (DFS) and RI at 7 years of recipients who had been in remission for 2 years since HCT (“DF2”, n=11,500) were 83.2% and 10.2%. DFS and RI at 10 years of recipients who had been disease-free for 5 years (“DF5”, n=7,093) were 91.5% and 2.9%. In multivariate analysis, disease, disease risk at transplant and patients’ age were powerful (p Adjusted for patients’ age, disease risk at transplant, year of transplant, donor-recipient sex match, ABO disparity, type of transplant and intensity of conditioning regimen, acute and chronic GVHD were included as time-dependent covariates in Cox model for relapse. In DF2, grade II-IV acute GVHD was associated with a reduced risk of relapse for patients with ALL (Hazard Ratio 0.75, 95% confidence interval 0.57-0.99, p=0.043) and AML (0.74, 0.57-0.97, p=0.029), and chronic GVHD for those with CML (0.65, 0.47-0.89, p=0.0067) and ML (0.37, 0.20-0.60, p=0.00067). In DF5, grade II-IV acute GVHD significantly reduced risk of relapse for recipients with AML (0.40, 0.18-0.85, p=0.018), and chronic GVHD for those with CML (0.56, 0.34-0.92, p=0.021). Conclusion Recipients of HCT for hematological malignancies who remain in remission for more than 2 or 5 years have favorable long-term survival. Our study demonstrates there are relatively small but persistent risks of relapse and underlying disease and disease risk at transplant are the major risk factors of late relapse. We also show acute and chronic GVHD are associated with a reduced risk of late relapse but the strength of this association differs between underlying diseases. Disclosures: No relevant conflicts of interest to declare.

Hemophagocytic lymphohistiocytosis (HLH) is often associated with malignant diseases, mainly B-cell or T/NK-cell lymphoma. However, to date, few studies have examined lymphoma-associated hemophagocytic syndrome (LAHS). Our aim was to clarify the risk factors and prognostic factors of LAHS. A total of 1,181 patients with non-Hodgkin lymphoma were analyzed at 12 institutions in Hokkaido prefecture between April 2007 and December 2011 to assess the incidence, prognosis, and risk factors of LAHS. To evaluate the risk factors for developing LAHS, patient characteristics including age, gender, and histopathology were compared between patients with and without LAHS. The cumulative incidence rate of LAHS was 3.0% (35/1,181). The mortality rate of patients with LAHS was 69% (24/35), which was significantly higher than that of patients without LAHS (29%, P Disclosures: No relevant conflicts of interest to declare.

Abstract 4544 Background: Current prognostic models, including the International Prognostic Index (IPI), incorporate both patient and tumor characteristics. In contrast, recent studies show that variables related to the host adaptive immunity and the tumor microenvironment are significant prognostic variables in cases of diffuse large B-cell lymphoma (DLBCL). Recently, Wilcox and co-workers reported that lymphopenia, defined as an absolute lymphocyte count (ALC) 630/μL, at diagnosis was associated with inferior survival in patients with DLBCL treated with CHOP/R-CHOP (Leukemia, 2011). The same group also reported that the ALC/AMC ratio at diagnosis can be a biomarker to predict the clinical outcome in DLBCL patients treated with R-CHOP (Porrate et al. ASH 2011). However, it remains to be determined if these parameters can predict the outcome of autologous peripheral blood stem cell transplantation (APBSCT) in patients with DLBCL in the first remission. Methods: We retrospectively examined the predictive value of the AMC and ALC in a cohort of 55 consecutive DLBCL patients who uniformly underwent APBSCT in their first remission at Hokkaido University Hospital and Sapporo City General Hospital. At presentation, all patients were at high risk (Coiffier et al. J Clin Oncol, 1991) (1997 to 2000), or high (H)/high-intermediate (HI) risk, in the age-adjusted IPI (aaIPI) (2001 to 2012). After six cycles of CHOP (before 2000, N=16) or R-CHOP (after 2001, N=39), all patients were treated with APBSCT, followed by the MCVC regimen, consisting of ranimustine, carboplatin, etoposide and cyclophosphamide. We performed a receiver operating characteristics (ROC) analysis to determine the optimal cut-off point for both the AMC and ALC in our patients, and values of 551/uL and 1,000/uL were set for the subsequent analyses. The disease free survival (DFS) and overall survival (OS) were estimated using the Kaplan–Meier method and two-tailed log-rank test. Results: Twenty-five patients were male and thirty were female. According to the aaIPI, 15, 31, and nine patients were classified as H, HI, and low (L)/low intermediate (LI), respectively. The median duration of follow-up after APBSCT was 85 months (range 1 to 179 months). At diagnosis, the median ALC was 1,095/μL (range 286–3,396/μL) and the median AMC was 551/μL (range 63–1,870/μL). The estimated 5-year OS and DFS for the entire cohort were 78% (95% confidence interval (CI) 64–88%) and 73% (95% CI 59–83%), respectively. In contrast to the previous study, the ALC did not predict an inferior OS or DFS in a univariate analysis of dichotomized variables. The estimated 5-year OS and DFS for those who had lymphopenia (ALC551/μL) had no significant impact on the 5-year OS, with rates of 88% for those who had an elevated AMC and 73% for those who did not. However, an elevated AMC was associated with a superior 5-year DFS of 88% compared to that of 59% in the cohort which did not have an elevated AMC (hazard ratio 3.18, 95% CI 1.10–11.41, p=0.03). Conclusions: Lymphopenia or an elevated monocyte number at diagnosis did not predict a poor outcome for high-risk patients with DLBCL when APBSCT was given in the first remission. Our results suggest that the dismal outcome obtained with CHOP/R-CHOP in high risk DLBCL patients who concomitantly had lymphopenia and elevated AMC could be overcome by an APBSCT in the first remission, although these results should be confirmed in a prospective study. Disclosures: No relevant conflicts of interest to declare.

Abstract 2021 Background: Postremission treatment for adult Philadelphia chromosome-negative acute lymphoblastic leukemia [Ph(-)ALL] in first remission remains elucidated and the role of autologous stem cell transplantation (auto-SCT) is still controversial in the application for Ph(-)ALL patients. We retrospectively analyzed the consolidative role of auto-SCT as a postremission therapy for adult Ph(-)ALL in first remission. Patients and Methods: Among 1962 adult Ph(-)ALL patients registered in the JSHCT database, we selected cases receiving auto-SCT with myeloablative preparative regimens in first complete remission from 1983 to 2009. Results: A total of 155 patients with 86 male patients were analyzed. The median age was 25 years (range: 16 to 74) and 79% of patients were less than 45 years of age at transplant. The number of patients having white blood cell (WBC) count We next performed comparison of auto-SCT with allogeneic stem cell transplantation (allo-SCT) in adult Ph(-)ALL patients. Patients undergoing allo-SCT were selected from the same JSHCT database. A total of 921 patients were identified as cases receiving allo-SCT with myeloablative preparative regimens in first complete remission. With a median follow-up of 4.95 years, the 4-year overall survival rate was 63% (95%CI: 51 to 59). Allo-SCT yielded better overall survival rate than that of auto-SCT (63% vs. 48% at 4 year, p=0.0004). In subset analyses, we limited analyses to the groups of patients younger than 45 years and receiving TBI regimens followed by auto-SCT (n=31) or allo-SCT (n=685). There were no differences in baseline patients' characteristics between the two groups except age [auto-SCT: 20 years (range: 16 to 40) vs. allo-SCT: 28 years (range: 16 to 44), p Conclusions: Our analyses demonstrated that auto-SCT produced excellent results showing a plateau in long-term survival and that clinical outcome of auto-SCT was comparable to that of allo-SCT in some part of patients. Candidates who obtained most benefit from auto-SCT was those younger than 45 years and well tolerated with TBI combined myeloablative conditioning regimens. Since optimal timing of postremission therapy is critical for adult Ph(-)ALL patients, auto-SCT in first complete remission could be one of promising treatment strategies for the patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3113 During pregnancy, women can develop both cytotoxic and regulatory immune cells against inherited paternal alloantigens of their fetus. Such immune cells reactive against fetal alloantigens can persist postpartum and may confer beneficial effects when the mother is used as a donor for hematopoietic cell transplantation (HCT) for her offspring. However, there have been few studies that aimed to evaluate the effects of parental donor type on outcomes after HCT performed with T-cell-replete grafts in a relatively large cohort of patients. Using datasets obtained fromthe Japan Society for Hematopoietic Cell Transplantation, we compared the outcomes of 477 patients who received T-cell-replete HCT from a maternal donor (n=305) or a paternal donor (n=172) between 1999 and 2009 as treatment for acute myeloid leukemia (n=203), acute lymphoblastic leukemia (n=213), chronic myelogenous leukemia (n=37), and myelodysplastic syndrome (n=24); in vivo T-cell depletion was employed in 48 patients. Patients who underwent HCT using ex vivo T-cell-depleted grafts were not included in the study. Cox proportional-hazard models were used to evaluate variables that may affect overall mortality, whereas Fine and Gray regression models were used to evaluate variables that may affect incidences of graft-versus-host disease (GVHD), relapse, and treatment-related death. Recipients of maternal donor HCT and paternal donor HCT had similar background characteristics with respect to age group, diagnosis, disease status, stem cell source, type of conditioning regimen, and type of GVHD prophylaxis. Proportions of male recipients (61% of maternal donor HCT vs 51% of paternal donor HCT, p=0.044) and patients given a graft with HLA mismatch in the GVH vector (70% vs 60%, p=0.031) were higher in maternal donor HCT compared with paternal donor HCT. Unadjusted overall survival at 3 years after transplantation were 49.6% after maternal donor HCT and 37.9% after paternal donor HCT (p=0.118). Multivariate analysis revealed a significantly lower oveall mortality after maternal donor HCT compared with paternal donor HCT [hazard ratio (HR) of maternal donor transplant vs paternal donor transplant, 0.70; p=0.008]; the use of a maternal donor was associated with a lower risk of treatment-related mortality (TRM) (HR, 0.62; p=0.018) and a higher incidence of chronic GVHD (HR, 1.43; p=0.036), whereas the incidence rates of grade 2–4 acute GVHD (HR, 1.06; p=0.690) and relapse (HR, 1.05; p=0.763) were similar between the two groups. Intriguingly, if the analysis is confined to 241 patients who developed grades 2 to 4 acute GVHD, the use of a maternal donor was associated with a lower TRM compared with the use of a paternal donor (HR, 0.55; p=0.028), suggesting protective effects of maternal grafts in the setting of acute GVHD. Of 159 patients who received grafts without mismatch at HLA-A, -B, and -DR antigens in the GVH vector, overall mortality (HR, 0.86; p=0.535) and TRM (HR, 0.98; p=0.956) were similar between the two groups. Of 318 patients who received grafts with 1 (n=202) or more (n=116) HLA antigen mismatch in the GVH vector, the use of maternal grafts was associated with a lower overall mortality (HR, 0.63; p=0.004) and a lower TRM (HR, 0.53; p=0.005) compared with the use of paternal grafts. Collectively, these results indicate that the use of an HLA-mismatched mother as a donor for T-cell-replete HCT can be a reasonable option in selected patients who lack a suitable conventional stem cell source. Disclosures: No relevant conflicts of interest to declare.

Abstract 3118 Introduction: Elderly patients with myelodysplastic syndromes (MDS) are often lack of suitable human leukocyte antigen (HLA)-matched related donors. Unfortunately, the data on the efficacy of allogeneic transplantation using alternative donor sources are limited. We retrospectively compared the outcomes of HLA 6/6 antigen-matched related bone marrow or peripheral blood progenitor cells (rBM/PBPC) transplantation (6/6rBM/PBPCT), HLA 8/8 allele-matched unrelated bone marrow (uBM) transplantation (8/8uBMT), HLA 7/8 allele-matched uBM transplantation (7/8uBMT), and HLA at least 4/6 antigen-matched single-unit umbilical cord blood (CB) transplantation (sCBT) for elderly patients with MDS. Method: The data were obtained from the Transplant Registry Unified Management Program, which includes data from the Japan Society for Hematopoietic Cell Transplantation, the Japan Marrow Donor Program, and the Japan Cord Blood Bank Network. Patients aged 50–70 years old at transplantation with MDS according to French-American-British (FAB) classification who received first allogeneic transplantation between January 1, 2001 and December 31, 2010 were included. Among 336 rBM/PBPC, 291 uBM, and 215 CB transplantation recipients with complete HLA data, 268 6/6rBM/PBPCT, 147 8/8uBMT, 90 7/8uBMT, and 211 sCBT recipients were included. Cox proportional-hazards regression model was used for adjusted comparisons of the donor sources on overall survival (OS). Fine and Gray proportional-hazards model was used for adjusted comparisons of the donor sources on non-relapse mortality (NRM), relapse, grade 2–4 acute graft-versus-host disease (aGVHD2–4), and neutrophil engraftment. Recipient age at transplantation (>=60 or 50–59), recipient sex (male or female), performance status (PS) at transplantation (2–4 or 0–1), FAB classification at transplantation (refractory anemia with excess blasts (RAEB) and RAEB in transformation (RAEBt), or refractory anemia (RA) and RA with ringed sideroblasts), latest cytogenetics classification according to International Prognostic Scoring System (good, intermediate, poor, or unevaluable), transplant conditioning regimen (reduced-intensity conditioning or myeloablative conditioning), and the year at transplantation (2001–2005 or 2006–2010) were included in the multivariate models. Result: Median follow-up was 2.9 years. The unadjusted 3-year OS rates for recipients of 6/6rBM/PBPCT, 8/8uBMT, 7/8uBMT, and sCBT were 50.7%, 58.2%, 46.2%, and 28.4%, respectively. Multivariate analysis revealed that the OS for sCBT recipients was significantly inferior to those for 6/6rBM/PBPCT (relative risk (RR) [95% confidential interval], 1.9 [1.5–2.5]; P= 60, male sex, PS > 1, FAB classification at transplantation as RAEB/RAEBt, and poor cytogenetics. The risk of relapse for sCBT recipients was comparable with that for 6/6rBM/PBPCT but significantly higher than those for 8/8uBMT (RR, 2.4 [1.5–3.8]; P Conclusion: For elderly patients with MDS, the outcome of sCBT recipient was inferior to those of 6/6rBM/PBPCT, 8/8uBMT, and 7/8uBMT recipients. Decreasing the risk of NRM, which would be brought by improving the neutrophil engraftment, and decreasing the risks of relapse are required to improve the outcome of sCBT. Disclosures: No relevant conflicts of interest to declare.

Abstract 3027 Background: Non-relapse mortality (NMR) may turn for the worse overall outcome of conventional myeloablative conditioning (MAC) allo-stem cell transplantation (SCT) for elderly patients and patients with comorbidities. Therefore, allo-SCT using reduced intensity conditioning (RIC) may provide opportunities to obtain a significant clinical effect without the adverse effects of intense myeloablative preparative regimens. Aims: In this Japanese nationwide survey we reported the outcome of 575 adult acute lymphoblastic leukemia (ALL) patients age at the first transplantation 45 years or more who underwent allo-SCT in CR and analyzed according to the type of conditioning regimen (MAC vs RIC) before allo-SCT registered in the JSHCT database between 2000 and 2009. Patients and Methods: The preparative regimen was classed as fludarabine-based RIC if it included non-myeloablative chemotherapy (total dose of busulfan ≤ 9 mg/kg or melphalan ≤ 140 mg/m2) with or without total body irradiation (TBI) ≤ 8 Gy (206 patients). Also, MAC included (total dose of busulfan > 9 mg/kg, melphalan > 140 mg/m2 or cyclophosphamide 120mg/kg) with or without TBI > 8 Gy (369 patients). Their median age was 51 years for MAC and 58 years for RIC (range: 45–70 years), with 280 males (180 vs 100) and 295 females (189 vs 106). There were 310 patients in the first complete remission (CR1) and 55 patients in the second CR (CR2) for MAC and 160 patients and 40 patients for RIC. Bone marrow from related or unrelated donors was transplanted in 343 patients (234 vs 109), as well as peripheral blood stem cell from related donors in 96 (47 vs 49) patients and cord blood in 127 (80 vs 47) patients. Results: Patients in the RIC group were older (median age, 58 vs 51 years, p Conclusions: This retrospective survey showed that conditioning intensity did not affect OS or DFS for adult ALL patients aged 45–70 years at transplantation. However, patients aged 55 years or more with HLA mismatch transplantation would be candidates for RIC rather than MAC. Disclosures: No relevant conflicts of interest to declare.

Abstract 3089 Adult T-cell leukemia/lymphoma (ATL) mainly occurs in HTLV-1 endemic areas such as the southwest island in Japan (Kyushu) and Caribbean countries. A recent report showed that the incidence of ATL was increasing in HTLV-I non-endemic areas (1). Although allogeneic stem cell transplantation (allo-SCT) has been considered to be the only curative treatment for ATL (2, 3), there has been no report about the treatment strategy for ATL occurring in HTLV-1 non-endemic area. We therefore conducted a retrospective analysis for all of the patients who developed ATL and received allo-SCT in an HTLV-1 non-endemic area of Japan, Hokkaido (a northernmost island). Clinical data for 56 patients who received allo-SCT were collected from 12 SCT centers in Hokkaido, Japan. The median age of the patients was 57 years (range: 37 – 69 years). Twenty-eight of the patients had acute type and 22 had lymphoma type. Median count of white blood cells and median levels of serum LDH and serum soluble interleukin-2 receptor (sIL-2R) at diagnosis were 10900/mL, 352 IU/L and 11153 mg/dL, respectively. After chemotherapies mainly using CHOP or VCAP–AMP–VECP regimens, twenty-three of the patents received allo-SCT in complete remission (CR), and the other patients received allo-SCT in non-CR (partial remission, n=16; primary refractory, n=9; relapse, n=23). Median levels of serum LDH and sIL-2R before the conditioning regimen were 218 IU/L and 1153 mg/dL, respectively. HCT-CI scoring was available in 42 of the patients, and the scores were 0 in 15 patients, 1 in 10 patients, 2 in 5 patients and more than 3 in 9 patients. Thirty-nine of the patients received bone marrow, 11 of the patients received peripheral blood stem cells and 6 of the patients received cord blood. Thirty of the patients received SCT from HLA-matched siblings, 22 of the patients received SCT from HLA-matched unrelated donors and 14 of the patients received SCT from HLA-mismatched donors. Seventeen patients received myeloablative conditioning and the other 39 patients received reduced-intensity conditioning. Fifty-three (95%) of the patients achieved neutrophil engraftment at median day of 16. Acute graft-versus-host disease (AGVHD) and grade II-IV AGVHD occurred in 40 (75%) and 31 (58%) evaluable patients, respectively, at median onset day of 29. Chronic GVHD (CGVHD) occurred in 24 (38%) evaluable patients at the median onset day of 168. After a median follow-up period of 48 months, 1-year overall survival (OS) and 5-year OS rates were 56.3% and 46.5%, respectively The survival curve reached a plateau at 22 months after SCT. Univariate analysis showed that year in which SCT was performed, male sex, high level of sIL-2R both at diagnosis and at SCT, and disease status (non-CR at SCT) were significant risk factors for overall survival. SIL-2R at SCT (P=0.02) was determined to be a significant risk factor for disease progression and male sex was marginally significant (P=0.06) by univariate analysis. Non-CR at SCT was marginally significant for transplant-related mortality (P=0.07). Worse survival for male patients and patients in non-CR at SCT were confirmed by using multivariate analysis with Cox's regression model [hazard ratio of 3.15 (95% confidence interval: 1.36–7.30) for male patients and hazard ratio of 2.70 (95% confidence interval: 1.01–7.24) for non-CR patients]. This is the first report on ATL patients in a non-endemic area who received allo-SCT, and we think that this report shows very important information for management of ATL patients in non-endemic areas. Disclosures: No relevant conflicts of interest to declare.

Abstract 1935 Background: Although RIST has become more popular for elderly patients with leukemia, its value for ALL patients is still uncertain. To clarify the clinical significance of RIST for elderly patients with ALL in remission and identify prognostic factors for recipients, we retrospectively surveyed ALL patients receiving RIST who were registered in the JSHCT database. Patients and Methods: This study included ALL patients aged ≥ 50 years who received fludarabine-based RIST as the first transplantation between 2000 and 2009. The preparative regimen was classed as fludarabine-based reduced intensity conditioning if it included non-myeloablative chemotherapy (total dose of busulfan ≤ 8 mg/kg or melphalan ≤ 140 mg/m2) with or without total body irradiation (TBI) ≤ 6 Gy. Results: There were 144 patients, including 118 in first complete remission (CR1) and 26 in CR2. Their median age was 59 years (range: 50–70 years), with 71 males and 73 females. Eighty-seven patients had Philadelphia chromosome-positive ALL. Conditioning regimens contained fludarabine combined with melphalan (n=70), busulfan (n=58), or cyclophosphamide (n=16). TBI plus chemotherapy was used in 90 patients. Bone marrow from related or unrelated donors was transplanted in 71 patients, as well as peripheral blood stem cell from related donors in 31 patients and cord blood in 42 patients. Primary graft failure occurred in 7 patients. Granulocyte and platelet engraftment was achieved after a median of 15 and 26 days, respectively. The incidence of grade II-IV and grade III-IV acute GVHD was 37% and 19%, respectively. After a median follow-up of 16 months (range: 1–83 months), 3-year overall survival (OS) was 53%. The cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) at 3 years was 29% and 31% respectively. According to univariate analysis, factors associated with worse 3-year OS included a high leukocyte count (≥ 30,000 /μl) at diagnosis (vs low leukocyte count: 35% vs 61%, p=0.004), CR2 at transplantation (vs CR1: 30% vs 58%, p=0.014), and grade III-IV acute GVHD (vs grade 0-II: 25% vs 63%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 3085 Background: A llogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for patients with acute myeloid leukemia (AML), however a major obstacle to success is represented by the relapse of the disease. We retrospectively analyzed prognostic factors to determine whether there are clinical features that predict relapse after transplantation. Patients and Methods: Clinical data were collected from the Transplant Registry Unified Management Program (TRUMP) in Japan. An individual patient data-based analysis was performed on patients with AML who achieved or continued complete remission (CR) after related allo-HCT between 1991 and 2009. A logistic regression model was used to analyze associations between relapse and clinical factors. Patients with CR1 or CR2 were defined as having a standard-risk disease, and patients with ≤CR3 or non-CR were defined as having a high-risk disease. Conditioning regimens included ≤ 8Gy in fractionated doses of total body irradiation, busulfan dose < 9mg/kg, melphalan dose ≤ 140 mg/m2 defined as a reduced-intensity conditioning (RIC) regimen. Results: We analyzed a cohort of 1, 960 patients with AML. The median age at allo-HCT was 42 years (range, 16 to 74 years). Among the 1, 960 patients, 778 patients (38%) relapsed after allo-HCT. The probabilities of survival at 5-year for all patients, standard-risk patients, and high-risk patients were 49%, 61%, and 31%, respectively. In a multivariate analysis, significant factors for no relapse were standard-risk disease at the time of allo-HCT (HR 0.366, 95%CI 0.279–0.480, p Conclusion: These results indicate that in patients with standard-risk AML at the time of HCT, chronic GVHD or CMV infection after HCT were crucial for reducing relapse. Immune modulation mediated by chronic GVHD or CMV infection may have played a role in preventing disease relapse. The use of donor lymphocyte infusion before relapse may reduce the recurrence for selected patients who do not develop chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 3093 Allogeneic hematopoietic stem cell transplantation (allo SCT) is potentially curative treatment for relapsed follicular lymphoma (FL) that remains non-curative disease even with modern immunochemotherapy. Number of potential candidate of allo SCT for FL has been increasing with the development of reduced intensity conditioning (RIC) regimens and increased donor availability due to use of hematopoietic stem cell from unrelated donor including cord blood (CB). However, short-term and long-term transplantation-related complications are still major obstacles in applying this treatment. Recently, EBMT and CIBMTR reported a prognostic score of allo SCT for FL (Ann Oncol 2011;22 :Suppl 4, iv94). However, this score awaits validation. To elucidate prognostic factors for OS after allo SCT for FL, we conducted a retrospective study using the national registry data of the Japan Society of Hematopoietic Cell Transplantation (JSHCT). In total, 472 cases of allo SCT for FL performed from 2000 to 2008 were identified. 220 (46.6%) were male and the median age at allo SCT was 50 yo (range: 27–75). Forty six (9.7%) patients were 60 yo or older. Eighty two (17.3%) patients had previous history of autologous transplantation (ASCT). Stem cell source was BM or PB from related donor in 215 (45.5%), unrelated donor BM in 180 (38.1%), and unrelated donor umbilical cord blood (CB) in 77 (16.3%). Conditioning regimen was myeloablative (MAC) in 20.7% and reduced intensity (RIC) in 79.3%, respectively. Patients undergoing MAC were younger than those with RIC (45 vs 50 yo, P Disclosures: No relevant conflicts of interest to declare.

Abstract 2306 Poster Board II-283 We previously reported excellent outcomes in adult patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic stem cell transplantation (allo SCT) conditioned with medium-dose VP-16 (VP, 30 mg/kg), cyclophosphamide (CY, 120 mg/kg) and fractionated total body irradiation (TBI, 12 Gy) (VP/CY/TBI). In this study, we retrospectively compared the outcomes of this regimen and the standard regimen of CY/TBI. Clinical data for patients who received the VP/CY/TBI regimen were collected from six SCT centers in Hokkaido, Japan, and data for patients who received the CY/TBI regimen were collected from the Japan Society for Hematopoietic Cell Transplantation database (the Transplant Registry Unified Management Program) and the Japan Marrow Donor Program database. Data for 35 patients and 515 patients who received VP/CY/TBI and CY/TBI, respectively, and who met all of the following criteria were analyzed: SCT having been performed between 1993 and 2007, first time for SCT, aged 15-59 years, diagnosed as having ALL/lymphoblastic lymphoma or acute biphenotypic leukemia, first or second complete remission (CR) at SCT, bone marrow (BM) or peripheral blood stem cells (PBSC) as stem cell source and HLA- phenotypically matched (A, B and DR loci) related donor (MRD) or unrelated donor (MUD). Patients who met at least one of following criteria were excluded: secondary SCT, Burkitt leukemia/lymphoma, cord blood as stem cell, secondary leukemia or T-cell depletion. The median age of the patients was 34 years (range: 15-59 years). Two hundred seventy-nine (50.7%) of the patients underwent transplantation from an MRD and 271 (49.3%) of the patients underwent transplantation from an MUD. Four hundred forty-two (80.4%) of the patients received BM and 105 (19.1%) of the patients received PBSC. One hundred fifty-five (28.2%) of the patients were Philadelphia chromosome-positive (Ph+) and 442 (80.4%) of the patients were in CR1 at SCT. Although patients who received VP/CY/TBI were younger (VP/CY/TBI: mean age of 29.6 years; CY/TBI: mean age of 34.1 years, P=0.03), other factors such as Ph chromosome positivity, SCT in CR2, and donor status were not different between the two regimen groups. Five hundred forty-two (98.5%) of the patients achieved neutrophil engraftment at median day of 16. Acute graft-versus-host disease (AGVHD) and grade II-IV AGVHD occurred in 336 (63.5%) and 277 (52.4%) evaluable patients, respectively, at median onset day of 21. Chronic GVHD (CGVHD) occurred in 215 (45.4%) evaluable patients at the median onset day of 120. Incidences of engraftment, AGVHD and CGVHD were not different between the two regimen groups. After a median follow-up period of 37.7 months, 2-year overall survival (OS) and 5-year OS rates were 91.0% and 82.2% in patients who received VP/CY/TBI, respectively, and 68.0% and 55.2% in patients who received CY/TBI, respectively. Survival curves reached plateaus at 47 months after SCT in patients who received VP/CY/TBI and at 82 months in patients who received CT/TBI. OS and disease-free survival (DFS) rates were significantly better in patients who received VP/CY/TBI regimen [OS: logrank P=0.003, DFS: logrank P Although our analysis has limitations due to its retrospective fashion, analysis of data for a large number of patients who were selected by precise eligibility criteria (conditioning regimen, disease status at SCT, donor status) provided reliable information that medium-dose VP/CY/TBI resulted in a superior survival rate and was more curative than the CY/TBI regimen for adult ALL patients. Figure 1. Overall survival after SCT according to the conditioning regimens. Blocked lines show survival curve and dotted lines show 95% confidential interval. Figure 1. Overall survival after SCT according to the conditioning regimens. Blocked lines show survival curve and dotted lines show 95% confidential interval. Disclosures: No relevant conflicts of interest to declare.

BACKGROUND. Synergistic or additive activities for rituximab and cladribine have been shown in preclinical studies. Indolent Non-Hodgkin’s lymphoma (I-NHL) tends to recur with shortening intervals of remission after standard chemotherapy. New modalities of treatment are necessary. Therefore, we evaluated the feasibility, efficacy, and toxicity of combined regimen that consisted of rituximab plus cladribine plus mitoxantrone (the RCM regimen) in the treatment of patients with relapsed or refractory I-NHL. METHODS. The RCM protocol consisted of rituximab at a dose of 375 mg/m2 on Day 1, cladribine at a dose of 0.09 mg/kg per day on Days 2 through 6, and intravenous mitoxantrone at a dose of 6 mg or 10 mg/m2 per day on Day 2. The RCM courses were repeated at 4-week intervals, for up to 4 cycles. RESULTS. Fourteen patients with I-NHL and one patient with mantle cell lymphoma entered in the study. The median age was 60 (range 47–77) and 8 were females. Histology was small lymphocytic lymphoma (n=1), follicular lymphoma (n=13), mantle cell lymphoma (n=1). Median time from diagnosis to RCM treatment was 3.6 (range 0.2–8.1) years and median number of prior treatment regimen was 2 (range 1–4). Twelve patients (80%) had recurrent disease after prior therapy including high dose therapy with autologous stem cell transplant, and 3 patients (20%) had refractory disease. Thirteen patients were treated on the RCM regimen, and 8 patients (61.5%) achieved a complete response, 3 patients (23.1%) achieved a partial response. Therefore, the overall response rate was 92.3%. Median time to response was 2.8 months (range 1.0–6.7). Median progression free survival of responders was 16.5 (range 1.3–25.5) months. The treatment revealed tolerability, with episodes of severe neutropenia (grade 3 and 4) observed in 12 patients (85.7%), episodes of grade 3 and 4 thrombocytopenia observed in 2 patients (15.4%). However, severe infections were not observed in any patients. CONCLUSIONS. The RCM regimen is highly effective and well tolerated modalities of treatment in heavily pretreated and relapsed or refractory patients with I-NHL.

DOCK180, which is an archetype of the CDM family protein, plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of small GTPase Rac as a guanine nucleoside exchange factor (GEF). The expression of DOCK180 is ubiquitous except in hematopoietic cells, while the expression of another CDM family protein, DOCK2, is strictly limited in hematopoietic cells. However, DOCK180 was recently induced in two types of hematopoietic cells, namely, macrophages and dendritic cells, during their differentiation from CD14 positive monocytes (Fujimoto et al. ASH 2005, abstract #3077). Therefore, the expression of DOCK180 was analyzed in various sorts of normal hematopoietic cells, including CD34 positive hematopoietic stem/progenitor cells (HSC/P). Real time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the relative levels of DOCK180 mRNA expression (defined in 293T cells as 1000). Consistent with the findings of a previous report, there was a negligible amount of DOCK180 mRNA and protein in the peripheral blood or bone marrow mononuclear cells from normal volunteers. On the other hand, purified CD34 positive cells from healthy human bone marrow (N=4) and G-CSF mobilized peripheral blood (N=4) expressed a high amount of DOCK180 mRNA (1302 ± 883 Units and 1455 ± 689 Units, respectively). Furthermore, the expression of DOCK180 protein was confirmed by Western blotting in these CD34 positive cells. To investigate the association of the DOCK180 expression and granulocytic differentiation, CD34 positive cells were cultured with stem cell factor, IL-3 and G-CSF in vitro to induce them to mature into a granulocytic lineage and thus lose their surface CD34 expression. Interestingly, there was a gradual reduction in the DOCK180 expression accompanied by a loss of the surface CD34 expression, thus suggesting that the DOCK180 expression may therefore be limited in CD34 positive, immature HSC/P. Since a large number of immature acute leukemia cells also express CD34 antigen on their surface, the expression of DOCK180 was investigated in CD34 positive leukemic cells from 25 acute leukemia patients. The subjects consisted of 16 patients with acute myeloid leukemia (AML: M0 2, M1 2, M2 4, M4 3, M7 1, MDS overt leukemia 2, secondary AML 2), 6 with acute lymphoblastic leukemia (ALL: Pro B 3, Ph1 positive 2, common 1), and 3 in blastic crisis of chronic myeloid leukemia (BC CML: 2 lymphoid BC and 1 myeloid BC). The leukemic cells were collected at the first onset of the disease from 19 patients and from 6 other patients at the time of relapse. While there was no difference in the expression of DOCK2 between leukemic and normal CD34 positive cells, the expression of DOCK180 in leukemic cells was significantly lower than that detected in normal CD34 positive cells (median 17 Units vs. 1096 Units, range 0–4746 Units and 279–2812 Units, respectively, p

Regulatory T cells (Treg) exert pivotal roles on the induction and maintenance of peripheral tolerance. Adoptive transfer of Treg is a promising strategy to control acute GVHD. However, in vitro expansion of Treg is costly, laborious and time-consuming (more than 3 weeks). As an alternative source, dendritic cells (DC) with regulatory function (DCreg) seem to be attractive, since DC are potent antigen presenting cells capable of regulating the initiation of various immune responses and can be generated in a relatively short term culture from peripheral blood (PB) monocytes. In the present study, we examined the feasibility of generating DCreg on a large scale from PB monocytes obtained by leukapheresis. PB mononuclear cells (range 1.8×109 to 3.1×109) were obtained from 5 healthy volunteers with informed consent by leukapheresis for 1–2 hours using a COBE Spectra apparatus. Immature DC were generated from isolated CD14+ monocytes by culturing with GM-CSF and IL4 with or without various concentrations of rapamycin for 5–7days. For maturation, immature DC were cultured with the addition of TNFα for 2 days. The yield of immature DC cultured with rapamycin (10ng/ml) was 8.4±3.6×107, constituting 4.2±1.8% of the initial PB mononuclear cells. FACS analysis showed that DC cultured with rapamycin (DC-rapa) had immature DC phenotype with CD14−, CD1a+, CD83−. But the surface expression of CD86, CD40, HLA-DR was much lower compared with control DC (DC-ctr) cultured without rapamycin. The reduction of the expression of CD86, CD40, HLA-DR was dependent of rapamycin dose and resistant to maturational stimulation of TNFα. DC-rapa showed very weak allostimulatory activity in a mixed lymphocyte culture (MLC) compared with DC-ctr, corresponding to the reduced expression of HLA-DR and costimulatory molecules. To further determine the types of cells involved, CSFE-labelled non-isolated CD4+ T cells were cultured with DC-rapa or DC-ctr for 7 days and the proliferating cells were identified by FACS analysis. DC-rapa inhibited the proliferation of CD4+CD25− alloreactive T cells by approximately 60% while the inhibitory effect of DC-rapa on CD4+ CD25+ cells was minimal. Neither the irradiation of DC nor the addition of IL2 altered the inhibition. Stimulation with both anti-CD3 and ani-CD28 moonoclonal antibodies coated on microbeads reversed the inhibition completely, suggesting the importance of the signals via T cell receptors and costimulatory molecules in the inhibition by DC-rapa. Double staining with anti-CD25 and anti-Foxp3 monoclonal antibodies revealed that DC-rapa increased the percentage and number of CD25+Foxp3+ Treg by approximately 2.6 times compared with DC-ctr during 7-day culture. In conclusion, these results demonstrated that immature DC induced with rapamycin have a potent immunoregulatory function including preferential expansion of Treg. To the best of our knowledge, this is the first demonstration that immunoregulatory DC can be generated on a large scale in relatively short term culture with rapamycin following leukapheresis. These findings have an important clinical implication to facilitate DC-based immunotherapy for acute GVHD.

Appearance of anti-hepatitis B surface antigen antibody (anti-HBs) and clearance of hepatitis B virus (HBV) from serum usually indicates resolution of hepatitis in patients infected with HBV. However, in most patients in whom HBV has been eliminated from serum, HBV DNA is still detectable in the liver using polymerase chain reaction. Reactivation of this dormant HBV in the liver is known as reverse seroconversion (RS). Previously, we reported that HBV-RS after allogeneic hematopoietic stem cell transplantation (allo-HSCT) was a frequent late-onset complication that can be predicted by careful monitoring of progressive disappearance of anti-HBs. RS hepatitis after allo-HSCT is thought to be a phenomenon caused by naive donor immunity after loss of recipient-oriented immunity against HBV. We speculated that vaccination could prevent reactivation of HBV in allo-HSCT recipients. Safety and efficacy of recombinant HBV vaccine in allo-HSCT recipients have already been confirmed. We studied HBV serological markers in 23 patients with anti-HBs and/or anti-HBc before allo-HSCT who were followed for more than 1 year. Patients’ characters are following; Age at HSCT 22 to 65 (median, 38) years; M:F ratio 14:9; Hematological disorders CML 6, AML 1, ALL 4, MDS 4, SAA 2, NHL 4, MM 1 and CAEBV 1; Serological markers anti-HBc(+) and anti-HBs(+) 17, anti-HBc(+) and anti-HBs(−) 3, anti-HBc(−) and anti-HBs(+) 3. No patients had a prior history of vaccination or HBV-specific immunoglobulin usage. All patients were negative for hepatitis B surface antigen (HBsAg) and were considered to have previous HBV infection. The follow-up period varied from 12 to 116 (median, 36) months. Eighteen patients were followed without intervention. Five patients were vaccinated with recombinant HBV vaccine by the standard three-dose protocol after cessation of immunosuppressant administration. RS was defined as disappearance of anti-HBs and appearance of HBsAg and HBV-DNA with or without clinical hepatitis. Progressive decreases in anti-HBs titer were observed in all pre-HSCT anti-HBs-positive recipients. In 18 of the 20 patients with pre-HSCT anti-HBs, anti-HBs titer decreased to less than the protective value (

Survivin, a member of the inhibitors of the apoptosis family, is overexpressed frequently in a variety of cancers and hematological malignancies, but not in normal tissues. Murine in vivo and human in vitro studies have suggested that immunotherapy of cancer patients using survivin peptide might be feasible. In the present study, we examined whether HLA-A24 restricted cytotoxic T lymphocytes (CTL) which recognize survivin peptide can be generated from peripheral blood of lymphoma patients. HLA-A24 positive four lymphoma patients and two healthy volunteers were enrolled. Three immunodominant 9-mer candidate peptides (2B, 3A, 3B) were selected on the basis of anchoring motif of peptide binding to HLA-A24 molecule. CD8 T cells from the patients and healthy volunteers were stimulated several times with autologous monocyte-derived dendritic cells pulsed with survivin or control HIV peptides and tested for peptide-specific cytotoxicity by an LDH-release assay. CTL generated with survivin 2B peptide lysed autologous monocytes pulsed with a relevant peptide. However, other survivin peptides did not elicit CTL response. Non-pulsed or HIV peptide-pulsed monocytes were not lysed. On the other hand, CTL generated with HIV peptide only lysed HIV peptide-pulsed monocytes. CTL did not lyse allogeneic monocytes regardless of the peptide pulse. Cytotoxic activity was inhibited by the pretreatment of target cells by anti-HLA class I, not by anti-HLA-DR monoclonal antibody, indicating that the lysis was HLA class I (A24) restricted. These cells did not lyse Daudi and K562, excluding the involvement of LAK or NK activity. Importantly, these survivin peptide-specific CTL showed cytotoxicity to the patient’s lymphoma cells and HLA-A24 positive lymphoma cells. Based on these preclinical data, we have just started a pilot clinical study to examine the safety and the efficacy of peptide vaccination to relapsed, chemotherapy-resistant malignant lymphoma patients who are HLA-A24 and survivin positive. A 46-year old male patient with diffuse large B-cell lymphoma has just completed two courses of four vaccinations at two-week intervals with survivin 2B peptide (1 μg subcutaneously) in an incomplete Freund’s adjuvant (Montanide ISA-51, SEPPIC Co. France). We observed a marked decrease in the size of extra-nodular surface and cervical lymphnodes following vaccinations without serious adverse events. Immunological evaluations using HLA-tetramer and T cell receptor clonality assays revealed an increase in survivin-specific CTL frequency after vaccinations. The in-vitro feasibility study and pilot clinical trial indicate that a vaccination with a survivin peptide is safe and might be a promising novel strategy for the treatment of lymphoma patients.

Innate immune cells such as natural killer (NK) cells play a crucial rule in antitumor immune responses. NK cells have functionally opposite receptors for activating and inhibiting signals to exert cytotoxicity. NKG2D is a C-type lectin-like activating receptor and is expressed in various immune cells, including NK cells, NKT cells, CD8+α/β+T cells, and γ/δ+T cells. NKG2D recognizes its ligands, MHC class I-related chain A and B (MICA, MICB). NKG2D ligands are not present on normal cells but are induced by various stresses such as viral infection. Moreover NKG2D ligands are expressed in many malignant cells including hematological malignancies. It has been suggested that involvement of NKG2D in NK or CD8+Tcell-mediated cytotoxicity correlates with the expression levels of ligands on target cells. Therefore induction of NKG2D ligands may lead to enhance the sensitivity to NKG2D-mediated cytotoxicity. In this study, we could enhance expression levels of MICA and MICB by treatment with the histone deacetylase inhibitor trichostatin A (TsA) in lymphoid leukemic cell line BALL1 and some primary patients’ lymphoid leukemic cells. Treatment of BALL1 and patients’ leukemic cells with TsA for 12 hr increased MICA and MICB mRNA expression at least by more than 2 fold by Real-time PCR. Treatment of BALL1 with TsA for 12 hr increased MICA and MICB protein expression on the cell surface by more than 2 fold by flow cytometry analysis. These results suggested that expression of MICA and MICB is partly regulated by histone acetylation. Chromatin immunoprecipitation assay revealed that treatment with TsA resulted in increased acetylation of histone H3 and decreased association with the counteracting enzymes of histone acetyltransferases HDAC1 at the MICA and MICB promoter in BALL1 cell and patients’ leukemic cells. To examine the impact of the cytolytic activity of NKG2D-expressing cells on leukemic cells in which expression of NKG2D ligands was induced by TsA treatment, we performed standard 4 hr 51Cr release assays using BALL1 cells and patients’ leukemic cells. Up-regulation of NKG2D ligands by TsA treatment led to enhance the susceptibility of BALL1 and patients’ leukemic cells by 2 or 3 times to the cytolytic activity of NKG2D-expressing cells. Blocking experiment using specific antibodies for MICA and MICB inhibited the NKG2D-expressing cell-mediated cytolytic activity against BALL1 cells. Our results suggest that regulation of NKG2D ligands expression by treatment with chromatin-remodeling drugs may be an effective strategy to enhance the susceptibility of leukemic cells to the cytolytic activity of NKG2D-expressing cells for immunotherapy.

It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR CD94/NKG2A heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94-expressing cells can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells resulting induction of graft-versus-leukemia (GVL) effect but does not attack normal cells with HLA class I expression resulting no enhancement of graft-versus-host disease (GVHD). On the other hand, CD4+ CD25+ regulatory T cells (Treg) contribute to suppress allogeneic immune responses and prevent transplant rejection and GVHD. In this study, we tried to expand CD94-expressing T cells and Treg cells from the same cord blood cells and then investigated their cytolytic characteristics and immunoregulatory function in order to develop a potential strategy of cell therapy for hematological malignancy. After CD4 enrichment by negative selection using magnetic cell sorting (MACS) (Miltenyi Biotec)(CD4-enriched fraction) from cord blood, CD4+ CD25+ cells were isolated by positive selection with anti-CD25 magnetic microbeads. We could get more than 1,000 fold expansion of CD94-expressing CD8 T cells from CD4-depleted fraction after 8 days culture with immobilized anti-CD3 monoclonal antibody (mAb) (1 μg/mL) and IL-15 (5 ng/mL). Isolated CD4+ CD25+ cells were cultured with anti-CD3/CD28 mAb-coated dynabeads and IL-15 (5 ng/mL) and we could get about 50 fold expansion of Treg cells for 8 days. These expanded Treg cells could suppress allogeneic mixed lymphocyte culture more than 80% (effector cells: Treg cells= 2:1) and expressed FoxP3 mRNA about 100 fold compared with isolated CD25-negative cells. Cytolytic activities of purified CD94-expressing cells (CD94 > 90%) detected by 4 hours 51Cr release assay against K562 were 68.8 ± 16.8 % (n=5). Coculture of CD94-expressing cells with expanded Treg cells (CD94-expressing cell: Treg cells= 1:1, preincubation 4 hours) did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells (66.1 ± 19.8 %, n=5). CD94-expressing CD8 T cells with cytolytic activity could be expanded from CD4-deplted fractions and Treg cells with immunosuppressive activity and increased expression level of FoxP3 mRNA could be expanded from CD4-enriched fractions of the same cord blood. Expanded these cytolytic CD94-expressing CD8 cells might be able to induce GVL effect without enhancing GVHD and Treg cells might be able to suppress allogeneic response including GVHD and graft rejection. Therefore, this strategy may be useful to differentiate lymphocytes in cord blood to two different kinds of effector cells exhibiting cytolytic or immunoreguratoly characters.

Objective: Tacrolimus, a commonly used potent immunosuppressive drug in hematopoetic stem cell transplantation (SCT), has large individual variation in concentrations and a relatively narrow therapeutic range. It is metabolized mainly by cytochrome P450 (CYP) 3A which has genetic polymorphisms influencing enzymatic activities; CYP3A5*1 and *3 genotypes represent expressor and non-expressor, respectively. In the present study, we investigated whether the tacrolimus concentration per dose is associated with CYP3A5 gene polymorphisms. Methods: 51 Japanese SCT patients (21 AML, 12 ALL, 9 CML, 3 NHL, 1 HL, 2 ATL, 1 MM, 2 AA), receiving tacrolimus were studied. DNA was extracted from peripheral blood samples. CYP3A5 genotype was analyzed by PCR-restriction fragment length polymorphism (RFLP) using specific primers and a restriction enzyme, SspI. Blood tacrolimus concentrations were measured twice a week by a commercially available EIA kit. Results: The frequency of the CYP3A5 *1/*1, *1/*3 and *3/*3 genotype was 7.8, 31.4, and 60.8% of 51 patients, respectively, and the genotype distribution was not different significantly from that of 121 Japanese healthy volunteers (2.5, 38.8, and 58.8%, respectively). The frequency of CYP3A5 genotypes of white Americans is reported to be 2.2, 12.0 and 86.0%, respectively, indicating the ethnic difference of distribution. The dose-adjusted concentrations of tacrolimus 5 to 7 days following SCT were significantly higher in CYP3A5 *3/*3 patients than in CYP3A5 *1/*1 and *1/*3 patients (691.7±298.0 ng/ml per mg/kg vs. 451.8±153.8 ng/ml per mg/kg, P=0.0017). The dose needed to achieve the target concentration (10 to 15 ng/ml) between 40 to 60 days following SCT, when the blood concentrations of tacrolimus tended to be stable in most patients, is significantly lower in CYP3A5 *3/*3 patients than in CYP3A5 *1/*1 and *1/*3 patients (0.008±0.005 mg/kg vs.0.016±0.006 mg/kg, P Conclusion: The tacrolimus concentration per dose in SCT patients is influenced by the CYP3A5 gene polymorphism. This is the first report showing the relationship between CYP3A5 gene polymorphism and the tacrolimus concentration in SCT patients to the best of our knowledge. Information of CYP3A5 genotype may be helpful to determine the initial tacrolimus dose adequate for each patient.