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Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the approach to patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). This study retrospectively analyzed patients treated with commercially available tisagenlecleucel at our hospital and evaluated its safety and effectiveness. Of the 21 patients evaluated, any grade and grade ≥3 cytokine release syndrome (CRS) occurred in 85.7% and 9.5% of the patients, respectively. A total of 66.7% received tocilizumab and 28.6% received glucocorticoids for the treatment of CRS. The complete response (CR) rate at 3 months was 61.9% (95% confidence interval [CI] 38.4-81.9). After a median follow-up of 6.3 months following CAR-T infusion, the progression-free survival (PFS) and overall survival rates at 6 months were 53.1% (95%CI 28.3-72.7) and 69.2% (95%CI 43.7-84.9), respectively. Severe cytopenia and hypogammaglobulinemia occurred frequently following CAR-T infusion. Eight patients (38.1%) had comorbidities that would have made them ineligible for leukapheresis in the JULIET trial. However, the presence of comorbidities at the time of leukapheresis had no significant effect on the rates of CR, PFS, and adverse events. Tisagenlecleucel for r/r DLBCL in the real-world setting showed high efficacy and manageable safety profile comparable with the pivotal trial.

Outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in nonremission acute myeloid leukemia (AML) are dismal [2-year overall survival (OS): 20–30%]. Though several risk classifications have been used, some factors are unavailable until the start of conditioning or transplantation. We analyzed prognostic gene mutations by targeted next-generation sequencing to identify predisposing factors for predicting OS at 1 month before transplantation. We enrolled 120 patients with nonremission AML who underwent first allo-HSCT between 2005 and 2018. Mutations were found in 98 patients; frequently mutated genes were FLT3-ITD, TP53, RUNX1, and WT1. TP53 mutation was detected in 21 patients and was the only predictor of poor OS. Multivariate analysis using Cox regression hazard model revealed primary AML, monosomal karyotype (MK), and TP53 mutation as independent factors for predicting poor OS. Based on these, patients were stratified into three groups. The low-risk group included patients with prior myeloid disorder without MK (n = 26). Among the rest, patients with TP53 mutation were assigned to the high-risk group (n = 19) and the rest into the intermediate-risk group (n = 75). Two-year OS in low-, intermediate-, and high-risk groups differed significantly (50.0%, 24.9%, and 0%, respectively). This suggests that the indication of allo-HSCT should be carefully judged for high-risk patients.

Chimeric antigen receptor (CAR) T-cell therapy is effective in treating relapsed and refractory B-cell non-Hodgkin lymphoma. However, because of the mortality risk associated with immune effector cell-associated neurotoxicity syndrome and pseudoprogression, patients with central nervous system (CNS) involvement are less likely to receive CAR T-cell therapy.We report a case of a 61-year-old, male patient with intravascular large B-cell lymphoma who suffered a CNS relapse after standard chemotherapy.A diagnosis of intravascular large B-cell lymphoma with CNS involvement was made.We treated the patient using CAR T-cell therapy following a conditioning regimen consisting of thiotepa and busulfan and autologous stem cell transplantation. Although he experienced grade 1 cytokine release syndrome, no other serious adverse events, such as immune effector cell-associated neurotoxicity syndrome or pseudoprogression, were observed.The patient achieved complete remission after the CAR T-cell infusion.CAR T-cell therapy following autologous stem cell transplantation is a viable option for relapsed/refractory lymphoma with CNS infiltration. Further clinical studies are warranted to verify its safety and efficacy.

Background: Additional cytogenetic abnormalities (ACA), the most frequent form of cytogenetic changes, are considered as a result of genetic instability and clonal evolution of leukemia cells. Recently, we described that ACA at the first relapse was associated with the significantly lower second complete remission (CR2) rate and poor survival in adult acute myeloid leukemia (AML) patients (Hematol Oncol. 2018;36:252-257). However, the prognostic impact of ACA after allogeneic stem cell transplant (allo-SCT) has not been elucidated in adult AML patients. Patients and methods: Of the 145 adult AML patients who underwent the first allo-SCT in CR2 between 1997 and 2019, 98 patients whose cytogenetic abnormality data both at diagnosis and at the first relapse were available were included in this study. Cytogenetic changes between at diagnosis and the first relapse were classified into four groups: (1) no change, (2) ACA was acquired at the first relapse, (3) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared at the first relapse, and (4) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared, and completely different ACA was acquired at the first relapse. In this study, groups 2 and 4 were defined as ACA acquisition. Overall survival (OS) was defined as the interval from the date of transplant to the date of death. Fisher's exact test was used to compare binary variables. The cumulative incidence (CI) of non-relapse mortality (NRM) and relapse were evaluated with Gray's test, considering relapse and NRM as a competing risk, respectively. OS was estimated with the Kaplan-Meier method and compared using the log-rank test. Factors associated with at least borderline significance (p < 0.20) in univariate analyses were subjected to multivariate analysis. The Cox and Fine-Gray proportional hazard model were used for multivariate analysis of prognostic and risk factors, respectively. Values of p < 0.05 were considered to indicate statistical significance. Results: Of the 98 patients included in this study, 57 were male, and 41 were female. The median age at transplant was 45 years (range, 17-71 years). The median duration of CR1 was 12.4 months (range, 1.3-70.3 months) and cytogenetic risk groups were good, intermediate, and poor in 26 (27%), 70 (71%), and two patients (2%), respectively. Donor types were related, unrelated, and cord blood in 23 (24%), 59 (60%), and 16 patients (16%), respectively, and 86 (87%) and 61 patients (62%) were conditioned with myeloablative and total body irradiation-containing regimens, respectively. According to the definition described above, 20 patients (20%) acquired ACA at the first relapse. There was no significant difference in baseline characteristics and transplant procedures between patients with and without ACA acquisition. The OS rates were not significantly different between two groups (55% vs. 72% at three years after transplant; p = 0.28). The CI of relapse was significantly higher in patients with ACA acquisition than those without ACA acquisition (59% vs. 15%; p < 0.01), while the CI of NRM were not significantly different between two groups (5% vs. 19%; p = 0.17). Multivariate analysis for OS revealed that age over 50 years (hazard ratio [HR] = 2.4; p < 0.01), but not ACA acquisition, was identified as an independent prognostic factor. ACA acquisition (HR = 4.7; p < 0.01) was extracted as an independent risk factor of relapse, while use of reduced intensity conditioning regimens (HR = 3.1; p = 0.03) and more than or equal to 1 of performance status at transplant (HR = 2.7; p = 0.04) showed independent risks of NRM. The similar OS rates between two groups might resulted from an offset of the lower relapse rate with the higher NRM rate in patients without ACA acquisition despite not reaching statistical significance. This increasing NRM rates in those without ACA acquisition was potentially associated with use of reduced intensity conditioning regimens in larger proportion (0% vs. 15%; p = 0.12). Conclusion: These findings suggested that ACA acquisition at the first relapse was associated with a higher risk of relapse even after allo-SCT in CR2 in adult AML patients. As AML cells with ACA acquisition was resistant to not only chemotherapy but also graft-versus-leukemia effect, innovative therapeutic strategy is warranted. Disclosures Handa: Janssen: Honoraria; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding; BMS: Honoraria; Chugai: Research Funding; Ono: Honoraria; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding.

A 51-year-old man underwent allogeneic bone marrow transplantation (BMT) for recurrent acute myeloid leukemia. Although the patient developed slight edema, pleural effusion, and cardiac effusion 6 months after BMT, his clinical condition improved with furosemide treatment. The patient was transfused with red blood cells for the management of anemia 8 months after BMT. He developed acute respiratory failure with pulmonary alveolar hemorrhage 80 min after the transfusion. He was diagnosed with transfusion-associated circulatory overload (TACO) due to the presence of acute pulmonary congestion and depressed left ventricular systolic function. Reduced circulatory load due to sufficient furosemide led to ventilator weaning 3 days later. Other causes of pulmonary alveolar hemorrhage were excluded, and the patient's condition improved by cardiac failure treatment only. This clinical course indicated that pulmonary alveolar hemorrhage would breakdown the blood vessels due to acute pulmonary congestion. Chemotherapy and prolonged anemia are high risks for cardiac failure in patients with hematological malignancies. Therefore, the possibility of cardiac failure is considered when patients with hematological malignancies have fluid retention, such as cardiac enlargement, edema, and pleural effusion. Moreover, the body fluids should be monitored before and after blood transfusion.

Recent studies have shown that CD25 expression at the time of diagnosis of acute myeloid leukemia (AML) may be associated with an unfavorable outcome. We focus on patients with AML without complete remission (CR) and examine the clinical correlation between surface CD25 expression at the time of transplant and subsequent transplant outcomes. We observed a significant difference in overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) between CD25 positive (+) (n = 22) and negative (-) groups (n = 44) (2-year OS; CD25 (+) group: 5% vs. CD25 (-) group: 40%, p

Increased levels of normal B cell precursors, termed hematogones (HGs), are observed in regenerating bone marrow after chemotherapy or hematopoietic stem cell transplantation (HSCT). Recent reports suggest that emergence of HGs is associated with better outcomes following allogeneic HSCT (allo-HSCT). We reviewed the emergence of HGs and the clinical features of 192 patients after allo-BMT. Patients undergoing allo-BMT from related donors were more likely to develop HGs at day 30 compared to unrelated donors. Furthermore, patients undergoing allo-BMT from HLA-mismatched donors were less likely to develop HGs at day 30. The emergence of HGs at day 30 was an independent prognostic factor among patients who underwent BMT. We found no difference in the relapse rate between HG-positive (+) and HG-negative (-) patients undergoing BMT. HG (-) patients had high non-relapse mortality, grade II to IV acute graft-versus-host-disease (GVHD), fungal infection, and lower IgG levels compared to HG (+) patients. The emergence of HGs at day 30 among patients undergoing BMT may be a very useful indicator of subsequent survival outcomes or acute GVHD in common clinical practice.

Although peripheral blood stem cell (PBSC) transplantations in an unrelated transplant setting have been performed since 2010, prior harvesting and cryopreservation of PBSCs from unrelated donors has not been approved in Japan. There are no restrictions with regard to related donors. Therefore, in April 2015, we conducted a nationwide survey and obtained written answers from 123 transplant hospitals throughout Japan. Our survey revealed that as much as 81.3% of transplant hospitals routinely perform prior harvesting and cryopreservation of PBSCs from related donors and that both cell processing and quality management of cryopreserved products have been appropriately conducted in line with domestic guidelines, although post-thaw quality control and storage period setting require further improvements. Moreover, informed consent obtained from both patients and donors with regard to cryopreservation of PBSCs was not always sufficient in some hospitals. We found that the average number of unused or discarded cryopreserved PBSCs is 1.09 per hospital, and the overall nonuse or discard rates of cryopreserved PBSCs were estimated to be as low as 2.67%.

Background Myelodysplastic syndromes (MDS), commonly seen in elderly patients, represent a heterogeneous group of clonal hematopoietic stem cell disorders caused by the accumulation of gene mutations. By contrast, congenital bone marrow failure syndromes and genetic predispositions associated with MDS are known in pediatric patients. However, little is known about the pathogenesis of MDS in adolescent and young adult (AYA) patients. Previous reports showed the patients with MDS aged under 40 or 41.5 years at allo-HSCT were associated with good survival compared to those among the older population (N Engl J Med. 2017;376:536-547, Blood. 2017;129:2347-2358). However, AYA-MDS is rare, and its clinical features and genetic abnormalities have not been analyzed enough. It is suspected that the clinical and genetic features of AYA-MDS patients might be different from those of elderly patients or pediatric patients. Therefore, we investigated the gene abnormalities of AYA-MDS patients and aimed to elucidate the genetic characteristics associated with the good outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We analyzed the patients younger than 50 years of age in order to reduce the variation of patient-related factors. Methods We analyzed the outcomes of all consecutive patients aged under 50 years who were diagnosed with MDS or acute myeloid leukemia evolving from MDS in our hospital between January 2005 and July 2018. The study was approved by the institutional review board, and patients gave written informed consent for the study, according to the Declaration of Helsinki. Cytogenetic analysis and genomic DNA extraction were carried out using diagnostic bone marrow samples. We performed targeted next-generation sequencing to identify mutations in 68 driver genes using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Overall survival (OS) was analyzed for all patients, and the Kaplan-Meier survival curve was used to assess OS using the log-rank test. Additionally, the cumulative incidence of relapse (CIR) was analyzed for patients who underwent allo-HSCT. Gray's test was used to evaluate the CIR. Results A total of 85 patients with MDS aged under 50 years (U40 between 15 and 39 years old: N=37, 40s between 40 and 49 years old: N=48) were analyzed. The median follow-up time of survivors was 2,041 days (range 176-5,085). There were no significant differences in patient characteristics between U40 and 40s. The 3-year OS of U40 were superior to 40s (79.9% vs. 58.1%, P=0.018), especially lower risk IPSS categories (3-year OS, 95.5% vs. 50.8%, P=0.002). In total, 69 of 85 patients (U40: N=31, 40s: N=38) had undergone allo-HSCT. U40 patients had lower percentage of bone marrow blasts at just before HSCT than 40s patients (over 10%, 12.9% vs. 36.8%, P=0.048), and better 3-year OS from HSCT in lower-IPSS (88.8% vs. 53.8%, P=0.024); but not in higher-IPSS (45.0% vs. 43.2%, P=0.834). In this cohort, at least one driver mutation was detected in 61% of allo-HSCT recipients. Frequently mutated genes (more than 10%) were ASXL1 and RUNX1; however, both of the genes did not have significant impact on the outcomes. While, only one patient in 40s had TP53 mutation. We detected 0.8 (range 0-3) and 1.8 (range 0-6) mutations at average in U40 and 40s, respectively (P=0.06). The proportions of the patients without any gene mutations were 52% in U40 and 30% in 40s. Transplanted patients with 0 or 1 mutation showed lower relapse rate than those with 2 or more mutations (3-year CIR, 23.3% vs. 45.2%, P=0.049). Conclusions The clinical outcomes of U40 patients with MDS were favorable than those in the 40s, especially in lower disease risk. The number of driver mutations in U40 tended to be lower than that in 40s. MDS in adult is regarded as a stem-cell aging disease with gene mutations; however, MDS-associated mutations were not detected in the half of U40. Moreover, TP53 mutation that is associated with extremely poor posttransplant survival was not detected in U40 patients. MDS patients with less than 2 mutations showed lower relapse rate, which maybe indicate genetic mutations have a great impact on transplant outcomes between 15 and 49 years old. Disclosures No relevant conflicts of interest to declare.

Background: Granulocyte-colony stimulating factor (G-CSF) is widely used to mobilize peripheral blood stem cells (PBSCs) in healthy donors. A few reports have shown that some healthy donors developed acute respiratory distress syndrome or capillary leak syndrome after more than several rounds of G-CSF administration or leukapheresis. Case Report: We report the case of a healthy donor for allogeneic stem cell transplantation who developed severe hypoxemia 1 h after only the first administration of G-CSF. The donor was administered 10 μg/kg G-CSF (lenograstim) subcutaneously for PBSC mobilization. 1 h after the first administration of G-CSF, the donor suddenly presented with dry cough and dyspnea. The oxygen saturation by pulse oximetry (SpO2) in the room air was 88%. An electrocardiogram and chest radiography revealed no abnormalities. We excluded other causes of severe hypoxemia and diagnosed the donor with hypoxemia due to G-CSF administration, which was subsequently terminated. The donor was administered 2 l/min oxygen via a nasal cannula and 100 mg hydrocortisone intravenously. He subsequently recovered, and SpO2 in the room air returned to 98% 10 h after hypoxemia. Conclusion: These respiratory symptoms might be related to anaphylactoid or hypersensitivity reaction. The donors should be observed for at least 1 h after the first administration of G-CSF.

Background Although the outcome after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for the patients with acute myeloid leukemia (AML) in complete remission has been improved, the prognosis of patients with active disease is still dismal, generally with 20 - 30% of overall survival (OS) at 2 years. Prognostic value of gene mutations detected by the next generation sequencing (NGS) for this extremely poor group remains to be evaluated. Methods A total of 120 patients with AML not in hematological remission who received the first allo-HSCT at our institute between April 2005 and December 2017 were enrolled. For each patient, genomic DNA was extracted from the frozen bone marrow sample harboring leukemic blasts which was preserved at the nearest available date before the initiation of conditioning regimen. Sequencing was performed using TruSight Myeloid Sequencing Panel® on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Charts were retrospectively reviewed on survival, relapse, and non-relapse mortality (NRM). The Kaplan-Meier method was used to assess OS using the log-rank test. Univariate and multivariate analysis were performed to identify potential prognostic factors. The Cox proportional hazards method was used for the multivariate analysis to assess OS. Gray's test and the Fine-Gray test were used to assess the cumulative incidence of relapse (CIR). Competing risks were relapse and NRM. Results Median follow-up of survivors was 1345 days (235 - 4888 days). Median age at transplant was 51 (range 21 - 71). Grafts were from bone marrow (n = 67, 55.8%), peripheral blood (n=42, 35.0%) and cord blood (n=11, 9.2%). Refined disease risk index (Blood. 2014;123:3664-71) scored high (n=61, 51.3%) and very high (n=58, 48.7%). OS at 2 years of the whole cohort was 27.3% (95% confidence interval [CI], 19.4% - 35.7%). There was no significant difference in OS between patients in primary induction failure and in relapse (OS at 2 years: 26.5% [n=50] vs 28.7% [n=70], p= 0.293). NGS analysis revealed TP53 loss-of-function mutation in 23 (19.2%) patients. Among all detected gene mutations, TP53 mutation was the most powerful predictor of poor OS after allo-HSCT (OS at 2 years: 13.5% vs 30.5% for TP53+ [n=23] vs TP53- [n=97], p= 0.0184). Consistent with previous reports, monosomal karyotype (MK, J Clin Ocnol 2010:26;4791-7) was significantly associated with positive TP53 mutation (13.3% of non-MK vs 37.9% of MK, p=0.006). Of note, all the patients (n=11) positive for both prognostic factors died within 1 year after allo-HSCT, whereas OS of the patients without either factor (n=78) was 33.6% at 2 years. Multivariate analysis on OS revealed MK, TP53 mutation, de novo AML (no prior history of MDS), ECOG performance status score 2 or more, C reactive protein 1.0 mg/dL or more, peripheral blood blast frequency of 1% or more at the initiation of conditioning regimen were independent prognostic factors for poor OS. Among them, to determine the prognostic factors critical for deciding the indication of allo-HSCT, we chose pre-transplant factors which were available around one months before transplant. From this point of view, multivariate analysis revealed independent prognostic factors for poor OS after allo-HSCT including MK (Hazard ratio [HR] 2.05; 95% confidence interval [CI] 1.30 - 3.25, p=0.00217), TP53 mutation (HR 1.72; 95% CI 1.04 - 2.86, p=0.035), and de novo AML (HR 1.67; 95% CI 1.04 - 2.86, p=0.036). Because the HR of these three factors were comparable, a score of 1 was assigned to each factor. OS at 2 years was 49.7%, 26.5% and 13.1% for patients with low (score 0, n=23), intermediate (score 1, n=63) and high (score 2 or 3, n=34) risk, respectively (p Conclusions NGS-analysis revealed the importance of TP53 mutation on transplant outcome of AML in non-remission. The combined score with the information on MK, de novo AML, and TP53 mutation stratified the patients into three groups, including low risk with 50% survival rate, and high risk with shorter survival after allo-HSCT for whom the other strategy might be optimal. Our results would require further validation in larger cohorts. Disclosures Harada: Celgene: Research Funding.

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3, conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered to irradiated mice, the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML), resulting in rapid death of the recipient mice. On the other hand, BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive, Sca-1-positive, and lineage-negative hematopoietic stem cells (KSLs), but not committed progenitors CMPs or GMPs, as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly, Hes1 was highly expressed in 8 of 20 patients with CML in blast crisis, but not in the chronic phase, and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML.

我々は，D抗原陰性者に対しD陰性の赤血球輸血を行なっていたにもかかわらず，抗D抗体価がクームス法で最高4,096倍と著しく上昇した症例を経験した．輸血に使用された血液製剤の遺伝子解析を行なったところ，Del型赤血球の特徴であるRHD遺伝子の1227番目がグアニンからアデニンに置換している製剤が含まれていたことが明らかとなった． したがって本症例はDel型赤血球の輸血により，D抗原の感作を受け，抗D抗体を産生したと考えられた．なお，輸血後明らかな溶血の所見は認められなかった． 通常のD陰性血の中にはDel型が含まれていることがあり，Del型赤血球輸血がD陰性者に抗D抗体を産生させる可能性があることは十分認識するべきであると思われた．

The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34+ cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-γ secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of γ-secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56+ cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56+ cells generated by culture with both Delta4 and IL-15 were CD7+ and cytoplasmic CD3+ from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.

背景·目的: わが国ではアルブミン製剤の使用量が多く，輸入に依存しているが，その使用実態に関しては必ずしも明らかではない．当院はアルブミン製剤を輸血部門で管理し，使用前に厚生労働省の適応基準に準拠することを確認しながら使用してきており，使用量が少ない．当院におけるアルブミン製剤の使用状況の特徴を明らかにするため，全国の総合病院7施設と比較した． 対象·方法: 当院および協力の得られた全国の7総合病院における入院患者で，平成17年4月の1カ月間にアルブミン製剤が使用された全症例を対象とし，患者背景およびアルブミン製剤の使用状況を調査した． 結果: 症例数およびアルブミン製剤使用本数は，当院およびその他7病院で，それぞれ，56例，180本，および444例，2,512本であった．アルブミン製剤の使用理由は，当院で循環動態の維持，7病院では低アルブミン血症が最も多かった．投与前の患者血清アルブミン値は，当院2.3±0.5g/dl，7病院2.8±0.7g/dl（p＜0.000001），1回のアルブミン使用量は当院23.5±15.3g，7病院44.7±59.3g（p＜0.000001），アルブミン投与1カ月後の死亡率は，当院16.1%，7病院14.6%（P=0.77）であった．全体的に見て投与前のアルブミン値が低いほど予後が悪い傾向を認めた． 考察: 当院のアルブミン製剤の使用量は7病院に比較して大幅に少なかった．これは使用前に輸血部門で適応を評価することによると思われた．また，低アルブミン血症は予後不良の指標になるが，アルブミン製剤の投与によっても予後は改善できない可能性が考えられた．

Objective: We performed a clinical trial of immunotherapy using autologous mature dendritic cells (DC) pulsed with autologous tumor lysate, for patients with metastatic renal cell carcinoma (RCC). Methods: Patients with refractory metastatic RCC were enrolled in the study. All of them received interferon (IFN)-α treatment after nephrectomy and were followed over 3 months prior to this study. Autologous monocyte-derived immature DC were pulsed with lysate from autologous primary tumor as the antigen and keyhole limpet hemocyanin (KLH) as immunomodulator, and cultured in the presence of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and prostaglandin (PG)E2 to generate mature DC. Mature DC were injected intradermally near bilateral inguinal lymph nodes of the patients. A delayed-type hypersensitivity (DTH) test and enzyme-linked immunospot (ELISPOT) assay were performed to evaluate the immunological response. After 4 months from first injection, the clinical effect was evaluated by diagnostic imaging. Results: The treatments were well tolerated without significant toxicity by the patients who were an average of 65.7 years old and had multiple metastases in the lung and other organs. One of the two patients developed a positive DTH reaction to tumor lysate and the other patient only to KLH. The patient with a positive DTH reaction to tumor lysate had stable disease in the clinical evaluation. Conclusions: We confirmed the safety of DC therapy in this clinical trial. The DTH test revealed that the DC therapy induced immunological response to RCC. On the other hand, it was necessary to reconsider the patient selection criteria.

Invariant natural killer T (iNKT) cells are thought to regulate anti-tumor immunity. Human iNKT (i.e. Valpha24+ NKT) cells have been reported to recognize CD1d on target cells and show cytotoxicity directly on the target cells in vitro. However, the anti-tumor effect of mouse iNKT (i.e. Valpha14+ NKT) cells has been repeatedly reported to be dependent on the activity of natural killer (NK) cells via interferon-gamma, with no evidence of direct cytotoxicity. In the present study, we report that in vitro cytolysis of EL-4 mouse lymphoblastic lymphoma cells by Valpha24+ NKT cells and in vivo eradication of these cells are both dependent on the level of CD1d expression on the tumor cell surface. These observations possibly suggest that direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells.

Summary. Human Vα24+ natural killer T (NKT) cells correspond to mouse Vα14+ NKT cells, both cell types use an invariant T-cell receptor-α chain and are activated by glycolipids in a CD1d-dependent manner. Mouse Vα14+ NKT cells have been reported to have an antitumour effect in vivo. Human Vα24+ NKT cells can kill a proportion of tumour cells in a CD1d-dependent manner in vitro. We report here that many human leukaemic T-cell lines express CD1d and can be directly killed by Vα24+ NKT cells. This killing activity was enhanced in the presence of α-galactosylceramide (α-GalCer), a ligand of Vα24+ NKT cells. Moreover, primary leukaemic T cells from five of eight T-cell acute lymphoblastic leukaemia (T-ALL) patients expressed CD1d and were good targets of Vα24+ NKT cells. This cytotoxicity was increased in the presence of α-GalCer. Our results suggest that T-ALL is a good candidate for Vα24+ NKT-cell-based immuno-cell therapy.

Approximately 50% of adult patients with acute myeloid leukemia (AML) do not show clonal chromosome aberrations at diagnosis, and although this group shows an intermediate prognosis, a minority of ...

Hemophagocytic syndrome (HPS) is a cytokine-related disorder characterized by sustained high-grade fever, pancytopenia, hepatomegaly, coagulopathy and hemophagocytosis in the marrow, spleen, or lymph nodes.1 The development of HPS is related to underlying immune dysregulation and inappropriate activation of macrophages. In adults, viral, bacterial, and fungal infections and/or malignant lymphoma are associated with HPS.1

We retrospectively analyzed the association between anti-host isohemagglutinin (IH) production and the development of acute GVHD. Of 189 patients who received minor or major/minor ABO-incompatible hematopoietic SCT (HSCT) at our hospital, 36 patients (19%) showed IH production. IH was detected before the onset of acute GVHD in 10, around the same time in 8, and after the onset of acute GVHD in 17 patients. The cumulative incidence of grade II–IV acute GVHD was significantly higher in the IH+ group compared with the IH− group (P

We performed a clinical trial of immunotherapy using autologous mature dendritic cells (DC) pulsed with autologous tumor lysate, for patients with metastatic renal cell carcinoma (RCC).Patients with refractory metastatic RCC were enrolled in the study. All of them received interferon (IFN)-alpha treatment after nephrectomy and were followed over 3 months prior to this study. Autologous monocyte-derived immature DC were pulsed with lysate from autologous primary tumor as the antigen and keyhole limpet hemocyanin (KLH) as immunomodulator, and cultured in the presence of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and prostaglandin (PG)E2 to generate mature DC. Mature DC were injected intradermally near bilateral inguinal lymph nodes of the patients. A delayed-type hypersensitivity (DTH) test and enzyme-linked immunospot (ELISPOT) assay were performed to evaluate the immunological response. After 4 months from first injection, the clinical effect was evaluated by diagnostic imaging.The treatments were well tolerated without significant toxicity by the patients who were an average of 65.7 years old and had multiple metastases in the lung and other organs. One of the two patients developed a positive DTH reaction to tumor lysate and the other patient only to KLH. The patient with a positive DTH reaction to tumor lysate had stable disease in the clinical evaluation.We confirmed the safety of DC therapy in this clinical trial. The DTH test revealed that the DC therapy induced immunological response to RCC. On the other hand, it was necessary to reconsider the patient selection criteria.

We conducted a multicenter study to evaluate a new enumeration method of hematopoietic progenitor cells (HPC) using an automated hematology analyzer, the XN-series model (Sysmex Corporation, Japan). This method appeared to be very promising in a pilot study in a single institution [Tanosaki R, et al. Int J Lab Hematol 2013]. We herein discuss the final results of our study. PB or PBSC samples were taken from G-CSF- and/or chemo-mobilized subjects. The samples were divided into two tubes, one for an in-house assay where HPC were counted in each institution and one was shipped to a central laboratory (SRL, Inc., Japan) where the CD34+ cell count was examined. Firstly, we examined the frequency of invalid data. Secondly, the correlation between HPC and CD34+ cells was investigated. Thirdly, cut-off HPC values were determined to predict certain CD34+ cell counts, i.e. 20 cells/µL for PB and 1,000 cells/µL for PBSC. Finally, for the clinical application, we estimated values which predicted more than certain CD34+ cell counts with 95% probability by constructing a two-dimensional normal distribution model. This study was approved by the IRB of each institution, and informed consent was obtained from each participant before apheresis. One hundred and nine subjects (46 healthy donors and 63 patients undergoing PBSCT) were registered in 5 institutions, and 954 HPC measurements from 614 samples were performed. Thirteen (4.4%) HPC values in 297 PBSC assays were estimated to be invalid, which was identified by an asterisk automatically applied to the corresponding result. Next, the data of the first assay from each sample where both the HPC and the central laboratory-based CD34+ count were available were extracted, and 470 HPC-CD34+ pairs from 102 subjects (43 healthy donors and 59 patients) were used for further analyses. The median age was 50.5 years (range, 3-78), 60 males and 42 females, and diagnoses were malignant lymphoma 28 patients, multiple myeloma 27, and others. It was revealed that there was one mismatched result in the PBSC samples (arrow in Fig 1B; HPC 35,768/µL, CD34+ 399/µL). When this value was removed, strong correlations between the HPC and CD34+ cell counts were observed in both the PB (r=0.94) and PBSC (r=0.97) samples (Fig 1). Strong correlations were observed also at each institution. The HPC cut-off values to distinguish CD34+ cell counts below or above 20/µL in PB and 1,000/µL in PBSC samples were determined to be 25 (AUC 0.931, Youden Index 0.713) and 921 (AUC 0.946, Youden Index 0.778), respectively, by a ROC analysis (Fig 2). Finally, HPC of 56/µL was determined to be the optimal value for predicting more than 20/µL of CD34+ cells in the PB samples with 95% probability. This study confirmed that there was a strong correlation between the HPC and CD34+ cell count in a larger number of patients with different diagnosis at different institutions. For the PBSC collection, HPC ≥ 25/µL suggested an appropriate timing, and HPC 56/µL indicated a more secure threshold. HPC may also be a useful marker to rapidly estimate the quantity of CD34+ cells in the apheresis product. In cases where the HPC value is invalid, this may be overcome by serially evaluating HPC in the clinical course. In conclusion, HPC may be a good surrogate or an alternative for the CD34+ cell count. Prospective studies are warranted to verify the usefulness of HPC in clinical practice. Disclosures No relevant conflicts of interest to declare.

Invariant NK T (iNKT) cells have an invariant TCR-α chain and are activated in a CD1d-restricted manner. They are thought to regulate immune responses and play important roles in autoimmunity, allergy, infection, and tumor immunity. They also appear to influence immunity after hemopoietic stem cell transplantation. In this study, we examined the role of iNKT cells in graft-vs-host disease (GVHD) and graft rejection in a mouse model of MHC-mismatched bone marrow transplantation, using materials including α-galactosylceramide, NKT cells expanded in vitro, and Jα18 knockout mice that lack iNKT cells. We found that host-residual iNKT cells constitute effector cells which play a crucial role in reducing the severity of GVHD, and that this reduction is associated with a delayed increase in serum Th2 cytokine levels. Interestingly, we also found that host-residual iNKT cause a delay in engraftment and, under certain conditions, graft rejection. These results indicate that host-residual iNKT cells attenuate graft-vs-host immunity rather than host-vs-graft immunity.

Human Valpha24+ natural killer T (NKT) cells have an invariant T-cell receptor-alpha chain and are activated in a CD1d-restricted manner. Valpha24+ NKT cells are thought to regulate immune responses and to play important roles in the induction of allograft tolerance. In this report, we analyzed the recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation and its correlation with graft-versus-host disease (GVHD). Patients who received a dose-reduced conditioning regimen, antithymocyte globulin- or CAMPATH-1H-containing conditioning regimen were excluded. NKT cells were reconstituted within 1 month after transplantation in peripheral blood stem cell transplantation recipients, while their numbers remained low for more than 1 year in bone marrow transplantation (BMT) recipients. The number of Valpha24+ NKT cells in BMT recipients with acute GVHD was lower than that in patients without acute GVHD, and both the CD4+ and CD4- Valpha24+ NKT subsets were significantly reduced. With regard to chronic GVHD, BMT recipients with extensive GVHD had significantly fewer Valpha24+ NKT cells than other patients. Furthermore, the number of CD4+ Valpha24+ NKT cells was also significantly reduced in patients with chronic extensive GVHD. Our results raise the possibility that the number of Valpha24+ NKT cells could be related to the development of GVHD.

Human Valpha24+ natural killer T (NKT) cells correspond to mouse Valpha14+ NKT cells, both cell types use an invariant T-cell receptor-alpha chain and are activated by glycolipids in a CD1d-dependent manner. Mouse Valpha14+ NKT cells have been reported to have an antitumour effect in vivo. Human Valpha24+ NKT cells can kill a proportion of tumour cells in a CD1d-dependent manner in vitro. We report here that many human leukaemic T-cell lines express CD1d and can be directly killed by Valpha24+ NKT cells. This killing activity was enhanced in the presence of alpha-galactosylceramide (alpha-GalCer), a ligand of Valpha24+ NKT cells. Moreover, primary leukaemic T cells from five of eight T-cell acute lymphoblastic leukaemia (T-ALL) patients expressed CD1d and were good targets of Valpha24+ NKT cells. This cytotoxicity was increased in the presence of alpha-GalCer. Our results suggest that T-ALL is a good candidate for Valpha24+ NKT-cell-based immuno-cell therapy.

A compact power amplifier (PA) module using a novel hybrid low-temperature co-fired ceramic (LTCC) substrate has been developed for 5-GHz wireless LANs (WLANs). Passive components were embedded in the multi-layer LTCC substrate, which was laminated with three types of dielectrics. The hybrid substrate makes it possible to reduce component area and minimize RF coupling among the components. These improvements allowed us to integrate matching and bias networks within a compact 5 × 5 × 1.4 mm3 (= 0.04 cc) module size. The PA module was accurately designed using a passive component library developed based on EM (electromagnetic) simulation. The fabricated module exhibited power performance sufficient for 5-GHz WLANs, i.e., - linear gain of 25 dB and output 1-dB compression power (P1dB) of 21 dBm with power added efficiency (PAE) of 19%. This is the first report of a PA module with fully embedded matching networks in an LTCC substrate for 5-GHz Wireless LANs.

This study was performed to determine if there is a relationship between apparent diffusion coefficient (ADC) and cellularity of bone marrow of the posterior ilium. Four groups of various marrow cellularity underwent diffusion-weighted echo-planar imaging: 1) adults with normal hypocellularity (21 patients); 2) adults with normal normocellularity (13 patients); 3) young children with normal hypercellularity (5 patients); and 4) adults with lymphoma-related hypercellularity (3 patients). In all adults, marrow cellularity was confirmed by uni-or bilateral bone marrow biopsies. In children, the iliac marrow was presumed hypercellular because of their ages. A total of 66 ADC values of bone marrow calculated from diffusion-weighted images with b-values of 30 and 300 seconds/mm(2) was evaluated. Hypercellular marrow (normal and lymphoma-related) showed the highest mean ADC, and hypocellular the lowest ADC. Statistically significant differences were found between three groups of normal marrow: hypocellular, normocellular, and hypercellular. There is a positive correlation between ADC and cellularity of bone marrow. J. Magn. Reson. Imaging 2001;13:757-760.

Background A rapid enumeration of CD34+ cells is expected to facilitate a safe and efficient PBSC collection for a successful transplantation. We previously reported a novel enumeration method of hematopoietic progenitor cells (designated as HPC) using an automated hematology analyzer, XN-series model (Sysmex Corporation, Japan), which does not require monoclonal antibody. This method seemed very promising in a pilot study in a single institute (ASH meeting #1922, 2011). Hence, we conducted a multicenter study to confirm the results in a larger number of subjects in various situations. In this study, we used an up-graded analyzer which had been installed with a revised analytical program. This new program employs an automatically-adjusted gating to fit the individual variety, whereas the former used a fixed gating for detecting HPC. We planned to divide the study into 2 parts; the first is to confirm the outcome of the previous single-institute study and to develop a model to predict CD34+ cell counts from HPC, and the second is to validate the model. Here we show the results of the first part of the study. Patients and Method PB or PBSC samples were taken from G-CSF- and/or chemo-mobilized subjects (25 patients undergoing PBSCT and 25 healthy donors) in 5 institutes. Samples were divided into 2 tubes; one is for in-house assay in which HPC and CD34+ cells were counted in each institute within a few hours without any manipulation, and another was shipped to the central laboratory (SRL, Inc., Japan) where CD34+ cell count was examined. HPC and central labo-assayed CD34+ cell counts were compared with each other and used in a correlation analysis. HPC and in-house CD34+ cells were used in the analysis of kinetics. This study was approved by IRB, and an informed consent was obtained from each participant before apheresis. Results Five PBSC samples (2.3%) were estimated to be invalid for HPC assay because of difficulty in identifying HPC, and were excluded from the analysis. There were good correlations between HPC and CD34+ cell counts in all samples (n=211; R2 0.9576, slope 1.0678, Fig.1), in PB (n=87; R2 0.7631, slope 0.8878) and PBSC (n=124; R2 0.9501, slope 1.0689), in autologous PBSCT patients (n=116; R2 0.9759, slope 1.0596) and donors (n=95; R2 0.7914, slope 1.1381). However, HPC and CD34+ cell counts differ more than 3 times around the critical concentrations for making decision in some samples. Concerning their kinetics in PB during mobilization, which was observed in 2 donors and 9 patients, HPC showed a very similar trend as CD34+ cells, and the timing of appearance and increase in PB was almost concordant in most cases (Fig. 2). Conclusions In this multicenter study, we confirmed not only there was a good correlation between HPC and CD34+ cell counts, but also both HPC and CD34+ cell counts in PB showed a very similar trend during mobilization, as was observed in our previous single-institute study. However, a few samples were estimated as invalid for HPC assay, and there were a few more samples in which HPC and CD34+ cell counts differ significantly. Therefore, it remains to be elucidated how much HPC could be helpful in the clinical setting for determining the optimal timing of collection, for predicting the number of CD34+ cells in the apheresis product, and for determining the optimal blood volume to be apheresed. We are currently underway of the later validation part of this study to evaluate these issues. Disclosures: Tanosaki: Sysmex Corporation: Automated hematology instruments were provided on loan and the cost for CD34+ cell counts were owed to Sysmex Corporation, Kobe, Japan in this study. Other.

Abstract 1150 Poster Board I-172 Introduction ABO-incompatibility is not generally considered to be a risk factor of hematopoietic stem cell transplantation (HSCT). However, in our single-institute study of 568 patients, the incidence of acute GVHD was higher in minor-ABO incompatible HSCT than others (45.3% vs 35.2%, p=0.019). We made a hypothesis that there is a potential association between donor-derived isohemagglutinin (IH) and the outcomes of minor-ABO incompatible HSCT. Patients and method There were 1052 patients undergone HSCT between 1988 and 2009 at Komagome hospital. We analyzed 130 of 1052 patients (12.4%), who had received minor- ABO incompatible HSCT. Blood type and anti-A/B antibody titers were evaluated at least weekly in first one month after HSCT, and were repeated monthly until blood type had completely changed to the donor type. Our analysis was especially focused on the impact of IH on the outcome of minor-ABO incompatible HSCT. Result Minor ABO-incompatible HSCT were 130 cases that included AML (n=40), ALL (n=28), CML (n=27), MDS (n=15), SAA (n=9), NHL (n=7), MM (n=2) and ATL (n=2). Median age was 37 (range: 16-67) years old. Seventy-seven patients were male, 53 were female. Stem cell sources were bone marrow (n=96), peripheral blood (n=16) and umbilical cord blood (n=18). Eleven cases were HLA-matched and 9 were HLA-mismatched transplants. Anti-host IgG and IgM IH had been detected in 20 of 130 transplants undergoing minor ABO-incompatible HSCT (15.4%). Median time to IH detection was 13 days after HSCT (12-39days). There was a higher incidence of IH production in the HLA-mismatched group than in the HLA-matched group (p=0.007).There was also a higher incidence of IH production in the unrelated transplants group than in the related transplants group. (p=0.021). None of 18 patients receiving umbilical cord blood transplantation showed evidence of IH production. The incidence of grade II-IV acute GVHD was significantly higher (90% vs 60%, p Discussion and Conclusion Our study showed that IH production had association with HLA-mismatched and unrelated transplants in minor-ABO incompatible HSCT. We also showed that early detection of IH was related with the onset of grade II-IV acute GVHD. These findings indicate that strong allo-immunity induces not only severe acute GVHD caused by T lymphocytes but also antibody-production by B lymphocytes. In conclusion, we suggest that early detection of IH has impact on severity of acute GVHD. Disclosures No relevant conflicts of interest to declare.

[Background] The Notch signaling pathway plays crucial roles in a variety of cellular functions, including cell proliferation, differentiation and apoptosis. When constitutively activated, Notch signaling has been proved to be oncogenic. It has been reported that human Notch1 activating mutations are observed in approximately 50% of human T-cell acute lymphoblastic leukemia (T-ALL) patients, and that the gamma-secretase inhibitor (GSI), which is known to block Notch signaling, induces cell cycle arrest in vitro in T-ALL cell lines with Notch1 activating mutations. Notch signaling has also been suggested to play a critical role in tumor angiogenesis. The purpose of this study is to examine whether GSI is potent in vivo in eradicating established tumors made of human T-ALL cell lines in xenograft models, and to elucidate the mechanism of action. [Methods] Human T-ALL cell lines (ALL-SIL, DND-41, TALL-1, HPB-ALL), which were sensitive to GSI in vitro, were implanted subcutaneously to SCID mice. After 3 to 6 weeks, when the tumor diameter reached to approximately 1 cm, the treatment with GSI, YO01027, a derivative of LY411575, by oral administration at 0.1, 1, 10 mg/kg/day was initiated and continued for at least 30 days. The tumor volume was measured to evaluate efficacy of treatment. Tumor vascular density was assessed sequentially until tumors began to shrink, by immunohistochemical staining with a vascular endothelial cell marker, CD31. In addition, we performed in vivo angiogenesis assay using Matrigel, either with or without GSI treatment of mice for 6 days. Hemoglobin content was measured by Drabkin’s method to evaluate neovascularization in Matrigel. [Results & Discussion] We found that the treatment with GSI resulted in partial (in HPB-ALL) or complete (in ALL-SIL, DND-41, and TALL-1) regression of subcutaneous tumors. When treated with YO01027 at 10 mg/kg/day, the growth of tumors made of HPB-ALL was suppressed to less than 50% compared to the growth without treatment. Tumors made of ALL-SIL, DND-41, and TALL-1 completely regressed by the treatment with YO01027 at 1 mg/kg/day in 2 to 3 weeks. Notably, it was demonstrated that tumor vascular density was significantly decreased before the tumors regress. In tumors made of ALL-SIL and DND-41, vascular density decreased more than 40% at the treatment days 5 to 8, suggesting that the inhibition of tumor angiogenesis was one of the mechanisms how GSI acts as a strong anti-tumor agent in vivo. In vivo angiogenesis assay showed significant (about 40% compared to the control) decrease in neovascularization in Matrigel by the treatment with YO01027 at 1 to 10 mg/kg/day. [Conclusion] Our observations indicate that the Notch signaling inhibitor, GSI, is highly effective in treating T-ALL with Notch1 activating mutations in our xenograft model, the efficacy of which might be attributable to (1) direct apoptosis of T-ALL cells and (2) inhibition of tumor angiogenesis.