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Treatment options are limited for patients with multiple myeloma (MM) in Japan who have received the 3 classes of drugs known as immunomodulatory drugs, proteasome inhibitors and anti-CD38 monoclonal antibodies, and are considered to be ‘triple-class exposed’ (TCE). The objective of this study was to understand the characteristics of this patient population and the treatments they received after becoming TCE. The study evaluated healthcare resources and costs and considered whether patients had received stem cell transplant as their first treatment, where cells that may differentiate into blood cells are administered. This study analyzed a database of anonymous patients' medical records collected from April 2008 through April 2021 in Japan. A total of 216 patients were included, of whom 194 never received stem cell transplantation. Patients who received treatment after being considered TCE received that first treatment for a median of 2 months. All patients who received treatment after TCE had a median overall survival of 15.8 months meaning half the patients were still alive after receiving treatment at 15.8 months. Patients who did not have a stem cell transplant after diagnosis had a median overall survival of 17.4 months. Healthcare resources were similar between groups, except those who did not have stem cell transplantation after diagnosis needed more medications. Both groups had total healthcare costs of ¥1.3 million per patient per month. These findings support the need for new treatments and future studies to understand how new treatments would help TCE relapsed or refractory multiple myeloma patients in Japan.

Minimal residual disease (MRD)-negative status in multiple myeloma (MM) is associated with favorable outcomes. Although EuroFlow next-generation flow (NGF) is a global standard for MRD detection, its operating cost is high. Therefore, it is desirable to develop a less expensive method with equivalent sensitivity to that of EuroFlow-NGF. In this study, we compared the analytical ability of our BML 10-color multiparameter flow cytometry (MFC) to that of EuroFlow-NGF. Bone marrow samples collected from 51 patients with MM were subjected to MRD detection using BML 10-color-MFC and EuroFlow-NGF. Our antibody panel consisted of CD38 multiepitope, CD138, CD45, CD56, CD19, CD27, CD81, CD117, cytoplasmic immunoglobulin (cIg) κ, and cIgλ in a single tube. The median percentages of total plasma cells, as per 10-color-MFC and EuroFlow-NGF, were 0.2148% and 0.2200%, respectively, with a good correlation between the methods (r = 0.950). The median percentages of myeloma cells determined via 10-color-MFC and EuroFlow-NGF were 0.0012% and 0.0007%, respectively, with a strong correlation (r = 0.954). Our 10-color-MFC demonstrated high sensitivity to detect MRD; the results showed a good correlation with those obtained using EuroFlow-NGF. Therefore, our cost-effective single-tube MFC (approximately 100 USD/sample) is a promising alternative method for the detection of MRD in patients with MM.

Objectives Bortezomib with lenalidomide and dexamethasone (VRd) is a standard induction regimen for transplant-eligible patients with newly diagnosed multiple myeloma (NDMM). However, some patients discontinue VRd because of severe adverse events, despite its high efficacy. We aimed to study the efficacy of modified dose of VRd (VRd lite) in transplant-eligible patients with NDMM. Methods Forty-eight transplant-eligible patients with NDMM were included. VRd lite was administered every 4 weeks. Bortezomib 1.3 mg/m2 was administered subcutaneously on days 1, 8, 15 and 22, and dexamethasone 20 mg was administered orally on the day of and the day after bortezomib administration. Lenalidomide was omitted on days 1, 8 and 15, which are the days of bortezomib administration. Results The overall response rate (ORR) after four cycles of VRd lite was 83%, including a complete response of 25%. Thirty-eight among the 45 patients who completed at least four cycles of VRd lite received autologous stem cell transplantation (ASCT). The ORR and very good partial response or better were upgraded to 100% and 74%, respectively, following ASCT. Conclusion Our strategy consisting of VRd lite followed by ASCT is, thus, a highly effective and well-tolerated regimen resulting in durable responses in patients with NDMM.

The combination of bortezomib, lenalidomide, and dexamethasone (VRD) is used as induction treatment in multiple myeloma; however, the optimum schedule for this regimen remains controversial. In this retrospective study, we compared the efficacy and tolerability of twice-weekly VRD (twVRD) and modified VRD-lite in transplant-eligible myeloma patients. Fifty-five patients (median age 61 years) were included; 22 received twVRD (bortezomib [1.3 mg/m2 on days 1, 4, 8, and 11] and lenalidomide [25 mg/body on days 1–14] over 21-day cycles) and 33 received modified VRD-lite (bortezomib [1.3 mg/m2 on days 1, 8, 15, and 22) and lenalidomide [15 mg/body on days 2–7, 9–14, 16–21] over 28-day cycles). Overall response, very good partial response, and complete response rates after VRD were 96.4%, 45.5%, and 20.0%, respectively (median follow-up period, 17.7 months). The 1-year progression-free survival (PFS) and overall survival rates were 95.8% and 98.2%, respectively. The response rate and PFS were similar between the groups, regardless of cytogenetic risk and age. The incidence of peripheral neuropathy ≥ grade 2 and thrombocytopenia ≥ grade 3 was higher in the twVRD group (27.2% vs. 0.0%, P = 0.003 and 27.2% vs. 0.0%, P = 0.003). In conclusion, modified VRD-lite had similar efficacy with, but better tolerability than, twVRD in transplant-eligible patients.

Plerixafor is increasingly used in combination with granulocyte-colony-stimulating factor (G-CSF) for peripheral blood stem cell collection. Although it is an expensive drug, its cost-benefit performance is not well investigated. Thus, we analyzed its cost-effectiveness in our hospital. A retrospective observational analysis was performed in patients who underwent stem cell collection between December 2013 and November 2018. A total of 203 patients were investigated and classified into three groups according to their pre-mobilization regimen: G-CSF alone, G-CSF and cyclophosphamide (G+CY), and G-CSF and plerixafor (G+plerixafor). The cost-effectiveness of apheresis of the collected cluster of differentiation (CD) 34+ cells was assessed based on two viewpoints: cost of drugs and cost of equipment. Due to the high cost of plerixafor, the cost of apheresis was higher in patients who received G+plerixafor. However, the difference narrowed when we calculated the cost to collect 2.0×10

Therapeutic strategies for multiple myeloma have dramatically changed in the last two decades, especially after the introduction of proteasome inhibitors. The first-in-class proteasome inhibitor, bortezomib, was approved by the US Food and Drug Administration in 2003. Since then, it has been a backbone therapy for not only relapsed or refractory myeloma patients but also newly diagnosed multiple myeloma patients. Second-generation proteasome inhibitors, such as carfilzomib and ixazomib, have been approved, and three proteasome inhibitors were incorporated into several regimens with other cytotoxic agents, such as alkylating agents, immunomodulatory drugs and monoclonal antibodies. Because each proteasome inhibitor shows different properties with respect to adverse events, understanding and managing each adverse event of proteasome inhibitors are necessary for the continuation of therapy with minimal interruption of treatment. This review summarizes the recent advances in proteasome inhibitors used in the treatment of multiple myeloma.

Although elotuzumab (ELO) is associated with improved outcomes in patients with relapsed/refractory multiple myeloma (MM), no data are available for the usage of ELO following allogeneic stem cell transplantation (allo-SCT). Here, we report two cases of relapsed MM treated with ELO in combination with lenalidomide (LEN) and dexamethasone (ELd) following allo-SCT. Case 1 had been treated with 11 lines of therapy followed by cord blood transplantation resulting in partial response. ELd was introduced 140 days post-transplantation and continued for eight cycles until disease progression. No worsening in graft-versus-host disease (GvHD) was observed under ELd treatment. Case 2 had received unrelated bone marrow transplantation due to primary refractory disease after undergoing six regimens. Carfilzomib-based maintenance therapy had to be discontinued owing to severe myelosuppression. Subsequently, ELd treatment was initiated 544 days following the allo-SCT, which led to an improvement in serum paraprotein level and amelioration in GvHD. In both cases, immunosuppressants were tapered off. Several studies have shown exacerbation of GvHD under LEN monotherapy following allo-SCT. However, an ELd regimen may be one of the safer options for treating post-allo-SCT relapse.

Because multiple myeloma is rare in young people, there are fewer reports on the same. Thus, its clinical aspects and prognosis remain unelucidated. We retrospectively evaluated 30 patients with multiple myeloma aged ≤ 45 years at diagnosis. We divided them into three groups based on their cytogenetic risks: standard risk (SR), high risk (HR), and unknown risk. The frequency of HR patients was 36.6%, the highest of the three groups, unlike the previous report. The median progression-free survival (PFS) was 35 months (SR vs. HR, 46 vs. 29 months), and the median overall survival (OS) was not reached (NR) (SR vs. HR, NR vs. 82 months). The OS was significantly worse, and the PFS also appeared inferior in HR patients. The International Staging System score was not associated with OS. Thus, young patients with myeloma appeared to have a higher frequency of HR features, suggesting that instead of age, the cytogenetic risk was a significant prognostic factor.

Failure of autologous peripheral blood stem cell collection (PBSCH) can affect the treatment modality for patients with hematological malignancies. The clinical efficacy of plerixafor in PBSCH was analyzed in our institution. The medical records of 61 patients were retrospectively reviewed. The use of plerixafor was determined according to the CD34

The BEAM regimen consisting of carmustine (BCNU), etoposide, cytarabine, and melphalan (MEL) is widely used before autologous hematopoietic stem cell transplantation (auto-HSCT) for lymphoma. However, intravenous BCNU is not available in Japan, and therefore, ranimustine (MCNU) has been used instead of BCNU (the MEAM regimen). We retrospectively analyzed the outcome of 79 adult patients who underwent auto-HSCT for lymphoma using this regimen in two centers, with 1- and 2-day dosing of MEL, respectively. Three-year overall survival (OS) and progression-free survival (PFS) probabilities were 77.3 and 56.5 % in the entire population and 71.7 and 58.0 % in patients with diffuse large B cell lymphoma. These outcomes were at least equivalent to those with the BEAM regimen. There was no regimen-related pulmonary toxicity. In a multivariate analysis, older age was the only factor that was significantly associated with for OS. In a comparison of the two MEL dosing schedules, while there was no significant differences in either OS or PFS, diarrhea was observed more frequently with 1-day dosing of MEL. In conclusion, the MEAM regimen appeared to be a promising conditioning regimen in auto-HSCT for lymphoma. A large prospective study is warranted to confirm the current findings.

Anti-thymocyte globulin (ATG) is a key drug in immunosuppressive therapy for patients with aplastic anemia. The mainstay of ATG therapy had been a horse ATG (hATG) formulation, Lymphoglobulin or ATGAM, but Lymphoglobulin was recently discontinued, and Thymoglobulin, a rabbit ATG (rATG) formulation, is currently used as the first-line drug in many countries, including Japan. However, a recent randomized clinical trial reported significantly unfavorable outcomes associated with the use of rATG regimens. We retrospectively analyzed clinical outcomes of adult patients with moderate to severe aplastic anemia who were treated with 3.5 mg/kg of Thymoglobulin (n = 22) or 15 mg/kg of Lymphoglobulin (n = 25) in our facility. The estimated overall response rates in the rATG and hATG groups were 64.6 versus 56.0 % at 6 months, and 76.4 versus 69.2 % at 12 months, respectively; and there was no statistical difference between the two groups (P = 0.32). Overall survival at 24 months was not significantly different: rATG 89.8 % versus hATG 96.0 % (P = 0.39). Early phase infection was observed in 37.5 % of cases in the rATG and 14.8 % in the hATG group, but the frequency was not statistically different (P = 0.107). Our data indicate that Thymoglobulin at a dose of 3.5 mg/kg is a viable alternative when hATG is not available.

We evaluated 121 patients with follicular lymphoma (FL) and analyzed the association between the soluble interleukin-2 receptor (sIL-2R) level at diagnosis and the cumulative incidence of transformation. By a receiver-operating characteristic analysis, we determined a cutoff value of sIL-2R for transformation at 4360 U/mL to classify patients into two groups. Patients in the high sIL-2R group showed a shorter progression-free survival (PFS) and shorter disease-specific survival (DSS) (p

Red blood cell distribution width (RDW) has been used for the differential diagnosis of anemia, but high RDW may also be associated with several human disorders. We evaluated the prognostic relevance of RDW in patients with light-chain (AL) amyloidosis. We retrospectively analyzed all patients with AL amyloidosis who were newly diagnosed at the Japanese Red Cross Medical Center between December 2011 and June 2018. RDW was evaluated in 94 patients; 48% (n = 45) of patients had a high RDW (≥ 13.8%) and 52% (n = 49) had a low RDW (

A 52-year-old man with chronic myelogenous leukemia (CML) received dasatinib after the failure of imatinib and nilotinib therapy. Two years after the initiation of dasatinib, he developed shortness of breath that gradually worsened. Chest X-ray and computed tomography scan showed pulmonary infiltrative shadows and bilateral pleural effusion. We performed a transbronchial lung biopsy and diagnosed organizing pneumonia caused by dasatinib treatment. Corticosteroid therapy was initiated after the discontinuation of dasatinib and all his symptoms were significantly improved. Because of the exacerbation of CML, the patient was treated with imatinib and then nilotinib; however, these drugs failed to decrease the leukemic cells. Re - administration of dasatinib in combination with corticosteroid therapy successfully controlled CML without recurrence of organizing pneumonia.

Twenty-nine patients with multiple myeloma were treated with carfilzomib, lenalidomide, and dexamethasone (KLd) therapy. A response better than partial response (PR) was observed in 72.4% patients with relapsed and/or refractory myeloma. Although 13.8% patients developed hypertension, none of them discontinued therapy as they could be managed by appropriate medication. A patient who had an elevated level of BNP prior to initiating KLd therapy developed heart failure. Results from this study demonstrate that KLd therapy is efficacious for treating patients with multiple myeloma; however, they should be carefully monitored for cardiotoxicity.

This retrospective analysis compared the efficacy of intensive induction therapy consisting of daunorubicin and cytarabine (DNR-AraC) to that of less-intensive therapy including low-dose cytarabine, aclarubicin and granulocyte colony-stimulating factor priming (CAG). Patients aged 60 years or older who were newly diagnosed as acute myeloid leukemia (AML) were analyzed. Sixty-four and 48 patients were treated with DNR-AraC and CAG, respectively. The complete remission rates, 3-year overall survival and event-free survival in the DNR-AraC group were significantly superior to those in the CAG group (65.6% vs. 29.2%, p

The early clearance of blast cells in peripheral blood (PB) during induction chemotherapy can predict the clinical outcome in acute leukemia. We retrospectively analyzed the kinetics of white blood cell (WBC) count, blast cell percentage (BCP), and blast cell count (BCC) in PB in 78 patients with de novo acute myeloid leukemia who underwent a uniform induction chemotherapy between December 2001 and December 2015 at Jichi Medical University. By a repeated-measures analysis of variance, the interaction of the decline in BCP with the achievement of complete remission (CR) was stronger than those of the decline in WBC or BCC. A receiver operating characteristic curve analysis for the achievement of CR showed that the areas under the curve for the decline in WBC, BCP, and BCC were 0.592, 0.703, and 0.634, respectively, and a decline in BCP of 9.25%/day within 4 or 5 days from induction chemotherapy was the optimal cutoff value. A multivariate analysis showed that a rapid decline in BCP (≥9.25%/day) was a significant predictive factor for CR, independent of the cytogenetic risk (p = 0.0096). A rapid decline in BCP during the first 5 days of induction chemotherapy may be a good predictor of CR. Copyright © 2015 John Wiley & Sons, Ltd.

IgE multiple myeloma (MM) is a rare subtype of MM characterized by an aggressive and poor prognosis. Although novel agents have improved the prognosis of MM, there are few case reports of IgE MM treated with these agents. A 53-year-old male patient presented with pain in the right rib and was diagnosed with IgE-κ MM. He was treated with multidrug chemotherapy, including bortezomib and lenalidomide, and underwent autologous stem-cell transplantation (ASCT). Finally, he achieved a complete response after the initiation of pomalidomide. In previous reports, majority of patients with refractory IgE MM treated with novel agents had a poor prognosis. In contrast, patients who were treated with novel agents from the beginning and underwent ASCT had a long-term survival. Overall, the use of novel agents as the first-line therapy is expected to improve IgE MM prognosis.

Background: Achievement of MRD-negative status in MM patients has been demonstrated to be associated with better outcome. EuroFlow MM-MRD has been a global standard for MRD detection by MFC. In Japan, EuroFlow is limited to research use only, because the dynamics of the population have raised concerns about increasing health care expenditure in Japan. Therefore, it is desirable to develop an inexpensive FCM method with equivalent sensitivity to EuroFlow for detecting MRD. Aims: In this study, we analyzed the number and percentage of total plasma cells (PC), abnormal PC with EuroFlow MM-MRD and 10-color-MFC in 50 MM patients. Patients and Methods: Bone marrow samples collected from 50 MM patients were subjected to MRD detection by EuroFlow MM-MRD and 10-color-MFC simultaneously. EuroFlow MM-MRD was performed according to EuroFlow standard operating procedure. 10-color MFC was performed at BML corporation. Briefly, CD38 multiepitope FITC, CD138 V450, CD45 V500-C, CD56 PE, CD19 APC-H7, CD27 APC R700, CD81 BV605, CD117 PE-Cy7, cIgk APC and cIgl PerCP-Cy5.5 of monoclonal antibody were used according to the ICCS(International Clinical Cytometry Society) guidelines. Data were analyzed with FACSuite ver 1.2.1(Becton Dickinson) using a FACSLylic(Becton Dickinson). Wilcoxon signed-rank test was used for the comparison of paired variables. Results: The representative example of 10-color-MFC were shown in figure 1. We first compared the percentage of normal plasma cells in bone marrow. The percentage of normal plasma cells analyzed with 10-color-MFC and EuroFlow were 0.287% (range 0.001-1.0322%) and 0.257%(range0.0002-1.37%), respectively. The good correlation between them was observed (r=0.509). The percentage of myeloma cells determined with 10-color-MFC and EuroFlow were 0.0705%(range 0-0.9086%) and 0.0940% (range 0-1.3000%), respectively. Quantification of abnormal plasma cells were highly similar between 2 tests (r=0.977, figure 2). To compare the percentage of abnormal plasma cells analyzed with 10-color-MFC and EuroFlow, p-value determined with Wilcoxon signed-rank test was 0.0069 in 50 sample. However, when total bone marrow cells were 3 million cells, limit of detection (LOD) and limit of quantification (LOQ) in 10-color MFC were defined as 0.001% and 0.0017%, respectively. The correlation in samples with less than 3 million of bone marrow cells was not good (p=0.424). Only when 3 million or higher number of bone marrow cells were analyzed, better correlation is thought to be observed (p=0.0001). High concordant analytical results were observed with an overall qualitative concordance of 95%. Conclusion: Our 10-color-MFC demonstrated high sensitivity to detect MRD with good correlation and also inexpensive (about 100 dollars as of July 2019). However, 3 million or higher number of bone marrow cells is inevitable to demonstrate good correlation. In the country where high medical expenditure is not acceptable, our inexpensive MFC is considered as good alternative methods to detect MRD in MM patients. Disclosures Tsukada: Takeda Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Sanofi: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Fujimoto Pharmaceutical: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Asahi Kasei Pharma Corporation: Honoraria; MOCHIDA PHARMACEUTICAL CO., LTD.: Honoraria. Suzuki:Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

CD25 has been reported to be highly expressed in leukemia stem cells and correlated with adverse outcomes in young patients with acute myeloid leukemia (AML). However, the significance of CD25 expression in elderly patients with AML has not yet been investigated.We retrospectively analyzed 154 newly diagnosed AML patients aged 60 years or over by flow cytometry.CD25-positive AML was characterized by high white blood cell counts, secondary AML, rare favorable karyotypes, and positivity for CD34 and CD7 antigens, compared with CD25-negative AML. CD25 positivity was significantly correlated with an inferior complete remission (CR), event-free survival (EFS), and overall survival. Multivariate analysis showed CD25 positivity to be a significant prognostic predictor of CR and EFS. A regimen of low-dose cytarabine and aclarubicin combined with granulocyte-colony-stimulating factor (CAG) led to higher CR rates in the CD25-positive AML patients than intensive chemotherapies. CD25 expression was increased at relapse and in the development of leukemic status from myelodysplastic syndrome or myeloproliferative neoplasm.An effective treatment strategy for elderly patients with CD25-positive AML has not been established. Further studies are needed to evaluate the effect of a CAG regimen and allogenic stem cell transplantation in patients.CD25 is an independent prognostic factor in elderly AML patients. Alternative therapies for CD25-positive elderly AML patients are needed.

A 51-year-old man was admitted due to a severe bleeding tendency. After he was diagnosed with immune thrombocytopenia (ITP), several therapies, including steroids, steroid pulse, vincristine and rituximab, were administered; however, the patient's bleeding symptoms were not sufficiently controllable with these treatments. Subsequently, a diffuse alveolar hemorrhage was observed. Treatment with a thrombopoietin receptor agonist, romiplostim, was initiated to prevent lethal hemorrhaging, although the efficacy of thrombopoietic receptor agonists in such emergency situations has not been elucidated. The initiation of romiplostim achieved prompt remission in platelets. This case suggests that combination therapy with romiplostim, rituximab and vincristine is effective in cases of newly diagnosed severe therapy-resistant ITP.

The prognosis of patients with systemic lymphoma with central nervous system (CNS) involvement is very poor and there is no established standard therapy. We retrospectively analyzed 18 patients (4 untreated and 14 relapsed) with systemic lymphoma with CNS involvement who received methotrexate and cytarabine-based multiagent chemotherapy (modified Bonn protocol). Complete and partial responses were achieved in 56 and 22% of the patients, respectively. The 1-year overall survival (OS) and progression-free survival (PFS) was 81.0 and 39.2%, respectively. Patients with parenchymal involvement showed a better 1-year PFS than those with either leptomeningeal involvement or both. In a multivariate analysis, poor performance status (PS) was the only independent prognostic factor for the 1-year OS and PFS (HR 10.8, 95% CI 1.09-108, p = 0.042; HR 20.8, 95% CI 2.39-181, p = 0.006, respectively). Grade 4 neutropenia and thrombocytopenia occurred in 17 patients each (94%), but there were no grade 4 nonhematopoietic adverse events. The modified Bonn protocol resulted in relatively favorable response and survival, and provided clinical benefits to patients with good PS, in particular. This study demonstrated that the modified Bonn protocol could be a feasible and encouraging treatment approach for lymphoma with CNS and systemic involvement.

The development of acute myeloid leukemia (AML) in patients with untreated chronic lymphocytic leukemia (CLL) is rare. We experienced a 65-year-old man who developed AML with aberrant CD7 expression and monoallelic CEBPA mutation during watchful waiting for CLL. He failed to achieve complete response (CR) by standard induction therapy for AML. We retrospectively reviewed 27 patients who developed AML with untreated CLL published between 1973 and 2016. The median age at diagnosis of AML was 68 years, and the median duration between the diagnoses of AML and CLL was 4.2 years. Diagnosis of AML and CLL was made simultaneously in 16 patients. The CR rate of AML was 42.9%, and the median survival was only 1.5 months after the diagnosis of AML. Patients who achieved CR tended to survive longer than those who did not. Our results demonstrated that the development of AML in patients with untreated CLL was associated with a poor response to chemotherapy and an extremely poor prognosis.

[Background] Thecombination therapy consisting of bortezomib, lenalidomide and dexamethasone (RVD) for newly diagnosed multiple myeloma has been one of the standard induction therapies in recent years. However, 9-23 % of patients discontinued treatments because of severe adverse events in the previous reports (SWOG S077, IFM2009). Thus, it has not been clarified ifthe dosing schedule of RVD is appropriate. Here, we investigated the efficacy and safety of modified RVD-lite for 45 transplant eligible patients with newly diagnosed multiple myeloma (NDMM). [Patients and methods] We retrospectively analyzed 45 transplant eligible patients with NDMM who received modified RVD-lite for induction therapy between February 2016 and March 2018. The median age was 58 years old (range 36~66), and 6 (13.3%) patients were AL amyloidosisassociated with multiple myeloma. Patients received bortezomib 1.3 mg/m2once weekly subcutaneous (SC) on days 1, 8, 15, 22, lenalidomide 15 mg/day on days 2-7, 9-14, 16-21 and dexamethasone 40mg on days 1, 8, 15, 22. The Revised International Staging System (R-ISS) wereI in 13 (28.9%), II in 30 (66.7%) and III in 2 (4.4%). High-risk cytogenetics, defined as the presence of deletion 17, t(4;14) and t(14;16) by FISH analysis, were identified in 5 (11.1%) patients. After 4 cycles of modifiedVRd-lite, we evaluated the efficacy and adverse events. [Results] The overall response rate (ORR) after four 28-day cycles of modifiedRVD-lite was obtained in 41 (91.1%), including sCR in 6 (13.3%) and CR in 5 (11.1%). SD and PD were observed in 2 patients (4.4%) and 1 patient (2.2%), respectively. One patient was not evaluated efficacy, because a patient changed modifiedRVD-lite to ixazomib, lenalidomideand dexamethasone therapy for grade 3 peripheral neuropathy. Thirty-eight of 45 patients (84.4%) received autologous stem cell transplantation (ASCT) after at least 4 courses of modifiedVRd-lite. The median number of CD34+cells/kg collected was 4.83 x 106(range, 1.1-11.9). All patients received melphalan at doses of 200 mg/m2. In these patients, response after ASCT were sCR in 15(41.7%), CR in 2 (5.6%), VGPR in 8 (22.2%) and PR in 8 (22.2%). Three of seven patients who did not received ASCT will receive ASCT ina few months. Among other 4 patients who did not receive ASCT, 2 patients chose other therapies without ASCT, and 2 patients could not receiveASCT because they showed no improvement in cardiac AL amyloidosis. Grade 3 or higher adverse events (AEs) were observed in 17 (37.8%). Most frequent grade 3 or higher AE was neutropenia (grade 3:17.8%, grade 4:6.7%). Only one patient (2.2 %) discontinued modifiedRVD-lite because of grade 3 peripheral neuropathy. [Conclusions] The ORR after 4 cycles ofmodifiedRVD-lite was high (91.1%). These results might be related to low rate of discontinuation of treatment. Also, the ORR after ASCT was comparable to the results previously published (N Engl J Med .2017 Apr 6:376(14):1311). AEs in modified RVD-lite were feasible and manageable in most patients. Our results suggest that modifiedRVD-lite is very feasible and effective treatment for patients with transplant eligible NDMM. Disclosures Suzuki: Sanofi Aventis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Ono: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; SRL.Inc: Employment.

We report a case of a 29-year-old male patient with a generalized adenovirus (AdV) infection after cord blood transplantation (CBT) for acute myelocytic leukemia with maturation at 2nd complete remission. Before engraftment, hemorrhagic cystitis was caused by AdV, which resulted in hydronephrosis, renal failure, and adenoviremia on day 34. Forced diuresis, hemodialysis, withdrawal of cyclosporin A, and administration of gamma-globulin or vidarabine were not effective and the patient died of pulmonary alveolar hemorrhage on day 67. At autopsy, old inflammatory change only was observed in the bladder section. In the lungs and kidneys, granular deposits in the nucleus and a high copy number of AdV-DNA were observed. Molecular diagnosis using PCR-restriction fragment length polymorphism analysis demonstrated that AdV with the serotype 14 caused the cystitis. However, retrospective genome typing using PCR sequencing revealed the infection of AdV serotype 35 in the kidneys, lungs, and serum. The present case suggested that Adv infection could not be always caused by a single AdV serotype, and suggested that multiple serotype infection was very difficult to treat. It is desired that a consensus regarding the treatment of AdV infections is established.

The combination of mitoxantrone (MIT), etoposide (ETP), and cytarabine (Ara-C) (MEC) is a frequently used salvage therapy for acute leukemia, but has been associated with severe myelosuppression. Therefore, we investigated the miniMEC regimen with reduced doses of AraC and MIT. Thirteen ALL and 44 AML patients, all relapsed or refractory, received miniMEC, which consisted of MIT at 8 mg/m2 for 3 d, ETP at 100 mg/m2 for 5 d, and Ara-C at 100 mg/m2 infused over 24 h for 7 d. CR + CRi was achieved in eight ALL patients (61.5%) and 16 AML patients (36.4%). Median duration of neutropenia was 30 d (range, 1–50). Thirty-one patients (54.4%) subsequently received allogeneic stem cell transplantation (SCT), and overall survival was significantly improved in this group (median OS 161 versus 481 d, p = 0.006). We concluded that miniMEC is a safe and effective bridging therapy to SCT.

Cyclosporin A (CsA) has been used most widely as an immunosuppressive agent for preventing graft-versus-host disease (GVHD). To explore the risk factors including CsA blood levels for grades II-IV acute GVHD, we retrospectively analyzed the data of patients who underwent allogeneic hematopoietic stem cell transplantation in our hospital between March 1989 and July 2001. Seventy-three patients (47 males and 26 females) received CsA and short-term methotrexate for GVHD prophylaxis. CsA 1.5 mg/kg was administered as a 3-h infusion twice daily from day 1 until the patient recovered from the toxic gastrointestinal complication. Methotrexate was given at a dose of 15 mg/m(2) on day 1 and 10 mg/m(2) on days 3, 6 and 11. Grades II-IV acute GVHD occurred in 18 patients (24.7%). Multivariate Cox regression analysis revealed that higher C(5) (the whole-blood CsA concentration at 5 h after the start of infusion) before the onset of acute GVHD reduced the onset of grades II-IV acute GVHD with a hazard ratio of 0.994 (95% confidence interval 0.989-0.999) for every increase of 1 ng/ml. Our data indicate that inadequate exposures of CsA can be a vital risk for developing acute GVHD. From our results, we consider that precise monitoring of CsA concentrations and adjustment of CsA dose using the concentration may be effective to prevent the onset of severe acute GVHD. To confirm this finding, further prospective study will be needed.

The early clearance of blast cells in peripheral blood (PB) during induction chemotherapy can predict the clinical outcome in acute leukemia. We retrospectively analyzed the kinetics of white blood cell (WBC) count, blast cell percentage (BCP), and blast cell count (BCC) in PB in 78 patients with de novo acute myeloid leukemia who underwent a uniform induction chemotherapy between December 2001 and December 2015 at Jichi Medical University. By a repeated-measures analysis of variance, the interaction of the decline in BCP with the achievement of complete remission (CR) was stronger than those of the decline in WBC or BCC. A receiver operating characteristic curve analysis for the achievement of CR showed that the areas under the curve for the decline in WBC, BCP, and BCC were 0.592, 0.703, and 0.634, respectively, and a decline in BCP of 9.25%/day within 4 or 5 days from induction chemotherapy was the optimal cutoff value. A multivariate analysis showed that a rapid decline in BCP (≥9.25%/day) was a significant predictive factor for CR, independent of the cytogenetic risk (p = 0.0096). A rapid decline in BCP during the first 5 days of induction chemotherapy may be a good predictor of CR. Copyright © 2015 John WileySons, Ltd.

Bcl11b −/− mice show developmental arrest at the CD44−CD25+ double-negative 3 (DN3) or immature CD8+single-positive stage of αβ T cell. We have performed detailed analysis of sorted subsets of Bcl11b−/− thymocytes, DN3 and CD44−CD25− double-negative 4 (DN4) cells. Surface expression of TCRβ proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRβ proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRβ or TCRαβ expression on the cell surface failed to promote transition from the DN3 to CD4+CD8+ double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b−/− DN3 thymocytes showed normal DNA rearrangements between Dβ and Jβ segments but limited DNA rearrangements between Vβ and DJβ without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b−/− DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vβ and Dβ gene locus. Analysis of Bcl11b−/− DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-xL and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRαβ in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.

We report here three cases of peripheral T-cell lymphoma unspecified (PTCL-US), which presented with bone marrow infiltration and hepatosplenomegaly and were successfully treated with high-dose chemotherapy (HDCT) and autologous peripheral blood stem cell transplantation (auto-PBSCT). The patients were all characterized by cytokine-induced symptoms such as fever, anasarca, cytopenia, poor general condition, and disseminated intravascular coagulation syndrome. Laboratory data showed extremely high levels of soluble interleukin-2 receptor, beta(2)-microglobulin, and ferritin. All three patients were negative for anti-adult T-cell leukemia antibody. In one patient, hemophagocytosis was revealed by a histological examination of the bone marrow. The International Prognostic Index was high for all three patients, and they all achieved complete remission after the intensive chemotherapy for remission induction. During complete remission, they were treated with HDCT [modified interleukin-converting enzyme regimen] followed by auto-PBSCT. The recovery of hematopoiesis after auto-PBSCT was prompt and sustained engraftment was obtained. No serious adverse effects other than myelosuppression were noted. One patient died due to cerebrovascular disease without relapse 18 months after auto-PBSCT. The other two patients are still alive and have not suffered from relapse. Our observations suggest that auto-PBSCT following HDCT may be an effective and safe therapeutic modality for high-risk PTCL-US patients characterized by hepatosplenomegaly and cytokine-induced syndrome.

A patient with extramedullary crisis from chronic myelogenous leukemia after allogeneic bone marrow transplantation is reported. A pathological neck lymph node observed after transplantation revealed pre-T lymphoblastic phenotype, and the fluorescence in situ hybridization (FISH) analysis showed recipient type sex chromosomes and bcr/abl fusion gene. The cells represented an additional translocation, t (6;8)(q25;q22). No rearrangements of the T-cell receptor (TCR) β, γ or δ chain genes were observed. The absence of TCR rearrangement indicated the clonogenic involvement of pluripotent hematopoietic stem cells by Philadelphia chromosome. Bone marrow specimens at that time showed donor type sex chromosomes and no bcr/abl-positive cells by FISH.

Bone marrow mononuclear cells from 93 patients with hematological malignancies after allogeneic hematopoietic stem cell transplantation (AHSCT) were analyzed using flow cytometry (FCM). The disease was acute myeloblastic leukemia (50 patients), acute lymphoblastic leukemia, and others. Conditioning was myeloablative (80 patients) or reduced intensity. The stem cell source was bone marrow (75 patients), peripheral blood stem cells, or cord blood. After AHSCT, granulocyte colony-stimulating factor was given to all patients. All patients showed engraftment of the donor cells. FCM was conducted on a median of 22 days after AHSCT. The gate was set around a granulocytic region consisting of immature granulocytes. The positivity rates of CD13, CD14, CD15, CD33, CD34, CD56, and HLA-DR in the cells were 59.9 ± 27.4%, 5.8 ± 8.8%, 98.3 ± 1.9%, 92.3 ± 12.4%, 2.6 ± 5.8%, 24.3 ± 16.7%, and 9.1 ± 6.6%, respectively. The greatest value of CD56 positivity was 73.1%. On the basis of CD56 expression, cases of more than 24% CD56 positivity were assigned to the CD56-high group (39 patients), while the rest were assigned to the CD56-low/negative group. There were no significant differences between the two groups in terms of disease status, sex, donor, hematopoietic stem cells, days of FCM analysis, or peripheral blood cell counts around the days of performing FCM. These results indicate that CD56 can be expressed in normal immature granulocytes at a variety of expression levels in regenerative bone marrow. Attention should be paid when evaluating aberrant antigen expression of CD56 in granulocytes.

To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions. The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery. We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model. CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone. Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding.

Myelodysplastic syndrome (MDS) is relatively common in the elderly, and aging of populations is progressing in developed nations, notably so in Japan. The major age group in Japan and Sado Island are distributed between 30 and 60 and between 50 and 80, respectively. The aim of this study was to analyze the features of MDS in the population of Sado Island to anticipate the characteristics of the disease in the near future. One-hundred and fifty-three patients (71 male, 82 female, 19–94 years old, median 73 years old) with de novo MDS between 1985 and 2005 were retrospectively evaluated. All patients were reclassified according to WHO-2001 criteria. The predictive power of the international prognostic scoring system and the WHO classification-based prognostic scoring system were evaluated. The major causes of death were leukemic transformation (38%) in refractory anemia with an excess of blasts and infection (48%) for total MDS. Age was another independent prognostic factor. Elderly patients exhibited a significantly poorer prognosis mainly due to infections such as pneumonia. Although novel remedies for MDS and hyperferremia have recently been developed, prevention of infection remains important in MDS, particularly for older patients.

Pharmacological study is predictably effective in establishing an optimal monitoring strategy for the usage of cyclosporine A (CsA) to prevent graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation recipients. Pharmacokinetic profiling of 33 recipients administered CsA twice daily by 3-h intravenous infusion revealed that levels peaked 2-3 h after the start of infusion, and an exponential decline of CsA concentrations after the termination of infusion was observed. The correlation between the area under the curve (AUC(0-12)) and CsA concentration at various time points after infusion revealed that C (2) and C (3) correlated best with AUC(0-12) (r (2) = 0.725), while the trough concentration correlated poorly. Ex vivo T cell stimulation followed by intracellular cytokine detection with flow cytometry revealed that the capacity of T cells to produce cytokines upon stimulation was inversely proportional to the CsA concentration, and reached a minimum at about 700 ng/mL with a marginal decrease above this concentration. Extrapolation using the regression equations of this study and the data from our retrospective study leads to the assumption that the dose adjustment of CsA based on maintaining the C (3) concentration above 800 ng/mL may effectively prevent acute GVHD. To confirm this assumption, a prospective clinical study is required.

A 26-year-old woman with acute lymphoblastic leukemia (ALL) relapsed three times after HLA-matched related bone marrow transplantation. Initially, ALL relapsed in the central nervous system (CNS) 1 year after transplantation. Then, ALL relapsed as a single bone tumor involving the CNS and pelvis 4 years after transplantation. Finally, multiple bone tumors in the pelvis and lumbar bones were found as well as spread to the bone marrow 5 years after transplantation. Bone marrow aspiration also showed ALL relapse. Flow cytometry analyses detected CD20-positive cells in the bone tumor. Though the initial bone tumor was resistant to hyper CVAD, radiation was effective and this patient achieved complete remission. At that time, the total radiation dose had already reached the upper limit. After the third relapse, bone marrow achieved complete remission with the administration of pirarubicin, vincristine, prednisolone, and L-asparaginase (arranged DVP-L), though this combination chemotherapy itself was not effective in multiple bone tumors. Thereafter, arranged DVP-L plus rituximab was administered, which resulted in significant tumor reduction. Biweekly rituximab administration as maintenance therapy has completely prevented the regrowth of bone tumors. Rituximab for relapsed CD20-positive ALL patients after stem cell transplantation could be beneficial.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal chromosomal translocation involving the gene for retinoic acid receptor alpha(RAR). Most APL patients have a t(15;17) translocation that generates the PML-RAR fusion gene, and such patients respond well to treatment with all-trans retinoic acid (ATRA). Some APL cases also involve rearrangements that fuse RAR to partner genes other than PML, including nucleophosmin (NPM), promyelocytic leukemia zinc finger (PLZF), nuclear mitotic apparatus (NUMA), and Stat5b, but the clinical characteristics of APL without PML-RAR have not been fully clarified. We describe a 64-year-old man with NPM-RAR-positive APL who was receiving hemodialysis therapy for chronic uremia. Complete remission was achieved with ATRA monotherapy and was maintained for 18 months with consolidation chemotherapy. These findings suggest that ATRA can be used to treat APL patients with NPM/RAR as well as APL with PML/RAR.

Recurrent chromosomal rearrangements at BCL11B are found in human hematopoietic malignancies mostly of T-cell origin. However, it is unclear how this disruption contributes to oncogenesis, because the majority of leukemias express BCL11B from an undisrupted allele. Here, we show that Bcl11b +/− p53 +/− mice exhibited greater susceptibility to lymphomas than Bcl11b +/+ p53 +/− mice but most lymphomas retained and expressed the wild-type Bcl11b allele. This strongly suggests that Bcl11b is haploinsufficient for suppression of thymic lymphoma development in mice of the p53 +/− background, a situation in which functional loss of only one allele confers a selective advantage for tumor growth. The haploinsufficiency is further supported by that Bcl11b +/− mouse embryos were impaired in thymocyte development and survival. These results indicate relevance of BCL11B aberration to human leukemogenesis.

Bcl11b(-/-) mice show developmental arrest at the CD44(-)CD25(+) double-negative 3 (DN3) or immature CD8(+)single-positive stage of alphabeta T cell. We have performed detailed analysis of sorted subsets of Bcl11b(-/-) thymocytes, DN3 and CD44(-)CD25(-) double-negative 4 (DN4) cells. Surface expression of TCRbeta proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRbeta or TCRalphabeta expression on the cell surface failed to promote transition from the DN3 to CD4(+)CD8(+) double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b(-/-) DN3 thymocytes showed normal DNA rearrangements between Dbeta and Jbeta segments but limited DNA rearrangements between Vbeta and DJbeta without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b(-/-) DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vbeta and Dbeta gene locus. Analysis of Bcl11b(-/-) DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-x(L) and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRalphabeta in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.

Signaling pathways such as the pre-TCR and Wnt pathways regulate alpha/beta T cell differentiation in thymus. Mice lacking an essential component of the pre-TCR exhibit arrest at the (CD4(-)CD8(-)) (CD44(-)CD25(+)) stage (DN3) of thymocyte development, and introduction of p53 deficiency into those mice abrogates this arrest, resulting in transition to the (CD4(+)CD8(+)) double-positive (DP) stage. This paper examines the effect of inactivation of p53 on thymocyte development in Bcl11b(-/-) mice that exhibit arrest at the DN3 or (CD4(-)CD8(+)) immature single-positive (ISP) stage. No DP thymocytes were detected in thymocytes of adoptive transfer experiments in scid mice that were derived from p53(-/-)Bcl11b(-/-) precursors but ISP thymocytes increased in the proportion and in the cell number approximately three times higher than those from Bcl11b(-/-) precursors. Consistently, the level of apoptosis decreased to the level of wild-type precursors. These results suggest that inactivation of p53 is sufficient for DN3 thymocytes to differentiate into the ISP, but not to DP, stage of thymocyte development in Bcl11b(-/-) mice. This provides evidence for a novel p53-mediated checkpoint that regulates the transition from the DN3 to ISP stage of thymocyte development.

The MZ93 cell line, established from a patient with CML, expressed CD4, CD7, CD13, CD25, CD33, CD34, CD56 and NKp46. The additional karyotype abnormality of the Ph-positive leukemia cells in vivo, 6p+, was also observed in MZ93. The early passages of MZ93 expressed CD3 in the cytoplasm, but the late passages did not. The cells did not express mature NK-markers as expected. The messenger RNAs of CD2 and NKp46 were detected and those of CD3varepsilon and CD3zeta were absent in the cells. Therefore, the cell line has the immunophenotype likely to NK and/or T cell precursor.

Background: Elderly patients with acute myeloid leukemia (AML) show unfavorable biologic characteristics and inferior clinical outcomes compared to younger patients. Because some elderly patients can benefit from intensive chemotherapy, the identification of prognostic markers is important for therapeutic decision-making. CD25 (interleukin 2 receptor alpha) has been reported to be highly expressed in leukemia stem cells and correlate with adverse outcomes in younger (less than 60 years) patients with AML. However, the significance of CD25 expression in elderly patients with AML has not yet been investigated. Methods: We retrospectively reviewed 154 newly diagnosed AML (de novo, secondary) patients aged 60 years or over (median: 69, range: 60-85 years), admitted between 2005 and 2013 to Jichi Medical University Hospital. Blast cells from bone marrow at diagnosis were identified by flow cytometry (FCM) using CD45/side scatter properties. CD25 expression in the blast region was analyzed by FCM combined with single- and two-color staining. Results: The two-color FCM analysis showed that CD25 was expressed in CD34, CD117, and CD13-positive blast cells. The median CD25 expression of the blast cells was 0.7% (range: 0-75.3%). On the basis of CD25 expression in bone marrow blasts from healthy volunteers (n=5; median, 3.2%; range, 1.5-5.6%), we defined CD25 expression >10% as positivity. In this setting, we identified CD25-positive blasts in 21 patients (14%). CD25+AML was characterized by the MDS/MPN history (30%) and, a low or zero frequency of favorable cytogenetics (0%), FAB M2 subtype (5%), and FAB M3 subtype (0%) compared with CD25-AML (n=133). Among the 118 patients who underwent induction chemotherapy, including 75 patients who received a combination of anthracyclin and cytarabine (Ara-C), 69 (58%) achieved complete remission (CR). CD25+AML was associated with an inferior CR rate (20 vs. 64%, respectively, P=0.003), inferior event-free survival (EFS) (1-year EFS, 0 vs. 33%, respectively, P< 0.001), and inferior overall survival (OS) (2-year OS, 13 vs. 38%, respectively, P=0.003) compared with CD25-AML. On multivariate analysis using the following factors: cytogenetics, age (over 75 years), MDS/MPN history, and intensity of induction chemotherapy, CD25-positivity was a significant risk factor for the CR rate (HR: 8.0, 95% CI: 1.7-36.6; P=0.008) and EFS (HR: 2.3, 95% CI: 1.1-4.7; P=0.021). For CD25+AML, the CR rate was higher with induction chemotherapy using a regimen of low-dose Ara-C combined with granulocyte colony-stimulating factor (50%) than that using a regimen of daunorubicine and Ara-C (0%). However, there was no significant difference in OS between patients with CD25+AML treated with induction chemotherapy and those who treated with best supportive care (2-year OS, 13 vs. 11%, respectively, P=0.29). There were no patients with CD25+AML received stem cell transplantation due to disease progression. Conclusion: This study demonstrated that CD25 is an independent prognostic factor for elderly AML patients. The prognosis of CD25+AML patients is extremely poor; therefore, it is necessary to develop alternative approaches, such as a regime including anti-CD25 antibody, therapy targeting leukemia stem cells, and nonmyeloablative stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Background Interleukin 2 (IL-2) is an important cytokine that controls the proliferation and differentiation of not only T- but also B-lymphocytes. Recently, we reported that CD25 (IL-2 receptor alpha chain, IL-2R) is expressed in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and high expression of CD25 in the two types of lymphoma is correlated with a poor prognosis following chemotherapy regimens containing rituximab (ASH annual meeting, 2011 118:2666, 2012 120:1543). We evaluated the clinical significance of CD25 expression in a larger series of different mature B-cell lymphomas (BCL). Patients and Methods Four hundred and thirty-seven newly diagnosed patients who were admitted to our hospital between 2002 and 2013 were retrospectively evaluated. Lymph node or related tissue biopsy samples of BCL were analyzed using flow cytometry, as follows: 182 patients, DLBCL; 92, FL; 48, chronic lymphocytic leukemia (CLL); 21, mantle cell lymphoma (MCL); 23, marginal zone lymphoma (MZL); 8, Burkitt lymphoma (BL); 18, B-cell lymphoma unclassifiable with features intermediate between BL and DLBCL (BL/DLBCL); 5, lymphoplasmacytic lymphoma (LPL); and 39, reactive lymphadenopathy with sufficient B-cells. CD25-positivity was defined as >20% of clonal B-cells in a gated region. Results CD25 expression in patients with MCL, CLL, MZL, and DLBCL was significantly higher than that in patients with reactive lymphadenopathy (P The highest median CD25 expression (41.5%) was observed in MCL. CD25 expression was higher in MCL than CD5+ BCL (CLL and CD5+ DLBCL) (median, 41.5 vs. 16.9%, respectively; P60%) MCL (n=9) were not treated with aggressive chemotherapy regimens such as Hyper-CVAD due to their age and characteristics, compared with those with CD25-low ( In patients with CLL, the range of CD25 expression was wide (0.4-90.7%), and 29 patients (60%) showed CD25-positivity (CD25+ CLL). CD25+ CLL showed higher soluble IL-2R (sIL-2R) levels and an inferior overall survival (OS) than CD25- CLL (median sIL-2R, 2,195 vs. 706 U/ml P=0.047; 5-year OS, 62.7 vs. 100%; P=0.037). There was a significant correlation between levels of CD25 and sIL-2R (r=0.53, P=0.0053). It is clinically important to distinguish between DLBCL and BCL involving MYC oncogene rearrangement (BL and BL/DLBCL, MYC+ BCL). The former showed higher CD25 expression than the latter (median, 10.2 vs. 2.1%, respectively, P=0.04). The progression-free survival rate (PFS) after rituximab containing chemotherapy was inferior in patients with CD25+ DLBCL (n=72) than those with CD25- DLBCL (n=110) and MYC+ BCL (5-year PFS, 49 vs. 70.4, 66.3%, respectively). In patients with DLBCL, central nerve system (CNS) involvement was observed in 15 patients (7 at diagnosis and 8 at relapse). CD25+ DLBCL showed a higher frequency of CNS involvement than CD25– DLBCL (13.8 vs. 4.5%, respectively, P=0.049). Regarding MZL, CD25 was highly expressed in nodal MZL, but it showed a low expression in splenic MZL. Regarding the sites of extranodal MZL, CD25 expression was lower in the thyroid than at other sites (median, 5.1 vs. 21.2%, respectively, P=0.37). There were some differences between CD25+ (n=9) and CD25- (n=14) MZL concerning the presence of B symptoms (33.3 vs. 0%, respectively) and advanced stage (66.6 vs. 35.7%, respectively). Conclusion CD25 expression using flow cytometry can potentially provide diagnostic and prognostic implications on BCL patient. The high expression of CD25 in MCL and CLL suggests the possibility of targeted anti-CD25 immunotherapy. These findings may shed light on the role of CD25 expression in B-cell lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

We report long-term results from a large animal model of in vivo selection. Nine years ago, we transplanted two dogs (E900 and E958) with autologous marrow CD34(+) cells that had been transduced with a gammaretrovirus vector encoding a conditionally activatable derivative of the thrombopoietin receptor. Receptor activation through administration of a chemical inducer of dimerization (CID) (AP20187 or AP1903) confers a growth advantage. We previously reported responses to two 30-day intravenous (i.v.) courses of AP20187 administered within the first 8 months post-transplantation. We now report responses to 5-day subcutaneous (s.c.) courses of AP20187 or AP1903 at months 14, 90, and 93 (E900), or month 18 (E958), after transplantation. Long-term monitoring showed no rise in transduced cells in the absence of drug. Retroviral insertion site analysis showed that 4 of 6 (E958) and 5 of 12 (E900) transduced hematopoietic cell clones persisted lifelong. Both dogs were euthanized for reasons unrelated to the gene therapy treatment at 8 years 11 months (E958) and 11 years 1 month (E900) of age. Three clones from E900 remained detectable in each of two secondary recipients, one of which was treated with, and responded to, AP1903. Our results demonstrate the feasibility of safely regulating genetically engineered hematopoietic cells over many years.

Cyclosporin A (CsA) is the most common immunosuppressive agent used for recipients undergoing allogeneic stem cell transplantation (SCT). CsA require therapeutic drug monitoring (TDM) for both its effectiveness and toxicity, but the schedule of administration and optimal blood levels vary among institutions. To establish better assessment of CsA effect on individual immune responsiveness, we analyzed the proportion of IFN-γ+ or IL-2+ cells among CD4+ lymphocytes using 3-color flow cytometric analysis before and after CsA administration. CsA is administered as 3-hour infusion or oral medication twice daily in equally divided doses. 83 peripheral blood samples from 8 allogeneic BMT recipients are analyzed. Cells were cultured in the presence of phorbol 12-myristate 13-acetate, ionomycin, and brefeldin A at 37°C for 4hrs and then stained for surface markers and intracytoplasmic IFN-γ and IL-2. Blood CsA levels were simultaneously measured. We found that the proportion of IFN-γ+CD4+ or IL-2+CD4+ lymphocytes were inversely proportional to blood CsA levels (Figure 1). To achieve profound inhibition of IL-2 production, high concentration of CsA is required (e.g. 600–800 ng/ml for less that 5 % IL-2+CD4+ cells). In vitro experiments were also conducted using lymphocytes from healthy donors with or without activation with CD3 and CD28 to show how the sensitivity to CsA is different in between activated and resting T cells. We found that higher CsA concentration is required for inhibiting IL-2 production from CD4+ lymphocytes activated with CD3 and CD28 than from resting CD4+ lymphocytes (Figure 2). In this regard, our 3 hour intravenous CsA infusion can be superior to continuous intravenous infusion in inhibiting activated T cells. Further analyses are required, but the simultaneous assessment of cytokine production among CD4+ T cells and blood CsA levels may be a useful index to estimate the degree of immunosuppression afforded by CsA and may enable us individualized therapeutic drug monitoring of this agent. Since more and more patients are undergoing cord blood transplantation (CBT) or reduced-intensity stem cell transplantation (RIST), this assessment may also be useful for the establishment of immunosuppressive therapy for those patients.