ALBERT

Description: Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule and expresses on activated T cells, B cells and myeloid cells. PD-L1, a primary ligand PD-1, is mainly expressed on T cells and primary B cell surfaces and plays a role in the differentiation and apoptosis of these cells, induces a coinhibitory signal in activated T cells, promotes T cells apoptosis, incompetence and functional exhaustion. The expression of PD-1 is high in patients with hematologic malignancies and a high expression of PD-L1 is found on hematologic malignancies cells. To further know the characteristics of PD-1 and PD-L1 in patients with MDS, we detected theexpression of PD-1 and PD-L1 in peripheral blood (PB) and bone marrow (BM) samples from 25 RAEB and 10 RARS patients using Real-time PCR. The PD1 and PD-L1 expression levels of 13 PB and 8 BM samples from normal individuals were as controls. The PD-1 levels of PB samples from 25 RAEB patients [42.40(4.5-173.96)%] were significantly higher than that from normal controls [32.32(19.45-41.38)%, P=0.026]. While the level of PD-1 in 10 RARS patients was comparable to that of normal controls and RAEB patients (P=0.401 and P=0.352). Compared to normal controls [23.72(3.23-39.2)%], the median PD-1 level of BM from 10 RAEB patients[36.81(12.14-151.52)%] showed an increasing tendency, but the difference was not statistically significant (P=0.062). PD-L1 expression levels of PB samples from RARS patients were significantly lower than that of normal controls (P=0.009). There were no significant difference between RAEB patients and normal controls about the PD-L1 expression level in PB and BM samples (P=0.248 and P=0.181) and between RAEB and RARS patients about PD-L1 expression level in PB samples (P=0.243). PD-1 level in BM samples from 11 RAEB patients with remission was (31.32±15.75)% and it was lower significantly than that before treatment [(59.94±47.44)%, P=0.034]. After progression or transformation, the expression level of PD1 (54.72±37.27)% increased again and was higher than that in remission (P=0.028).There were also no significant difference on PD-L1 expression among before treatment, bone marrow remission and progression or transformation (P〉0.05). In 8 RAEB patients transformed to AML after treatment, PD-1 level has a decreasing tendency(P =0.05)and the change of PD-L1 has no significant difference (P〉0.05). In conclusion, PD1 mRNA level increased significantly in patients with RAEB and the changes of PD-1 was associated with the evolution of the disease after treatment with demethylating agents. The level of PD1 may be used as an indicator to determine the efficacy, but the changes of PD-L1 was not found regularity. Disclosures No relevant conflicts of interest to declare.

Description: Comparison of the 5 year Medical Outcome and Quality of Life of Patients with Chronic Myeloid Leukemia treated with Imatinib or Allogeneic Transplant Jianyu Weng1*, Lisi Huang1*, Peilong Lai1, Zesheng Lu1, Ping Wu1, Suijing Wu1, Chengwei Luo1, Wei Ling1, Chenxin Deng1, Xin Huang1, Suxia Geng1, Xin Du1? 1Department of Hematology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, P.R. China. *These authors contributed equally to this work.? Corresponding author: Xin Du, MD, Ph.D. 106. Zhongshan Er Rd, Guangzhou, 510080, P.R. China E-mail: miyadu@hotmail.com Phone: (86)-20-83827812-62122 Fax: (86)-20-83889772 Abstract To evaluate and compare the long-term medical outcome and health-related quality of life (HRQOL) of patients with CML in chronic phase (CML-CP) who received imatinib or an allogeneic hematopoietic stem cell transplant (allo-HSCT) using retrospective analysis and an embedded cross-sectional study. CML-CP patients who received imatinib or transplantation from January 4, 2004 to October 10, 2011 in the Department of Hematology at Guangdong General Hospital (GGH) were enrolled in this study. A total of 131 patients, including 90 and 41 in the imatinib and allo-HSCT groups, respectively, were enrolled. The two groups including HRQOL investigation were well matched for gender, marital status, employment status, educational background, and Sokal score at diagnosis. There were no significant differences in the five-year EFS, PFS and OS between the two groups. The HRQOL was measured using EORTC QLQ-C30. Those questions include five multi-item function scales (Physical, Role, Emotional, Cognitive and Social Functioning), a combined Global Health Status/QOL scale, and a number of individual items (appetite loss, dyspnea, diarrhea, constipation, sleep disturbances, and financial impact). In terms of HRQOL, with the exception of social functioning (64.9 vs. 77.5, P=0.035), there was no significant difference in many of the HRQOL scores between the imatinib and transplant groups: global HRQOL, role function, physical function, emotional function and cognitive function. However, symptoms including nausea/vomiting (14.8 vs. 3.2, P=0.013), diarrhea (18.4 vs. 2.5, P=0.001), and financial difficulty (56.9 vs. 33.3, P=0.023) more negatively impacted the imatinib group (P

Description: Chronic graft-versus-host-disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with highly variable clinical presentations. The pathophysiology of cGVHD remains to be further understood. The utilization of murine models to study cGVHD has contributed to the understanding of cGVHD, and highlights its mechanistic complexity. Here, we report a new murine cGVHD modle with obvious lung tissue damage in these mice resulted in the development of bronchiolitis obliterans (BO), which is pathopneumonic for cGVHD. Recipients 8 weeks BALB/c (H-2d) female mice, which irradiated with 700cGy dose of linear accelerator, then were injected with bone-marrow cells (1×107) and spleen cells (5×106) from (C57BL/6 x BALB/c) F1 donors (H-2bd haplotype) male mice. The observed items post-transplantation included hematopoietic reconstruction, implant, and general condition. Clinical scores were assessed every 3 days after +14 d. At + 135 d, mice were killed to evaluate the pathological changes of major target organs. Mice in transplantation groups were in hematopoietic reconstruction at +7 d, and all survived to the end point (+135 d). Chromosomes of recipient mice were in full donor chimera. Clinical scores of cGVHD group have been more than 0.6 since +90 d. These mice develop pathologic manifestations in several organs including lung, skin, and liver. Biopsy-proven BO incidence was 100%, and pathological scores of cGVHD group were 5.33±1.55, which significantly higher than those of transplantation control groups (P 〈 0.05). To date, most models involve parent-into-F1 combinations the transfer of MHC-mismatched cells resulted in a phenotype that resembles clinical systemic lupus erythematosus (SLE), termed SLE-cGVHD. However, some of the models do not use any pretransplantation conditioning and no obvious lung tissue damage. We exchanged parent for recipients, and provide a total boby irradiation as pretransplantation conditioning. New MHC-mismatched murine cGVHD model was easily obtained, and more relevancy to the clinical features of cGVHD. This model provides an ideal study model of clinical cGVHD pathogenesis and treatment strategies. NOTES: The project was sponsored by grants from National Natural Science Foundation (No. 81270648 and 30972790), National Public Health Grand Research Foundation (No.201202017), and Guangdong Natural Science Foundation (No. S2012010009560). Disclosures: No relevant conflicts of interest to declare.

Description: Acute graft-versus-host disease (aGVHD) is still an important complication after allogeneic hematopoietic stem cell transplantation. Dendritic cells (DCs) are crucial for initiating cell-mediated adaptive immune responses or maintaining immune tolerance, which play key role on inducting of GVHD. We have reported a subset of dendritic cells (DCs), CD8α+ Jagged2high CD11bhigh regulatory dendritic cells (DCregs), which also express MHC molecules and low lever of costimulatory molecules, induce the production of regulatory T cells and inhibit the proliferation function of lymphocyte. In this study, we examined the role of the CD8α+ Jagged2high CD11bhighregulatory dendritic cells (DCregs) in murine major histocompatibility complex (MHC)-incompatible model of aGVHD in alloHSCT. BALB /c (H-2d) mice bone marrow cells (BMCs) were isolated and were induced to DCs generation by 4 cytokines, SCF (100ng/mL), IL-4 (20ng/mL), Flt3 ligand (25ng/mL), and GM-CSF (5ng/mL) for 3 days. The new DCs were selected by flow cytometry, and were co-cultured with Bone marrow mesenchymal stem cells (BMSCs) for 10 days. Characteristics of such regulatory dendritic cells and immune tolerance features were detected before and after co-culture by FCM analysis, ELISA kit, and mixed lymphocyte reaction. Male 8-week-old C57BL / 6 (H-2b) mice were used as donor, and female 8-week-old BALB / c (H-2d) were used as recipient. The recipient mice received SSD100, 30×30 radiation field, 700cGy total body irradiation (TBI) pretreatment, and grouped as follows: 1.normal control group, 2.TBI control group, 3. transplantat control group, 4. aGVHD group: 1 × 107 BMC and 1 × 107 spleen cells (SPC) were injected through caudal vein 5. DCregs groups: 1 × 107 BMC, 1 × 107 spleen cells (SPC) and 1 × 106DCregs were injected through caudal vein. Evaluate the severity of GVHD according to clinical manifestations (weekly the GVHD ratings), the survival time and histopathological examination. The results showed that novel DCs were transformed into CD8α+Jagged2highCD11bhigh Dcregs. The expression of CD86, CD80, CD40, and MHC-II were evidently decreased, while that of CD205, Jagged1 and Jagged2 were significantly increased on DCregs (P

Description: Background: Treatment for acute myeloid leukemia (AML) has remained relatively unchanged over the past few decades. Standard-dose cytarabine and idarubicin (7+3 IA) or daunorubicin (7+3 DA) induction regimen have been recommended for AML induction therapy by global guidelines. Mitroxantrone, a type II topoisomerase inhibitor, disrupts DNA synthesis and DNA repair, has been recommended as a salvage treatment for refractory/relapsed AML or induction therapy for elderly patients. Objective: To compare the the clinical response and adverse events of Idarubicin regimen ( IA) with mitoxantrone and cytarabine (MA) in the treatment of newly acute myeloid leukemia (AML) . Methods: This retrospective study evaluated 164 patients with newly diagnosed AML who received either IA (idarubicin 10mg/M2, d1-3; cytarabine 200mg/d, d1-7) or MA regimen (mitoxantrone 10mg/M2, d1-d3; cytarabine 200mg/d, d1-7) as induction therapy from September 2010 to November 2017 at Guangdong General Hospital. The primary end point of this study was complete response and complete response with incomplete blood count recovery (CR/CRi), with secondary end points were adverse event rates and days of granulocyte and platelet recovery. Results: A total of 164 patients , 90 patients were males and 74 females, the median age was 41 (range:14~64) years old. There were 88 patients received IA regimen and 76 patients received MA regimen. There was no significant difference in clinical features and molecular biological characteristics in two groups (P〉0.05). The CR/CRi rate was 72.3% and 64.0% (P=0.263) in IA and MA group after the first induction regimen, respectively. And the accumulated CR/CRi rate was 85.9% and 75.7% in two group, respectively (P=0.109). The common adverse reactions in the two groups were myelosuppression and infection , but with no statistical difference (P〉0.05) in the incidence and grade of serious. The grade 4 and grade 5 neutropenia were 95.3% vs.98.7% and 4.7% vs. 1.3%, P〉0.05 in IA and MA group respectively. And thrombocytpenia were 72.9% vs.63.2% and 4.7% vs. 1.3%, P〉0.05. There was no significant difference in the incidence and severity of gastrointestinal, cardiovascular, respiratory, skin, liver and kidney injury between the two groups (P〉0.05). The median days of intravenous antibiotics (including antifungal drugs), neutrophil recovery, platelet recovery and the units of platelet and red blood cell suspension transfusion had no statistical difference in two groups (P〉0.05). Conclusion: This retrospective study implied mitoxantrone with standard-dose cytarabine (3+7 MA) regimen has similar efficacy and outcome to the idarubicin with standard-dose cytarabine (3+7 IA) regimen for newly diagnosed AML, without increasing the incidence of adverse event rates. Disclosures No relevant conflicts of interest to declare.

Description: To investigate the effects of Artesunate treatment on chronic graft-versus-host disease (cGVHD). Recipient BALB/cJ mice received 8 × 106 bone marrow cells with 8 × 106 spleen cells from B10.D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after transplatation. The clinical scores, survival times and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice was evaluated by flow cytometry. In addition, we cultured the CD4+ T cells from the spleens of mice in vitro, and stimulated these cells with artesunate, the frequency of Th17 and Tregs in these splenocytes was evaluated by flow cytometry. Artesunate administration diminishes clinical and histopathological damage and improved the survival of cGVHD mice. Artesunate contributed to Tregs development (4.45±0.04 vs 8.40±0.23; 6.62±0.24 vs 10.48±0.48; p

Description: Background: Our preliminary research found that STAT3, IL-17A, and IL-21 expressed in cGVHD patients. So, we provide the blocking STAT3 signal to the induction of Treg cells differentiation, and to provide experimental basis on new targets of cGVHD immunotherapy. Methods: 1. Mice spleen CD4+ CD62L+ naiveT cells were separated by immune magnetic bead and then activated for 72 h. After 96 h infection with STAT3-shRNA and negative control lentivirus, the Th subgroup proportion were measured by flow cytometry. Th related cytokines levels test by Luminex. Real time quantitative PCR was to detect STAT3 and Th subgroup related transcription factor mRNA levels. CD117+ mouse bone marrow stem progenitor cells were sorted by flow cytometry, and transfected by STAT3-shRNA. Inhibition of STAT3 gene in mRNA level was measured at 96 h. Cell proliferation activity was test with CCK8 kit, and cell apoptosis rate determined by flow cytometry. Differentiation of CD117+ cells was induced by 2.2% of methyl cellulose and different cytokines. 2. BALB/c female mice, after the linear accelerator 700cGy of whole body irradiation, accepted miHA mismatched male B10. D2 mice bone marrow cells and spleen cells (8 x 106, 1:1). Randomly assigned 6 mice of cGVHD clinical score of 0.6 or above to each group. After STAT3-shRNA or negative control lentivirus treatment, the observe end point was 58th day after transplantation. The clinical and pathologic scores compared. Th17 and Treg cells measured by flow cytometry. Th related cytokines measured by Luminex. Purpose genes in blood and protein expression levels in target organs were found by Q-PCR and western blot test, respectively. Results: 1. The Th17 / Treg ratio of shRNA group was significantly decreased than that in the NC group (P 〈 0.05). Except for the Foxp3 gene, other purpose genes, including T-bet, Gata3, RORγt, TGFβ, Notch1, and Jagged2 mRNA levelsin interference group were cut. GM-CSF, IFN-gamma, beta, IL- 3, IL-2, IL-4, IL-6, TNF alpha, IL-17, IL-22a, IL-27, and IL-9 factor expression levels were significant difference between shRNA and negative control group (P 〈 0.05). There was no significant difference of cell proliferation activity, early apoptosis rate, and differentiation ability in STAT3-shRNA treated CD117+ bone marrow, compared with negative control group and blank control group (P 〉 0.05). 2. After 50th day, shRNA treatment group appear hair recovery, energy recovery, weight gain, shortness of breath better, mean of cGVHD score decreased. At the 58th day, clinical scores of cGVHD between shRNA treatment group and the negative control group overall mean difference was statistically significant (P 〈 0.05). cGVHD pathological score of lungs in shRNA treat group reduced (P 〈 0.05). STAT3mRNA levels in peripheral blood, phosphorylated STAT3, and STAT3 expression level of lung declined than control groups. The proportion of Th17 / Treg cells of spleen was significant reduced in shRNA group, compared with negative control group (P 〈 0.05). Conclusion: 1. STAT3 knocking down in naïve CD4 + Th cells induced the increased Treg cells, and the decreased Th17 cells. IL-2 confirmed to promote the growth of Treg cells. It speculated that blocking STAT3 might bring Th9 cells differentiation. STAT3 blocking in CD117+ stem progenitor cells have no significant effect on the proliferation, apoptosis and differentiation, validation the safety of STAT3-shRNA. 2. STAT3-shRNA treatment cGVHD mice in vivo achieved curative effect. The main target organs was the lung, which might be closely related to the fall in the proportion of Th17 /Treg. STAT3 may be used as a new target for immunotherapy of cGVHD. Acknowledgment The project was sponsored by grants from National Natural Science Foundation of China (No. 30972790; No.81270648; No.81370665; No.81300446), Provincial Natural Science Foundation of Guangdong (No. S2012010009560), Provincial Science and Technology Planning Project of Guangdong (No.2013B021800186; No.2013B021800201), and Science and Technology Planning Project of Guangzhou (No. 201400000003-4, 201400000003-1). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4709 Objective: Acute graft-versus-host disease (aGVHD) confines the wider application of allogeneic bone marrow transplantation (allo-BMT), but recently studies indicate that it is possible to reduce the incidence and severity of aGVHD while preserving the GVT by using bortezomib. In current study, we explored the changes of T cell subsets after allo-BMT administrated with bortezomib immediately, in order to establish the mechanism about bortezomib attenuation aGVHD. Materials and Methods: BALB/c mouse were injected of 0.5 mL PBS containing C57BL/6 2×107 nucleated BM cells plus 1×107 splenocytes followed a single dose of lethal total body irradiation (TBI, 0.7 Gy/min, 8.0 Gy) with or without bortezomib at 1.0 mg/kg. The level of CD4+ CD25+ Foxp3+ regulator T cells is quantified by flow cytometry, and the cytokine level of IL-2 and IL-4 is quantified by ELISA. Results: Bortezomib remarkably reduce aGVHD severity and prolonged the surviving time. Along with bortezomib injection, the level of CD4+ CD25+ Foxp3+ regulator T cell is significantly increased, the cytokine level of IL-2 is decreased but IL-4 is increased. Conclusion: Bortezomib inhibit aGVHD through shifting the combination of T cell subpopulations with up regulation CD4+CD25+ Foxp3+ regulator T cells lead to reset Th1/Th2 cytokine balance. Disclosures: No relevant conflicts of interest to declare.

Description: Xiaomei Chen and Jianyu Weng contributed equally to this study. The outcomes ofrelapsed or refractory acute myeloid leukemia (RR-AML) are poor and effective salvage regimens are urgently needed. We present a study of 14 patients with RR-AML (median age 42years, range 18-65years; male n=12, female n= 2) treated with CLAT regimen, which consisted of cladribine 5mg/m² per day i.v. 2-3 hour on days 1-5, cytarabine 1.0g/m² per day i.v. 4 hours after cladribine on days 1-5, topotecan 1.25mg/m² per day i.v. 4 hours after cytarabine on days1-5 and G-CSF 300ug per day subcutaneous injection on days until neutrophile granulocyte recovery. Total of fourteen patients were included into the study from June 2013 to June 2015, Two (14.3%) patients were relapsed and twelve (85.7%) patients were refractory, 4 of 14 patients were relapsed or refractory after allogeneic-HSCT. Two patients died of invasive fungal infection before the assessment. Seven patients (58.3%) achieved complete remission (CR), and one patient (8.3%) achieved partial remission (PR), the rest patients (33.3%) did not respond (NR). The overall response rate was 66.7%. Following CLAT treatment, four patients with CR underwent allogeneic hematopoietic stem cell transplantation (HSCT) or microtransplantation. The median relapse-free survival (RFS) for RR-AML patients receiving CLAT regimen was 8.6 (range 2-16) months. Thirteen patients developed grade 4 granulocytopenia and thrombocytopenia, the median duration was 13(range 2 to 21) days and12 (range 2 to 21) days, respectively. The most common non-hematological side effects included nausea, vomiting, diarrhoea, and were grade 1/2. The CLAT regimen seems promising for the treatment of patients, and it was well tolerated. This regimen offers an alternative treatment for those patients with RR-AML who have received severe intensive treatment, especially with anthracycline-containing chemotherapy. The project was sponsored by grants from National Natural Science Foundation of China (No. 30972790; No.81270648; No.81370665; No.81300446) Provincial Natural Science Foundation of Guangdong (No. S2012010009560) Provincial Science and Technology Planning Project of Guangdong (No.2013B021800186; No.2013B021800201), and Science and Technology Planning Project of Guangzhou (No. 201400000003-4, 201400000003-1). Disclosures No relevant conflicts of interest to declare.

Description: Background: Hypomethylating agents (HMAs), including decitabine and azacitidine, have improved outcomes for patients with myelodysplastic syndrome (MDS). However, not all patients benefit from this therapy, and no reliable prognostic tool can predict differential likelihood of benefit from HMAs. Methods: To investigate predictive factors of treatment response to HMAs in patients with MDS, we analyzed the baseline biomarkers, clinical variables and TP53 mutations of 52 newly diagnosed Chinese patients with MDS who received decitabine or azacitidine (1-23 cycles, median number cycles: 7) Results: In the 46 assessable patients who received more than 2 cycles of standard treatment, a total of 22 patients had an overall response after treatment, and 24 patents showed no response. The results showed that there was no significant difference in response by treatment regimen (P=0.147) or source of sample (P=0.413). Furthermore, age (P=0.382), sex (P=0.595), IPSS risk groups (P=0.117) and cytogenetic abnormalities (P=0.935) were not associated with response rate. However, in univariate analysis of the overall cohort (n=40) who received more than 3 cycles of standard treatment, the response rate was significantly higher for those patients who had platelet count doubling after 2 cycle of treatment (P=0.013), but there was no statistically difference after 1 cycle(P=0.376). Moreover, a total of 5 patients (11%) had TP53 mutations, overall response rate to HMAs was 100% in patients with TP53 mutation, which is significantly higher than that of patients with wild type cases (p = 0.013). Conclusions: These findings indicate that patients with TP53 mutation or achieving platelet count doubling after 2 cycle of HMAs respond well. Disclosures No relevant conflicts of interest to declare.