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: 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: Abstract 4673 Refractory chronic GVHD (cGVHD) is an important complication after allogeneic hematopoietic SCT and is prognostic of poor outcome. Bone Marrow Stromal Cells (MSCs) are involved in tissue repair and modulating immune responses in vitro and in vivo. MSCs as salvage treatment for refractory cGVHD have been reported in our previous study, however, the possible mechanism have yet not to be determined. Between November 2006 and November 2010, 18 patients were diagnosed with refractory cGVHD, 8 patients were treated with in vitro expanded BM-derived MSCs as a compassionate treatment for refractory cGVHD, 10 patients that did not receive BMSCs treatment were control group. The median MSC dose given was 0.6×106/kg body weight. MSCs were harvested fresh from culture and administered to the patients by intravenous infusions over 30 minutes. The median time of MSC administrations was 3 (range, 2–6). The response was assessed monthly after BMSCs treatment, and the total follow-up period was 6 months. The organ response and the overall response were used to determine the therapeutic efficacies of MSC for refractory cGVHD. The expression of the Jagged2 gene of peripheral blood mononuclear cells in patients at the assessment points were analyzed using the TaqMan real-time polymerase chain reaction, with ABL mRNA expression levels as an internal reference. After BMSCs treatment, a total of 6 patients (75%) had an overall response (PR n=6), and 2 patients had a minor partial response (mPR n=2). The expression levels of Jagged2 mRNA in these cases at the diagnosis of refractory cGVHD were significantly increased, compared with none cGVHD patients (23.94%±18.68% vs 3.76%±1.50%, P 〈 0.05), and the copies of Jagged2 mRNA in BMSC treatment responsed patients' peripheral blood were significantly reduced (5.15%±3.25%, P 0.05). Our pilot study showed that Jagged2 gene reproduction upregulated when the cGVHD is active, so, dynamic monitoring of Jagged2 mRNA expression may have the potential effect on predicting the activity of chronic graft-versus-host disease. Mechanism of Bone marrow stromal cells to treat refractory cGVHD may be related to down-regulation of donor T cells Notch ligand Jagged2 gene expression, which suppression of T cell Notch signaling pathway activation, thus inducing immune tolerance. Disclosures: No relevant conflicts of interest to declare.

Description: Background: In the human T-cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4, siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, which may be related to PHTF1 gene expression, and the FEM1B and Apaf-1 genes may be downstream of PHTF1. In this study, we analyzed the expression level of PHTF1 and related genes in patients with ALL to clarify the role of the PHTF1-FEM1b-Apaf-1 pathway in hematologic malignancies. Methods: Fifteen newly diagnosed and untreated patients with AML, fourteen newly diagnosed and untreated patients with CML in chronic phase, twenty-two newly diagnosed and untreated patients with ALL, and six newly diagnosed and untreated patients with CLL were recruited. Peripheral blood mononuclear cells (PBMCs) from ten healthy individuals (HIs) served as controls. PBMCs were separated using the Ficoll-Hypaque gradient centrifugation method. All procedures were conducted in accordance with the guidelines of the Medical Ethics committees of the Health Bureau of Guangdong Province, China.Real-time PCR was used to determine the gene expression level of PHTF1 in hematologic malignancies. The PHTF1, BCL11B, FEM1B and Apaf-1 gene expression levels and correlations were analyzed in patients with primary ALL and healthy individuals (HIs). Results: PHTF1 overexpression was found in recently diagnosed AML (p

Description: Abstract 1147 Poster Board I-169 In the past twenty years, allogeneic hematopoietic stem cells transplantation (Allo-HSCT) has been accepted as the most effective treatment for many hematologic malignancies. However, the successful rate of allo-HSCT has been limited by transplantation-related mortality and malignancies relapse, no matter using traditional intensity conditioning or reduced-intensity conditioning. In this study, we presented ninety-two patients with hematopoietic malignancies received fludarabine combinasion with modified Bu/Cy (FABC) conditioning regimen before allogeneic hematopoietic stem cell transplantation. Ninety-two patients with hematological malignancies (58 males, 34 females) ranged in age from 14 to 50 (median 28) years. These patients were diagnosed with acute lymphoblastic leukemia (ALL, n=30), acute myelogenous leukemia(AML, n=24), chronic myelogenous leukemia (CML, n=33; CP, n=27; CML-AP, n=5; CML-BC, n=1), myelodysplastic syndrome ( MDS, n=3), chronic myelomonocytic leukemia (CMML, n=1), and one patient coexisted chronic myelomonocytic leukemia and T lymphoblast cell lymphoma. Fifty-five (59.8%) patients were at high risk. From June 2004 to October 2008, 92 patients gave their informed consent and received conditioning regimen with fludarabine-based modified Bu/Cy (FABC conditioning regimen) in allo-HSCT. The FABC regimen consist of cytarabine 2.0 g/ m2 on day -9, busulphan (Bu) 3.2 mg/kg per day for intravenous on days -8 to day-6, followed by cyclophosphamide (Cy) 60 mg/kg per day on days -5 and day-4, combined fludarabin 30 mg/m2 per day for three consecutive days, on days -6 to day-4, and Me-CCNU (1-(2-Chloroethyl)-3-(4-ethylnitrobiphenyl Cylohexyl4)-1- Nitrosourea) 250 mg/m2 on day -3. Graft-versus-host-disease(GVHD) prophylaxis consisted of cyclosporine A, short-term MTX and Mycophenolate Mofefil (MMF 1.0g/day, on d-8 to d-1). Anti-T-lymphocyte globulin (2.5 mg·kg-1·d -1, on d-3 to d-1) was added to patients with mismatched sibling or unrelated donors. Follow-up was performed on 30 December, 2008. Ninety-two patients engrafted successfully, the median time for ANC 〉0.5×109/L was 12 (8 to 22) days, and for BPC 〉 20×109/L was 12 (7 -32) days. Detected by short tandem repeat (STR)-PCR, complete donor chimerism was comfirmed in all patients on day +21 or day+30. The incidence of acute GVHD was 25% (23/92), and grades 3 to 4 acute GVHD developed in 8 (8.7%) of 92 patients with in 100 days after HSCT. Chronic GVHD developed in 40(47.6%) of 84 patients who were alive more than 100 days after HSCT, and the incidence of extensive cGVHD was 35.7%(30/84). The transplant related mortality (TRM) was 19.6% (18/92), mainly from severe infection (n=7), acute or chronic GVHD (n=5), transplant associated-microangiopathy (n=2), diffusion alveolar hemorrhage (n=2), and post-transplant lymphoproliferative disorders (n=2). With a median follow-up of 16.2(1.5 to 54.5) months, 70 (76.1%) of the 92 patients were alive and 67(72.8%) were disease-free. The probabilities of OS at 1 year and 2 years was 80% and 72.5%, and DFS was 79.1% and 71.4%, respectively. These results suggest that the fludarabine-based modified Bu/Cy conditioning regimen (FABC) should reduce severe acute GVHD and accelerate hematopoietis resconsition without increasing chronic GVHD and lower leukemia relapse rates even in high-risk patients. Footnotes Corresponding author Disclosure 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.

Description: Background: The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates for overall response and complete remission. We aimed to investigate the effects of the combination of DAC and homoharringtonine (HHT) in the SKM-1 and Kg-1a cell lines and explore their associated mechanisms. Methods: Cell viability was estimated by MTS assay. Cell apoptosis were detected by flow cytometry analysis and PI/Annexin V staining. Western blot and quantitative reverse transcription-polymerase chain reaction assays were performed to detect the expression of apoptosis-related genes including caspase-3, caspase-9, BCL-2 and BCL-XL and DNA methyltransferases(DNMT) including DNMT1, DNMT3A and DNMT3B. Changes in LINE-1 gene methylation were assessed by quantitative polymerase chain reaction after bisulfite conversion. The effects of the combinations were estimated using CalcuSyn software. Results: The combination of DAC and HHT showed synergistic effects for inhibiting cell viability in SKM-1 and KG-1a cell lines. This combination can enhance inhibition of colony formation and apoptosis induction of DAC alone in SKM-1 cell line. However, in Kg-1a cells, this combination only enhanced the apoptosis induction of DAC alone. For SKM-1 cells, further study found that different doses of DAC and HHT alone have different effects on the expression of the apoptosis-related genes caspase-3, caspase-9, BCL-XL and BCL-2. The combination of 0.4 μM DAC and 5 nM HHT treatment significantly increased the mRNA expression of caspase-3 (P=0.0005) and caspase-9 (P=0.0075) and decreased the expression of BCL-2 (P=0.0331), but has no significantly effect on the BCL-XL (P=0.3436) compared with 0.4 μM DAC alone. However, 4 μM DAC plus 50 nM HHT had no significant effects on the mRNA and protein expression of the apoptosis-related genes examined compared with 4 μM DAC alone (P〉0.05). Low-dose DAC induced greater hypomethylation than higher doses of the drug. Whereas HHT had no demethylation effects, and also had no enhancement effects with DAC on the hypomethylation and mRNA and protein expression of DNMT1, DNMT3A and DNMT3B in SKM-1 cells. Conclusion: The combination of DAC and HHT has synergistic effects on cell viability inhibition and enhancement effects on cell apoptosis in SKM-1 and KG-1a cell lines. But this combination did not enhance the hypomethylation effect of DAC.These data suggest that DAC used in combination with HHT has clinical potential in the treatment of MDS and AML. Disclosures No relevant conflicts of interest to declare.

Description: Refractory extensive chronic graft-versus-host disease (GVHD) after allogeneic stem-cell transplantation (SCT) is associated with high mortality [Margolis J., SeminOncol 2000].However, conventional therapies including steroids are often unsuccessful in those patients with multiorgan involvement and are associated with significant therapy-related complications and poorly life quality. Mesenchymal stem cells (MSCs) have immunomodulatory effects [Tse WT et al., Transplantation 2003; Spees JI et al.,Proc Natl Acad Sci USA 2003]. Recently MSCs have been given intravenously to treat seven steroid resistant acute GVHD patients and one patient with chronic GVHD. MSCs effects in chronic GVHD is rarely known, although this successfully experience suggests that MSCs have been well tolerated and had a powerful immunosuppressive effects on acute GVHD. [Katarina Le Blanc et al., Lancet 2004; Olle Ringden., Transplantation 2006 ]. Here, we present our experience of using MSCs for treatment of Thirteen patients with refractory chronic GVHD. Between May 2005 and March 2007, thirteen patients (8 male, 5female) with hematological malignancies with a median age of 26(range:15 to 40) years who had received peripheral stem cells from sibling donors. All patients developed steroid resistant or extensive chronic GVHD, with progressive involvement of the skin(13), liver(10), oral mucosa(12),ocular glands(12), and thrombocytopenia (1) when the immunosuppressive agents were taped after five to twenty-four months. The MSC dose was median 1.0 ×106 cells/kg body weight of the recipient. In all, thirteen patients had at least received one dose, seven patients received more than two doses. MSC donors were in seven cases HLA-identical siblings, six unrelated mismatched donors. No side-effects were seen after MSCs infusions. All patients have responded after follow-up of the median time 15 months. One patient with moderate cGVHD had a complete responses, and discontinued all of the immunosuppressive agents without relapse more than 18.4 months after MSC infusion. Three moderate and two patients with severe chronic GVHD improved to mild degree, and six severe turned to moderate degree. Complete resolution was seen in gut(2/3), liver(5/10), skin(5/13), oral(6/12) and eye(2/12). One patient responded in skin, liver, oral mucose and eye, but developed in lung (bronchiolitis obliterans, BO) score of 2 which are considered severe chronic GVHD. Mean follow-up periods was 27m (rang: 14 to48m), Leukemia free survival(LFS)rate were 85%(11/13), and the overall survival (OS)rate were 92.3%(12/13). Our experience suggests that MSC infusion is a safe and effective adjunct therapy for refractory extensive chronic GVHD with resistance to conventional therapy. But more prospective, controlled studies with MSCs for treatment of GVHD should be performanced to evaluate this new treatment exactly.

Description: Abstract 4501 Chronic graft-versus-host disease (GVHD) is a major complication of allogeneous hematopoietic stem cell transplantation, and it is regarded as a type of Th2 disease and seems to be a T-cell-mediated disorder in which immune tolerance to self-antigens is broken. Mesenchymal stem cells (MSCs) are multipotent cells which can extenuate refractory (GVHD) have been recently reported; however, the clinical data on MSC relieves chronic GVHD are generally lacking, and the machanisms remain to be investigated. One area of recent interest and controversy is the role of regulatory T cells (CD4+/CD25high T cells) in chronic GVHD. We examined the lymphocyte subsets and regulatory T cells of patients with refractory chronic GVHD were treated with basic immunosuppression plus MSCs derived from volunteer donors. Between April 2005 and October 2008, 19 patients were treated with their basic immunosuppression plus in vitro expanded bone-marrow-derived MSC as a compassionate treatment for resistant chronic GVHD. The median MSC dose given was 0.6×106/kg per body weight. Flow cytometric analysis was perfromed to analysis lymphocyte composition and activation at the time before MSC infusion(pre-MSC) and 3 months after MSC infusion (post-MSC). The study was approved by the Ethics Committee of the Guangdong General Hospital. All patients and the MSC donors provided written informed consent. In the present study, there were no significant changes in the proportion of T cells, B cells, NK cells and activation in no-response patients between pre-MSC and post-MSC. In the patients with response, the total lymphocyte was greater post-MSC transfusion than pre-MSC transfusion, and the proportion of CD4+ T cells were increased CD+8 T cells were decreased (the P value was 0.001 and 0.018, respectively). However, the proportion of CD4+CD25+ T cell and CD8+CD25+ T cells hadn't significantly changed post-MSC transfusion compared to pre-MSC infusion in patients with response. The T lymphocyte subsets of CD8+CD28+ cells, which are active CTLs that participate transplantation immunity and rejection reaction, were down-regulated when the chronic GVHD improved post-MSC infusion (P=0.023). By contrast, CD8+CD28- cells, which regulate cells to induce immune tolerance and inhibit antibody production and imbalance in the production of Th1 and Th2 cytokines, were increased in patients responsed to MSC infusion (P=0.025), and the ratio of CD8+CD28- cells to CD8+CD28+ was increased. Importantly, the increase of CD8+CD28- T cells and decrease of CD8+CD28+ T cells with improvement of cGVHD indicate that these factors may be involved in the development of chronic GVHD and that MSC suppresses chronic GVHD via modulation of the ratios of CD8+CD28- to CD8+CD28+ T cells. However, further immunological studies specifically addressing this issue are needed to confirm this possibility. This work was supported by Science and Technology Planning Project of Guangdong Province (2006B36005003,2007A032100003) and Science and Technology Planning Project of Guangzhou (2006Z2-E4071,2008A1-E4011-4,2008A1-E4011-5). Disclosures: No relevant conflicts of interest to declare.