ALBERT

Description: Backgroud and objective: Myeloproliferative neoplasms (MPNs) are a group of clonal haematological disorders that characteristic with a multipotent haematopoietic stem cell transformation, often associating with the progression of myelofibrosis in the evolution course of disease. Progressive myelofibrosis finally turned out a high risk factor of transformation to leukemia and bone marrow failure. Cancer Associated Fibroblasts (CAFs) are recently thought to be a critical mediator in several hematological malignancies tumor microenvironment and associate with fibrosis. Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that promote the crosslinking of collagens or elastins in the extracellular matrix and mediate epithelial-mesenchymal transition (EMT). Here, we want to verify CAFs mediating myelofibrosis and explore the potential role of LOXL2 association with CAFs in simulated vivo microenvironment. Patients and methods:For bone marrow specimens, normal samples (n=19) and patients with polycythemia vera (PV) (n=21), essential thrombocythemia (ET) (n=32), and primary myelofibrosis (PMF) (n=9) were contrasted. Markers of CAFs including α-smooth actin(α-SMA), fibroblast activation protein(FAP), transforming growth factor-β1(TGF-β1) and LOXL2 were detected by quantitative reverse transcription-PCR(RT-PCR). we also detected α-SMA, FAP, LOXL2 and reticulin protein by western blot and immunohistochemical staining. For cell lines, α-SMA and FAP were measured after cocultured mesenchymal stem cell(MSCs) with recombinant human lysyl oxidase homolog 2 Protein(rhLOXL2) in hypoxic niche for 24, 48, 72 and 96 hours, respectively. Results: Markers of CAFs displayed a differential pattern of expression in MPNs especially in PMF(P

Description: Large heterogeneity in bleeding phenotype is observed among patients with severe hemophilia A. Some severe patients do not require regular replacement therapy, so to address the factors influencing phenotypic Heterogeneity in Patients with Severe Hemophilia A seems particularly important. We first initiate a cohort study 0f 274 patients with severe hemophilia A who were treated at the hemophilia treatment centre at Nanfang hospital, then 209 patients were enrolled and their bleedings were recorded, finally 12.9% (12/209) of the patients were found to be mild phenotypes, which annual bleeding rate(ABR)were less than 6 times. We then use Flow-cytometric analysis to detect the platelet-derived microparticles (PMPs) among patients with mild phenotypes, patients with severe phenotypes (ABR more than 24) and the normal control group, megamix beads (0.5μm; BioCytex, Marseille, France), CD42a-PE, CD42b-PE, CD41b-PE (GPIX, platelet MPs [PMPs], BD, NJ, USA) were involved, then the results showed that there was no difference in the level of PMPs between hemophliac patients and the normal control group (P=0.317), however, the level of PMPs in patients with mild phenotypes were significantly higher than patients with severe phenotypes (3.65±1.38 vs 1.13±0.64, P

Description: Aggressive natural killer cell leukemia (ANKL) is a rare and highly aggressive subtype of mature NK-cell neoplasms. Similar with extranodal NK/T-cell lymphoma, nasal type (ENKL), another subtype of NK-cell neoplasm, ANKL is also an Asian-prevalent and Epstein-Barr virus (EBV)-related neoplasm. In contrast, our knowledge of ANKL, especially about EBV biological behavior in this rare leukemia, lags far behind that of ENKL and other EBV-related hematopoietic malignancies, such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and post-transplant lymphoproliferative disorder (PTLD). Dissection of the virus-host crosstalk in ANKL could contribute to better understanding the mechanism and finding out effective therapy for this neoplasm. In the present study, we investigated EBV-associated biological behavior in serial ANKL patients, including the clinical presentation, EBV genomic DNA, EBV antigens expression, cytogenetic-molecular aberrations, and leukemia-associated microenvironment. A total of 28 ANKL patients were collected upon review of the clinical database in Nanfang hospital. Different items of EBV infection evidence consisted of EBV viremia (n=9), EBV genomic DNA (n=20), and EBER/EBNA/LMP1/LMP2A expression (n=23). EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) was the predominant clinical feature. Bone marrow smear was infiltrated with large granular lymphocyte (LGL) with LMP1/LMP2a-positive bulb, indicating the presence of EBV viral inclusions bodies. Positron emission tomographic (PET)-computed tomographic (CT) scan revealed bone marrow, liver and spleen as the most frequently involved organs, compared with nose and nasopharynx in ENKL. Cytogenetic analysis demonstrated 7q10-32 (n=4) was the “hotspot” of chromosome aberrations in ANKL. PCR analysis with EBNA-2/LMP1 specific primers on reserved DNA samples (n=20) revealed ANKL cells were infected with type-1 EBV strain with wide-type LMP1 (n=20), compared with 30bp-deleted LMP1 gene in ENKL. Integrated mutation analysis (n=20) identified recurrent mutations in Src homology 2 (SH2) domains of STAT5a (n=7) and p16inka (exon 3/4, n=20), but no mutation in SH2 domains of ID2, STAT1, and STAT3. Immunochemical (IHC) analysis on formalin-fixed paraffin-embedded tissues (n=23) revealed latency type-3 EBV expression in ANKL cells, with latency antigens of EBER, EBNA, LMP-1, and LMP-2. Furthermore, LMP-1/LMP-2-positive leukemia/lymphoma-associated macrophages (LAMs, n=23) were enriched in ANKL microenvironment. Notably, EBV-positive LAMs were significantly associated with poor prognosis and disease progression. Univariate analysis revealed significant difference (p

Description: Background Allogeneic hematopoietic stem cell transplantation is an effective method for treatment of hematological malignancies, while GVHD and graft rejection are main complications, which seriously affect patients' survival rates and quality of life. Aim Establishing allo-transplantation mice model with mRFP and GFP transgenic mice, to simulate clinical hematopoietic stem cell transplantation and explore the mechanism of stem cell homing and GVHD. Methods 1) Thirteen C57BL/6 GFP transgenic mice, used as recipients, were irradiated with 7 Gy. Each mouse was injected through caudal vein with 2*106 bone marrow cells isolated from FVB mRFP transgenic mice. 2) Symptoms like weight loss, depilation, diarrhea were observed as GVHD manifestation while survival rates were evaluated. Routine blood test and FACS were performed at different time points to confirm hematopoiesis reconstitution. 3) Mice were perfused with paraformaldehyde under anesthesia to fix the tissue, while pathological examination and real-time PCR were performed for studying donor and recipient cells interactions in different organs. 4) Semi-solid decalcification was used to treat the femora before observing under confocal microscope directly or after making frozen section, three-dimensional reconstruction were made to observe the cellular interaction, especially for cells within the bone marrow. Result 1) Depilation, wrinkled skin, hunchback and sharp decline of weight were observed in 8/13 mice. Routine blood test implicated hematopoietic reconstitution. FACS showed 86.1%±7.8% mRFP+ cells in peripheral blood of recipients. 2) mRFP+ cells were found distributing throughout the body's organs. mRFP+ Lymphocyte infiltration and inflammatory exudate were seen especially in the small intestine, lung, liver and skin (Fig.1). GFP+ cells were found surrounding mRFP+ cells in the bone marrow of the femora decalcified with semi-solid decalcification. Their interactions can be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction by confocal microscope (Fig.2). Discussion Owing to RFP on donors' cells and GFP on recipients' cells, together with our novel protocol named semi-solid decalcification, we can visually observe the donor and recipient cells' location, ratio and cellular interaction, as well as morphological changes. Within various tissues especially for such tissues as bone marrow and lung, the details between cells can be studied lively by fluorescence microscope and confocal microscope. In recipient mice with GVHD, donor cells can be found in various target tissues such as intestine, lung, liver and skin. Gene marked cells with fluorescence protein can benefit morphological, immunological, cytogenetic and molecular studies in recipients after HSCT. Conclusion The allo-transplantation model with mRFP and GFP transgenic mice is powerful in study of Stem Cell Homing and Donor-Recipient Cellular Interaction. The cellular interaction can be easily observed by three-dimensional reconstruction after semi-solid decalcification, especially for bone marrow and lung. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Bronchiolitis obliterans syndrome (BOS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a devastating complication with limited therapeutic options. We aimed to assess the efficacy and safety of mesenchymal stem cells (MSCs) in BOS after allo-HSCT. Methods: Eighty-one allo-HSCT recipients with BOS diagnosed within 6 months were enrolled in this multicenter prospective cohort study. The choice of prednisone and azithromycin combined with or without MSCs was based on patient preferences (MSC n=49, non-MSC n=32). The primary endpoint was response rate at 3 months, defined as the proportion of patients achieving FEV1 improvement or steroid sparing. This trial was registered at ClinicalTrials.gov, NCT02543073. Results: Response rate was 35/49 patients (71%, 95% confidence intervals [CI] 59 to 84%) (FEV1 improvement n=10, steroid sparing n=25) and 14/32 (44%, 95% CI 27 to 61%) (FEV1 improvement n=3, steroid sparing n=11) in the MSC and non-MSC group, respectively (p=0.013). The adjusted odds ratio of response in the MSC group compared with the non-MSC group was 3.32 (95% CI 1.21-9.11; p=0.02). The addition of MSCs was associated with a better difference for change in FEV1 rate of decline, compared to non-MSC group (53 ml/months, 2 to 103; p=0.040). The 3-year overall survival post-diagnosis was 70.6% (95% CI 55.9 to 85.3%) and 58.2% (95% CI 36.1 to 78.5%) in the MSC and non-MSC group, respectively (p=0.21). Clinical improvement was accompanied by a significant increase of interleukin (IL)-10-producing CD5+ B cells. The incidences of infection and leukemia relapse were no significant difference between two groups. Multiple infusions of MSCs were well-tolerated with no serious adverse events. Interpretation: MSCs might be an effective and safe therapy for BOS patients after allo-HSCT. Our study strengthens evidence for clinical practice of MSC therapy in BOS. These data also offer insight into potential biological mechanisms of MSC treatment and support further investigation in larger randomized controlled trials. Disclosures No relevant conflicts of interest to declare.

Description: Background Internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutations have been reported in 20%-30% of patients with acute myeloid leukemia (AML). FLT3-ITD-positive AML patients have an inferior survival, primarily due to lower complete remission (CR) rate and higher relapse rate. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves the outcomes of some FLT3-ITD-positive AML, a significant number will suffer disease recurrence after allo-HSCT. Sorafenib, an inhibitor of multiple kinases including FLT3, has shown promising activity in FLT3-ITD-positive AML. Recent studies have shown that sorafenib monotherapy or in combination with chemotherapy are effective in attaining CR, but they do not have significant improvement in relapse. Currently, prophylactic use of sorafenib after allo-HSCT has been rarely reported, and whether it can improve outcomes of FLT3-ITD-positive AML remains unclear. Methods A total of 18 patients with FLT3-ITD-positive AML undergoing allo-HSCT from January 2012 to March 2014 at our single institute were enrolled in this retrospective study. Seven patients were in first CR at the time of transplantation and all received standard conditioning: busulfan 4 mg/kg/d P.O. or 3.2 mg/kg/d I.V. (on days -7 to -4), cyclophosphamide 60 mg/kg/d (on days -3, -2). Eleven patients were not in CR (NR) at the time of transplantation and all received intensified conditioning: fludarabine 30 mg/m2/d (on days -10 to -6), cytarabine 2.0 g/m2/d (on days -10 to -6), total body irradiation 4.5 Gy/d (on days -5, -4), cyclophosphamide 60 mg/kg/d (on days -3, -2), etoposide 15 mg/kg/d (on days -3, -2). Sorafenib was used from day 30 to 180 post-transplantation. The initial dose of sorafenib was 400 mg orally twice daily and was adjusted in case of suspected toxicity or resistance (dose range, 200-800 mg daily). Results Of the 18 patients, 7 received prophylactic sorafenib, including 3 in CR and 4 in NR pre-transplantation; 11 did not receive prophylactic sorafenib, including 4 in CR and 7 in NR pre-transplantation. There were no significant differences in disease status pre-transplantation, HLA-typing and the source of donors between the two groups (all P〉0.05). The eleven patients in NR at the time of transplantation all achieved CR and had complete chimerism by day +30 post-transplantation. With a median follow up of 433 (range, 124-765) days post-transplantation, 7 patients with prophylactic sorafenib all had no recurrence and survived. Of the 11 patients without prophylactic sorafenib, 5 experienced leukemia relapse at a median time of 92 (range, 49 to 335) days post-transplantation, including hematologic in 4 and central nervous system (CNS) in 1. The patient with CNS relapse achieved CR after radiotherapy and donor lymphocyte infusion (DLI), and was still alive now. Of the 4 patients with hematologic relapse, one abandoned treatment and 3 received treatment, including sorafenib combined with chemotherapy and DLI. Two of the three patients achieved short CR after treatment and finally all of them died of relapse within half a year. With a median follow up of 343 (range, 135-863) days post-transplantation, 6 patients survived and 5 died in the 11 patients without prophylactic sorafenib. Causes of death included leukemia relapse (n=4) and chronic graft-versus-host disease (n=1). The 1-year cumulative incidence of leukemia relapse was 0.0% and 50.9% in patients with and without prophylactic sorafenib (P=0.046). The 1-year overall and disease-free survival were 100.0% and 100.0% in patients with prophylactic sorafenib, compared with 49.9% and 42.4% in those without prophylactic sorafenib (P=0.066, P=0.028). The main side effect of sorafenib was rash, and none of patients experienced bone marrow suppression. Of the 10 patients with sorafenib treatment, 6 had hand-foot skin reaction and/or rash. Anti-allergy therapy was ineffective, and glucocorticoid was usually required. In 4 of the 6 patients, the skin rash covered gradually after reducing the dose of sorafenib and the use of glucocorticoid. Other two patients even required drug discontinuation, but sorafenib was tolerated after the reuse. Conclusions Early prophylactic use of sorafenib has an acceptable toxicity profile and could reduce relapse of FLT3-ITD-positive AML. Disclosures Liu: It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017): Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding. Xuan:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding.

Description: Background and Introduction: A consensus of opinion exists among hemophilia doctors that the optimal management of patients with severe hemophilia during childhood is the administration of regular prophylactic infusions of factor concentrate. However, considering patient burden and costs, it is equally important to evaluate for how long prophylaxis is required. So we initiated a study to demonstrate the feasibility and benefits of the low-dose secondary prophylaxis for adults with severe hemophilia. Methods: Patients with severe hemophilia A (F VIII 〈 1%) born between January, 1961, and January, 1988, who were treated at the hemophilia treatment centre at Nanfang hospital and treatment data available were eligible for enrollment in the study. The main exclusion criteria were a history of FVIII inhibitor (titer ≥ 0.6BU [Bethesda unit]), detectable FVIII inhibitors at screening (titer ≥ 0.4 BU), chronic liver disease, immunodeficiency, another hemostatic defect and the need for major surgery. An observational cohort study, using each patient as its own control, was conducted on adult patients with severe hemophilia switched from on-demand treatment to low dose secondary prophylaxis from January 2012 to December 2013. Enrollment began from December 2011 to January 2012. During the prophylaxis period, all participants received FVIII (including rFVIII Kogenate and pd FVIII Green Cross, Liaocheng, China) 10 IU kg-1, twice a week. Breakthrough acute bleedings were treated according to the regular practice and guidelines. The study was approved by the Ethic Committee of Nanfang hospital, and informed consent was obtained from all patients before participation. Results: The study was completed over a 2-year period (January 2012 to December 2013). Twenty adults with severe hemophilia performed on-demand treatment in preceding 5 years were enrolled. Switching to prophylaxis reduced the annual number of total and joint bleeds (median 24 vs. 8.5 and median 21 vs. 8.5; P 〈 0.001, respectively). The relative reduction in median annual number of total bleeds was 64.6% for subjects treated with prophylaxis compared with during on-demand treatment. Annual number of non-joint bleeds were also significantly lower (P 〈 0.001) on prophylaxis compared with during on-demand treatment (median 2 vs. 1 P 〈 0.001). After prophylaxis, the FISH score increased from median 22.5 (range 15-28) to median 24 (range 15-28) (P=0.016). Meanwhile, after two-years of prophylaxis, the number of target joints didn’t increase (median 8.5, range: 2-14; median 8.5, range: 2-14; P=0.317, respectively). The Health-related Quality of life (HRQoL) in patients with severe hemophilia were measured by the SF-36. The SF-36 score showed better results in terms of overall perceived health status between patients with On-demand period and with Prophylaxis period (P 〈 0.01) and all the seven domains like Physical Function, Role physical, Bodily Pain, General health, Vitality, Social function and Role emotional were improved after prophylaxis (P 〈 0.05), while Mental health did not differ significantly between the two groups (P=0.267). Conclusion: The most important finding in this pilot study is its confirmation that low-dose secondary prophylaxis even in short-term does provide substantial benefits in controlling haemorrhage and improving daily activities/function, as well as improve HRQoL in China. Our study clearly established that low-dose prophylaxis can be provided efficiently for adults in a number of haemophilia treatment centers in China. Disclosures No relevant conflicts of interest to declare.

Description: Keywords: Hemophlia A, Phenotype; Heterogeneity; Flowcytometry ; Mircoparticle Background: The clinical phenotype for hemophlia A are typically recognized sub-clinical, mild, moderate and severe according to the residue FVIII level of the plasma. For recent years, there are evidences indicate that the residue FVIII level is not the only factor relate to the clinical phenotype of patients with severe hemophlia A. Platelets are an important role in hemostasis and platelet-derived microparticles (PMPs) are known to mediate a prothrombotic state in patients, we hypothesized that PMPs contributes to the clinical phenotype heterogeneity of hemophilia A patients. Methods: Thirty-one patients with severe hemophlia A (FVIII:C

Description: Objective To explore the regimen related toxicity (RRT) and the effects of super-intensified conditioning regimen combined with inducing graft versus leukemia (GVL) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for refractory leukemia which can’t obtain complete remission (CR) pre- transplantation. Methods 18 patients who did not obtain CR before transplantation received super-intensity conditioning regimen protocol(experimental group), and 62 patients with acute leukemia who obtained CR or chronic myeloid leukemian (CML) who were in the chronic phase before transplantation received total body irradiation (TBI) plus cyclophosphamide (CTX) or modified BuCY (hydroxyurea, busulfan, Ara-C, CTX) protocol (control group). Cyclosporin A was reduced rapidly and gradually or donor lymphocytic infusion (DLI) was used to induced GVL if acute graft versus host disease (GVHD) did not happened in patients with refractory leukemia at 30 days post-transplantation. The incidence and mortality of RRT during transplantation, and the rate of CR, GVHD and leukemia relapse after transplantation was investigated. Kaplan-Meier survival analysis model was used to estimate the disease-free survival (DSF) rate at 3 years post-transplantation. Results Except one patient in experimental group and two patients in control group died of transplant-related complications, all the other patients obtained hematopoietic reconstitution.’ the total RRT incidence were both 100% in two groups. The RRT of stomach intestine were most common in all the organs and the RRT incidence of experimental group and control group was 83.3% and 85.5%, respectively, in stomach intestine. The RRT incidence was 44.4% and 62.9% in oral cavity and 16.7% and 33.9% in bladder, respectively, in the experimental group and control group. There was no significance and P value was 0.823, 0.172 and 0.244, respectively, in the RRT incidence of stomach intestine, oral cavity and bladder between the two groups. The RRT mortality was 0 and 5%, respectively, and was not different (P=0.341) in the experimental and control group. Except one patient died of infection, all the other patients obtained CR in patients who were treated with supper-intensified conditioning regimen. The incidences of acute or chronic GVHD were 58.8% and 40.0% or 92.6% and 55.8%, respectively, in the experimental and control group. The incidence of leukemia relapse was 11.8% and 18.3%, respectively, in the two groups. The DSF at 3 years after transplantation was 61.2±12.3% and 65.0±7.4% (P=0.6311), respectively, in the two groups. Conclusion The consecutive super-intensified conditioning regimen combined with inducing GVL post transplantation protocol can increase the rate of CR and DFS, and dose not increase RRT incidence and mortality in allo-HSCT for the refractory leukemia which can’t obtain CR pre- transplantation.

Description: Backgroud: To evaluate the efficacy of preemptive therapy for preventing EBV related PTLD based on the duration of EBV-emia and the viral loads in the patients undergoing haplo-HSCT, a prospective multicenter study was conducted. Methods: Rituximab or rituximab followed by adoptive cellular therapy was applied for preemptive therapy if EBV-DNA in blood was positive twice consecutively with a rising trend over one log in virus loads or positive consecutively six times without a dropping trend over one log at least within three weeks or not turning negative consecutively eight times within four weeks. Results : Of the 408 patients with acute leukemia enrolled in this study, 133 occurred EBV-emia, including 19 PTLD and 6 end-organ diseases. The patients with EBV-emia had inferior three-year overall survival (OS) and higher non-relapse mortality (NRM) to those without EBV-emia (OS: 58.0%±5.0% vs. 71.0%±3.0%, =0.042; NRM: 27.9%±3.9% vs. 18.9%±2.4%, =0.050, respectively). Compared with non-preemptive group, the negative conversion of EBV-emia was significantly higher (100% vs. 76.1%, =0.002) and the three-year incidence of PTLD was markedly lower in preemptive group (1.3% vs. 21.7%, =0.000). The median time from EBV-emia to EBV diseases onset was 15 days and the durative time of EBV-emia was positively correlated with PTLD occurrence (=0.419, =0.024). Conclusion s: Rituximab preemptive therapy could reduce the incidence of PTLD. Earlier preemptive therapy should be advocated once the EBV-emia is positive persistently regardless of EBV-DNA levels (NCT01883180). Disclosures No relevant conflicts of interest to declare.