ALBERT

Description: T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates 〉3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.

Description: Introduction Hepatic veno-occlusive disease (VOD) and thrombotic microangiopathy (TMA) are important and serious thrombotic complications after hematopoietic stem cell transplantation (HSCT), but the early diagnosis remains difficult since their clinical manifestations are similar to those of other transplantation-related complications. Our aim in this study is to illustrate the early alteration of hemostatic parameters in recipients of hematopoietic stem cell transplantation and then determine its value in transplantation-related thrombotic complications and other post-HSCT clinical settings, such as acute graft-versus-host disease (aGVHD) and infection. Methods Plasma from 95 patients undergoing HSCT was collected prior to conditioning therapy and then weekly until five weeks after HSCT. Hemostatic parameters were evaluated prospectively in our institution. 1. Plasminogen activator inhibitor-1(PAI-1), tissue-plasminogen activator(t-PA), protein C(PC), von Willebrand factor(vWF)and thrombomodulin(TM)were investigated by enzymimmunoassay. Other hemostatic parameters such as activated partial thromboplastin time(APTT), prothrombin time(PT), fibrinogen (Fg), antithrombin III(AT III) and D-dimer(D-Di) were measured with hemagglutinin equipment in the same time. 2. According to the different settings after transplantation, three groups of transplant associated complications were classified as thrombus group (VOD n=5, TMA n=1), aGVHD group (n=29) and infection group (n=19). Systemic analyses were carried out for the hemostatic parameters and transplantation-related thrombotic complications or other clinical settings. Results Significant increase was observed in the levels of fibrinogen, t-PA and PAI-1 after transplant, while Protein C and ATIII decreased significantly(P0.05). All the patients with three different complications presented with significantly increased PAI-1 and lower level of Protein C compared with those who had no complication (P0.05). However, 6 patients with thrombotic complications (VOD5, TMA1) extremely showed elevated PAI-1 levels after the clinical onset of thrombotic complications by comparison with highest post-HSCT values in the aGVHD patients or infection patients (P

Description: We aimed to evaluate the effects of arsenic trioxide (ATO, Trisenox) on STI571 (Gleevec, imatinib mesylate)-induced apoptosis of a chronic myelogenous leukemia (CML) cell line, K562 cells. Cell prolifration and colony-forming assays were performed to determine the cytotoxicity of ATO alone and in combination with STI571. Apoptosis was analyzed by morphological changes, apoptosis rate and cell cycles. An Elisa assay was used to detect the levels of cytosolic cytochrome c (cyt c) and caspase-3 in K562 cells exposed to ATO and STI571 at graded concentrations. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay the transcriptional levels of Bcl-XL and Bcr-Abl genes in K562 cells. Results showed that both the colony-forming assay and cell proliferation assay demonstrated additive to synergistic effects of ATO on STI571-induced apoptosis in K562 cells, a Bcr-Abl positive cell line. Caspase-3 was activated during apoptosis and there was an increase in cytosolic accumulation of cytochrome c. Treatment of K562 cells with STI571 alone led to down-regulation of transcriptional levels of Bcl-XL at 12 hours and Bcr-Abl at 96 hours after drug administration. Treatment with ATO alone only led to reduce the mRNA levels of Bcl-XL, but not Bcr-Abl. Combined treatment with ATO and STI571 down regulated the transcripts of Bcl-XL at 12 hours and Bcr-Abl 72 hours after drug administration. We conclude that ATO enhanced cytotoxic and proapoptotic actions of STI571 could be mediated by the down-regulation of Bcr-Abl and Bcl-XL genes in K562 cells. Therefore ATO in combination with STI571 could be a promising therapy for CML.

Description: Targeted therapy with the Bcr-Abl tyrosine kinase inhibitor Imatinib Mesylate can induce high response rates in chronic myelogenous leukemia (CML) patients. However, evidences that discontinuation of imatinib mesylate inevitably exerts rapid loss of response and some patients with imatinib mesylate monotherapy virtually occur potential relapse suggest that the modification of treatment strategy is critical. We have previously demonstrated that the combination of trisenox (arsenic trioxide) with the tyrosine kinase inhibitor imatinib mesylate or genistein appears to induce markedly more cell apoptosis than imatinib mesylate alone through downregulating Bcl-XL and Bcr-Abl in Bcr-Abl gene transfected HL-60 cells. We here report the preliminary results of a pilot study comparing imatinib mesylate plus trisenox with imatinib mesylate alone for frontline treatment of CML patients. Up to date 56 patients were enrolled in this clinical trail. All patients (required to be 18 years or older with Bcr-Abl positive CML in chronic phase within three month of diagnosis) were divided into two groups, i.e., monotherapy group and combined therapy group. 42 patients entering monotherapy received imatinib mesylate 400mg daily and 14 patients entering combined therapy imatinib mesylate 400mg daily plus trisenox 10mg daily for one week and then twice a week. We compared treatment results of both groups including complete hematologic response(CHR), major/complete cytogenetic response(MCR/CCR),--defined as 1–35% Ph+ and 0% Ph+ metaphases respectively and major/complete molecular response(MMR/CMR),--defined as ≥ 3 log reduction and negative expression in Bcr-Abl transcript numbers assayed by RQ-PCR respectively. The median follow-up for patients in both groups lasted 36 months. In the combined therapy group, the median age of patients is 42 years old (range, 22–61), the CHR, MCR, CCR, MMR and CMR is 92.8%, 64.3%, 42.9%, 35.7% and 21.4%, respectively. While in the imatinib mesylate monotherapy group, the median age of patients is 46 years old (range, 23–65), the CHR, MCR, CCR, MMR and CMR is 85.7%, 59.5%, 40.5%, 33.3% and 19%, respectively. Although combination therapy of imatinib with trisenox is not significantly superior to imatinib mesylate monotherapy in efficacy, no resistance case happens in the combination therapy group, in the imatinib mesylate monotherapy group, imatinib resistance occurs in 4 patients. In addition, the safety and tolerability of a combination of imatinib mesylate and trisenox is good. This result indicates that the combination of imatinib and trisenox to treat chronic myeloid leukemia may be promising in avoiding the occur of imatinib resistance.

Description: Abstract 1433 BACKGROUND High levels of 2-HG in serum were found among AML cases with IDH1/IDH2 mutations. However, the impact of 2-HG levels on biological characteristics and clinical outcomes has not been systematically evaluated in a large cohort of AML patients. METHODS Serum 2-HG levels in 699 patients with hematologic malignancies, including 405 AML patients, were measured by GC-TOFMS. Clinical prognostic power of 2-HG and genetic risk factors associated with 2-HG were also evaluated in AML patients. RESULTS Among all patients with hematopoietic malignancies investigated, high serum 2-HG levels were observed only in AML group. 64 out of 405 (15.8%) AML patients displayed aberrantly higher levels (7.14±2.11μg/ml) of 2-HG. Compared to cases with normal 2-HG (3.65±1.02μg/ml), these patients showed a higher prevalence in AML-M0/M1 subtypes, a closer correlation with mutated IDH1/2 genes, a distinct gene expression profile and an aberrant DNA methylation status in bone marrow blasts. Additionally, the patients with high 2-HG were associated with higher serum levels of α-ketoglutarate (α-KG) and glutamate, suggesting presence of impaired metabolic pathway involved in the biosynthesis of 2-HG. Univariate and multivariate analyses indicate that high level of 2-HG is among the most significant negative indicators for complete remission (CR) rate, overall survival (OS) and event-free survival (EFS) either in all AML cases or in cases with cytogenetically normal AML (CN-AML). CONCLUSIONS Serum 2-HG is an independent prognostic marker in AML. Patients with high 2-HG had significantly higher frequency of IDH1/IDH2 gene mutations and unfavorable prognosis compared to those with normal 2-HG. Disclosures: No relevant conflicts of interest to declare.

Description: Objective: To explore the possible immune pathophysiology of CD4+T regulatory (CD4+Treg) in acquired aplastic anemia (AA). Methods: According to the markers on membrane, cytokines, and effective mechanisms of CD4+Treg, the levels of CD4+CD25+Treg,CD4+CTLA-4+Treg,CD4+ICOS+Treg,CD4+PD-1+Treg,CD3+CD8−IL-10+Treg,CD3+CD8−TGF-β1+Treg,CD3+CD8−IL-Treg in the bone marrow of 23 cases with AA at active phase, 10cases with AA at recovery phase, 15 normal controls, were measured, and the relationship between CD4+Treg and priming immune factor-CD28 or effective immune factor-IFN-γ were also evaluated respectively. Results: [circ1]In the group of AA at active phase, at recovery phase, and normal controls, CD4+CD25+Treg: (3.36±2.05)%, (3.49±1.71)%, (2.40±0.77)%, CD4+CTLA-4+ Treg: (1.43±0.67)%, (3.46±2.26)%, (2.45±1.30)%, CD4+ICOS+Treg: (2.04±1.79)%, (2.95±2.23)%, (2.37±1.68)%, CD4+PD-1+Treg: (1.40±1.32)%, (2.34±0.87)%, (2.07±1.64)%, CD3+CD8−IL-10+Treg: (2.14±1.19)%, (2.37±1.20)%, (2.04±1.25)%, CD3+CD8−TGF-β1+Treg: (1.83±0.99)%, (1.52±0.73)%, (2.15±1.17)%, CD3+CD8−IL-4+Treg: (2.46±1.65)%, (2.53±1.61)%, (1.99±1.27)%; costimulatory of CD3+CD4+CD28+ was: (39.84±10.89)%, (22.72±9.08)%, 31.40±10.83)%, the Th1cells CD3+CD8−IFN-γ+ were: (11.13±4.96)%, (5.39±4.29)%, (4.21±2.11)%. [circ2] Contrast to normal controls, while CD4+CTLA-4+Treg of AA at active phase deceased markedly (p=0.05); [circ3] Contrast to normal controls, the ratio of membrane costimulatory of CD28/ICOS CD28/CTLA-4, CD28/PD-1 were all increased significantly (p

Description: We analyzed donor chimerism(DC) in unfractionated(UF) and CD3+ cells from a secondary acute myeloid leukemia patient who relapsed after reduced-intensity allogeneic hematopoitic stem cell transplantation(RIST) and had undergone donor lymphocyte infusion(DLI). Sequential and quantitative chimerism was performed by multiplex PCR amplification of STR markers (STR-PCR). The peripheral blood(PB) every 3 days and bone marrow every week were collected for chimerism analysis. The patient presented hematological relapse 26 months after the first RIST. There were 31.5% blast cell in the bone marrow smear and presented additional abnormal chromosome [44,XY,-7,dic(16,17)]. While UF donor chimerism from PB and CD3+ fraction donor chimerism from PB and BM were always full donor chimerism (range, 96.4%~98.1%). However UF chimerism from BM was 64.9% at the same time point. Afterwards the patient received 4 times of dose-escalated donor lymphocytes infusion (DLI) with CD3+ cell from 1*107/kg to 1.0*108/kg. Unfortunately the patient hasn’t response to this therapy. The blast cell in BM smear augment from 31.5% to 51.5%. UF chimerism from BM ranged from 55.2 to 46.5%. But UF DC from PB and CD3+ fraction DC from PB and BM ranged from 95.5% to 98%. In conclusion, Our result suggest that in this AML patient the donor T-cell chimerism and unfractionated chimerism in PB can not predict relapse and response to donor lymphocyte infusion. and the reason is not clear.

Description: Objective To explore the efficiency and safety of Low-dose Cytosine arabinoside and aclarubicin in combination with granulocyte colony-stimulating factor (CAG regimen) for patients with refractory and relapsed adult acute lymphocytic leukemia (ALL). Methods We treated 10 patients of refractory and relapsed adult ALL with CAG regimen (median age 28 years, range 17–58, male 5, female 5). The regimen consisted of low-dose cytosine arabinoside (10mg/m2 q12h, day1to14), aclarubicin (10mg/day, day1to8), and granulocyte colony-stimulating factor (G-CSF) (200 μg/m2/day). Result 8/10 patients (80%) achieved complete remission (CR) after one course, including 4 out of 5 refractory and 4 out of 5 relapsed patients. 1 patient who relapsed after the first course, then achieved CR again after the second course. 6 of the 8 complete remission received consolidation therapy with the CAG regimen or other regimens. Toxic effects were very rare and mainly consisted of neutropenia and thrombocytopenia due to myelosuppression; 8 of all patients had neutropenia or thrombocytopenia that exceeded WHO grade II. However, severe non-hematologic toxicity included nausea and vomiting (WHO grade ≥III) was characteristically rare. Conclusion CAG regimen is an effective and safety in the treatment of refractory and relapsed ALL.

Description: Objective To illustrate the alteration of hemostatic parameters in recipients of hematopoietic stem cell transplantation (HSCT) and then determine its value in transplantation-related complications. Methods 45 patients receiving HSCT were evaluated prospectively in our institution. Hemostatic parameters were investigated prior to conditioning therapy and then weekly until five weeks after HSCT by enzymimmunoassay. Results Significant increase was observed in the levels of fibrinogen, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) after transplant, while Protein C decreased significantly. No significant change existed in prothrombin time (PT), activated partial thromboplastin time (APTT) and antithrombin III (ATIII) levels. Six patients with acute grades II–IV graft-versus-host disease (aGVHD) presented with significantly lower level of Protein C compared with those who had grades 0–I aGVHD, PAI-1 level didn’t change apparently as well as fibrinogen and t-PA in the same time. However, three patients with veno-occlusive disease (VOD) extremely showed elevated PAI-1 levels after the clinical onset of VOD by comparison with mean post-HSCT values in the non-VOD patients; similar result was found in the patient who developed thrombotic microangiopathy (TMA). Conclusion Our results suggest hemostatic imbalance is one important manifestation during HSCT, reflecting prothrombotic states and endothelial damage, which may be caused by the conditioning regimen and/or transplantation-related complications. The extreme elevation of PAI-1 alteration may be useful to recognize the development of VOD and TMA, while Protein C diminution facilitates the early diagnosis of aGVHD.