Guangsen Zhang

Treatment of lycorine on SCID mice model with human APL cells

Abstract In our previous study, lycorine, a natural alkaloid extracted from Amaryllidaceae, exhibited anti-leukemia effects in vitro. To determine whether lycorine has an anti-tumor effect in vivo, a series of experiments were carried out in this study. HL-60 cells (5×106) were inoculated i.v. into severe combined immuno-deficiency (SCID) mice after these mice had been irradiated (total body receiving 200cGy χ irradiation). Treatment was given once a day from day 2 to 6, and from day 14 to 18. Lycorine (5 or 10mg/kg/day i.p.) was found to decrease the percentages of immature granular leukocytes and of monocytes among the peripheral blood cells, and the mean survival time of both lycorine-treated groups was longer than that of the control group. Compared with the asynchronous and cytosine arabinoside- (Ara-C) treated (20mg/kg/day i.p.) group, treatment with lycorine was more effective. Lycorine was also found to alleviate the infiltration of tumor cells into the liver, bone, and marrow. When SCID mice inoculated with HL-60 cells were then treated with lycorine, no severe adverse effects were observed. This study revealed that lycorine, when tested in the human leukemia xenograft models, appears to exhibit anti-tumor activity in vivo and is a useful therapy against acute promyelocytic leukemia.

Use of all-trans retinoic acid in combination with arsenic trioxide for remission induction in patients with newly diagnosed acute promyelocytic leukemia and for consolidation/maintenance in CR patients.

In the present study, 90 patients with newly diagnosed acute promyelocytic leukemia (APL) were studied for all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) combination treatment in remission induction and postremission therapy. In addition, 20 APL patients who had achieved complete remission (CR) with an ATRA-based regimen received ATRA/As2O3 combination for consolidation and maintenance were also enrolled. The results showed that ATRA/As2O3 combination therapy yielded a high CR rate of 93.3% and a significantly shorter time to enter CR (median: 31 days; range: 18–59 days) compared to the ATRA-based regimen (n = 72; median: 39 days; range: 25–62 days). With the ATRA/As2O3 combination for CR maintaining, regardless of the way by which CR was attained, the relapse-free survival was significantly better than with an ATRA plus cytotoxic chemotherapy regimen (92.9 ± 3.2% vs. 72.4 ± 7.6%, for the 3-year Kaplan-Meier estimate of relapse-free survival). The drug toxicity profile showed that with the use of As2O3, the incidence of hepatotoxicity was obviously high during remission induction but decreased significantly during postremission treatment. We conclude that APL patients may benefit from the early use of the combination of ATRA and As2O3, in either remission induction or consolidation/maintenance.

Decreased 5-hydroxymethylcytosine levels are associated with TET2 mutation and unfavorable overall survival in myelodysplastic syndromes

Abstract The clinical significance and mechanisms of TET2 are not well defined in myeloid malignancies. We detected TET2 mutations and assayed its catalyzing conversion product 5-hydroxymethylcytosine (5-hmC) in 61 Chinese patients with MDS. Ten patients were identified to have TET2 mutations (16.4%). 5-hmC levels in patients with MDS with TET2 mutations were significantly lower than in those without mutations, and CD34+ cells of patients with MDS exhibited a lower 5-hmC content than that of controls. TET2 expression and 5-hmC in patients with MDS with P15 methylation were both significantly lower than in those without P15 methylation. We did not observe a correlation between TET2 mutations and overall survival (OS) in MDS. Interestingly, we found that patients with MDS with higher 5-hmC levels or in lower risk groups of the Revised International Prognostic Scoring System (IPSS-R) had a longer overall survival, suggesting that 5-hmC levels may be a new molecular marker for prognostic assessment of MDS and that revised IPSS criteria are also applicable to the risk categories of Chinese patients with MDS.

Deregulation of Mitochondrial ATPsyn-β in Acute Myeloid Leukemia Cells and with Increased Drug Resistance

The mechanisms underlying the development of multidrug resistance in acute myeloid leukemia are not fully understood. Here we analyzed the expressions of mitochondrial ATPsyn-β in adriamycin-resistant cell line HL-60/ADM and its parental cell line HL-60. Meanwhile we compared the differences of mitochondrial ATPsyn-β expression and ATP synthase activity in 110 acute myeloid leukemia (AML, non-M3) patients between relapsed/refractory and those in remission. Our results showed that down-regulation of ATPsyn-β expression by siRNA in HL-60 cells increased cell viability and apoptotic resistance to adriamycin, while up-regulation of mitochondrial ATPsyn-β in HL-60/ADM cells enhanced cell sensitivity to adriamycin and promoted apoptosis. Mitochondrial ATPsyn-β expression and ATP synthase activity in relapsed/refractory acute myeloid leukemia patients were downregulated. This downregulated ATPsyn-β expression exhibited a positive correlation with the response to adriamycin of primary cells. A lower expression of ATPsyn-β in newly diagnosed or relapsed/refractory patients was associated with a shorter first remission duration or overall survival. Our findings show mitochondrial ATPsyn-β plays an important role in the mechanism of multidrug resistance in AML thus may present both a new marker for prognosis assessment and a new target for reversing drug resistance.

An intron mutation in the ACVRL1 may be associated with a transcriptional regulation defect in a Chinese family with hereditary hemorrhagic telangiectasia.

Purpose To identify a novel pathogenic gene mutation present in a Chinese family with hereditary hemorrhagic telangiectasia (HHT) and to determine if an intron mutation may influence the transcriptional activity of the ACVRL1 gene. Methods HHT family members were ascertained following the presentation of proband and involved subjects. All family members (n = 5) and 113 healthy individuals were genotyped for the variant in intron 6 c.772+27G>C of ACVRL1 gene. The genomic structure of ACVRL1 in affected HHT patients and healthy individuals was determined by long range PCR and sequencing. The expression of ACVRL1 mRNA and protein in patients with HHT was evaluated using real-time polymerase chain reaction and immunoblot analysis. Luciferase activity assay and electrophoretic mobility shift assay (EMSA) were performed to uncover the mechanism of intron-related transcriptional regulation. Results Only one novel mutation in intron 6 (c.772+27G>C) of ACVRL1 gene, no other mutation, abnormal splice, gross genomic deletion or rearrangement was found in this HHT2 family. Compared with healthy individuals, ACVRL1 mRNA and protein were significantly decreased in affected HHT2 individuals. Luciferase activity assay demonstrated that the transcriptional activity of the mutated ACVRL1 was significantly lower than that of the wild-type of intron 6; EMSA results showed that intron 6 c.772+27G>C mutation was able to inhibit the binding of transcriptional factor Sp1. Conclusions A novel intron mutation in ACVRL1 gene is associated with familial HHT2. The mechanisms may be involved in the down-regulation of ACVRL1 gene transcription.

GTF2I-RARA is a novel fusion transcript in a t(7;17) variant of acute promyelocytic leukaemia with clinical resistance to retinoic acid

PAF is an employee of Xencor, Inc. AK, KAB, HF, MRS, and AY have no conflicts of interest to disclose. Anita Kumar Kristie A. Blum Henry C. Fung Mitchell R. Smith Paul A. Foster Anas Younes Memorial Sloan-Kettering Cancer Center, New York, NY, Ohio State University, Columbus, OH, Rush University Medical Center, Chicago, IL, Fox Chase Cancer Center, Philadelphia, PA, Xencor, Inc., Monrovia, CA, and MD Anderson Cancer Center, Houston, TX, USA. E-mail: younesa@mskcc.org

Coexistence of JAK2V617F mutation and BCR-ABL1 transcript in two Chinese patients with chronic myelogenous leukemia.

phils, 9% basophils, 4% nucleated red cells and 5% lymphocytes, Hb of 6.1 g/dl and PLT of 718 ! 10 9 /l. The neutrophil alkaline phosphatase score was 16.5 (reference range: 30.0–70.0). A bone marrow biopsy showed megakaryocytic hyperplasia and marked marrow reticulin fibrosis. There was a positive JAK2V617F mutation according to an allele-specific PCR assay. The BCR-ABL1 specific fusion signal was detected in bone marrow cells by fluorescence in situ hybridization (FISH), and the percentage of positive BCR-ABL1 was 90.9% (60/66 cells). Major BCR-ABL1 transcripts were found above the threshold levels ( BCR-ABL1/ABL1 ratio: 0.11%) by quantitative realtime PCR. The patient was diagnosed as having coexistent myelofibrosis and chronic-phase CML. Because of economic factors, the patient chose HU therapy at a dose of 1.0 g/day, thalidomide at 100 mg per night and aspirin (100 mg/day). Afterwards, due to a severe lung infection complicated by type I pulmonary insufficiency, the patient refused further treatment and was discharged voluntarily. Patient 2 was a 46-year-old female being evaluated for acute diarrhea who was found to have thrombocythemia in April 2010. Physical examination was unremarkable. Her Hb was 7.3 g/dl, PLT was 2,273 ! 10 9 /l and WBC was 17.1 ! 10 9 /l, with a differential count of 73.3% neutrophils, 1.5% eosinophils and 1.3% basophils. The neutroEssential thrombocythemia (ET), myelofibrosis, polycythemia vera (PV) and chronic myeloid leukemia (CML) belong to the category of myeloproliferative dis orders (MPDs). Although these diseases share some similar clinical features and clonal neoplastic proliferation in bone marrow, the BCR-ABL1 fusion transcript is still an important molecular marker in CML, while the mutation of the Janus kinase 2 gene (JAK2V617 F) has been reported to be associated with Philadelphia chromosome (Ph)-negative MPDs [1] . The general viewpoint is that a positive BCRABL1 transcript and the JAK2V617F mutation are exclusive to each other. Here, we report two patients with CML in whom the JAK2V617F mutation was positive. Patient 1 was a 77-year-old male who presented with splenomegaly. Twelve years prior to this presentation, he was diagnosed with PV, but he did not receive any special treatment. Then, in 2005, he had increased platelet counts, and bone marrow aspiration yielded a dry tap. The patient was administered hydroxyurea (HU) and aspirin irregularly. In 2007, he suffered a cerebral infarction and was found to have an increase in WBC of 26 ! 10 9 /l and was given HU at a dose of 2.0 g/day for more than 1 month. Because of pronounced splenomegaly, the patient was referred to our hospital in May 2010. Physical examination revealed remarkable splenomegaly (2.5 cm under the umbilicus). Laboratory tests showed a WBC of 19.9 ! 10 9 /l, with a differential count of 1% myeloblasts, 2% promyelocytes, 5% myelocytes, 3% metamyelocytes, 5% eosinoReceived: June 23, 2011 Accepted after revision: August 9, 2011 Published online: October 19, 2011

Thalidomide Effects in Patients with Hereditary Hemorrhagic Telangiectasia During Therapeutic Treatment and in Fli-EGFP Transgenic Zebrafish Model

Background:Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by recurrent epistaxis, mucocutaneous telangiectasia, and arteriovenous malformations. The efficacy of traditional treatments for HHT is very limited. The aim of this study was to investigate the therapeutic role of thalidomide in HHT patients and the effect in FLI-EGFP transgenic zebrafish model. Methods:HHT was diagnosed according to Shovlin criteria. Five HHT patients were treated with thalidomide (100 mg/d). The Epistaxis Severity Score (ESS), telangiectasia spots, and hepatic computed tomography angiography (CTA) were used to assess the clinical efficacy of thalidomide. The Fli-EGFP zebrafish model was investigated for the effect of thalidomide on angiogenesis. Dynamic real-time polymerase chain reaction assay, ELISA and Western blotting from patients peripheral blood mononuclear cells and plasma were used to detect the expression of transforming growth factor beta 3 (TGF-&bgr;3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment. Results:The average ESS before and after thalidomide were 6.966 ± 3.093 and 1.799 ± 0.627, respectively (P = 0.009). The “telangiectatic spot” on the tongue almost vanished; CTA examination of case 2 indicated a smaller proximal hepatic artery and decreased or ceased hepatic artery collateral circulation. The Fli-EGFP zebrafish model manifested discontinuous vessel development and vascular occlusion (7 of 10 fishes), and the TGF-&bgr;3 mRNA expression of five patients was lower after thalidomide therapy. The plasma VEGF protein expression was down-regulated in HHT patients. Conclusions:Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-&bgr;3 and VEGF in HHT patients. It also leads to vascular remodeling in the zebrafish model.

Thrombotic thrombocytopenic purpura as initial and major presentation of multiple myeloma

To the Editor, Thrombotic thrombocytopenic purpura (TTP) is a rare but fatal disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic disturbances, renal abnormalities and fever. It is now recognized that congenital and acute acquired TTP are due to a deficiency of von Willebrand factor (vWF) cleaving protein, also known as ADAMTS13, which inhibits vWF-dependent platelet aggregation [1]. Acquired thrombotic microangiopathy comprises about 40 % of all cases of TTP, including pregnancy, drugs, HIV infection, hematopoietic stem cell transplantation, autoimmune diseases or malignancies. The malignancies associated with TTP are usually solid tumors. TTP as first manifestation of multiple myeloma is very rare. To our knowledge, there is only one documented case in the literature [2]. Here we report a case of multiple myeloma, which initially presented with TTP. A 61-year-old male presented to local county hospital with a 2-month history of fatigue, jaundice and chest pain. Laboratory tests showed white blood cell of 4.9 9 10/L, hemoglobin (Hb) of 74 g/L, platelet count of 53 9 10/L, total bilirubin (TBIL) of 100.1 lmol/L (reference range 5.0–16.7 lmol/L) and direct bilirubin (DBIL) of 11.8 lmol/ L (0–5.7 lmol/L). On the third day after admission, he developed confusion, disorientation and delirium at night. Subsequently, he was transferred to our hospital for further diagnosis and treatment on December 8, 2010. Physical examination showed pale face, jaundice and somnolent with loss of orientation. No focal neurological deficit was present. Laboratory tests showed Hb of 75 g/L, platelet count of 52 9 10/L, reticulocytes of 11 %, TBIL of 103.9 lmol/L (reference range 5.1–17.1 lmol/L), DBIL of 51.8 lmol/L (0.0–6.0 lmol/L), serum lactate dehydrogenase (LDH) of 447.8l/L(reference range 109.0–245.0l/L) and blood urea nitrogen (BUN) of 8.2 mmol/L (reference range 2.90–7.14 mmol/L), creatinine(Cr) of 144.8 mmol/L (reference range 40.0–133.0 mmol/L). Serum protein electrophoresis was normal. Serum immunoglobulins, calcium and urinary Bence Jones protein was in normal range. The coagulation tests (prothrombin time, activated partial thromboplastin time, fibrinogens) and blood ammonia were within normal limits. Direct Coomb’s test was negative. Tests for HIV, Hepatitis B, Hepatitis C and autoimmune serologies were negative. Cranial computed tomography and magnetic resonance imaging remained normal. Bone marrow aspiration showed increased plasma cells of 50 % with matureappearing. His blood smear displayed schistocytes of 64/1,000. ADAMTS-13 activity was 29.9 % (reference 40–140 %). A diagnosis of TTP was made. Plasmapheresis was emergently initiated followed by fresh frozen plasma infusions of 300 mL daily with concomitant 10 mg intravenous dexamethasone per day for 6 days, then the patient was on regimen of oral prednisone of 45 mg per day with a gradual reduction. Dramatic response was observed after three plasma exchanges with 1.5 times of total plasma volumes. Clearness of mind, improvement in his chest pain, reticulocytes decreased to 3.9 % and TBIL, DBIL, LDH became normal. His platelet count was 70 9 10/L, and Hb was 80 g/L. Because the presence of plasma cell infiltration in the patient’s bone marrow biopsy, the diagnosis of multiple myeloma was highly suspected. Tests for monoclonal paraprotein were repeated performed, and the results were Xiang Xiao and Hai-ying Zhong contributed equality to this study.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Ameliorates Hippocampal Synaptic Impairment after Transient Global Ischemia

Recent studies have found that administration of stem cells or extracellular vehicles (EVs) derived from stem cells exert neuroprotective effects after transient global ischemia. However, the underlying mechanisms of this effect remain unclear, especially at the level of synaptic functions. In this study, we compared the suppressive effects on cyclooxygenase-2 (COX-2) upregulation by EVs derived from bone marrow mesenchymal stem cells (BMSC-EV), adipose tissue MSC (AdMSC-EV) and serum (serum-EV). Then we examined whether BMSC-EVs could restore functional integrity of synaptic transmission and plasticity. Mice were randomly assigned to four groups: sham, sham with EV treatment, ischemia and ischemia with EV treatment. EVs were administered by intracerebroventricular injection (ICVI). We examined the consequence of transient global ischemia on pre- and post-synaptic functions of the hippocampal CA3-CA1 synapses at basal level, and long-term potentiation (LTP), an activity-dependent form of synaptic plasticity. Then we tested the therapeutic effects of EVs on these synaptic deficits. Meanwhile, Morris water maze (MWM) test was performed to examine the efficacy of EVs in rescuing ischemia-induced impairments in spatial learning and memory. EV treatment significantly restored impaired basal synaptic transmission and synaptic plasticity, and improved spatial learning and memory compared with the control group. In addition, EVs significantly inhibited ischemia-induced pathogenic expression of COX-2 in the hippocampus. EVs exert ameliorating effects on synaptic functions against transient global cerebral ischemia, which may be partly attributed to suppression of COX-2 pathogenic expression.