c-myb hyperactivity leads to myeloid and lymphoid malignancies in zebrafish.

The c-MYB transcription factor is a key regulator of hematopoietic cell proliferation and differentiation, and dysregulation of c-MYB activity often associates with various hematological disorders. Yet, its pathogenic role remains largely unknown due to lack of suitable animal models. Here, we report a detail characterization of a c-myb-gfp transgenic zebrafish harboring c-Myb hyperactivity (named c-mybhyper). This line exhibits abnormal granulocyte expansion that resembles human myelodysplastic syndrome (MDS) from embryonic stage to adulthood. Strikingly, a small portion of c-mybhyper adult fish develops acute myeloid leukemia-like or acute lymphoid leukemia-like disorders with age. The myeloid and lymphoid malignancies in c-mybhyper adult fish are likely caused by the hyperactivity of c-myb, resulting in the dysregulation of a number of cell-cycle-related genes and hyperproliferation of hematopoietic precursor cells. Finally, treatment with c-myb target drug flavopiridol can relieve the MDS-like symptoms in both c-mybhyper embryos and adult fish. Our study establishes a zebrafish model for studying the cellular and molecular mechanisms underlying c-Myb-associated leukemogenesis as well as for anti-leukemic drug screening.

Cell-structure specific necrosis by optical-trap induced intracellular nuclear oscillation.

A drug-free procedure for killing malignant cells in a cell-type specific manner would represent a significant breakthrough for leukemia treatment. Here, we show that mechanically vibrating a cell in a specific oscillation condition can significantly promote necrosis. Specifically, oscillating the cell by a low-power laser trap at specific frequencies of a few Hz was found to result in increased death rate of 50% or above in different types of myelogenous leukemia cells, while normal leukocytes showed very little response to similar laser manipulations. The alteration of cell membrane permeability and cell volume, detected from ethidium bromide staining and measurement of intracellular sodium ion concentration, together with the observed membrane blebbing within 10min, suggest cell necrosis. Mechanics modelling reveals severe distortion of the cytoskeleton cortex at frequencies in the same range for peaked cell death. The disruption of cell membrane leading to cell death is therefore due to the cortex distortion, and the frequency at which this becomes significant is cell-type specific. Our findings lay down a new concept for treating leukemia based on vibration induced disruption of membrane in targeted malignant cells.

Establishment of a congenital amegakaryocytic thrombocytopenia model and a thrombocyte-specific reporter line in zebrafish.

Mutations in the human myeloproliferative leukemia (MPL) protein gene are known to cause congenital amegakaryocytic thrombocytopenia (CAMT). The prognosis of this heritable disorder is poor and bone marrow transplantation is the only effective treatment. Here, by using the TALEN (transcription activator-like effector nuclease) technology, we created a zebrafish mpl mutant to model human CAMT. Disruption of zebrafish mpl lead to a severe reduction in thrombocytes and a high bleeding tendency, as well as deficiencies in adult hematopoietic stem/progenitor cells. We further demonstrated that thrombocytopenia in mpl mutant zebrafish was caused by impaired Tpo/Mpl/Jak2 signaling, resulting in reduced proliferation of thrombocyte precursors. These results indicate that mpl mutant zebrafish develop thrombocytopenia resembling the human CAMT. To utilize fully zebrafish to study thrombocyte biology and thrombocytopenia disorders, we generated a transgenic reporter line Tg(mpl:eGFP)smu4, in which green fluorescent protein (GFP) expression was driven by the mpl promoter. Detailed characterization of Tg(mpl:eGFP)smu4 fish confirmed that the thrombocyte lineage was specifically marked by GFP expression. In conclusion, we generated the first transmissible congenital thrombocytopenia zebrafish model mimicking human CAMT and a thrombocyte-specific transgenic line. Together with Tg(mpl:eGFP)smu4, mpl mutant zebrafish provide a useful tool for drug screening and study of thrombocytopoiesis.

Next-generation sequencing with a myeloid gene panel in core-binding factor AML showed KIT activation loop and TET2 mutations predictive of outcome.

Clinical outcome and mutations of 96 core-binding factor acute myeloid leukemia (AML) patients 18-60 years old were examined. Complete remission (CR) after induction was 94.6%. There was no significant difference in CR, leukemia-free-survival (LFS) and overall survival (OS) between t(8;21) (N=67) and inv(16) patients (N=29). Univariate analysis showed hematopoietic stem cell transplantation at CR1 as the only clinical parameter associated with superior LFS. Next-generation sequencing based on a myeloid gene panel was performed in 72 patients. Mutations in genes involved in cell signaling were associated with inferior LFS and OS, whereas those in genes involved in DNA methylation were associated with inferior LFS. KIT activation loop (AL) mutations occurred in 25 patients, and were associated with inferior LFS (P=0.003) and OS (P=0.001). TET2 mutations occurred in 8 patients, and were associated with significantly shorter LFS (P=0.015) but not OS. Patients negative for KIT-AL and TET2 mutations (N=41) had significantly better LFS (P<0.001) and OS (P=0.012) than those positive for both or either mutation. Multivariate analysis showed that KIT-AL and TET2 mutations were associated with inferior LFS, whereas age ⩾40 years and marrow blast ⩾70% were associated with inferior OS. These observations provide new insights that may guide better treatment for this AML subtype.

Quantification of circulating Epstein-Barr virus DNA in NK/T-cell lymphoma treated with the SMILE protocol: diagnostic and prognostic significance.

Circulating Epstein-Barr virus (EBV) DNA is a biomarker of EBV-associated malignancies. Its significance in natural killer/T-cell lymphoma treated with the novel regimen SMILE was investigated. EBV DNA was quantified with a World Health Organization EBV standard in 910 plasma samples collected during 230 courses of SMILE in 56 patients. Median presentation EBV DNA was 1900 (0-1.4 × 10(7)) IU/ml. Presentation EBV DNA was significantly associated with tumor load and treatment response. To examine lymphoma chemosensitivity, EBV DNA changes after SMILE were evaluated. EBV DNA after SMILE (I) significantly correlated with tumor load and treatment response. Two dynamic parameters were further analyzed: negative EBV DNA after SMILE (I) and EBV DNA change patterns during treatment (A: persistently undetectable; B: persistently detectablepresentation). Negative EBV DNA after SMILE (I) and pattern A EBV DNA change significantly correlated with lower tumor load and superior outcome. Multivariate analysis involving presentation features, international prognostic index (IPI), Korean prognostic score and EBV DNA parameters showed that negative EBV DNA after SMILE (I) had the most significant impact (P<0.001) on overall survival and pattern A EBV DNA change had the most significant impact (P=0.002) on disease-free survival. Presentation EBV DNA, IPI and Korean prognostic scores were not independent prognostic factors.

Antifungal drug usage in haematologic patients during a 4-year period in an Asian university teaching hospital.

BACKGROUND: Invasive fungal disease (IFD) is an important problem complicating the therapy of haematologic patients. AIM: This study aimed to provide data on the epidemiology of IFD in an Asian teaching hospital, as well as the prescription practice of antifungal drugs. METHOD: We conducted a retrospective review of 275 haematologic patients who were prescribed antifungal drugs in a 4-year period (2007-2010), of whom 130 (47%) had undergone haematopoietic stem cell transplantation. RESULTS: Antifungal prophylaxis with either fluconazole or itraconazole was given in 214 patients (78%). There were 414 prescriptions of antifungal drugs (including liposomal amphotericin B, voriconazole, caspofungin, micafungin, anidulafungin), of which 361 prescriptions were empirical. There were 14 patients with proven IFD, 11 of whom had breakthrough infection while on itraconazole prophylaxis. Interestingly, seven of these cases were due to infection by itraconazole-sensitive candida. CONCLUSION: These results provide important epidemiologic data necessary for the formulation of strategies for prevention and treatment of IFD in Asian patients.

FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia.

Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is a gain-of-function mutation common in acute myeloid leukaemia (AML). It is associated with inferior prognosis and response to chemotherapy. Single base mutations at the FLT3 tyrosine kinase domain (TKD) also leads to a gain of function, although its prognostic significance is less well defined because of its rarity. The clinical benefits of FLT3 inhibition are generally limited to AML with FLT3-ITD. However, responses are transient and leukaemia progression invariably occurs. There is compelling evidence that leukaemia clones carrying both ITD and TKD mutations appear when resistance to FLT3 inhibitors occurs. Interestingly, the emergence of double ITD and TKD mutants can be recapitulated in vitro when FLT3-ITD+ leukaemia cell lines are treated with mutagens and FLT3 inhibitors. Furthermore, murine xenotransplantation models also suggest that, in some cases, the FTL3-ITD and TKD double mutants actually exist in minute amounts before treatment with FLT3 inhibitors, expand under the selection pressure of FLT3 inhibition and become the predominant resistant clone(s) during the drug-refractory phase. On the basis of this model of clonal evolution, a multipronged strategy using more potent FLT3 inhibitors, and a combinatorial approach targeting both FLT3-dependent and FLT3-independent pathways, will be needed to improve outcome.

Mental health recovery for psychiatric inpatient services: perceived importance of the elements of recovery.

OBJECTIVES. To develop a questionnaire for measuring the perceived importance of the elements of mental health recovery in psychiatric inpatients in Hong Kong and to test the psychometric properties of the questionnaire. METHODS. Thematic content analysis of identified literature on mental health recovery was performed to identify the elements related to mental health recovery. A questionnaire was developed to assess the perceived importance of the identified elements. An expert panel was set up to evaluate the content validity and patient focus group's face validity of the questionnaire. Participants were recruited from medium-stay and rehabilitation wards of Castle Peak Hospital. RESULTS. A total of 101 psychiatric inpatients completed the questionnaire, the majority of whom suffered from schizophrenia (75%). Having meaning in life was rated by 91% of the participants as an important element of recovery, followed by hope (86%) and general health and wellness (85%). Cronbach's alpha for internal consistency was 0.91. Explorative factor analysis yielded 7 factors and intraclass correlation coefficients revealed a fair-to-good test-retest reliability. CONCLUSIONS. The results supported the psychometric properties of the questionnaire for measurement of mental health recovery and serve as a basis for the future development of recovery-oriented services in the psychiatric inpatient settings in this locality.

Indolent T-cell large granular lymphocyte leukaemia after haematopoietic SCT: a clinicopathologic and molecular analysis.

Four women and three men after allogeneic (n=4) and autologous (n=3) haematopoietic SCT (HSCT) were observed to have an increase in T-cell large granular lymphocytes (T-LGLs) of CD3+CD8+ phenotype for a median of 41 (15-118) months. Clonal rearrangement of the T-cell receptor gene was verified by two PCR techniques and direct DNA sequencing, confirming that the cases were neoplastic and therefore classifiable as T-LGL leukaemia. In the allogeneic HSCT cases, T-LGL leukaemia was derived from donor T cells in three patients, as shown by DNA chimerism analysis, and recipient T cells in one patient who had graft failure previously. None of the patients showed cytopenia, autoimmune phenomenon or organ infiltration, which were features typical of de novo T-LGL leukaemia. Six patients had remained asymptomatic with stable large granular lymphocyte counts. One patient died from cerebral relapse of the original lymphoma. T-LGL leukaemias occurring post-HSCT are distinct from de novo T-LGL leukaemia and may have a different pathogenesis and clinical course. Patients did not require specific treatment, and the disease remained stable for long periods.

Effects of azithromycin in bronchiolitis obliterans syndrome after hematopoietic SCT--a randomized double-blinded placebo-controlled study.

Bronchiolitis obliterans syndrome (BOS) is an important complication after hematopoietic SCT (HSCT). Recent observations suggested that azithromycin might improve lung function in BOS after HSCT. We conducted a randomized double-blinded placebo-controlled study on azithromycin in patients with BOS after HSCT. The treatment group (n=10) received oral azithromycin 250 mg daily while the control group (n=12) received placebo daily for 12 weeks. Respiratory symptoms were assessed by the St George Respiratory Questionnaires and spirometry at baseline (drug commencement), 1, 2, 3 (drug cessation) and 4 months (1 month after drug cessation). There was no significant difference in the baseline demographic characteristics between the treatment and the control groups in age, gender, time from HSCT to BOS, time since diagnosis of BOS, chronic GVHD, baseline respiratory symptom scores and baseline forced expiratory volume in 1 s (FEV(1)). Throughout and after 3 months of treatment, there were no significant changes in respiratory symptom scores and FEV(1) measurements between the treatment and the control groups. In conclusion, there was no significant benefit of 3 months of oral azithromycin on the respiratory symptoms and lung function in patients with relatively late BOS after HSCT in this randomized placebo-controlled study.

A DEAB-sensitive aldehyde dehydrogenase regulates hematopoietic stem and progenitor cells development during primitive hematopoiesis in zebrafish embryos.

Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs), its function during hematopoiesis was unclear. Here, we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB, 1 µmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl, lmo2), erythropoiesis (gata1, α- and ß-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all, 14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos, of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs, but none of them, even in combined aldh1a2 and aldh1a3 knockdown, recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion, DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined.

[Rosiglitazone and all-trans retinoic acid inhibit human myeloma cell proliferation via apoptosis signaling pathway modulation].

OBJECTIVE: To investigate the effects of artificial ligand of peroxisome proliferators activated receptors (PPARs), rosiglitazone (RGZ) and all trans-retinoic acid (ATRA) on human myeloma cell line growth in vitro and in vivo. METHODS: U266 and RPMI 8226 cells were treated with different concentration of RGZ in the presence or absence of ATRA and the results were studied by 3H-TdR thymidine incorporation (for cells proliferation), Annexin V-PI staining and caspase-3 activity assay (for cells apoptosis), RT-PCR (for FLIP, XIAP and survivin mRNA expression), and tumor formation test in BALB/c nude mice. RESULTS: Exposure to RGZ induced proliferation inhibition in a dose-dependent manner in both U266 (r = 0.991, P < 0.01) and RPMI 8226 cells (r = 0.961, P < 0.01). A combination of RGZ with ATRA could enhance the inhibition effect (P < 0.001 in U266, P < 0.01 in RPMI8226). 10 micromol/L of RGZ induced apoptosis of (9.8 +/- 1.7)% in U266 cells and (10.7 +/- 3.3)% in RPMI8226 cells, in a time and dose dependent manner and combined with ATRA intensified the apoptosis induction effects (P < 0.01 in both cell lines). The FLIP, XIAP and survivin mRNAs were expressed in both cell lines and their levels decreased significantly after cultured with RGZ. The addition of RGZ + ATRA in the culture further decreased the levels. Caspase-3 activity increased substantially with the increase of RGZ concentration and the addition of RGZ + ATRA in the culture medium showed similar synergism effect on caspase-3 activation (P < 0.01). The xenograft of U266 cells in BALB/c nude mice were inhibited by RGZ and so did more by the combination of RGZ and ATRA (P < 0.01). CONCLUSION: The down-regulation of FLIP, XIAP and Survivin induced by RGZ can activate caspase-3, whereby induced apoptosis and proliferation inhibition in myeloma cells. ATRA can enhance these effects of RGZ.

The role of survivin2 in primitive hematopoiesis during zebrafish development.

Survivin is an inhibitor of apoptosis and its role in embryonic development is not completely understood. In zebrafish, survivin undergoes gene duplication. Survivin1 (sur1) has been shown to mediate angiogenesis but not hematopoiesis. In this study, we examined survivin2 (sur2) with particular reference to its role in primitive hematopoiesis during zebrafish development. sur2 was expressed predominantly in the intermediate cell mass (ICM, site of primitive hematopoiesis). Morpholino (MO) targeting at intron1-exon2 junction of sur2 significantly reduced green fluorescent protein(+) (erythroid) cell population in transgenic Tg (gata1:gfp) embryos at 18 h post-fertilization (h.p.f.; wild type: 4.49+/-0.15%; Sur2(MO) embryos: 2.22+/-0.12%, P=0.02). Molecular targeting was confirmed by reverse transcription-PCR and MO specificity by successful sur2 mRNA rescue. sur2 MO also downregulated genes associated with hematopoietic stem cells (scl, lmo2), erythroid (gata1, alpha- and beta-embryonic hemoglobins) as well as early (pu.1) and late (mpo, l-plastin) myelomonocytic lineages at 12 and 18 h.p.f. This was associated with an increase in apoptosis in the ICM and alteration of cell-cycle status of erythroid cells. Both effects were caspase dependent. In conclusion, sur2 is important in maintaining hematopoietic stem and lineage committed cells during zebrafish development, by virtue of its antiapoptotic activity in a caspase dependent and cell autonomous fashion.