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.

Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential.

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43), acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases, a high SSC(lo)ALDH(br) cell population was identified (ALDH(+)AML) (median: 14.89%, range: 5.65-48.01%), with the majority of the SSC(lo)ALDH(br) cells coexpressing CD34(+). In another 29 cases, there was undetectable (n=23) or rare (< or =5%) (n=6) SSC(lo)ALDH(br) population (ALDH(-)AML). Among other clinicopathologic variables, ALDH(+)AML was significantly associated with adverse cytogenetic abnormalities. CD34(+) BM cells from ALDH(+)AML engrafted significantly better in NOD/SCID mice (ALDH(+)AML: injected bone 21.11+/-9.07%; uninjected bone 1.52+/-0.75% vs ALDH(-)AML: injected bone 1.77+/-1.66% (P=0.05); uninjected bone 0.23+/-0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSC(lo)ALDH(br) population (median: 2.92%, range: 0.92-5.79%), but none of the ALL cases showed this fraction. In conclusion, SSC(lo)ALDH(br) cells in ALDH(+)AML might denote primitive LSC and confer an inferior prognosis in patients.

Safety of vaccinating sibling donors with live-attenuated varicella zoster vaccine before hematopoietic stem cell transplantation.

Reactivation of varicella zoster virus (VZV), clinically manifested as herpes zoster (HZ) is a common complication after hematopoietic stem cell transplantation (HSCT). The optimum prophylaxis for this disease has not been defined. In this study, we examined the effects of vaccinating donors with a live-attenuated vaccine with particular reference to their immune responses and the outcome of HSCT patients. Forty prospective HLA-matched sibling donors were vaccinated before HSCT. There were humoral immune responses in both sero-positive (P<0.01) and sero-negative (P=0.058) donors. Cellular immune response was assayed in 26 donors. Significant correlation was observed between cellular immune responses as enumerated by thymidine incorporation and interferon gamma secretion (P<0.001) and the latter was used in subsequent analyses. Significant response was observed in sero-negative (6/26) and a group of sero-positive (13/26) donors while 7/26 sero-positive donors showed no response. Thirty-four HSCT were performed. These patients have a lower, albeit insignificant, risk of HZ compared with historical controls and only 3/34 patients developed single dermatomal HZ at 6, 9 and 28 months after HSCT. No patients developed VZV-related mortality. Vaccinating donors with live-attenuated VZV vaccine was safe, but whether it confers a significant protection to the patients would require further study.

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.

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.

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.

Polyoma BK virus and haemorrhagic cystitis in haematopoietic stem cell transplantation: a changing paradigm.

Haemorrhagic cystitis (HC) is a distinct clinical disorder of multiple aetiologies. It is characterized by painful haematuria due to haemorrhagic inflammation of the urinary bladder mucosa. In allogeneic haematopoietic stem cell transplantation (HSCT), HC occurring before engraftment is mostly transient and self-limiting, whereas that after engraftment is severe and sometimes life-threatening. Pre- and post-engraftment HC represent distinct disorders with different aetiologies and treatment implications. Recent data suggest that reactivation of the polyoma BK virus (BKV) plays a pivotal role in post-engraftment HC. Urotoxicity of the conditioning regimen and alloimmune reaction accompanying graft-versus-host disease (GVHD) upon engraftment are also important pathogenetic factors. Based on data from BKV studies, we propose that HC may be divided into three phases. In the first phase, the conditioning regimen damages uroepithelial cells, providing a milieu for BKV replication. In the second phase, unchecked uroepithelial BKV replication leads to BK viruria. In the last phase after engraftment, alloimmunity against BKV-infected uroepithelial cells leads to HC. The quinolone antibiotics suppress BKV replication in vivo and in vitro, suggesting that their prophylactic use may prevent the occurrence of HC.

Clinicopathological features and risk factors of clinically overt haemorrhagic cystitis complicating bone marrow transplantation.

Haemorrhagic cystitis (HC) is an important complication after bone marrow transplantation (BMT). Overt HC (grade > or =2, gross haematuria, clot retention and impairment of renal function), clinically more important than mild and occult HC (grade 1, microscopic haematuria), leads to substantial morbidity and occasional mortality. We retrospectively analyzed 32 cases of clinically overt HC from a series of 236 BMT patients. Significant risk factors included the use of busulphan during conditioning, allogeneic BMT and acute GVHD. Logistic regression showed GVHD to be the most important risk factor. According to the time of engraftment, HC could be divided into pre- and post-engraftment subtypes. Pre-engraftment HC was brief, not more severe than grade 2, and subsided with supportive treatment. In contrast, post-engraftment HC was protracted, often of grade > or =3, associated with severe GVHD, and required surgical intervention in many cases. Polyoma BK viruria, but not adenoviruria, could be demonstrated in both types of HC. The increased severity and association with GVHD of post-engraftment HC suggested that attack of urothelium by immunocompetent cells, possibly directed against BK viral antigens, might play a pathogenetic role.

Macrophage migratory inhibitory factor (MIF) expression in acute graft-versus-host disease (GVHD) in allogeneic hemopoietic stem cell transplant recipients.

Graft-versus-host disease (GVHD) is a major complication after hemopoietic stem cell transplantation (HSCT), but its pathogenesis remains uncertain. Macrophage migratory inhibitory factor (MIF) is an important mediator in the allo-immune reaction during renal transplantation, yet its role in hemopoietic stem cell transplantation (HSCT) remains unexplored. This study investigated the potential role of MIF in acute graft-versus-host disease (aGVHD) following allogeneic HSCT. Forty-six randomly selected patients undergoing autologous or allogeneic HSCT were studied. Immunohistochemistry and in situ hybridization were performed to examine tissue MIF mRNA and protein expression on skin and colonic biopsy specimens. The associated T cell and macrophage activation was also studied by immunohistochemical studies. A semi-quantitative method was used to assess MIF staining, as well as T cell and macrophage staining. Serial blood samples were analyzed by ELISA for serum MIF levels. Immunohistochemistry and in situ hybridization performed in 15 skin and 19 colonic biopsies from 17 patients who developed moderate to severe aGVHD showed a significant increase in MIF mRNA and protein expression compared with normal controls (seven skin and five colonic biopsies). MIF was localized within the epidermis and the vascular area of skin, but diffusely expressed in the entire thickness of colon. Macrophage and T lymphocyte infiltration was confined to areas of strong MIF expression. Serial analysis by ELISA showed that only patients who developed aGVHD (n = 19) exhibited an increase (two- to three-fold) in serum MIF during HSCT, but not in the allogeneic HSCT recipients without aGVHD (n = 7) or those who received autologous HSCT (n = 8). In 14 out of 19 patients, serum MIF peaked before the onset of aGVHD. Local and systemic up-regulation of MIF expression is associated with the occurrence of acute GVHD. Its pathogenetic role remains to be further determined.

Antirecipient helper and cytotoxic T-cell frequencies in bone marrow transplantation.

We assayed helper T-lymphocyte precursor frequencies (HTLPf), interferon (IFN)-gamma-producing cell frequencies (IFN-gammaPf) and CTL precursor frequencies (CTLPf) to see if they could predict the severity of acute graft-versus-host disease (aGVHD) and disease relapse after transplantation. In all, 48 bone marrow transplantation (BMT) patients and their HLA-identical sibling (n=29) or matched unrelated donors (MUD) (n=19) were recruited. HTLPf, IFN-gammaPf and CTLPf were measured using a limiting dilution assay (LDA). Patients were followed prospectively to assess the severity of aGVHD and the status of the primary disease after BMT. High (>5 x 10(-6)) HTLPf, CTLPf and IFN-gammaPf were significantly associated with the occurrence and severity of aGVHD in patients who received transplants from HLA-identical sibling. Among patients receiving BMT from MUD, HTLPf and CTLPf, but not IFN-gammaPf, were associated with aGVHD. Five patients had disease relapse post-BMT and the risk was not significantly associated with HTLPf, CTLPf or IFN-gammaPf. Patients with high (>5 x 10(-6)) HTLPf, IFN-gammaPf or CTLPf before BMT are at higher risk of developing aGVHD after transplantation from both matched sibling donors and MUD. Whether these parameters can predict disease relapse would have to be investigated with a larger cohort of patients.

Liver graft-versus-host disease after donor lymphocyte infusion for relapses of hematologic malignancies post allogeneic hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) is the commonest complication after donor lymphocyte infusion (DLI). In 19 patients undergoing DLI for relapses of hematologic malignancies post hematopoietic stem cell transplantation (HSCT), 11 developed GVHD, of whom nine had isolated liver involvement, and two had liver and skin involvement. The clinical diagnosis of liver GVHD was hepatitic in six patients (55%) and classical in five patients (45%). Patients with GVHD post-DLI showed a different clinical pattern when compared to a cohort of 106 cases of GVHD post-HSCT, in having significantly more isolated liver involvement (9/11 vs 17/106, P<0.001), and less skin (2/11 vs 80/106, P<0.001) and gut (0/11 vs 28/106, P<0.001) involvement. However, liver GVHD post-DLI and post-HSCT had comparable patient characteristics, underlying diseases, clinical subtypes (classical and hepatitic) and response to treatment.

Candida tropicalis fungaemia in adult patients with haematological malignancies: clinical features and risk factors.

Candida tropicalis fungaemia is a serious opportunistic infection. Eighteen consecutive patients with C. tropicalis fungaemia diagnosed within a five-year period were studied retrospectively. All patients had haematological malignancies treated by chemotherapy or bone marrow transplantation (BMT). Antifungal prophylaxis included nystatin (20 mg daily) for patients receiving chemotherapy, and fluconazole (200 mg daily) for patients undergoing BMT. Sixteen patients had refractory and advanced haematological malignancies. All patients were neutropenic, had central venous catheters, and were receiving treatment with broad-spectrum antibiotics at the time of fungaemia. Septic shock with skin emboli were the most common presenting features. In seven cases, fungaemia was preceded by a positive culture of C. tropicalis in the urine. Concomitant bacteraemia was found in 11 cases, of which six cases were due to Staphylococcus aureus. The overall mortality rate was 56%. The predominance of chemotherapy-treated patients developing fungaemia in this series might be attributable to the omission of fluconazole prophylaxis. The clinicopathologic features and risk factors identified in this study may help design better treatment strategies for this often-lethal complication.

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.

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.

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.

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.