Potential benefits of a virtual, home-based combined exercise and mindfulness training program for HSC transplant survivors: a single-arm pilot study.

PURPOSE: Impaired quality of life (QOL) including reduced physical fitness is a recognized late effect of hemopoietic cell transplantation (HCT). Guided exercise and mindfulness-based stress management (MBSM) programs have shown promise, mainly in the inpatient setting. We aimed to examine the feasibility of a virtual, home-based, combined exercise and MBSM program. METHODS: Patients attending post-HCT clinic were invited to participate in this single-arm pre-post study. Eligibility criteria included age 18-75 years, > 6 months post allogeneic HCT. Consented participants attended an in-person session, followed by weekly exercise and MBSM training for 6 weeks via videoconferencing. Assessments were performed pre-training, and at 3-, 6- and 12-months and compared using a linear mixed effects model. RESULTS: 21 of 24 patients consenting to the study completed the program (median age 56 years [IQR 46-62], median time post-HCT 37 months [IQR 26-46]). Six-minute walk test scores were significantly higher at 3 (mean difference 79.6, 95%CI 28-131, ES 0.55) and 12 months (mean difference 48.4, 95%CI 13-84, ES 0.33) compared to baseline. Sit-to-stand test was significantly higher at 3 (mean difference 4.4, 95%CI 1.4-7.4, ES 0.68) and 12 months (mean difference 3.9, 95%CI 0.24-7.6, ES 0.61). Dominant hand grip was significantly stronger at 3 (mean difference 0.16, 95%CI 0.04-0.28, ES 0.45), and 12 months (mean difference 0.21, 95%CI 0.08-0.24, ES 0.62). Significantly higher FACT-BMT total (mean difference 6.9, 95%CI 1.5-12.4, ES 0.49) and FACT-G scores (mean difference 5.2, 95%CI 1.4-9.1, ES 0.48) were found at 3 months. Over 80% of participants rated the virtual combined modal program highly and no adverse events were reported. CONCLUSION: A 6-week virtual, home-based exercise and MBSM program was an acceptable, and potentially effective intervention for sustained improvement of some physical capacity and QOL outcomes in HCT survivors. Virtual-based healthcare service is highly relevant particularly during pandemics. To our knowledge, this study has the longest follow-up observation period for Internet based combined modality training program reported to date and warrants additional investigation. Trial Registration Research protocol approved by St Vincent's Hospital Ethics Committee (HREC 12/SVH/175), approved 27/09/2012, trial commenced 24/05/13 and the first participant 07/06/13. Retrospectively registered with ANZCTR (ACTRN12613001054707) 23/09/2013.

Sustained immunotolerance in multiple sclerosis after stem cell transplant.

OBJECTIVE: Autologous haematopoietic stem cell transplantation (AHSCT) has the potential to induce sustained periods of disease remission in multiple sclerosis (MS), which is an inflammatory disease of the central nervous system (CNS) characterised by demyelination and axonal degeneration. However, the mechanisms associated with durable treatment responses in MS require further elucidation. METHODS: To characterise the longer term immune reconstitution effects of AHSCT at 24 and 36 months (M) post-transplant, high-dimensional immunophenotyping of peripheral blood mononuclear cells from 22 MS patients was performed using two custom-designed 18-colour flow cytometry panels. RESULTS: The higher baseline frequencies of specific pro-inflammatory immune cells (T-helper-17 (Th17) cells, mucosal-associated invariant T-cells and CNS-homing T-conventional (T-conv) cells observed in MS patients were decreased post-AHSCT by 36M. This was accompanied by a post-AHSCT increase in frequencies and absolute counts of immunoregulatory CD56hi natural killer cells at 24M and terminally differentiated CD8+ CD28- CD57+ cells until 36M. A sustained increase in the proportion of naïve B-cells, with persistent depletion of memory B-cells and plasmablasts was observed until 36M. Reconstitution of the B-cell repertoire was accompanied by a reduction in the frequency of circulating T-follicular helper cells (cTfh) expressing programmed cell death-1 (PD1+ ) at 36M. Associations between frequency dynamics and clinical outcomes indicated only responder patients to exhibit a decrease in Th17, CNS-homing T-conv and PD1+ cTfh pro-inflammatory subsets at 36M, and an increase in CD39+ T-regulatory cells at 24M. INTERPRETATION: AHSCT induces substantial recalibration of pro-inflammatory and immunoregulatory components of the immune system of MS patients for up to 36M post-transplant.

Effect of donor age on adult unrelated donor haemopoietic cell transplant outcome: the Australian experience.

BACKGROUND: Results have been varied regarding the effect of donor age on the outcome of unrelated donor haemopoietic cell transplantation (HCT). AIMS: To determine the influence of donor age on adult unrelated donor HCT outcome in Australia. METHODS: Patients were included in the study if they were aged 16 years or above and underwent first allogeneic unrelated donor HCT in Australia for the indications of acute lymphoblastic leukaemia (ALL), acute myelogenous leukaemia (AML), chronic myelogenous leukaemia (CML) or myelodysplastic syndromes (MDS) between the years of 2001 and 2014 inclusive. The main outcome measure was overall survival (OS), which was tested against independent variables using univariate Kaplan-Meier methods and multivariate Cox regression. RESULTS: A total of 1158 unrelated donor HCT were represented in the data. Cumulative incidences of engraftment, transplant related mortality (TRM), acute graft-versus-host disease (GvHD), chronic GvHD and relapse were not significantly affected by donor age. OS probability at 5 years post-transplant was 48.3%. In multivariate analysis of OS, year of transplant 2001-2007, recipient age 40 years or greater, poor risk disease, human leukocyte antigen (HLA) match less than 6/6 and poor performance status at transplant (Karnofsky scale) were independently significant adverse OS risk factors. Donor age was not a significant risk factor for OS in univariate or multivariate analysis. CONCLUSIONS: The conclusion from this study was that donor age (up to 59 years) did not influence post-transplant outcome among adult unrelated donor HCT performed in Australia for haematologic malignancies.

Long-Term Suppression of Circulating Proinflammatory Cytokines in Multiple Sclerosis Patients Following Autologous Haematopoietic Stem Cell Transplantation.

Autologous haematopoietic stem cell transplantation (AHSCT) is a therapeutic option for haematological malignancies, such as non-Hodgkin's lymphoma (NHL), and more recently, for autoimmune diseases, such as treatment-refractory multiple sclerosis (MS). The immunological mechanisms underlying remission in MS patients following AHSCT likely involve an anti-inflammatory shift in the milieu of circulating cytokines. We hypothesised that immunological tolerance in MS patients post-AHSCT is reflected by an increase in anti-inflammatory cytokines and a suppression of proinflammatory cytokines in the patient blood. We investigated this hypothesis using a multiplex-ELISA assay to compare the concentrations of secreted cytokine in the peripheral blood of MS patients and NHL patients undergoing AHSCT. In MS patients, we detected significant reductions in proinflammatory T helper (Th)17 cytokines interleukin (IL)-17, IL-23, IL-1ß, and IL-21, and Th1 cytokines interferon (IFN)γ and IL-12p70 in MS patients from day 8 to 24 months post-AHSCT. These changes were not observed in the NHL patients despite similar pre-conditioning treatment for AHSCT. Some proinflammatory cytokines show similar trends in both cohorts, such as IL-8 and tumour necrosis factor (TNF)-α, indicating a probable treatment-related AHSCT response. Anti-inflammatory cytokines (IL-10, IL-4, and IL-2) were only transiently reduced post-AHSCT, with only IL-10 exhibiting a significant surge at day 14 post-AHSCT. MS patients that relapsed post-AHSCT exhibited significantly elevated levels of IL-17 at 12 months post-AHSCT, unlike non-relapse patients which displayed sustained suppression of Th17 cytokines at all post-AHSCT timepoints up to 24 months. These findings suggest that suppression of Th17 cytokines is essential for the induction of long-term remission in MS patients following AHSCT.

Tolerance regeneration by T regulatory cells in autologous haematopoietic stem cell transplantation for autoimmune diseases.

Autologous haematopoietic stem cell transplantation shows increasing promise as a therapeutic option for patients with treatment-refractory autoimmune disease, particularly systemic sclerosis and multiple sclerosis. However, this intensive chemotherapy-based procedure is not always possible due to potential treatment toxicities and comorbidities. The biological mechanisms of how this procedure induces long-term remission in autoimmune disease are increasingly understood. The focus of this review is on recent research findings on the role of CD4+ T regulatory cells (Tregs) in resetting the immune system leading to the eradication of the autoimmune disease after transplantation. Discovery of the precise mechanisms of this process will allow development of novel Treg-based therapies and thus avoid the need for intensive chemotherapy-based treatment for these autoimmune diseases in the future.

Favorable Outcome of Lung Transplantation for Severe Pulmonary Graft Versus Host Disease: An Australian Multicenter Case Series.

BACKGROUND: Severe pulmonary chronic graft versus host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Few treatments influence outcome, with 5-year overall survival as low as 13%. Lung transplantation (LTx) has been reported in small numbers of patients worldwide. METHODS: We investigated the outcomes of LTx performed for this indication at 2 large Australian LTx centers. RESULTS: Eighteen patients (aged 10-64 y; median, 29.6 y) received bilateral deceased lung transplants for pulmonary chronic GVHD between 2002 and 2017. LTx was performed at a median of 8.6 years after allogeneic stem cell transplantation (range, 2-23 y) with a median interval of 16 months from the time of transplant unit review to LTx. There were 2 early infective deaths and 3 further deaths from pulmonary infection and lung allograft rejection. There were no primary disease relapses. At a median follow-up of 5 years, the 5-year overall survival post-LTx is 80% and comparable to the Australia and New Zealand registry data of 64% for LTx performed for all indications. CONCLUSIONS: From one of the largest series of deceased LTx for this indication, we conclude that it is a feasible option for selected patients with severe pulmonary GVHD. The outcomes appear superior to that of non-LTx-based therapies and similar to the survival of the general LTx population. Establishing guidance on referral triggers, patient eligibility, organ selection, prophylaxis of allograft rejection, and supportive care would assist hematopoietic and lung transplant units in optimizing resource allocation and patient outcomes.

Safety and activity of ibrutinib in combination with nivolumab in patients with relapsed non-Hodgkin lymphoma or chronic lymphocytic leukaemia: a phase 1/2a study.

BACKGROUND: Preclinical studies have shown synergistic antitumour effects between ibrutinib and immune-checkpoint blockade. The aim of this study was to assess the safety and activity of ibrutinib in combination with nivolumab in patients with relapsed or refractory B-cell malignant diseases. METHODS: We did a two-part, open-label, phase 1/2a study at 21 hospitals in Australia, Israel, Poland, Spain, Turkey, and the USA. The primary objective of part A (dose escalation) was to assess the safety of daily oral ibrutinib (420 mg or 560 mg) in combination with intravenous nivolumab (3 mg/kg every 2 weeks) to ascertain a recommended phase 2 dose in patients with relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, or diffuse large B-cell lymphoma. Dose optimisation was investigated using a modified toxicity probability interval design. The primary objective of the part B expansion phase was to establish the preliminary activity (the proportion of patients who achieved an overall response) of the combination of ibrutinib and nivolumab in four cohorts: relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, diffuse large B-cell lymphoma, and Richter's transformation. All participants who received at least one dose of treatment were included in the primary analysis and analyses were done by disease cohort. This trial is registered with ClinicalTrials.gov, number NCT02329847. The trial is ongoing. FINDINGS: Between March 12, 2015, and April 11, 2017, 144 patients were enrolled in the study. Three patients died before receiving study treatment; thus, 141 patients were included in the analysis, 14 in part A and 127 in part B. One dose-limiting toxicity (grade 3 hyperbilirubinaemia) was reported at the 420 mg dose in the diffuse large B-cell lymphoma cohort, which resolved after 5 days. The combination of ibrutinib and nivolumab led to overall responses in 22 (61%) of 36 patients with high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma, 13 (33%) of 40 patients with follicular lymphoma, 16 (36%) of 45 patients with diffuse large B-cell lymphoma, and 13 (65%) of 20 patients with Richter's transformation. The most common all-grade adverse events were diarrhoea (47 [33%] of 141 patients), neutropenia (44 [31%]), and fatigue (37 [26%]). 11 (8%) of 141 patients had adverse events leading to death; none were reported as drug-related. The most common grade 3-4 adverse events were neutropenia (40 [28%] of 141 patients) and anaemia (32 [23%]). The incidence of grade 3-4 neutropenia ranged from eight (18%) of 45 patients with diffuse large B-cell lymphoma to 19 (53%) of 36 patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma; incidence of grade 3-4 anaemia ranged from five (13%) of 40 patients with follicular lymphoma to seven (35%) of 20 patients with Richter's transformation. The most common serious adverse events included anaemia (six [4%] of 141 patients) and pneumonia (five [4%]). The most common grade 3-4 immune-related adverse events were rash (11 [8%] of 141 patients) and increased alanine aminotransferase (three [2%]). INTERPRETATION: The combination of ibrutinib and nivolumab had an acceptable safety profile and preliminary activity was similar to that reported with single-agent ibrutinib in chronic lymphocytic leukaemia or small lymphocytic lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. The clinical response in patients with Richter's transformation was promising and supports further clinical assessment. FUNDING: Janssen R&D.

Prospective phase II clinical trial of autologous haematopoietic stem cell transplant for treatment refractory multiple sclerosis.

BACKGROUND: Autologous haematopoietic stem cell transplantation (AHSCT) has been explored as a therapeutic intervention in multiple sclerosis (MS) over the last two decades; however, prospective clinical trials of the most common myeloablative conditioning regimen, BEAM, are limited. Furthermore, patient selection, optimal chemotherapeutic regimen and immunological changes associated with disease response require ongoing exploration. We present the outcomes, safety and immune reconstitution (IR) of patients with active, treatment refractory MS. METHODS: This study was a single-centre, phase II clinical trial of AHSCT for patients with active relapsing remitting (RRMS) and secondary progressive MS (SPMS). Patients underwent AHSCT using BEAM (carmustine, etoposide, cytarabine, melphalan)+antithymocyte globulin chemotherapeutic regimen. OUTCOMES: The primary outcome was event-free survival (EFS); defined as no clinical or radiological relapses and no disability progression. Multiparameter flow cytometry was performed for evaluation of post-transplant IR in both MS and lymphoma patients receiving the same chemotherapy regimen. RESULTS: Thirty-five patients (20 RRMS, 15 SPMS) completed AHSCT, with a median follow-up of 36 months (range 12-66). The median Expanded Disability Status Scores (EDSS) was 6 (2-7) and patients had failed a median of 4 (2-7) disease modifying therapies. 66% failed treatment with natalizumab. EFS at 3 years was 60%, (70% RRMS). Sustained improvement in EDSS was seen in 15 (44%) of patients. There was no treatment-related mortality. A sustained rise in CD39+ T regulatory cells, immunosuppressive CD56hi natural killer cells and ablation of proinflammatory mucosal-associated invariant T cells was seen for 12 months following AHSCT in patients with MS. These changes did not occur in patients with lymphoma receiving the same chemotherapy for AHSCT. CONCLUSIONS: The EFS in our MS cohort is significantly greater than other high-efficacy immunosuppressive therapies and similar to other AHSCT studies despite a more heavily pretreated cohort. TRIAL REGISTRATION NUMBER: ACTRN12613000339752.

Autologous hematopoietic stem cell transplant for autoimmune diseases: evolution,evidence of efficacy, and real-world economics.

Despite an increase in the development of biological therapies for autoimmune disease (AID), a proportion of patients remain treatment refractory, resulting in long term morbidity and increased rates of mortality. Furthermore, maintenance biologic therapies are associated with treatment-related side effects, significant financial cost,and restricted access, which is of particular relevance in the developing world. Although it carries a significant 'front loaded' cost both financially and regarding adverse events, autologous hematopoietic stem cell transplantation (AHSCT) represents a potential single therapeutic intervention, which in the appropriate patient, condition, and transplant center, may offer sustained disease remission resulting in improved overall survival, disease relapse-free survival, improved quality of life, and decreased financial burden. Emerging Phase II and III trial and registry data, to which our center has been a significant contributor over the past two decades, are providing invaluable evidence as to which AIDs are most likely to receive a sustained benefit from AHSCT and which conditioning regimens are preferable. Similar to trends for the treatment of malignant disease, AHSCT for AID may find a place in both developed and developing countries as nations become more familiar with the transplantation process. If this occurs, benchmarking by key regulatory bodies, collaboration between medical specialties, and the development of experienced 'centers of excellence' will be key to enhance safety and benefit to patients and society at large.

Regenerating Immunotolerance in Multiple Sclerosis with Autologous Hematopoietic Stem Cell Transplant.

Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system where evidence implicates an aberrant adaptive immune response in the accrual of neurological disability. The inflammatory phase of the disease responds to immunomodulation to varying degrees of efficacy; however, no therapy has been proven to arrest progression of disability. Recently, more intensive therapies, including immunoablation with autologous hematopoietic stem cell transplantation (AHSCT), have been offered as a treatment option to retard inflammatory disease, prior to patients becoming irreversibly disabled. Empirical clinical observations support the notion that the immune reconstitution (IR) that occurs following AHSCT is associated with a sustained therapeutic benefit; however, neither the pathogenesis of MS nor the mechanism by which AHSCT results in a therapeutic benefit has been clearly delineated. Although the antigenic target of the aberrant immune response in MS is not defined, accumulated data suggest that IR following AHSCT results in an immunotolerant state through deletion of pathogenic clones by a combination of direct ablation and induction of a lymphopenic state driving replicative senescence and clonal attrition. Restoration of immunoregulation is evidenced by changes in regulatory T cell populations following AHSCT and normalization of genetic signatures of immune homeostasis. Furthermore, some evidence exists that AHSCT may induce a rebooting of thymic function and regeneration of a diversified naïve T cell repertoire equipped to appropriately modulate the immune system in response to future antigenic challenge. In this review, we discuss the immunological mechanisms of IR therapies, focusing on AHSCT, as a means of recalibrating the dysfunctional immune response observed in MS.

Activity and Capacity Profile of Transplant Physicians and Centers in Australia and New Zealand.

We conducted a study to analyze and report on indicators of hematopoietic cell transplant (HCT) physician time use and HCT center output measures. HCT centers in Australia and New Zealand (A&NZ) were invited to provide demographic and time use details for physicians participating in HCT patient care (HCT physicians). Resource details for adult and pediatric centers were included. From a total of 46 centers that were invited to participate, completed data were received from 37 centers (80%) representing 185 HCT physicians, with a median age of 48 (range, 33 to 72), of whom 31% were women. Just over half of HCT physicians cited prior work experience in large overseas HCT centers (97, 52%) and over one-third (79, 43%) possessed postgraduate qualifications other than specialist training. Total annual mean HCTs per HCT physician full-time equivalent (FTE) were 14.2 for centers performing both allogeneic and autologous HCT, 6.6 for autologous only centers, and 10.6 for all centers. For all HCT physicians surveyed the mean proportion of time spent on HCT related tasks was 31.7%. In A&NZ, for centers that perform both allografts and autografts, there was a mean of 4.0 allogeneic HCT annually per HCT bed, compared with 2.6 for the United States and 7.1 allogeneic HCT annually per HCT physician FTE (United States, 6.3). Projections of the A&NZ HCT physician workforce indicated that the numbers of HCT physicians are likely to stay within the region of 170 to 190 for the next 10 years, whereas HCT activity will likely continue to climb steadily. Healthcare and government authorities should be prepared to enable and support greater HCT activity in A&NZ in the future.

Dealing with the spiralling price of medicines: issues and solutions.

Escalating cost of medicines is rapidly becoming a serious threat to patients and health systems. This trend has been documented to impact patient outcomes adversely. As clinicians and tax payers, it is our responsibility to be aware of the potential detrimental effects spiralling costs have on our patients, our community and our health system and to mitigate these effects by exposing this issue to our respective professional societies, representatives of the pharmaceutical companies that we interact with, government regulatory bodies and to patients who we are caring for. Only through understanding and constructive actions will we be able to provide the best quality of care to our patients and continue to enjoy universal healthcare in our country.

Pro-survival role of protein kinase C epsilon in Philadelphia chromosome positive acute leukemia.

Durable responses to imatinib monotherapy are rarely seen in aggressive forms of Philadelphia chromosome positive (Ph+) leukemias. To investigate the possible cause of treatment failure we examined the role of protein kinase C epsilon (PKCE), an oncogene highly implicated in the development of solid tumors and resistance to chemotherapy. We found high levels of PKCE transcripts in Ph+ acute lymphoblastic leukemia (ALL) cells from patients and cell lines, and imatinib resistant chronic myeloid leukemia, which were also less responsive to imatinib-induced apoptosis than Ph+ cells with lower PKCE expression. Furthermore, the siRNA-mediated knockdown or peptide inhibition of PKCE in Ph+ cells increased imatinib-induced apoptosis while overexpression of PKCE reduced imatinib-induced apoptosis, with concomitant increase in the pro-survival factor AKT. Our results suggest PKCE plays a protective role against apoptosis induced by BCR-ABL inhibition in Ph+ leukemias with high PKCE expression, such as Ph+ ALL.

A Review of Hematopoietic Cell Transplantation in Australia and New Zealand, 2005 to 2013.

This report describes hematopoietic cell transplantation (HCT) activity and outcome in Australia and New Zealand during the years 2005 to 2013. In 2013, 1018 autologous, 221 allogeneic with related donors, and 264 allogeneic with unrelated donors HCT were performed in 40 centers in Australia, with corresponding figures of 147, 39, and 47 in 6 centers in New Zealand. Annual numbers of HCT in 2013 increased, compared to 2005, by 25% in Australia and by 52% in New Zealand. The majority of both allogeneic and autologous HCT used peripheral blood as the stem cell source for all years studied. Major indications for transplantation were acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), plasma cell disorders, and non-Hodgkin lymphoma (NHL). Overall survival probabilities at 5 years after transplantation for adult (16+) allogeneic first HCT recipients were 54.2% for ALL, 46.0% for AML, 48.4% for myelodysplastic syndromes, and 58.6% for NHL. Consistent patterns over time include a steady increase in HCT, particularly for older recipients, relatively constant numbers of allografts using cord blood, and a recent increase in the number of allografts with 2 or more HLA-mismatched related donors.

Gene profiling reveals association between altered Wnt signaling and loss of T-cell potential with age in human hematopoietic stem cells.

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T-cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9-DL1 in vitro co-culture system to promote T-cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T-lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T-lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T-cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T-progenitor cell subset derived during in vitro T-lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age-related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T-cell generation and immune reconstitution following clinical transplantation.

MicroRNA-155 as an inducer of apoptosis and cell differentiation in Acute Myeloid Leukaemia.

BACKGROUND: Acute myeloid leukaemia (AML) is characterised by the halt in maturation of myeloid progenitor cells, combined with uncontrolled proliferation and abnormal survival, leading to the accumulation of immature blasts. In many subtypes of AML the underlying causative genetic insults are not fully described. MicroRNAs are known to be dysregulated during oncogenesis. Overexpression of miR-155 is associated with some cancers, including haematological malignancies, and it has been postulated that miR-155 has an oncogenic role. This study investigated the effects of modulating miR-155 expression in human AML cells, and its mechanism of action. RESULTS: Analysis of miR-155 expression patterns in AML patients found that Fms-like tyrosine kinase 3 (FLT3)-wildtype AML has the same expression level as normal bone marrow, with increased expression restricted to AML with the FLT3-ITD mutation. Induction of apoptosis by cytarabine arabinoside or myelomonocytic differentiation by 1,23-dihydroxyvitaminD3 in FLT3-wildtype AML cells led to upregulated miR-155 expression. Knockdown of miR-155 by locked nucleic acid antisense oligonucleotides in the FLT3-wildtype AML cells conferred resistance to cytarabine arabinoside induced apoptosis and suppressed the ability of cells to differentiate.Ectopic expression of miR-155 in FLT3-wildtype AML cells led to a significant gain of myelomonocytic markers (CD11b, CD14 and CD15), increase in apoptosis (AnnexinV binding), decrease in cell growth and clonogenic capacity.In silico target prediction identified a number of putative miR-155 target genes, and the expression changes of key transcription regulators of myeloid differentiation and apoptosis (MEIS1, GF1, cMYC, JARID2, cJUN, FOS, CTNNB1 and TRIB2) were confirmed by PCR. Assessment of expression of apoptosis-related proteins demonstrated a marked increase in cleaved caspase-3 expression confirming activation of the apoptosis cascade. CONCLUSIONS: This study provides evidence for an anti-leukaemic role for miR-155 in human FLT3-wildtype AML, by inducing cell apoptosis and myelomonocytic differentiation, which is in contrast to its previously hypothesized role as an oncogene. This highlights the complexity of gene regulation by microRNAs that may have tumour repressor or oncogenic effects depending on disease context or tissue type.

Flow cytometry demonstrates differences in platelet reactivity and microparticle formation in subjects with thrombocytopenia or thrombocytosis due to primary haematological disorders.

BACKGROUND: Traditional methods for the assessment of platelet function require a minimum number of platelets. As flow cytometry is independent of platelet number, we measured platelet activation and microparticle formation in thrombocytopenia and thrombocytosis. MATERIALS AND METHODS: Blood was obtained from normal subjects or subjects with immune thrombocytopenic purpura (ITP), myelodysplasia (MDS) or essential thrombocythaemia (ET). Platelet activation and microparticle formation were assessed in resting and agonist stimulated samples. RESULTS: Platelet activation was significantly decreased in MDS in agonist-stimulated platelets when compared to normals and ITP, however increased microparticle-to-platelet ratios were found. Absolute platelet-derived microparticle counts were significantly higher in ET when compared to normals, but there was no significant difference in microparticle-to-platelet ratios between ET and normals. CONCLUSIONS: Decreased platelet activation was demonstrated in MDS when compared to normal subjects and ITP. Platelet-derived microparticle counts are increased in ET, reflecting increased platelet counts rather than an increase in platelet reactivity. Flow cytometric analysis of platelets may aid the diagnosis and management of these conditions.

A population-based analysis of the effect of autologous hematopoietic cell transplant in the treatment of multiple myeloma.

This population registry-based study followed all cases of myeloma diagnosed in New South Wales, Australia, during 2002-2005 and compared survival outcomes of those who proceeded to autologous hematopoietic cell transplant (HCT) with those who did not. Data available consisted of demographic details and survival, and did not include disease details or treatment type or response. Of 708 patients, 270 (38%) had a HCT. The 5-year overall survival (OS) of HCT recipients was significantly better than for those who did not proceed to HCT (62% vs. 54%, p = 0.003). HCT was a significant favorable risk factor for OS, while age over 60 was an adverse risk factor. However, for patients alive at 1 year from diagnosis, there was no significant difference in survival between HCT and non-HCT patients, suggesting that worse disease biology and/or coexisting morbidities were likely to be major reasons for the poorer outcome for non-HCT patients.

Low concentration detergent sclerosants induce platelet activation but inhibit aggregation due to suppression of GPIIb/IIIa activation in vitro.

INTRODUCTION: Sclerotherapy is associated with thromboembolic and ischemic neurological adverse events but the effects of sclerosants on platelet function are unknown. The aim of this study was to investigate the in vitro effects of detergent sclerosants Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on platelet activation and aggregation. MATERIALS AND METHODS: Whole blood and platelet rich plasma samples were incubated with sclerosants. Platelet and platelet microparticle (PMP) counts were measured by flow cytometry. Platelet activation was examined by ELISA for soluble factors (sP-selectin, von Willebrand factor, sCD40L and serotonin) and by flow cytometry for membrane-bound markers (CD62p, CD63) and cytoplasmic calcium. Platelet aggregation was assessed by PFA-100®, light transmission and impedance (Multiplate®) aggregometry, and by flow cytometry for glycoprotein (GP) Ib and GPIIb/IIIa subunits, heterodimer expression and activation (PAC-1 binding). RESULTS: Both agents lysed platelets at high concentrations (≥ 0.1%) but induced platelet activation at lower concentrations as evident by a rise in membrane-bound and soluble markers, cytoplasmic calcium and release of phosphatidylserine+PMP. Agonist-stimulated platelet aggregation was inhibited by both sclerosants. Membrane expression of GPIb and GPIIb/IIIa individual subunits or heterodimer was not affected by sclerosants but the activation of GPIIb/IIIa was suppressed. CONCLUSION: Low concentration sclerosants activated platelets and released microparticles but inhibited platelet aggregation due to suppression of GPIIb/IIIa activation.