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Allogeneic haematopoietic-cell transplantation (allo-HCT) is a potentially curative therapy for high-risk myelodysplastic syndrome (MDS). Reduced-intensity conditioning (RIC) is usually associated with lower non-relapse mortality (NRM), higher relapse rate and similar overall-survival (OS) as myeloablative-conditioning (MAC). Fludarabine/treosulfan (FT) is a reduced-toxicity regimen with intense anti-leukaemia activity and a favourable toxicity profile. We investigated post-transplant outcomes in 1722 MDS patients following allo-HCT with FT (n = 367), RIC (n = 687) or MAC (n = 668). FT and RIC recipients were older than MAC recipients, median age 59, 59 and 51 years, respectively (P < 0·001) but other disease characteristics were similar. The median follow-up was 64 months (1-171). Five-year relapse rates were 25% (21-30), 38% (34-42) and 25% (22-29), after FT, RIC and MAC, respectively, (P < 0·001). NRM was 30% (25-35), 27% (23-30) and 34% (31-38, P = 0·008), respectively. Five-year OS was 50% (44-55), 43% (38-47), and 43% (39-47), respectively (P = 0·03). In multivariate analysis, FT was associated with a lower risk of relapse (HR 0·55, P < 0·001) and better OS (HR 0·72, P = 0·01). MAC was associated with higher NRM (HR 1·44, P = 0·001). In conclusion, FT is associated with similar low relapse rates as MAC and similar low NRM as RIC, resulting in improved OS. FT may be the preferred regimen for allo-HCT in MDS., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

CD4(+) T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN), suggesting that T cells play an important role in their pathogenesis. Treatment with JAK inhibitors (JAKi) results in improvements in MPN-associated constitutional symptoms as well as reductions in splenomegaly. However, effects of JAKi on T cells in MPN are not well established and the baseline immune signature remains unclear. We investigated the frequency and function of CD4(+) T cell subsets in 50 MPN patients at baseline as well as during treatment with either ruxolitinib or fedratinib in a subset. We show that CD4(+)  CD127(low)  CD25(high)  FOXP3(+) T regulatory cells are reduced in MPN patients compared to healthy controls and that this decrease is even more pronounced following JAKi therapy. Moreover, we show that after 6 months of treatment the number of T helper (Th)-17 cells increased. We also describe a functional 'silencing' of T helper cells both in vivo and in vitro and a blockade of pro-inflammatory cytokines from these cells. This profound effect of JAKi on T cell function may underlay augmented rates of atypical infections that have been reported with use of these drugs., (© 2015 John Wiley & Sons Ltd.)

Sweet syndrome (SS) is an acute febrile neutrophilic dermatosis. It has been associated with malignant disease, especially acute myeloid leukaemia (AML), infections, autoimmune disorders and drugs, particularly granulocyte colony-stimulating factor (GCSF). No cause is found in the rest, which are labelled idiopathic. We describe 15 patients with SS, which we believe represent 'immune dysregulation' secondary to myelodysplastic syndrome (MDS). We initially identified 31 patients with SS in a cohort of 744 patients with MDS and 215 with AML seen over a 6-year period (2004-10). The cause in 16 patients could be attributed either to administration of GCSF or chemotherapy. The eruption was brief and resolved spontaneously or following withdrawal of GCSF. Fifteen patients however, had a chronic debilitating illness dominated by the skin eruptions. Diagnosis of chronic relapsing SS was delayed because the pathology was not always typical of classical neutrophil-rich SS and included lymphocytic and histiocytoid infiltrates and bone marrow was not always performed because the relevance of the eruption to MDS was often not immediately appreciated. All these patients had 'low risk' MDS, diagnosed at a median of 17 months (range 0-157) following the diagnosis of SS. We describe a chronic debilitating episodic clinically distinctive skin eruption with features of SS but not always definitive histopathology often associated with immunological abnormalities affecting other systems related to underlying low risk MDS., (© 2015 John Wiley & Sons Ltd.)

Invasive fungal disease (IFD) is difficult to diagnose. We investigated the incidence of IFD and risk factors using the revised European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG) definitions. Patients (N = 203) undergoing intensive therapy with expected neutropenia ≥10 d were recruited prospectively and followed for a median (range) of 556 (12-730) d. Baseline chest computerized tomography (CT) was performed pre-therapy. Twice-weekly surveillance with galactomannan (GM) was combined with targeted β-d-glucan (BDG) testing on patients with possible IFD or who were GM-positive. Tissue diagnosis was obtained whenever possible. The cumulative incidence of proven/probable IFD among the 202 evaluable cases after 2 years follow-up was 21%, including 14 proven and 30 probable IFDs. Using either GM or BDG as the sole biomarker (plus host and clinical evidence) the apparent overall incidence of proven/probable IFD was 11% and 16%, respectively. Combined GM/BDG detected all biopsy-proven mould IFD. Baseline CT abnormalities were found in 76/202 (38%) patients. Baseline CT abnormalities and Karnofsky score <90, monocytopenia >10 d and bacteraemia were independent risk factors associated with greater than twofold increased IFD risk. This combined diagnostic approach identified a high incidence of IFD and important risk factors in this cohort., (© 2014 John Wiley & Sons Ltd.)

Low endogenous erythropoietin levels and limited red blood cell transfusion history can predict response to erythropoiesis-stimulating agents in anaemic patients with myelodysplastic syndromes (MDS). The relationship between endogenous thrombopoietin (THPO) levels and platelet response to romiplostim is unknown. Variables including baseline endogenous THPO levels, transfusion needs, and platelet response were analysed in a randomized trial of 250 thrombocytopenic, lower-risk MDS patients (International Prognostic Scoring System low/intermediate-1). A predictive scoring system was developed based on log-likelihood ratios and logistic coefficients. Patients with HI-P (haematological improvement - platelets) responses had lower mean baseline THPO levels (P = 0·0497) and were more likely to have <6 platelet units transfused in the past year (P = 0·0027), as did patients with platelet responses ≥50% of weeks on romiplostim (P = 0·001 and P = 0·0037, respectively). A model for predicting response to romiplostim was developed and validated in a separate MDS cohort (N = 72). Patients in low-, intermediate-, and high-response groups had response rates of 17·4%, 29·6%, and 50·7%, respectively, for HI-P, and 17·4%, 33·8%, and 65·2%, respectively, for ≥50% response. For thrombocytopenic patients with lower-risk MDS, lower baseline THPO levels (<500 pg/ml) and limited platelet transfusion history predicted a greater likelihood of a subsequent platelet response to romiplostim., (© 2014 John Wiley & Sons Ltd.)

The efficacy and tolerance of azacitidine in higher-risk myelodysplasia with hypocellular bone marrow (BM) are unknown. This post hoc AZA-001 trial analysis assessed whether baseline BM cellularity affected the overall survival (OS) advantage demonstrated with azacitidine versus conventional care regimens (CCR). Baseline BM biopsies of <30% cellularity were considered hypocellular with data evaluable from 299 patients (azacitidine n = 154, CCR n = 145); 13% (n = 39) hypocellular, 87% (n = 260) non-hypocellular. Patient characteristics were balanced between cellularity and treatment groups. Most patients (90-100%) had 2-3 cytopenias at baseline. Median (range) azacitidine treatment cycle lengths were 35·5 (28-54) and 33·0 (15-75) d in hypocellular and non-hypocellular groups, respectively. At 33 months, median OS was not reached (NR) [95% confidence interval (CI): 19·2, NR] in hypocellular patients receiving azacitidine versus 16·9 months (95% CI: 11·1, 19·3) with CCR (P = 0·001); and in non-hypocellular patients, it was 21·1 months (95% CI: 16·2, 34·7) versus 15·3 months (95% CI: 9·3, 17·6) (P = 0·012). Azacitidine tolerance was similar regardless of cellularity. Grade 3-4 thrombocytopenia and neutropenia occurred similarly in hypocellular patients treated with azacitidine versus CCR (80% vs. 92% and 88% vs. 75%). Azacitidine OS results are consistent with those from AZA-001, regardless of cellularity, and demonstrate its safety and efficacy in higher-risk myelodysplasia with hypocellular BM., (© 2014 John Wiley & Sons Ltd.)

The advent of novel genomic sequencing technologies has aided the identification of somatically acquired genetic abnormalities up to 80% of myelodysplastic syndrome (MDS) patients. Novel recurrent genetic mutations in pathways such as RNA splicing, DNA methylation and histone modification and cohesion complexes, underscore the molecular heterogeneity seen in this clinically varied disease. Functional studies to establish a causative link between genomic aberrations and MDS biogenesis are still in their infancy. The deluge of this molecular information, once validated on a larger cohort, will be incorporated into prognostic systems and clinical practise, and also hopefully aid in MDS therapeutics, especially in guiding targeted therapy., (© 2013 Crown copyright. British Journal of Haematology © 2013 John Wiley & Sons Ltd.)

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein., (© 2013 Crown copyright. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

The response of the tumour microenvironment to anti-cancer drugs can influence treatment efficacy. Current drug-screening methodologies fail to distinguish and quantify simultaneously the concomitant effect of drugs on the tumour stroma and cancer cells. To overcome this limitation we have developed a fluorescence-based experimental model that employs mCherry-labelled stromal cells (e.g. bone marrow fibroblastic stromal cells) co-cultured in direct contact with enhanced green fluorescent protein-labelled tumour cell lines for accurate assessment of proliferation and viability in both cell compartments and adhesion of tumour cells. Additionally, we used fluorescence-based image analysis to determine morphological changes that correlate with cell function (e.g. morphology of the actin cytoskeleton and nuclearity of osteoclasts to predict their bone resorption activity). Using this platform we have revealed that dexamethasone induces HS5 fibroblast proliferation and contact with multiple myeloma cells via a process involving Src/c-Abl kinases. Osteoclasts also inhibited dexamethasone-induced apoptosis in myeloma cells while retaining their normal morphology and functionality in bone resorption. Myeloma resistance to dexamethasone mediated by HS5 cells and osteoclasts was reversed by treatment with the Src/c-Abl inhibitor dasatinib but not with bortezomib. This new experimental platform provides a more precise screening of new therapeutics for improved efficacy of tumour cell killing within the bone marrow microenvironment., (© 2012 Blackwell Publishing Ltd.)

Graft-versus-leukaemia (GvL) and graft-versus-host disease (GvHD) are both caused by alloreactive lymphocytes. We previously reported that GvHD correlated with higher numbers of effector CD4 T cells and Natural Killer cells early after allogeneic transplantation using a regimen comprising fludarabine, busulphan and alemtuzumab. Here, we assessed immune cell subset recovery in these patients in the context of early myeloid malignant disease relapse. Despite the close relationship between the GvL and GvHD immune responses, rapid recovery of lymphocyte subsets was not associated with protection from disease relapse. These results indicated that GvL may be weak in this treatment setting for patients with myelodysplastic syndromes and acute myeloid leukaemia. Consistent with low GvL activity, we previously reported that mixed T cell chimaerism had no detrimental effect on relapse in this treatment setting and instead correlated with better outcome because of reduced GvHD incidence. We now report that patients with significantly higher lymphocyte numbers prior to transplantation subsequently maintained the mixed T cell chimaeric state. This pre-transplant profile, together with absence of the early post-transplant signature indicative of GvHD predisposition, could potentially be used to identify patients suitable for early withdrawal of immunosuppression and prophylactic donor leucocyte infusion to boost GvL activity.

In the phase III AZA-001 trial, low-dose cytarabine (LDara-C), the most widely used low-dose chemotherapy in patients with higher-risk myelodysplastic syndrome (MDS) who are ineligible for intensive treatment, was found to be associated with poorer survival compared with azacitidine. This analysis further compared the efficacy and the toxicity of these two drug regimens. Before randomization, investigators preselected patients to receive a conventional care regimen, one of which was LDara-C. Of 94 patients preselected to LDara-C, 45 were randomized to azacitidine and 49 to LDara-C. Azacitidine patients had significantly more and longer haematological responses and increased red blood cell transfusion independence. Azacitidine prolonged overall survival versus LDara-C in patients with poor cytogenetic risk, presence of -7/del(7q), and French-American-British subtypes refractory anaemia with excess blasts (RAEB) and RAEB in transformation. When analyzed per patient year of drug exposure, azacitidine treatment was associated with fewer grade 3-4 cytopenias and shorter hospitalisation time than LDara-C in these higher-risk MDS patients.

Matched unrelated donor stem cell transplantation (MUD-SCT) provides the only curative option for patients with follicular lymphoma (FL) who fail conventional therapies and do not have a sibling donor. The purpose of this study was to analyse the outcome of patients with FL treated with MUD-SCT included in the European Group for Blood and Marrow Transplantation registry. 131 patients treated with reduced-intensity conditioning (RIC, n = 87) or conventional myeloablative (CONV, n = 44) MUD-SCT between 2000 and 2005 were included. Median time from diagnosis to MUD-SCT was 47 months and the median number of previous therapeutic regimens was 4 (previous autograft: 47%). RIC recipients were significantly older, with a longer interval from diagnosis to MUD-SCT and had failed a previous autograft more frequently than CONV recipients. Non-relapse mortality (NRM) was 24% and 30% at 100-d and 1-year, respectively. After a median follow-up of 36 months, 17% of the patients developed disease progression, the 3-year progression-free survival (PFS) being 47%. Three-year overall survival (OS) for the whole series was 51%. On multivariate analysis, RIC regimens were associated with at lower NRM and a significantly longer PFS and OS. This retrospective study demonstrated that MUD-SCT results, even in heavily pre-treated populations, in a meaningful PFS and OS.

Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46.6% to 90.4%) and allogeneic AML cells (from 11.8% to 20.1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0.4% to 22.5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.

Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3(+) CD4(+) IL-17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS (P < 0.01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4(+)CD25(high) FoxP3(+) regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease (P < 0.005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis (P < 0.01). Tregs from MDS patients suppressed interferon-gamma (IFN-gamma) secretion by effector CD4(+) T cells but had no effect on interleukin (IL)-17 production. In addition, the serum levels of IL-7, IL-12, RANTES and IFN-gamma are significantly elevated in low risk MDS, while inhibitory factors, such as IL-10 and soluble IL-2 receptor, are significantly higher in high risk disease. The 'unfavourable' Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.

Van Den Berghe established 5q- syndrome as a discrete clinical entity in 1974 when he described patients with macrocytic anaemia, thrombocytosis, dyserythropoiesis, hypolobulated megakaryocytes and an interstitial deletion within chromosome 5q. With del(5q) as the sole cytogenetic abnormality, 5q- syndrome represents an opportunity to define precisely the molecular defect(s) underlying the pathogenesis of this disease. The commonly deleted region in 5q- syndrome, which is distinct from that in patients with complex cytogenetic changes that include del(5q), includes the ribosomal protein S14 locus and it has been proposed that that loss of an RPS14 allele accounts for the 5q- syndrome phenotype. However, this hypothesis fails to explain the growth advantage of the 5q- syndrome clone and it is evident that ribosomal protein defects are not specific to 5q- syndrome, as they are found in other bone marrow failure syndromes. Lenalidomide therapy leads to normalization of both haematological and cytogenetic parameters in the majority of 5q- syndrome patients. This review examines the potential role of several genes, including RPS14, in the pathogenesis of the 5q- syndrome and recent advances in clinical management, with particular emphasis on the role and mechanism of action of lenalidomide.

Haemopoietic cell transplantation (HCT) with reduced-intensity conditioning (RIC) has been associated with delayed disappearance of host anti-A and anti-B isohaemaglutinins and hindrance of donor erythropoiesis in major ABO mismatched transplants. Erythroid recovery, disappearance of recipient type and appearance of donor-type isohaemaglutinins was compared in 84 patients undergoing RIC and 50 patients with standard-conditioning (SCo) HCT. All patients received alemtuzumab as part of their conditioning. The incidence of immune-mediated anaemia and red cell transfusion usage were also compared. Immune factors affecting post-transplant erythroid kinetics showed little variance between different conditioning regimens. Disappearance of recipient isohaemaglutinins and emergence of donor red cells proceeded at similar rates in RIC and SCo transplants; the effects of ABO mismatch were marginal. Pure red cell aplasia, alloimmune haemolysis and autoimmune haemolytic anaemia were not more common in RIC transplants. We believe that alemtuzumab played a critical role in dampening immune reactions of both the host and the donor. Patients in both conditioning groups had similar post-transplant erythroid burst-forming unit (BFU-E) counts; BFU-E chimaerism analysis showed that 90-100% progenitors were of donor origin. However, transfusion requirements were significantly higher in the SCo group, due at least partly to earlier onset of bone marrow hypoplasia.

The role of haematopoietic stem cell transplantation (HSCT) in relapsed follicular lymphoma remains controversial. This study analysed 126 patients with relapsed, advanced stage follicular lymphoma who received BEAM (BCNU [carmustine], cytarabine, etoposide, melphalan)-alemtuzumab allogeneic HSCT (BEAM-allo) (n = 44) or BEAM-autologous HSCT (BEAM-auto) (n = 82). The BEAM-allo group had a younger median age (48 years vs. 56 years, P < 0.001) but received a higher median number of therapies pretransplant (P = 0.015) compared with the BEAM-auto group. There was a higher non-relapse mortality (NRM) in the BEAM-allo group compared with the BEAM-auto group at 1 year (20% vs. 2%, P = 0.001). Older age and heavily pretreated patients were associated with a higher NRM and poorer survival in the BEAM-allo group. There was, however, a significantly lower relapse rate (20% vs. 43%, P = 0.01) at 3 years with BEAM-alemtuzumab, with no relapses after 2 years, compared with a continued pattern of relapse in the autologous group. No difference in overall survival (OS) (P = 0.99) or disease-free survival (DFS) (P = 0.90) was identified at 3 years, whereas a plateau in OS and DFS with crossing of the survival curves in favour of BEAM-allo group was observed. Furthermore, the ability to re-induce remissions with donor leucocytes provides additional benefit in favour of allogeneic HSCT.

This prospective study evaluated the kinetics of lymphoid (CD3) engraftment in 110 patients with acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) after allogeneic transplantation and conditioning with fludarabine, busulphan and alemtuzumab, using ciclosporin for post-transplant immunosuppression. Declining donor CD3 chimaerism beyond day+100 was treated with pre-emptive donor lymphocyte infusion (pDLI). The median age of patients was 53.0 years (range: 19-72 years), and the median follow-up was 690 d (range:168-1470 d). Patients achieving full CD3 donor chimaerism (FDC, n = 46) by day+100 had a significantly inferior disease-free survival (DFS) and overall survival (OS) compared to patients with mixed donor chimaerism (MDC, n = 59). Twenty patients had stable MDC and did not require pDLI. Patients attaining early FDC had a higher transplant-related mortality compared to those who maintained stable levels of MDC (P = 0.02), with no difference between the FDC and pDLI groups (P = 0.07). There was no difference in relapse between all three groups (P = 0.21). On multivariate analysis, only CD3 chimaerism status at day+100 and disease status at transplantation had a significant effect on DFS and OS. In patients with AML/MDS undergoing alemetuzumab based-RIC HSCT, prolonged MDC beyond day+100 is associated with an improved OS. Future studies need to be directed towards establishing the underlying factors that dictate T-cell engraftment, expansion and homing post-transplantation.

The outcomes of 55 consecutive haemato-oncology patients admitted to the intensive care unit (ICU) were retrospectively analysed. Twenty-eight patients were admitted following haemopoietic stem cell transplantation (HSCT). Thirty-nine patients were admitted with respiratory failure, and all patients required respiratory support. Seventeen patients survived to be discharged from ICU, with an actuarial 1-year survival of 18%. Overall survival between patients who received intensive chemotherapy and those who underwent allogeneic HSCT was not significantly different (19% vs. 10%, P = 0.19). None of the nine myeloablative HSCT recipients survived (median survival: 9 d). Six of the 15 reduced-intensity conditioned HSCT recipients survived beyond 1 year (median survival: 1050 d, range: 438-1437).

This prospective study evaluated the outcomes of 75 successive patients receiving a FBC (fludarabine, busulphan, alemtuzumab) reduced-intensity conditioning (RIC) regimen for myelodysplastic syndromes (MDS) using volunteer unrelated donors(VUD). The prognostic significance of a variety of clinical variables including the recently described haematopoietic cell transplantation co-morbidity index (HCT-CI) was assessed. The median age of the cohort was 52.0 years (range: 19-68 years) with a median follow-up of 1038.5 d. Forty-nine patients (65%) had an International Prognostic Scoring System stage of > or = Intermediate-2, 35 (46%) had intermediate or poor risk cytogenetics, and 23 patients(31%) were human leucocyte antigen-mismatched. The actuarial 3-year overall survival (OS) and disease-free survival (DFS) was 43% [95% confidence interval (CI): 37-49] and 41% (95%CI: 35-47) respectively, and the cumulative incidence of extensive chronic graft-versus-host disease was 22%. On multivariate analysis, presence of either one class II mismatch or a two-antigen mismatch adversely influenced transplant-related mortality, DFS and OS. In addition, disease status at transplantation and the haematopoietic cell transplantation-specific comorbidity index were independent variables for overall survival. In contrast, both advanced age and pre-transplant cytogenetic status did not significantly affect overall outcomes. RIC regimens using VUD was associated with durable long-term survival even in older patients with MDS, and the use of a pre-transplant comorbidity index may help to improve patient selection for transplantation.

Conventional allogeneic stem cell transplantation (SCT) for myelodysplastic syndrome (MDS) is associated with excessive procedure-related mortality. The outcome following volunteer-unrelated donor (VUD) or sibling allogeneic SCT was therefore evaluated in 23 MDS patients conditioned with reduced-intensity regimens (fludarabine/busulphan/Campath-1H) because of advanced age (48 vs 37 years, P = 0.002) and/or co-morbidity (19 vs 3, P < 0.0001) which precluded conventional transplantation, and compared with 29 treated with standard protocols [busulphan/cyclophosphamide (Bu/Cy); Bu/Cy/total-body irradiation/Campath-1G]. Graft-versus-host disease (GVHD) prophylaxis comprised of cyclosporine/methotrexate. One hundred per cent donor engraftment (variable number tandem repeat analysis/cytogenetics/fluorescence in situ hybridization) was achieved in 18/19 (95%) evaluable patients receiving reduced-intensity regimens, although six (32%) have subsequently shown mixed chimaerism. Reduced-intensity conditioning was associated with significantly reduced duration of aplasia, less mucositis, fever, antibiotic, analgesia, parenteral nutrition use, less acute and chronic GVHD, and lower early procedure-related mortality [two (9%) vs nine (31%), P < 0.05]. Six patients relapsed (two standard, four reduced-intensity) and two (reduced-intensity) experienced late graft failure. The 2 year actuarial overall/disease-free survival (OS/DFS) was 48/39% in the reduced-intensity arm and 44/44% in the standard group. The 2 year non-relapse mortality was 31% and 50% respectively. In VUD recipients, OS was superior in the reduced-intensity arm (49%vs 34%). Predictors of DFS included good/intermediate-risk karyotype, low/intermediate-1 International Prognostic Scoring system score, human leucocyte antigen compatibility and attainment of complete remission. Our data demonstrates that VUD or sibling allogeneic SCT following reduced-intensity conditioning is feasible in high-risk MDS patients considered unsuitable for standard transplantation and is associated with comparable 3.5 year DFS to those receiving conventional regimens.

We have compared the phenotypic and functional characteristics of dendritic cells (DC) generated in vitro from the peripheral blood mononuclear fraction of myelodysplastic syndrome (MDS) patients (four refractory anaemia, four refractory anaemia with excess of blasts) with DCs generated in a similar way from eight healthy donors. After 10 d of culture in the presence of GM-CSF and IL-4, reduced numbers and percentages of DCs were obtained in MDS subjects. MDS DCs exhibited significantly lower expression of CD1a, CD54, CD80 and MHC class II molecules. Their ability to stimulate T lymphocytes in an allogeneic mixed leucocyte reaction was reduced in comparison to normal subjects. Furthermore, MDS DCs also showed a reduced receptor-mediated endocytosis as demonstrated by FITC-dextran uptake. Simultaneous fluorescence in situ hybridization (FISH) and immunophenotypic analysis demonstrated that MDS DCs have the same cytogenetic abnormality of the malignant clone. Taken together these findings indicate that, in MDS, DCs are part of the malignant clone and exhibit a deficient antigen uptake and presentation.

We performed flow cytometric analysis of CD34+ cell apoptosis in 59 patients with myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) secondary to MDS (MDS-AML) using annexin V-FITC, which binds to exposed phosphatidylserine on apoptotic cells. Apoptosis was significantly increased in FAB subtypes RA, RARS and RAEB (<10% blasts) (56.5% (15.1-86.5%)) compared to normal controls (18.5% (3.4-33.4%), P<0.0001) and RAEB-t/MDS-AML (16% (2.1-43.2%), P<0.0001). There was no correlation between % apoptosis, Full blood count or cytogenetics in any disease category. Two-colour cytometric analysis of permeabilized CD34+ cells stained with antibodies to Bcl-2, Bcl-X (anti-apoptotic), Bax and Bad (pro-apoptotic), demonstrated significantly higher ratios of pro- v anti-apoptotic proteins in early MDS (2.47 (1.19-9.42) compared to advanced disease (1.14 (0.06-3.32), P=0.0001). Moreover, using repeated measures of variants (ANOVA), we found that variations between individual Bcl-2-related proteins differed significantly according to disease subtype (P<0.0005). Our results confirm that CD34+ cell apoptosis was significantly increased in MDS subtypes RA and RARS and fell with disease progression. Early MDS was also associated with a significantly higher CD34+ cell pro- v anti-apoptotic Bcl-2-family-protein ratio than advanced disease. Furthermore, patterns of expression of individual Bcl-2 related proteins differed significantly between different disease categories. However, no correlation between pro- v anti-apoptotic Bcl-2-family-protein ratios and the degree of apoptosis was observed.

In myeloid malignancies, T-cell and NK function has been shown to deteriorate with transformation from pre-leukaemia to advanced disease. Immune dysfunction in solid tumours has been attributed to abnormal signal transduction, possibly through altered expression of intracellular components of the TCR/CD3 complex (e.g. CD3-zeta), receptors on NK cells and their associated protein tyrosine kinases (PTKs; p56lck, p59fyn and ZAP-70). Using a flow cytometric method to detect dual-expression of surface proteins and intracellular components of the TCR/CD3 complex, we have studied 46 patients with myeloid malignancies. CD3-zeta expression was abnormal in 64% of patients, and was more prominent in those with advanced disease. Three patients with reduced CD3-zeta were analysed both pre- and post-treatment, and recovery of CD3-zeta expression was associated with successful remission induction (expression of PTKs was variable and reduced levels were seen all disease stages). The results of this study suggest that loss of signalling proteins is not a result of direct contact of leukaemic cells with lymphocytes per se or the extent of the leukaemia burden, but to a specific property of some myeloid malignancies, which is more frequently acquired with greater malignant transformation.

Nineteen patients with high-risk myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML) received fludarabine, cytarabine, granulocyte-colony stimulating factor (G-CSF), and idarubicin chemotherapy (de novo MDS/MDS-AML, nine; relapsed/refractory MDS/AML, seven; therapy-related MDS, three). Median age was 44 years and median disease duration 10 months. 16/19 (84%) patients had abnormal cytogenetics with seven (37%) harbouring abnormalities of chromosome 7. 18/19 (94.7%) patients responded to FLAG-idarubicin with 12 (63%) achieving complete remission (CR) (< 5% blasts and normal cytogenetics). 7/9 (78%) patients with de novo MDS/MDS-AML achieved CR compared to 5/10 (50%) with alternative diagnoses. Response was associated with age < 50 years, disease duration < 3 months, and cytogenetics other than abnormalities of chromosome 7. Haemopoietic regeneration was rapid in most patients and there were no toxic deaths. Nine patients received a second course of chemotherapy, three have proceeded to allogeneic bone marrow transplant and three to autologous blood stem cell/bone marrow transplantation. Follow-up is short (median 10 months). 12/19 (63%) patients remain alive and 5/12 (42%) have relapsed at a median 5 months following CR achievement. FLAG-idarubicin was well tolerated. High rates of morphological and cytogenetic remission, especially in de novo MDS, offer a window of opportunity for assessment of autologous BMT in this group of diseases where no treatment except alloBMT has led to prolongation of survival.

Abnormalities of chromosome 5 and 7 are frequently found in primary MDS. Cases with familial monosomy 7 are well recognized, but there are no reports of familial MDS with deletion of 5q. We describe two sisters, aged 38 and 36 years, both of whom had MDS and interstitial deletion of 5q. The occurrence of this chromosomal abnormality reinforces the concept of tumour suppressor gene hypothesis in some cases with familial MDS.

Recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) is under investigation for the treatment of a wide range of haematological disorders. At commonly used doses of > 120 micrograms/m2/d, extramedullary toxicity is common. We report the effects of low-dose (LD) rhGM-CSF in patients with chronic neutropenia related to HIV infection, myelodysplastic syndrome and idiopathic neutropenia. Nine patients with a mean pre-treatment neutrophil count of 0.6 x 10(9)/l (range 0.2-1.4 x 10(9)/l) received daily rhGM-CSF at doses of between 5 and 15 micrograms/m2. Eight patients responded with a mean post-treatment ANC of 3.2 x 10(9)/l (range 1.9-4.6 x 10(9)/l). There was no significant therapy-related morbidity. We conclude that in chronic neutropenia, LD rhGM-CSF is an acceptable treatment which has important cost/benefit implications.

Peripheral blood cytopenias present a major problem in the management of patients with HIV infection. Their pathophysiology is likely to be multifactorial, although there is controversy as to whether haemopoietic progenitors are a target for HIV. In order to investigate the haemopoietic defect in HIV infection, we looked at bone marrow culture characteristics of marrow from eight HIV+ patients compared to normal controls. We performed long-term liquid culture (LTC) and colony forming assays for granulocyte-macrophage (CFU-GM) and granulocyte, erythroid, megakaryocyte, macrophage (CFU-GEMM). In LTC we found normal stromal appearance and haemopoietic focus formation. There was no difference in colony assays of CFU-GM and CFU-GEMM between HIV+ and normal controls. Colonies taken from CFU-GM and CFU-GEMM were analysed for HIV DNA sequences, and we were able to detect HIV DNA in colonies from all HIV+ patients. Our results indicate that despite infection of haemopoietic progenitor cells by HIV, bone marrow function is preserved. This suggests that HIV-related cytopenias may be due to alternative mechanisms not present in our in vitro system.

We describe the clinicomorphological features in 33 cases of primary myelodysplastic syndrome classified according to the FAB classification which presented to a single centre over a 12 year period. Presenting features were typically related to pancytopenia although hepatosplenomegaly and granulocytic sarcomas were far more prevalent than in the adult population. Morphological assessment of the peripheral blood and the bone marrow showed seven patients had refractory anaemia (RA), 13 patients had RA with excess of blasts (RAEB), nine patients had RAEB in transformation (RAEB-t) and four patients had chronic myelomonocytic leukaemia (CMML). The overall mean survival was short (9.9 months) in all the subgroups and the leukaemic transformation rate was high. None of the patients scored 0-1 according to the Bournemouth Scoring System; four patients scored 2 whereas 29 patients scored 3 to 4. We conclude that unlike adults, the myelodysplastic syndromes in children run an aggressive clinical course, irrespective of the FAB subtype, and the pathogenesis of these diseases in paediatric practice warrants scientific scrutiny. Intensive chemotherapy such as the one used in de novo-AML lead to complete remission in some children and these early results suggest that this should be the treatment of choice in paediatric MDS.