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Del(5q) myelodysplastic syndromes defined by the International Prognostic Scoring System as low- or intermediate-1-risk (lower-risk) are considered to have an indolent course; however, recent data have identified a subgroup of these patients with more aggressive disease and poorer outcomes. Using deep sequencing technology, we previously demonstrated that 18% of patients with lower-risk del(5q) myelodysplastic syndromes carry TP53 mutated subclones rendering them at higher risk of progression. In this study, bone marrow biopsies from 85 patients treated with lenalidomide in the MDS-004 clinical trial were retrospectively assessed for p53 expression by immunohistochemistry in association with outcome. Strong p53 expression in ≥ 1% of bone marrow progenitor cells, observed in 35% (30 of 85) of patients, was significantly associated with higher acute myeloid leukemia risk (P=0.0006), shorter overall survival (P=0.0175), and a lower cytogenetic response rate (P=0.009), but not with achievement or duration of 26-week transfusion independence response. In a multivariate analysis, p53-positive immunohistochemistry was the strongest independent predictor of transformation to acute myeloid leukemia (P=0.0035). Pyrosequencing analysis of laser-microdissected cells with strong p53 expression confirmed the TP53 mutation, whereas cells with moderate expression predominantly had wild-type p53. This study validates p53 immunohistochemistry as a strong and clinically useful predictive tool in patients with lower-risk del(5q) myelodysplastic syndromes. This study was based on data from the MDS 004 trial (clinicaltrials.gov identifier: NCT00179621)., (Copyright© Ferrata Storti Foundation.)

Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukemia and myelodysplastic syndrome correlated with chromosomal instability and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity through disruption of DNA repair function. Acute myeloid leukemia cell lines (n=12) and primary cell samples (n=18), and bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients (n=63) were profiled for microsatellite instability using fluorescent fragment polymerase chain reaction. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, Western blotting and gene expression analysis was used to study the functional consequences of mutations. Acute myeloid leukemia cell lines (4 of 12, 33%) and primary samples (2 of 18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukemia samples and cell lines demonstrated a downregulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11 of 56 (20%) patients with de novo high-risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4 of them (36%) possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP inhibitor sensitivity, whilst CtIP differentially regulated the expression of homologous recombination modulating RecQ helicases, WRN and BLM. In conclusion, microsatellite instability dependent mutations in DNA repair genes, CtIP and MRE11 are detected in myeloid malignancies conferring hypersensitivity to PARP inhibitors. Microsatellite instability is significantly correlated with chromosomal instability in myeloid malignancies.

Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients' survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4(+) T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4(+) T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4(+) T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3(+)/interleukin-17(+) cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4(+) T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1-0.8) and 2.1 (P<0.04, 95% CI, 1.1-4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease evolution with emerging oncogenic mutations adversely affecting patients' outcome, implicating spliceosome mutations as founder mutations in myelodysplastic syndromes.

Background: A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care., Design and Methods: Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease., Results: Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO(+) CD27(-)) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25(high) Foxp3(+)) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease., Conclusions: Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.

The monocyte is still the most difficult cell to identify with confidence in the peripheral blood or in the bone marrow in healthy individuals as well as in patients with infections, and in those with leukemic proliferations. The goal of this study was to establish morphological definitions so that monocytes, including immature monocytes, could be separated from the spectrum of monocyte precursors. Cells from peripheral blood or bone marrow were selected to provide a large panel of normal and leukemic cells at different maturational stages and were submitted to 5 experts, who had previously reached a consensus, on the basis of microscopy, in defining 4 subtypes: monoblast, promonocyte, immature monocyte, mature, monocyte. They achieved a good concordance rate of 76.6% and a high kappa rate confirming that the criteria for defining the 4 subtypes could be applied consistently. It has now to be established whether these monocyte subtypes correlate with immunological or molecular markers and are clinically relevant.

Unlabelled: Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors., Design and Methods: Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents., Results: PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.

Background: A stored cord blood donation may be a valuable source of hemopoietic stem cells for allogeneic transplantation when a matched sibling donor is not available. We carried out a study to define the optimal size of a national cord blood bank for the UK., Design and Methods: We calculated the actual numbers of possible donors and the chance of finding at least one donor for 2,000 unselected and for 722 non-North Western European patients for whom searches had been initiated as a function of three levels of HLA matching (4, 5 and 6 out of 6 alleles by HLA-A, -B low and -DRB1 high resolution HLA typing) according to various donor bank sizes., Results: With a bank size of 50,000, 80% of patients will have at least one donor unit available at the 5 out of 6 HLA allele match level (median 9 donors per patient), and 98% will have at least one donor at the 4 out of 6 allele match level (median 261). Doubling the size of the bank yields at least one donor for only an additional 6% of patients at the 5 of 6 allele match level. Moreover, for non-North Western European patients a 50,000 unit bank provides a donor for 50% at the 5 allele match level, and for 96% at the 4 allele match level., Conclusions: A bank containing 50,000 units is optimal for the UK and larger banks would only marginally increase the chance of finding suitable units.

Poly ADP-ribose polymerase inhibitors have been shown to target cells with homologous recombination DNA repair defects. We report that poly ADP-ribose polymerase inhibitors induces apoptosis in cells deficient in other key DNA repair components. Chromosomal instability disorders, Fanconi Anemia and Bloom's syndrome have dysfunctional DNA repair and an increased likelihood of leukemic transformation. PI addition to Fanconi Anemia and Bloom's syndrome cells resulted in significant apoptosis. Furthermore, poly ADP-ribose polymerase inhibitors induced apoptosis in DNA repair signaling defective ATM(-/-) and NBS(-/-) fibroblasts. Immunocytochemistry showed homologous recombination was abrogated in NBS(-/-) and ATM(-/-) fibroblasts, compromised in Fanconi anemia and normal in Bloom's syndrome cells in response to poly ADP-ribose polymerase inhibitors. Strikingly, poly ADP-ribose polymerase inhibitors increases non-homologous end joining repair activity, whilst non-homologous end joining deficient cells are extremely sensitive to poly ADP-ribose polymerase inhibitors. These data suggest poly ADP-ribose polymerase inhibitors target cells with DNA repair and signaling defects rather than solely defects in homologous recombination improving the potential of poly ADP-ribose polymerase inhibitors therapy in a wider range of cancers.

The classification of myelodysplastic syndromes is based on the morphological criteria proposed by the French-American-British (FAB) and World Health Organization (WHO) groups. Accurate enumeration of blast cells, although essential for diagnosis of myelodysplastic syndrome and for assignment to prognostic groups, is often difficult, due to imprecise criteria for the morphological definition of blasts and promyelocytes. An International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) of hematopathologists and hematologists expert in the field of myelodysplastic syndrome reviewed the morphological features of bone marrows from all subtypes of myelodysplastic syndrome and agreed on a set of recommendations, including recommendations for the definition and enumeration of blast cells and ring sideroblasts. It is recommended that (1) agranular or granular blast cells be defined (replacing the previous type I, II and III blasts), (2) dysplastic promyelocytes be distinguished from cytologically normal promyelocytes and from granular blast cells, (3) sufficient cells be counted to give a precise blast percentage, particularly at thresholds that are important for diagnosis or prognosis and (4) ring sideroblasts be defined as erythroblasts in which there are a minimum of 5 siderotic granules covering at least a third of the nuclear circumference. Clear definitions and a differential count of a sufficient number of cells is likely to improve precision in the diagnosis and classification of myelodysplastic syndrome. Recommendations should be applied in the context of the WHO classification.

Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T-cell lymphoma characterized morphologically by lymphadenopathy with a polymorphic infiltrate, marked vascular and follicular dendritic cell proliferation. Patients usually present with advanced disease and the overall prognosis is poor. While intensive chemotherapy has been shown to induce complete remissions in 50-70% of patients, the majority of patients subsequently relapse. Herein we report the case of a 32 year old man with AITL who was refractory to conventional chemotherapy, but achieved a remarkable sustained response to treatment with thalidomide and dexamethasone. Thalidomide may be an effective therapeutic agent against AITL, and given the poor prognosis of AITL, prospective clinical studies with either thalidomide or one of the thalidomide analogues are warranted.

Background and Objectives: Primary graft failure is a serious complication following hematopoietic cell transplants, particularly when using unrelated donors. We analyzed factors affecting primary graft failure in recipients of hematopoietic cell transplants from unrelated donors, which were performed using reduced intensity conditioning., Design and Methods: This was a retrospective analysis of 144 patients whose transplants took place between March 1998 and October 2004. The data were analyzed in January 2005., Results: The median age of the patients was 51 years. The diagnoses were varied. Conditioning regimens were fludarabine, melphalan, campath (n=80), fludarabine, busulphan, campath (n=38), fludarabine, BEAM, campath (n=9) and other (n=17). The donor was 10/10 allele matched in 95/144 (66%) cases; 94 donated bone marrow and 50 peripheral blood stem cells. The 3-year probability of overall survival was 43%. The median follow-up was 724 days (range: 91-1651 days). Of evaluable patients, 7/140 (5%) failed to achieve myeloid engraftment. Primary graft failure was significantly associated with the use of a mismatched donor (6/47,13% versus 1/93, 1%, p=0.006), as well as: bone marrow as the source of stem cells (p=0.046), chronic myeloid leukemia compared to other diagnoses (p=0.022), and a female rather than a male donor (p=0.019). In multivariate analysis chronic myeloid leukemia, HLA mismatched and/or female donors remained significantly associated with primary graft failure. Single HLA mismatches were tolerated, however in multiply mismatched grafts, overall survival was worse (p=0.005); transplanted-related mortality (p=0.005) and chronic graft-versus-host disease (p=0.025) were increased., Interpretation and Conclusions: These data have implications for the choice of donor and stem cell source in transplants performed using reduced intensity conditioning regimens, suggesting that the use of bone marrow, female donors and HLA-mismatched grafts increase the risk of primary graft failure, and should be avoided in certain situations.