Mitochondrial DNA mutations in patients with myelodysplastic syndromes

We undertook to systematically analyze the entire mitochondrial genome by gene amplification and direct sequencing in 10 patients with myelodysplasia; results were compared with concomitantly studied 8 healthy volunteers as well as mtDNA sequences in a standard database. Nucleotide changes that were present in our healthy controls as well as those in published databases were counted as polymorphisms. Overall, there was no increase in the number of mtDNA genes harboring polymorphisms or "new" mutations between our patients and healthy controls, although there were a few more mtDNA changes resulting in amino acid changes in myelodysplasia (9 in 8 controls versus 16 in 10 patients). Thirty new mutations, all nucleotide substitutions, were found among the 10 patients, distributed throughout the mitochondrial genome; 5 mutations resulted in amino acid changes. None of the mutations in controls produced amino acid changes. We were not able to confirm previously described mutations in sideroblastic anemia or "hot spots" in the cytochrome c oxidase I and II genes. Our data do not support a major role for mitochondrial genomic instability in myelodysplasia, and they fail to reproduce previous reports of significant or widespread mitochondrial mutations in this disease. Modest changes in mutation numbers and mitochondrial microsatellites may be evidence of increased mutagenesis in mtDNA, or, more likely, a reflection of limited clonality among hematopoietic stem cells in this bone marrow failure syndrome.     

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that small deletions in SRSF2 cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. We also verified aberrant splicing of key genes USP9X, USP24 (deubiquitinating enzymes), LUC7L2 (splice factor) and EED (PRC2 component) in MDS harboring small deletions of SRSF2. Pathway analysis revealed that mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations. Our study reveals splicing signatures of each splice factor mutation and identifies shared and distinct sets of mis-spliced genes and affected biological processes in different spliceosome mutant MDS.     

Current therapeutic approaches for patients with myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a heterogeneous spectrum of disorders requiring selective therapy based on patients’ specific clinical features, predominantly their prognostic subgroups, age and performance status. Guidelines for management of patients with MDS have been generated by a number of national panels. This review focuses on evidence‐based data supporting therapeutic approaches, which have also been recommended by the US National Comprehensive Cancer Network MDS Panel, with discussion of accessibility of recommended drugs in the US and in other countries. For lower risk disease (International Prognostic Scoring System Low and Intermediate‐1) therapy is aimed at haematological improvement whereas for higher risk disease (Intermediate‐2 and High) treatment focuses on altering disease natural history. Recent information regarding additional clinical and biological features has provided useful parameters for assessing disease prognosis that aid risk‐based management decisions. The rationale for use of low versus high intensity therapies with these agents, including allogeneic haematopoietic stem cell transplantation, is discussed in detail.     

Hypoalbuminemia is an independent prognostic factor for overall survival in myelodysplastic syndromes

We hypothesized that hypoalbuminemia is an independent prognostic factor in patients with myelodysplastic syndromes (MDS). We analyzed records of 767 patients treated at Moffitt Cancer Center between January 2001 and December 2009 to evaluate the relationship between serum albumin (SA) at the time of presentation and overall survival (OS). Patients (median age of 69 years) were stratified into three groups based on SA concentration (≤3.5, 3.6–4.0, and >4.0 g/dL). Two‐thirds of the patients had low or intermediate‐1 International Prognostic Scoring System (IPSS)‐based risk for MDS. Median OS by SA concentration of ≤3.5, 3.6–4.0, and >4.0 g/dL was 11, 23, and 34 months, respectively (P < 0.005), whereas rate of acute myeloid leukemia progression was highest in patients with low SA (≤3.5 g/dL). The SA level offered prognostic discrimination for outcomes within the lower and higher IPSS risk groups, as well as with the MD Anderson risk model. In multivariable analysis, SA was a significant independent co‐variate for OS after adjustment for IPSS, age, serum ferritin, and transfusion dependence (hazard ratio = 0.8; 95% CI 0.6–0.9; P = 0.004). Our findings indicate that hypoalbuminemia is an independent prognostic biomarker that may serve as a surrogate representative of disease biology or comorbidities in patients with MDS. Am. J. Hematol., 2012. © 2012 Wiley Periodicals, Inc.     

NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.     

Evaluation of apoptosis as a prognostic factor in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hypercellular bone marrow. This is thought to be as a result of the apoptosis of haematopoietic bone marrow cells resulting in ineffective haematopoiesis. To clarify the relationship between prognosis and apoptosis and/or cell proliferation in the bone marrow, we studied 51 cases with MDS. Bone marrow biopsies were stained immunohistochemically for MIB-1 (marker for proliferating cells) and CD34 (marker for stem cells). Apoptosis was visualized by detection of DNA fragmentation using TdT incorporation of nucleotides on 3' ends of DNA (TUNEL technique) and expressed as the apoptotic rate. MDS patients included 32 with refractory anaemia (RA), one RA with ringed sideroblasts (RARS) patient, seven RA with excess of blasts (RAEB) patients, eight patients with RAEB in transformation (RAEB-t) and three patients with chronic myelomonocytic leukaemia (CMMoL). In addition, we also studied six cases with acute myeloid leukaemia (AML) arising from MDS (AML-MDS) and ten control subjects. Fatal pancytopenia was the cause of death in 19 out of 51 patients. The apoptotic rate was higher in MDS patients (5.5%) than in control subjects (0.6%) and AML-MDS patients (0.4%). The percentage of MIB-1 positive cells was higher in MDS and AML-MDS than in control. The percentage of CD34-positive cells was higher in AML-MDS, RAEB, RAEB-t and CMMoL patients than control subjects and RA patients. Our findings indicate the activation of both the proliferative and apoptotic rates in MDS. Poor prognosis correlated significantly with higher apoptotic rates, but not with percentages of MIB-1 and CD34-positive cells. Our results suggest that apoptosis might be a useful prognostic factor and inhibition of apoptotic mechanisms may induce leukaemic transformation in MDS.     

Serum stem cell factor concentration in patients with myelodysplastic syndromes

Summary. Stem cell factor (SCF) is characterized by its capacity to synergize dramatically with other haemopoietic growth factors in in vitro erythroid, myeloid, and lymphoid progenitor culture systems. We have measured serum SCF concentrations by enzyme immunoassay in 85 patients with myelodysplasia (MDS). Serum samples were taken in 1988–89 and in 1991–92 and stored at -20°C. Mean serum SCF concentration in the MDS patients was 2.81 ng/ml (range 0.6–8.0). This was significantly lower (P=0.0001) than the values for 234 normal subjects: mean 3.30 ng/ml (range 1.3–8. 0). No significant relationship between SCF concentration and peripheral blood counts, bone marrow parameters, red cell transfusion status, survival or FAB subtype was found, although a trend of decreasing SCF concentration from refractory anaemia through sideroblastic anaemia and chronic myelomonocytic leukaemia to refractory anaemia with excess blasts was seen. The reduced SCF serum concentration in some patients with myelodysplasia suggests a rationale for therapy with recombinant SCF in these patients.     

Hemochromatosis-associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional iron-overload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P = 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P = 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls.     

A therapeutic trial with low-dose cytarabine in myelodysplastic syndromes and acute leukemia

The myelodysplastic syndromes are a group of bone marrow stem cell disorders which were considered refractory to chemotherapy until recently. Low-dose cytarabine was given to 6 patients with symptomatic myelodysplastic syndromes and 2 patients with acute leukemia. 5 patients responded to therapy, 3 of whom with refractory anemia achieved normalization of peripheral blood counts. Therapy was well tolerated, myelosuppression was the predominant side effect. This preliminary trial demonstrates that patients with symptomatic myelodysplastic syndromes, particularly patients with refractory anemia may be benefited by low-dose cytarabine therapy.     

TP53 overexpression is an independent adverse prognostic factor in de novo myelodysplastic syndromes with fibrosis

Bone marrow (BM) fibrosis is associated with poor prognosis in patients with de novo myelodysplastic syndromes (MDS). TP53 mutations and TP53 (p53) overexpression in MDS are also associated with poor patient outcomes. The prevalence and significance of TP53 mutations and TP53 overexpression in MDS with fibrosis are unknown. We studied 67 patients with de novo MDS demonstrating moderate to severe reticulin fibrosis (MDS‐F). Expression of TP53 was evaluated in BM core biopsy specimens using dual‐colour CD34/TP53 immunohistochemistry with computer‐assisted image analysis. Mutation analysis was performed using next‐generation sequencing, or Sanger sequencing methods. TP53 mutations were present in 47·1% of cases. TP53 mutation was significantly associated with TP53 expression (P = 0·0294). High levels of TP53 expression (3 +  in ≥10% cells) were associated with higher BM blast counts (P = 0·0149); alterations of chromosomes 5 (P = 0·0009) or 7 (P = 0·0141); complex karyotype (P = 0·0002); high‐ and very‐high risk IPSS‐R groups (P = 0·009); and TP53 mutations (P = 0·0003). High TP53 expression independently predicted shorter overall survival (OS) by multivariate analysis (P = <0·001). Expression of TP53 by CD34‐positive cells was associated with shorter OS and leukaemia‐free survival (P = 0·0428). TP53 overexpression is a predictor of poor outcome in patients with MDS‐F.     

SF3B1 mutations in patients with myelodysplastic syndromes: The mutation is stable during disease evolution

The SF3B1 mutation can be detected in patients with myelodysplastic syndrome (MDS), but the report regarding the association of this mutation with other genetic alterations and its stability during disease progression is limited. In this study, SF3B1 mutations were identified in 10% of total cohort of 479 MDS patients and 61.8% of 34 patients with refractory anemia with ring sideroblasts (RARS). SF3B1 mutations were closely associated with older age, higher platelet counts, lower lactate dehydrogenase levels, good‐risk cytogenetics, and mutations of DNMT3A, but inversely related to ASXL1 mutations. Most SF3B1‐mutated patients had concurrent other genetic alterations, including DNMT3A and RUNX1 mutations. There was no prognostic difference between patients with SF3B1 mutations and those without. Sequential studies in 417 samples from 142 patients demonstrated that all SF3B1‐mutated patients retained the same mutations during disease evolution with the exception of two patients who lost the mutation after allogeneic hematopoietic stem cell transplantation, whereas none of the SF3B1‐wild patients acquired a novel mutation during clinical follow‐ups. In conclusion, the patients with SF3B1 mutations had distinct clinic‐biologic features. SF3B1 mutations, accompanied with other genetic alterations, especially DNMT3A mutations, may play a role in the development of MDS, but have little role in disease progression. Am. J. Hematol. 89:E109–E115, 2014. © 2014 Wiley Periodicals, Inc.     

Rare occurrence of DNMT3A mutations in myelodysplastic syndromes

Gene mutations and epigenetic changes have been shown to play significant roles in the pathogenesis of myelodysplastic syndromes. Recently, mutations in DNMT3A were identified in 22.1% of patients with acute myeloid leukemia. In this study, we analyzed the frequency and clinical impact of DNMT3A mutations in a cohort of 193 patients with myelodysplastic syndromes. Mutations in DNMT3A were found in 2.6% of patients. The majority of mutations were heterozygous missense mutations affecting codon R882. Patients with DNMT3A mutations were found to have a higher rate of transformation to acute myeloid leukemia. When assessing the global methylation levels in patients with mutated versus unmutated DNMT3A and healthy controls no difference in global DNA methylation levels between the two groups was seen. Our data show that in patients with myelodysplastic syndromes, DNMT3A mutations occur at a low frequency and may be a risk factor for leukemia progression.