Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines

The aim of this study was to investigate whether the suppressor of cytokine signalling (SOCS)-1 can act as a tumour suppressor when functioning as a negative regulator of the Janus family tyrosine kinases (JAKs), which have been reported to play important roles in leukaemogenesis. For this purpose, we carried out molecular analysis of the SOCS-1 gene in human acute myeloid leukaemia (AML) and human haematopoietic cell lines. Sequencing alterations in the coding region were found in two of 90 primary AML samples and one of 17 cell lines. Hypermethylation of the SOCS-1 gene was also observed in 72% of primary cases and 52% of cell lines and aberrant methylation strongly correlated with reduced expression. Transfection of SOCS-1 into Jurkat cells harbouring the mutation and methylation suppressed cell growth at a low serum concentration. These findings indicate that SOCS-1 is frequently silenced in haematopoietic malignancies, mainly as a result of hypermethylation, and suggest that SOCS-1 may be able to function as a tumour suppressor.     

Recent advances in the pathogenesis and treatment of juvenile myelomonocytic leukaemia

Myeloid neoplasms derive from the pathological clonal expansion of an abnormal stem cell and span a diverse spectrum of phenotypes including acute myeloid leukaemia (AML), myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). Expansion of myeloid blasts with suppression of normal haematopoiesis is the hallmark of AML, whereas MPN is associated with over-proliferation of one or more lineages that retain the capacity to differentiate, and MDS is characterized by cytopenias and aberrant differentiation. MPD and MDS can progress to AML, which is likely due to the acquisition of cooperative mutations. Juvenile myelomonocytic leukaemia (JMML) is an aggressive myeloid neoplasm of childhood that is clinically characterized by overproduction of monocytic cells that can infiltrate organs, including the spleen, liver, gastrointestinal tract, and lung. JMML is categorized as an overlap MPN/MDS by the World Health Organization and also shares some clinical and molecular features with chronic myelomonocytic leukaemia, a similar disease in adults. While the current standard of care for patients with JMML relies on allogeneic haematopoietic stem cell transplant (HSCT), relapse is the most frequent cause of treatment failure. This review outlines our understanding of the genetic underpinnings of JMML with a recent update on the discovery of novel CBL mutations, as well as a brief review on current therapeutic approaches.     

Elevated TRIB2 with NOTCH1 activation in paediatric/adult T-ALL

TRIB2 is a potent oncogene, elevated in a subset of human acute myeloid leukaemias (AML) with a mixed myeloid/lymphoid phenotype and NOTCH1 mutations. Although rare in AML, activating NOTCH1 mutations occur in 50% of all T cell acute lymphoblastic leukaemias (T-ALL). TRIB2 is a NOTCH1 target gene that functions in the degradation of key proteins and modulation of MAPK signalling pathways, implicated in haematopoietic cell survival and proliferation. This study showed that TRIB2 expression level is highest in the lymphoid compartment of normal haematopoietic cells, specifically in T cells. Analysis of TRIB2 expression across 16 different subtypes of human leukaemia demonstrated that TRIB2 expression was higher in ALL phenotypes versus all other phenotypes including AML, chronic lymphocytic leukaemia (CLL), myelodysplastic syndrome (MDS) and chronic myeloid leukaemia (CML). A T cell profile was distinguished by high TRIB2 expression in normal and malignant haematopoiesis. High TRIB2 expression was seen in T-ALL with normal karyotype and correlated with NOTCH signalling pathways. High TRIB2 expression correlated with NOTCH1/FBXW7 mutations in a paediatric T-ALL cohort, strongly linking NOTCH1 activation and high TRIB2 expression in paediatric T-ALL. The relationship between TRIB2 and T cell signalling pathways uniquely identifies leukaemia subtypes and will be useful in the advancement of our understanding of T cell and ALL biology.     

Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia: molecular classification and treatment options

Myelodysplastic syndromes (MDS) and the mixed myelodysplastic/myeloproliferative disorder juvenile myelomonocytic leukaemia (JMML) are rare haematopoietic stem cell diseases in children. While MDS-initiating events remain largely obscure, a growing body of clinical, genetic and laboratory evidence suggests that JMML is, at least in part, caused by aberrant signal transduction resulting from mutations of components of the RAS signalling pathway. To date, haematopoietic stem cell transplantation cures more than half of children diagnosed with MDS or JMML. Research on genetic conditions predisposing to MDS in young age, such as inherited syndromes with bone marrow failure, may present important insights into MDS pathogenesis.     

Expression of the Evi-1 gene in haemopoietic cells of children with juvenile myelomonocytic leukaemia and normal donors

Activation of the Evi-1 gene was first described to be associated with the transformation of murine myeloid leukaemias and has previously been detected in cases of human acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) in blast crises and in myelodysplastic syndromes. In this study we determined the frequency and the level of Evi-1 expression in juvenile myelomonocytic leukaemia (JMML) and in normal haemopoiesis. Using RT-PCR and Southern blot hybridization mRNA of Evi-1 could be detected in bone marrow (BM) and peripheral blood (PB) mononuclear cells (MNC) of normal donors. In JMML 12/20 patients examined expressed elevated levels of Evi-1 compared to normal controls. In these samples over-expression of the gene was correlated with a higher percentage of blasts ( P  = 0.02). Expression levels in BFU-E and CFU-GM derived colonies from BM of JMML patients were lower than those in the corresponding MNC samples. Analysis of CD34⁺ and CD34⁻ cells demonstrated that Evi-1 is primarily expressed in the CD34⁺ cell population of both JMML and normal donors. These findings suggest that Evi-1 expression is linked to the early stages of haemopoiesis. Studies on the regulation of Evi-1 expression in CD34⁺ cells will elucidate its function in progenitor cells and clarify its possible role in the pathogenesis of JMML.     

Methylation status of the p15 and p16 genes in paediatric myelodysplastic syndrome and juvenile myelomonocytic leukaemia

Aberrant DNA methylation is frequently observed in adults with myelodysplastic syndrome (MDS), and is recognized as a critical event in the disease's pathogenesis and progression. This is the first report to investigate the methylation status of p15 and p16, cell cycle regulatory genes, in children with MDS (n = 9) and juvenile myelomonocytic leukaemia (JMML; n = 18) by using a methylation-specific polymerase chain reaction. The frequency of p15 hypermethylation in paediatric MDS was 78% (7/9), which was comparable to that in adult MDS. In contrast, p15 hypermethylation in JMML was a rare event (17%; 3/18). In JMML, clinical and laboratory characteristics including PTPN11 mutations and aberrant colony formation were not different between the three patients with hypermethylated p15 and the others. Aberrant methylation of p16 was not detected in children with either MDS or JMML. Since p15 and p16 genes were unmethylated in two children with JMML, in whom the disease had progressed with an increased number of blasts, a condition referred to as blastic crisis, we infer that the aberrant methylation of these genes is not responsible for the progression of JMML. The results suggest that demethylating agents may be effective in most children with MDS and a few patients with JMML.     

Mutational analysis of the tumour suppressor gene MMAC1/PTEN in malignant myeloid disorders

The candidate tumour suppressor gene MMAC1/PTEN located at chromosome 10q23.3 has been reported to be frequently mutated in a number of solid tumours. Less is known about its status in leukaemia. In the present study we first analysed 13 leukaemia cell lines for mutations and homozygous deletions in MMAC1/PTEN using PCR and denaturing gradient gel electrophoresis (DGGE). We identified an intragenic deletion including MMAC1/PTEN exons 2-5 in an acute myelocytic leukaemia cell line, HL-60 blast, and an insertion of four nucleotides in exon 5 in an acute monocytic leukaemia cell line, U937. Analysis of 59 patients with acute myeloid leukaemia (AML), 26 patients with myelodysplastic syndromes (MDS) and 10 patients with chronic myeloid leukaemia (CML) only revealed a polymorphic base substitution in codon 44 in one AML patient, suggesting that mutations in the MMAC1/PTEN gene are infrequent genetic aberrations in myeloid leukaemia.     

Farnesyltransferase inhibitors and their potential role in therapy for myelodysplastic syndromes and acute myeloid leukaemia

Novel strategies are required for treatment of acute myeloid leukaemia (AML) and higher risk myelodysplastic syndrome (MDS) patients who are not eligible for intensive chemotherapy and/or allogenic stem cell transplantation. As activating RAS mutations are frequent in these diseases, one novel approach, consisting of interfering with isoprenylation of RAS proteins by farnesyltransferase inhibitors (FTIs), has been proposed. Clinical phase II studies with the oral FTIs tipifarnib and lonafarnib in previously untreated AML, MDS and chronic myelomonocytic leukaemia yielded rather encouraging results while results in relapsed and/or refractory AML were disappointing. Results of a phase III trial in untreated AML in the elderly with tipifarnib were also disappointing. Clinical responses were not related to RAS mutations, suggesting additional actions of FTIs on other molecular targets. The combination of existing FTIs with other treatments, such as chemotherapy (in AML) and hypomethylating agents (in MDS and AML), is under investigation. Ongoing studies will also determine if gene profiling analysis may help to identify patients that will respond to FTIs.     

Epigenetic dysregulation of the Jak/STAT pathway by frequent aberrant methylation of SHP1 but not SOCS1 in acute leukaemias

SOCS1 and SHP1 are negative regulators of the Jak/STAT signalling pathway that is implicated in leukaemogenesis. We studied if aberrant methylation of SOCS1 and SHP1 might be involved in the pathogenesis and prognostication of acute leukaemias by methylation-specific polymerase chain reaction (MSP). At diagnosis, methylation of SHP1 occurred more frequently in acute myeloid leukaemia (AML) (n=26, 52%) than acute lymphoblastic leukaemia (ALL) (n=6, 24%) (p=0.02). Methylation of SOCS1 was absent in both AML and ALL patients. SHP1 methylation was not associated with specific clinicopathologic features and had no prognostic impact on AML patients. Frequent methylation of SHP1, but not SOCS1, may be important in the pathogenesis, but not prognosis, of acute leukaemias.     

WT1 gene expression: useful marker for minimal residual disease in childhood myelodysplastic syndromes and juvenile myelo‐monocytic leukemia?

The WT1 gene is considered to be highly expressed in patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia and is thought to play a key role in maintaining the viability of leukemia cells. However, little is known about the WT1 gene expression levels in pediatric patients with juvenile myelo-monocytic leukemia (JMML) and myelodysplastic syndromes (MDS). We studied WT1 expression in diagnostic bone marrow (BM) and peripheral blood (PB) samples of 90 patients with JMML, low grade MDS, advanced MDS and myelodysplasia-related AML in BM (n = 20) and PB (n = 18) samples of normal healthy volunteer donors.     

Megakaryopoiesis in vitro in myelodysplastic syndromes and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.     

Primary myelodysplastic syndrome in children--clinical, hematological and histomorphological profile from a tertiary care centre in India

We describe the clinical, hematological and histomorphological features in children of primary myelodysplastic syndrome (MDS) seen at the All India Institute of Medical Sciences over three years (Jan 2001-Jan 2004). Twenty-one patients of primary MDS aged 17 year or less were classified using the latest proposed WHO classification for Pediatric MDS. The median age was 9 years with male predominance (80%). Pallor was present in all the cases while fever and bleeding diathesis was present in more than 50% of the cases. Morphological assessment of the peripheral blood showed macrocytosis in 50%, pancytopenia in 15% and blast cells in 45% of cases. A complete analysis of clinical features in conjunction with the bone marrow profile revealed 8 cases of refractory cytopenia (RC), 3 cases of refractory anemia with excess blasts (RAEB), 5 cases of refractory anemia with excess blasts in transformation (RAEB-T), 4 cases of Juvenile myelomonocytic leukemia (JMML) and a solitary cases of acute myeloid leukemia (AML) in Downs syndrome.These children were followed up from 1-36 months (mean 15 months). Three patients of RAEB-T progressed to AML within 3-4 months. RC had the best prognosis and all are alive and under regular follow up. The solitary case of AML of Downs syndrome died 1.5 months after initial diagnosis. All 3 cases of RAEB are under regular follow-up and doing well. Three cases of RAEB-T died (all had progressed to AML); the remaining 2 cases were lost to follow up. Of the 4 cases of JMML 1 died within 6 months of diagnosis; the other 3 cases are under regular follow up of whom 1 has a progressively increasing blast count.We conclude that the latest proposed WHO classification for Pediatric MDS can be successfully applied to all cases of primary MDS.     

Somatic point mutations in RUNX1/CBFA2/AML1 are common in high-risk myelodysplastic syndrome, but not in myelofibrosis with myeloid metaplasia

OBJECTIVE: Acquired somatic point mutations in RUNX1/CBFA2/AML1 have recently been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Given the importance of core-binding factor in megakaryocytic differentiation and platelet production, as well as the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), we hypothesised that RUNX1 gene mutations might be common in MMM. In addition, it is unclear whether patients with MDS-associated acquired alpha thalassaemia (ATMDS), a special subgroup with a very high incidence of point mutations in the ATRX gene, have an especially high incidence of RUNX1 mutations. METHODS: We analysed samples from 78 patients for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC): 26 with MMM and 52 with MDS, including 18 with ATMDS. RESULTS: We found five RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 or a history of treated AML, but none in MMM patients. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18, 11.1%) when compared with MDS without thalassaemia (3/34, 8.8%; P = 0.58). CONCLUSION: RUNX1 point mutations are common in high-risk MDS, but not in MMM. DHPLC is a useful technique for high-throughput analysis of RUNX1 mutation status in myeloid disorders, and may be complementary to screening via other methods.     

Absence of N-RAS point mutations in peripheral blood cells of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinurea

Summary. The myelodysplastic syndromes (MDS) have a significant frequency of evolution into acute myeloid leukaemia (AML). Approximately 30% of MDS patients show activating mutations of the N-RAS proto-oncogene, and these patients are at increased risk of leukaemic evolution. Long-term survivors of aplastic anaemia (AA) and paroxysmal nocturnal haemoglobinurea (PNH) are also at significant risk of developing AML. We have screened peripheral blood DNA from 42 AA patients and 15 PNH patients for the presence of N- RAS point mutations. No mutations were detected in these samples, indicating that the mechanisms of evolution into AML may be different from those in MDS.     

多基因甲基化在胰腺癌发病机制中的作用

目的:检测基NRASSF1A,p16,SOCS-1和 hMLH1在胰腺癌中甲基化的作用．方法:采用饱和氯化钠法提取肿瘤组织(n= 21)和瘤旁组织(n=21)DNA．采用甲基化特异 PCR对上述基因甲基化状态进行分析．结果:在癌旁组织中,RASSF1A,p16,SOCS-1 和hMLH1甲基化频率分别为36．4％,13．6％, 13．6％和4．5％;在胰腺癌中分别为59．1％, 40．9％,31．8％和18．2％．P16甲基化频率在两种组织间具有显著性差异(x2=4．13,P<0．05)． 45．5％胰腺癌被检出存在2个或2个以上基因甲基化,明显高于癌旁组织(9．1％,x2=7．33, P<0．01)．31．8％胰腺癌没有检出任何一种基因甲基化．结论:多基因甲基化是部分胰腺癌发展过程中一种早期事件,在这部分胰腺癌的发病中起重要作用．     

Methylation of the p15(INK4B) gene in myelodysplastic syndrome: it can be detected early at diagnosis or during disease progression and is highly associated with leukaemic transformation

To investigate the time sequence of occurrence of p15(INK4B) gene methylation in myelodysplastic syndrome (MDS) and its correlation with leukaemic transformation and survival of patients, the methylation status of the p15(INK4B) promoter region was analysed in 50 patients and was serially studied in 22 of them. Of the 50 patients, 17 (34%) showed p15(INK4B) gene methylation, first demonstrated at diagnosis or during follow-up. When FAB subtypes at the time of study were used in the analysis, the incidence of (p15INK4B) methylation in each risk group of MDS remained stable throughout the course: 0% for low-risk MDS [refractory anaemia (RA) and RA with ring sideroblasts] and from 23% at diagnosis to 30% for high-risk MDS [RA with excess of blasts (RAEB), RAEB in transformation and chronic myelomonocytic leukaemia] respectively. The incidence of p15(INK4B) methylation rose to 60% at initial study and, finally, to 75% in cases of acute myeloid leukaemia (AML) evolved from MDS. Most patients (69%) with p15(INK4B) methylation showed disease progression to AML; it could be detected before, at the time or after the diagnosis of leukaemic transformation. p15(INK4B) methylation in MDS patients implicated a shorter survival time in univariate analyses, but its prognostic significance disappeared in multivariate analyses. In conclusion, p15(INK4B) methylation can be detected early at the diagnosis of MDS or acquired during disease progression. It may play an important role in the pathogenesis of some high-risk MDS and is related to leukaemic transformation of MDS.     

Progression of myelodysplastic syndrome: allelic loss on chromosomal arm 1p

Myelodysplastic syndrome (MDS) is a common neoplasm of haematopoietic pluripotent stem cells. Although one third of MDS patients evolve to acute myeloid leukaemia (AML), little is understood about the mechanisms responsible for this progression. We have previously detected the frequent loss of heterozygosity (LOH) on the short arm of chromosome 1 in blast crisis of chronic myelocytic leukaemia. In this study, we examined the chromosomal arm 1p for allelic loss in the progression of MDS to AML, using 17 microsatellite markers spanning chromosome 1 in 20 patients who progressed from MDS to AML. DNA was extracted from slides of bone marrow smears. In each patient, DNA from MDS was analysed alongside DNA from AML. Allelic loss on 1p was observed in six of the 20 individuals (30%). Serial cytogenetic information was available in five of the six patients with LOH on 1p; no deletions in this region were detected. Three samples showed LOH at all informative loci on 1p. The other three samples showed LOH on at least one but not all loci on 1p with consensus regions of LOH located distal to D1S253 (1p36.3) and probably proximal to D1S496 (1p32-). Our results suggest that tumour suppressor genes that play an important role in the progression of MDS to AML may reside in at least two different regions on 1p.