Low expression of the putative tumour suppressor gene gravin in chronic myeloid leukaemia, myelodysplastic syndromes and acute myeloid leukaemia

The putative tumour suppressor gene gravin is down-regulated in several solid tumours and is implicated in tumorigenesis. We have evaluated the expression levels of the gravin gene in the CD34(+)/blast cells of a range of myeloid malignancies as compared with controls using real-time quantitative polymerase chain reaction (PCR). Gravin was markedly down-regulated in 41 of 41 patients with acute myeloid leukaemia (AML), nine of 10 patients with myelodysplastic syndromes (MDS) and 33 of 33 patients with chronic myeloid leukaemia (CML), of whom 24 were in blast crisis (BC). We have shown that gravin is consistently down-regulated in the CD34(+)/blast cells of myeloid malignancies and may play a role in the molecular pathogenesis of these disorders.     

A phase 2 trial of high dose lenalidomide in patients with relapsed/refractory higher‐risk myelodysplastic syndromes and acute myeloid leukaemia with trilineage dysplasia

Limited therapies exist for patients with refractory and relapsed (RR ) higher‐risk myelodysplastic syndromes (HR ‐MDS ) and acute myeloid leukaemia with trilineage dysplasia (AML ‐TD ). High dose (HD ) lenalidomide (50 mg) has activity as frontline therapy in elderly AML but there is limited data in the RR setting. This phase II trial included patients with RR HR ‐MDS or AML ‐TD at 2 doses of lenalidomide (15 or 50 mg) on days 1–28 of 42‐day cycles. The primary endpoint was response rate using the 2006 International Working Group criteria. Overall survival (OS ) was estimated by Kaplan–Meier methods. Of 27 patients enrolled, 59% had HR ‐MDS and 31% AML ‐TD . No patient had isolated del5q; 41% had poor‐risk karyotype. Of 9 patients treated at 15 mg, 56% completed ≥2 cycles with no responses. Of 18 patients treated at 50 mg, 39% completed ≥2 cycles and 11% responded but all experienced grade 3/4 neutropenic fever/infection. The 60‐day mortality rate was 30%. Median OS was 114 days with 19% surviving ≥1 year. The study was terminated due to lack of robust clinical activity. In conclusion, lenalidomide at 15 mg is ineffective in RR myeloid malignancies. Continous high dosing schedules are poorly tolerated and minimally active. Further evaluation should be considered in upfront intensive chemotherapy‐ineligible patients.     

A review of therapy-related myelodysplastic syndromes and acute myeloid leukaemia (t-MDS/AML) in Irish patients: a single centre experience

Objectives: To demonstrate the incidence, characteristics, treatment and outcomes of patients with therapy-related myelodysplastic syndromes and therapy-related acute myeloid leukaemia (t-MDS/AML) in a tertiary referral centre.

Methods: Patients meeting the diagnostic criteria for t-MDS/AML from 2003 to 2014 were reviewed to analyse their diagnostic features, details of antecedent disorder and treatment, approach to management and survival.

Results: 39 patients who developed t-MDS/AML were identified with incidence of 8.7%. Median age and gender distribution were similar to de novo MDS but t-MDS/AML patients had greater degree of cytopenia and adverse karyotypes. Time to development of t-MDS/AML was shortest for patients with antecedent haematological malignancy compared to solid tumours and autoimmune disorders (46, 85 and 109 months). Patients with prior acute leukaemia had the shortest latency and poor overall survival. Treatment options included best supportive care (56%), Azacitidine (31%) or intensive chemotherapy/allogeneic transplant (13%). Median OS of all patients was 14 months. Survival declined markedly after two years and 5-year OS was 13.8%. Longer survival was associated with blast count <5% at diagnosis, previous haematological disorder, lower risk IPSS-R and a normal karyotype. Four out of five patients who received intensive therapy/transplant remain alive with median OS of 14 months. Median OS of Azacitidine-treated group was 11 months.

Discussion: t-MDS/AML patients showed unique characteristics which influenced their treatment and outcomes. IPSS-R may be useful in risk-adapted treatment approaches and can predict outcomes. Survival remains poor but improved outcomes were seen with allogeneic transplantation. Azacitidine may be effective in patients unfit for intensive therapies.     

Aberrant methylation of the negative regulators RASSFIA, SHP-1 and SOCS-1 in myelodysplastic syndromes and acute myeloid leukaemia

Mutations in the receptor tyrosine kinase (RTK/RAS) signalling pathway frequently provide a proliferative signal in myeloid malignancies. However, the role of RASSF1A, SHP-1 and SOCS-1, negative regulators of RTK/RAS signalling, has not been extensively investigated in the myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). This study employed methylation-specific polymerase chain reaction (MS-PCR) to determine if aberrant promotor methylation of RASSF1A, SHP-1 and SOCS-1 is involved in the pathogenesis of myeloid malignancies. Patients with MDS (n = 107), AML (n = 154) and juvenile myelomonocytic leukaemia (JMML, n = 5) were investigated, together with 15 normal controls. Primers were located in the promotor region of each gene as well as within exon 2 of SOCS-1. Methylation of RASSF1A was found in five of 55 (9%) MDS cases, but not in any of 57 AML cases studied. RASSF1A methylation was present in one case (20%) of JMML. SHP-1 methylation was present in 13 of 121 (11%) AML cases but was not found in MDS or JMML. SOCS-1 promoter methylation was present in eight of 74 (11%) MDS patients but was not seen in JMML or AML. Importantly, RAS mutations and RASSF1A and SOCS-1 methylation were mutually exclusive indicating that approximately 30% of MDS cases had a defect of the RTK/RAS pathway and its negative regulation. Finally, SOCS-1 exon 2 methylation may not be pathogenetically relevant, since it was detected in samples from normal individuals and did not correlate with promotor methylation.     

Molecular pathways in myelodysplastic syndromes and acute myeloid leukemia: relationships and distinctions-a review

The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are both hematopoietic stem cell disorders. However, while leukemic stem cells have been revealed by clonal tracking experiments, dysplastic stem cells have never been demonstrated by xeno-transplantation assays because of poor engraftment problems. These engraftment difficulties may be due to the unique nature of MDS genetic lesions that are truly able to recapitulate the disease phenotype. MDS and AML of younger patients harbour clonal yet different chromosomal markers, whereas MDS and AML of the elderly present similar defects. Potential involvement of tumor suppressor genes in MDS has been hypothesized but never confirmed, while cooperation between class I and class II mutations has been identified in AML. The reciprocal interactions between stromal cells and neoplastic clones are disrupted in both MDS and AML. In early MDS, stromal and neoplastic cells produce high levels of inhibitory cytokines, whereas in advanced MDS and AML they produce high levels of anti-apoptotic molecules.     

Cancer in relatives of children with myelodysplastic syndrome, acute and chronic myeloid leukaemia

Familial myelodysplastic syndrome (MDS) has been claimed to account for as many as one third of children with MDS, especially among those showing monosomy 7. The present study is the first to provide population-based estimates of the risk of haematological and other malignancies in relatives of children with MDS. The study was extended to include children with acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML). The index group consisted of 46 children with MDS, 62 with AML, and eight with CML, which is thought to represent all myeloid leukaemias in Danish children, 1980–91. By linkage to the Central Population Register we identified parents (230), siblings (231), grandparents (151), aunts and uncles (132) and cousins (140). Information on the cancer incidence was obtained from the Danish Cancer Registry. 27 cancers were observed versus 26.7 expected (relative risk 1.0). Leukaemia in relatives was observed in only one family. None of 11 children with MDS and monosomy 7 had family members affected by leukaemia. We found no evidence of an increased overall risk of cancer in the relatives. The risk of familial MDS may be considerably lower than previously estimated.     

Delayed attainment of full donor chimaerism following alemtuzumab-based reduced-intensity conditioning haematopoeitic stem cell transplantation for acute myeloid leukaemia and myelodysplastic syndromes is associated with improved outcomes

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.     

Platelet doubling after the first azacitidine cycle is a promising predictor for response in myelodysplastic syndromes (MDS), chronic myelomonocytic leukaemia (CMML) and acute myeloid leukaemia (AML) patients in the Dutch azacitidine compassionate named patient programme

The efficacy of azacitidine in the treatment of high‐risk myelodysplastic syndromes (MDS), chronic myelomonocytic leukaemia (CMML) and acute myeloid leukaemia (AML) (20–30% blasts) has been demonstrated. To investigate the efficacy of azacitidine in daily clinical practice and to identify predictors for response, we analysed a cohort of 90 MDS, CMML and AML patients who have been treated in a Dutch compassionate named patient programme. Patients received azacitidine for a median of five cycles (range 1–19). The overall response rate (complete/partial/haematological improvement) was 57% in low risk MDS, 53% in high risk MDS, 50% in CMML, and 39% in AML patients. Median overall survival (OS) was 13·0 (9·8–16·2) months. Multivariate analysis confirmed circulating blasts [Hazard Ratio (HR) 0·48, 95% confidence interval (CI) 0·24–0·99; P = 0·05] and poor risk cytogenetics (HR 0·45, 95% CI 0·22–0·91; P = 0·03) as independent predictors for OS. Interestingly, this analysis also identified platelet doubling after the first cycle of azacitidine as a simple and independent positive predictor for OS (HR 5·4, 95% CI 0·73–39·9; P = 0·10). In conclusion, routine administration of azacitidine to patients with variable risk groups of MDS, CMML and AML is feasible, and subgroups with distinct efficacy of azacitidine treatment can be identified.     

Rapid and sensitive screening for CEBPA mutations in acute myeloid leukaemia

The presence of CCAAT/enhancer binding protein alpha (CEBPA) gene mutations in patients with cytogenetically normal acute myeloid leukaemia (CN-AML) confers a favourable prognosis. Routine screening of all CN-AML patients for CEBPA mutations is therefore important for individual risk-adapted post-remission therapy and requires a fast and easy screening method. CEBPA mutations are distributed over the entire CEBPA gene and the functional and clinical consequences of the different mutations are still largely unknown. Therefore, we developed a multiplex polymerase chain reaction-based fragment length analysis mutation screening method for the entire CEBPA coding region. We initially evaluated our method by analysing 120 CN-AML samples both by fragment analysis and nucleotide sequencing and reached a sensitivity of 100% and a specificity of 90%. 349 CN-AML samples were subsequently screened for CEBPA mutations by fragment length analysis. Among a total of 469 CN-AML patient samples, 58 CEBPA mutations were detected in 38 CN-AML patients (8.1%). In conclusion, we established a fast and sensitive CEBPA mutation screening method suitable for inclusion in routine AML diagnostics.     

Dysplastic peripheral blood polymorphs link acute myeloblastic leukaemia in elderly to the myelodysplastic syndromes.

We studied dysplastic features in peripheral blood polymorphs from 80 patients with acute leukaemia. Thirty-seven patients with de novo acute myeloblastic leukaemia (AML) were compared to 26 patients with AML that had developed after a myelodysplastic phase (MDS-AML), and 17 cases of acute lymphoblastic leukaemia (ALL). Cytoplasmic hypogranulation in neutrophils, measured as a score value (G-score; normal range: 255-300), and the percentage of pelgeroid polymorphs (ppp; normal range: 0.5%) were studied retrospectively by reviewing the diagnostic peripheral blood smears. The mean G-score was decreased in MDS-AML (178 +/- 67.9), and in de novo AML (212 +/- 65.1), but not in ALL (275 +/- 24.3). When de novo AML patients were divided by age, the elderly (greater than 60 yr) had significantly (p = 0.0001) lower mean G-score than the younger (less than 45 yr) ones; 156 +/- 64.8 v 243 +/- 41.4. This age-related difference became accentuated when only patients with extreme hypogranulation (G-score less than 150) were studied. Elderly de novo AML patients also had significantly (p = 0.0057) higher mean ppp. By studying the degree of polymorph dysplasia in the peripheral blood, it seems possible to identify a subset of dysplastic elderly AML patients, who might have passed a (preleukaemic) MDS phase unnoticed.     

Clinical significance of nuclear non-phosphorylated beta-catenin in acute myeloid leukaemia and myelodysplastic syndrome

Wnt signaling activates the canonical pathway and induces the accumulation of non-phosphorylated beta-catenin (NPBC) in the nucleus. Although this pathway plays an important role in the maintenance of haematopoietic stem cells as well as in oncogenesis, the significance of nuclear NPBC remains unclear in malignant haematopoiesis. This study examined the expression of nuclear NPBC in bone marrow specimens from 54 and 44 patients with de novo acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS), respectively. On immunohistochemistry with an anti-NPBC antibody, the nuclei were positively stained in 22 and 18 of AML and MDS specimens, respectively. Staining of nuclear NPBC was associated with AML subtypes (M6 and M7), low complete remission (CR) rate, and poor prognosis. Nuclear NPBC was also associated with a high score when using the International Prognostic Scoring System (IPSS) for MDS and with −7/−7q and complex karyotypes. These findings suggest that in situ detection of nuclear NPBC by immunohistochemistry could provide new insights into the pathogenesis and prognosis of AML and MDS.     

Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal disorders of the haematopoietic stem cell and primarily involve cells of the myeloid lineage. Using cDNA microarrays comprising 6000 human genes, we studied the gene expression profiles in the neutrophils of 21 MDS patients, seven of which had the 5q- syndrome, and two acute myeloid leukaemia (AML) patients when compared with the neutrophils from pooled healthy controls. Data analysis showed a high level of heterogeneity of gene expression between MDS patients, most probably reflecting the underlying karyotypic and genetic heterogeneity. Nevertheless, several genes were commonly up or down-regulated in MDS. The most up-regulated genes included RAB20, ARG1, ZNF183 and ACPL. The RAB20 gene is a member of the Ras gene superfamily and ARG1 promotes cellular proliferation. The most down-regulated genes include COX2, CD18, FOS and IL7R. COX2 is anti-apoptotic and promotes cell survival. Many genes were identified that are differentially expressed in the different MDS subtypes and AML. A subset of genes was able to discriminate patients with the 5q- syndrome from patients with refractory anaemia and a normal karyotype. The microarray expression results for several genes were confirmed by real-time quantitative polymerase chain reaction. The MDS-specific expression changes identified are likely to be biologically important in the pathophysiology of this disorder.     

Array-based DNA methylation profiling in acute myeloid leukaemia

Methylation in the promoter region of many genes is involved in regulating gene expression patterns. Using the Illumina GoldenGate^© methylation assay, we examined the methylation status of 1505 CpG‐sites from 807 genes in 32 samples from patients with acute myeloid leukaemia (AML) at diagnosis, nine at relapse and 15 normal controls and performed additional pyrosequencing and semiquantitative methylation specific polymerase chain reaction (MSP) of the GNMT promoter in 113 diagnostic AML samples. We found a gain of overall methylation in AML samples with a further increase at relapse. Regional hypermethylation as assessed by array analysis could be confirmed by both MSP and pyrosequencing. Additionally, large‐scale methylation analysis identified interesting candidate genes. Cluster analysis indicated that cytogenetic subgroups seemed to be characterized by additional distinct epigenetic modifications and that basic DNA methylation patterns remain at relapse. Therefore, promoter hypermethylation is a frequent event in AML and is accentuated at relapse. Array‐based methylation analysis determined distinct methylation profiles for non‐malignant controls and AML samples with specific chromosomal aberrations and can identify target genes for further evaluation.     

Identification of relapse-associated gene mutations by next-generation sequencing in low-risk acute myeloid leukaemia patients

Recommended genetic categorization of acute myeloid leukaemias (AML) includes a favourable-risk category, but not all these patients have good prognosis. Here, we used next-generation sequencing to evaluate the mutational profile of 166 low-risk AML patients: 30 core-binding factor (CBF)-AMLs, 33 nucleophosmin (NPM1)-AMLs, 4 biCEBPα-AMLs and 101 acute promyelocytic leukaemias (APLs). Functional categories of mutated genes differed among subgroups. NPM1-AMLs showed frequent variations in DNA-methylation genes (DNMT3A, TET2, IDH1/2) (79%), although without prognostic impact. Within this group, splicing-gene mutations were an independent factor for relapse-free (RFS) and overall survival (OS). In CBF-AML, poor independent factors for RFS and OS were mutations in RAS pathway and cohesin genes, respectively. In APL, the mutational profile differed according to the risk groups. High-risk APLs showed a high mutation rate in cell-signalling genes (P = 0·002), highlighting an increased incidence of FLT3 internal tandem duplication (ITD) (65%, P < 0·0001). Remarkably, in low-risk APLs (n = 28), NRAS mutations were strongly correlated with a shorter five-year RFS (25% vs. 100%, P < 0·0001). Overall, a high number of mutations (≥3) was the worst prognostic factor RFS (HR = 2·6, P = 0·003). These results suggest that gene mutations may identify conventional low-risk AML patients with poor prognosis and might be useful for better risk stratification and treatment decisions.