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Recent studies have suggested that mutations in the mitochondrial genome (mtDNA) may play a role in the development and response to treatment for human cancer. The aim of this study was to investigate whether mtDNA variations have any prognostic relevance, to clarify the spectra of mtDNA variation and to determine whether there was any correlation to known prognostic factors in acute myeloid leukemia (AML). To elucidate this, we sequenced the entire mtDNA in 56 AML patients and 14 control subjects. When analyzing the biologic impact of the non-synonymous variations in the mtDNA coding genes, we found an inferior disease-free survival for patients exhibiting variations in the two most important catalytic genes of the complex IV of the oxidative phosphorylation complexes (OXPHOS), that is, the cytochrome c oxidase subunit I and the cytochrome c oxidase subunit II (hazard ratio 2.6, P = 0.03; multivariate analysis). In addition, the most frequent variation was the T16311C in the control region, which was found in 11 (20%) of the 56 patients. This observation was confirmed in another cohort of 173 diagnostic AML samples. In this expanded group, the T16311C variation tended to be associated with chromosomal abnormalities.

Udgivelsesdato: 2009-Nov HOXA4 gene expression is a predictor for outcome in normal karyotypic acute myeloid leukaemia (AML) patients. Given that Meis1 is a co-factor for Hox genes, we hypothesized that the combined expression of HOXA4 and MEIS1 might add prognostic information in these patients. When diagnostic samples from 246 AML patients were divided into three main groups based on gene expression levels of HOXA4 combined with MEIS1 we found that within the group of patients exhibiting low levels of HOXA4, those with a high expression of MEIS1 had a significantly worse outcome than those exhibiting low MEIS1 expression (P = 0.025). Moreover, this prediction was independent of cytogenetics, mutational status of the NPM1 and FLT3 genes as well as upon WBC and age. To evaluate the possible contribution of regulatory events underlying these observations, 157 patient samples were subjected to promoter hypermethylation analysis. We observed that 77% were HOXA4- and 15%MEIS1 hypermethylated and that this epigenetic alteration was highly correlated to the gene expression level (MEIS1: P = 0.001; HOXA4: P = 0.007). Finally, we found a higher expression level and a higher frequency of hypermethylation of HOXA4 among patients with NPM1 mutations. In conclusion, our data show that the combination of low HOXA4 and low MEIS1 gene expression is a favourable predictor for outcome in all AML patients and that the expression levels are governed by the methylation state of these genes.

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.

Introduction: The CEBPA gene encodes a transcription factor, CCAAT/enhancer binding protein (C/EBP)α. Expression of the wildtype protein is essential for the lineage specific differentiation of myelocytic haematopoietic precursors into mature neutrophils. Eight percentage of all AML patients harbour at least one mutation in this gene, increasing up to 15% in the group, where standard karyotypic analysis do not reveal chromosomal aberrations. Objective: We designed a method, which discriminates as little as single base insertions or deletions accounting for 90% of all CEBPA mutations. The TAD2C length polymorphism was also identified using this set up. Patients and Methods: Diagnostic bone marrow or peripheral blood from 446 adults and 39 children diagnosed with AML from 1980 to 2006 was analysed for mutations by PCR and capillary gel electrophoresis. Results: We analysed pretreatment samples from 485 AML patients and 57 healthy volunteers and identified sequence variations in 35/446 adults and 1/39 children. We were immediately able to distinguish N- and C-terminal insertions and deletions as well as normally occurring polymorphisms. Abnormal PCR products were reprocessed and analysed by direct sequencing. We found stringent accordance between the two methods and reached the same frequency of mutations and polymorphisms as known from the literature. Conclusion: We conclude that capillary gel electrophoresis can be used as an accurate and high throughput diagnostic procedure for mutational status in the CEBPA gene identifying not only the same mutational frequency as in the published reports but also the TAD2C polymorphism in addition.

Partial tandem (PTD) and internal tandem duplications (ITD) of the MLL or FLT3 genes respectively, have been demonstrated in acute myeloid leukemia (AML). While occurrence of each of these PTD/ITD seem to confer an unfavorable prognosis, the literature contains only sparse information of the occurrence and the prognosis of simultaneous PTD/ITD of these genes. We have therefore attempted to determine the presence and its consequence in AML and with the further aim of characterizing such patients with respect to other genetic aberrations and to prototype variables in this disease. We analyzed blast cells from 250 adult patients treated at the same institution during a 15-year period for FLT3 ITD and MLL PTD and the duplications were found in 24% and 4%, respectively. The four co-duplicated cases (2%) did not differ with respect to sex, age, FAB-type, or immunophenotype, promoter methylation of p15, E-cadherin (CDH1), Estrogen receptor, MDR1, expression of apoptosis-related or multidrug resistance-related genes, though a trend toward decreased gene expression of MDR1 was observed. Two of the patients had a normal karyotypic analysis, while the remaining two showed aberrations in chromosome 11, one with trisomy 11 and the other with a der (11). The extensive molecular characterization of FLT3/MLL coduplicated patients presented here indicates that, even though they do not differ molecularly from the groups of patients with single ITDs, their prognosis and overall survival is universally poor. More patients are needed to determine whether coduplication has independent clinical implications compared to patients with single ITD/PTD.