Susanne Schnittger

Dominant-negative mutations of CEBPA , encoding CCAAT/enhancer binding protein-α (C/EBPα), in acute myeloid leukemia

The transcription factor C/EBPα (for CCAAT/enhancer binding protein-α; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPα DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.

Acute myeloid leukemias with reciprocal rearrangements can be distinguished by specific gene expression profiles

Acute myeloid leukemia (AML) is a heterogeneous group of genetically defined diseases. Their classification is important with regard to prognosis and treatment. We performed microarray analyses for gene expression profiling on bone marrow samples of 37 patients with newly diagnosed AML. All cases had either of the distinct subtypes AML M2 with t(8;21), AML M3 or M3v with t(15;17), or AML M4eo with inv(16). Diagnosis was established by cytomorphology, cytogenetics, fluorescence in situ hybridization, and reverse transcriptase–PCR in every sample. By using two different strategies for microarray data analyses, this study revealed a unique correlation between AML-specific cytogenetic aberrations and gene expression profiles.

Morphologic Dysplasia in De Novo Acute Myeloid Leukemia (AML) Is Related to Unfavorable Cytogenetics but Has No Independent Prognostic Relevance Under the Conditions of Intensive Induction Therapy: Results of a Multiparameter Analysis From the German AML Cooperative Group Studies

PURPOSEnOn the basis of cytomorphology according to the French-American-British (FAB) classification, we evaluated the prognostic impact of dysplastic features and other parameters in de novo acute myeloid leukemia (AML). We also assessed the clinical significance of the recently introduced World Health Organization (WHO) classification for AML, which proposed dysplasia as a new parameter for classification.nnnPATIENTS AND METHODSnWe analyzed prospectively 614 patients with de novo AML, all of whom were diagnosed by central morphologic analysis and treated within the German AML Cooperative Group (AMLCG)-92 or the AMLCG-acute promyalocytic leukemia study.nnnRESULTSnPatients with AML M3, M3v, or M4eo demonstrated a better outcome compared with all other FAB subtypes (P <.001); no prognostic difference was observed among other FAB subtypes. The presence or absence of dysplasia failed to demonstrate prognostic relevance. Other prognostic markers, such as age, cytogenetics, presence of Auer rods, and lactate dehydrogenase (LDH) level at diagnosis, all showed significant impact on overall and event-free survival in univariate analyses (P <.001 for all parameters tested). However, in a multivariate analysis, only cytogenetics (unfavorable or favorable), age, and high LDH maintained their prognostic impact. Dysplasia was not found to be an independent prognostic parameter, but the detection of trilineage dysplasia correlated with unfavorable cytogenetics.nnnCONCLUSIONnOur results indicate that cytomorphology and classification according to FAB criteria are still necessary for the diagnosis of AML but have no relevance for prognosis in addition to cytogenetics. Our results suggest that the WHO classification should be further developed by using cytogenetics as the main determinant of biology. Dysplastic features, in particular, have no additional impact on predicting prognosis when cytogenetics are taken into account.

Molecular characterization of acute leukemias by use of microarray technology

Accurate subclassification of leukemia and the identification of prognostic determinants are essential to guide therapy and to improve patients outcome. According to present standards, pre‐therapeutic assessment depends on a combination of different methods. We aimed to expand the molecular characterization of different acute leukemia subtypes to identify new genome‐wide diagnostic markers. Total RNA from 90 adult patients suffering from acute lymphoblastic leukemia (ALL, n = 25) and acute myeloid leukemia (AML, n = 65) was extracted at diagnosis and high density oligonucleotide microarrays were used to analyze the expression profiles of 12,000/22,000 genes in all specimens (Affymetrix U95Av2/U133A). All cases were thoroughly characterized by individual combinations of cytomorphology, cytogenetics, multiparameter immunophenotyping, and molecular genetics. The expression signature of a small set of differentially expressed genes was sufficient to accurately discriminate eight clinically relevant acute leukemia subgroups. Underlying chromosomal aberrations or immunophenotypical characteristics were strictly correlated with a distinct gene expression pattern for AML with t(8;21), t(15;17), t(11q23)/MLL, or inv(16) as well as for precursor B‐ALL with t(9;22), t(8;14), or t(11q23)/MLL and precursor T‐ALL. These data support a possible future application of microarray technology for classification of the acute leukemias.

Acute myeloid leukemia with a complex aberrant karyotype is a distinct biological entity characterized by genomic imbalances and a specific gene expression profile.

Acute myeloid leukemia (AML) with a complex aberrant karyotype is a distinct biological entity. It is characterized by: (1) a sharp increase in incidence above age 50; (2) a characteristic pattern of chromosomal gain and, especially, loss, that is, of 5q14q33, 7q32q35, and 17p13, translating into reduced expression of genes in these regions; (3) a unique gene expression pattern including up‐regulation of genes involved in DNA repair; (4) a high incidence of TP53 deletions and/or mutations; and (5) an overall unfavorable prognosis. Further unraveling the biology of AML with a complex aberrant karyotype by gene expression profiling may provide deeper insights into the pathogenesis of as well as the reasons for chemoresistance in this AML subtype. These data may be the basis for developing targeted therapeutic strategies to increase the cure rate in patients with AML and a complex aberrant karyotype.

Loss of genetic material is more common than gain in acute myeloid leukemia with complex aberrant karyotype: A detailed analysis of 125 cases using conventional chromosome analysis and fluorescence in situ hybridization including 24-color FISH

Patients with acute myeloid leukemia (AML) and a complex aberrant karyotype have a poor outcome despite intensive antileukemic treatment. The aim of this study was to analyze in detail the genetic abnormalities in this subgroup of AML. Therefore, 125 AML cases with complex aberrant karyotype detected by G‐banding were examined in addition with 24‐color FISH and FISH with locus‐specific probes for EGR1 (5q31), D7S522 (7q31), and TP53 (17p13), given that these regions are known to be commonly deleted in AML with a complex aberrant karyotype. The number of chromosome abnormalities per case varied from 3 to 30 (median 10). A gain of a whole chromosome was observed 131 times, with +8 (n = 30), +10 (n = 11), and +22 (n = 10) being the most frequent trisomies. A loss of a whole chromosome occurred 128 times. The chromosomes most often lost were 7 (n = 25), 18 (n = 24), and 17 (n = 17). Structural aberrations, leading to a gain or loss of chromosomal material, were detected 104 times and 433 times, respectively. Aberrations including only two chromosomes that seemed to be balanced were found only 19 times. Losses resulting from structural abnormalities most frequently involved 5q (n = 100), 17p (n = 47), and 12p (n = 29), whereas gains of 11q (n = 21), 21q (n = 19), and 8q (n = 11) were observed. Using locus‐specific probes, deletions of the EGR1 locus (5q31), of 7q31, and the TP53 gene were observed in 103 (82%), 57 (46%), and 66 (53%) cases, respectively. In conclusion, in AML with a complex aberrant karyotype, loss of chromosomal material was observed much more often than gain. Unbalanced rearrangements leading to loss of chromosomal material are much more frequent than loss of whole chromosomes. These data suggest that in AML with a complex aberrant karyotype, loss of tumor‐suppressor genes is a more important mechanism of leukemogenesis than activation of oncogenes, and that gene‐dosage effects may play a significant role in the pathogenesis of this AML subtype.

FLT3 Length Mutations as Marker for Follow-Up Studies in Acute Myeloid Leukaemia

Length mutations within the FLT3 gene (FLT3-LM) can be found in 23% of acute myeloid leukaemia (AML) and thus are the most frequent mutations in AML. FLT3-LM are highly correlated with AML with normal karyotype and other cytogenetic aberrations of the prognostically intermediate group. This group is supposed to be a mixed group of AML with differences in the underlying pathogenesis. For more individualized treatment options it would be helpful to better characterize this large AML group not only by molecular mutations but also use these markers for the definition of minimal residual disease (MRD). However, so far the cytogenetically intermediate AML has been lacking suitable markers for PCR-based MRD detection like the fusion genes in the prognostically favourable subgroups. The suitability of the FLT3-LM as a target for PCR-based MRD was discussed controversially as it seemed to be a rather unstable marker. Thus, we aimed at the evaluation of FLT3-LM as a marker for residual disease in a large cohort of AML. Paired samples of 97 patients with AML at diagnosis and at relapse were analyzed. It could be shown that in only four cases a loss of the length mutation was detected. This is in the range of other well-characterized AML relapsing with a different geno- and/or phenotype. In contrast, a change in the ratio of the mutated allele in comparison to the wild-type allele was frequently observed. In detail, the FLT3-LM showed a tendency to accumulate during disease progression and was found more frequently at relapse than at diagnosis. In addition, 45 patients were analyzed at different time points during and after therapy. Using conventional PCR it clearly could be shown that for most of the patients positive at presentation FLT3-LM is a reliable PCR marker for monitoring treatment response. Even an early detection of relapse was possible in some cases.

Stability of leukemia‐associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: Comparison with cytomorphologic, cytogenetic, and molecular genetic findings

Multiparameter flow cytometry is increasingly used to monitor minimal residual disease in patients with acute myeloid leukemia to identify leukemic cells by leukemia‐associated aberrant immunophenotypes (LAIPs). Changes in LAIPs during the course of the disease may be a limitation for this approach.

Distinct gene expression patterns associated with FLT3- and NRAS-activating mutations in acute myeloid leukemia with normal karyotype

In acute myeloid leukemia (AML), constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplications (FLT3-ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (FLT3-TKD), as well as point mutations of the NRAS gene (NRAS-PM) are among the most frequent somatic gene mutations. To elucidate whether these mutations cause aberrant signal transduction in AML, we used gene expression profiling in a series of 110 newly diagnosed AML patients with normal karyotype. The different algorithms used for data analysis revealed highly concordant sets of genes, indicating that the identified gene signatures are specific for each analysed subgroup. Whereas samples with FLT3-ITD and FLT3-TKD could be separated with up to 100% accuracy, this did not apply for NRAS-PM and wild-type samples, suggesting that only FLT3-ITD and FLT3-TKD are associated with an apparent signature in AML. The set of discriminating genes included several known genes, which are involved in cell cycle control (CDC14A, WEE1), gene transcription (HOXB5, FOXA1), and signal transduction (SMG1). In conclusion, we showed that unique gene expression patterns can be correlated with FLT3-ITD and FLT3-TKD. This might lead to the identification of further pathogenetic relevant candidate genes particularly in AML with normal karyotype.

Conventional cytogenetics of myeloproliferative diseases other than CML contribute valid information.

In chronic myeloproliferative disorders other than CML (CMPD) recurrent cytogenetic abnormalities occur, but specific patterns of chromosomal aberrations in the specific entities have so far not been detected. Thus, the value of conventional cytogenetics in the routine diagnostic setting of CMPD remains to be clarified. We performed a cytogenetic study on 409 patients with different CMPD [polycythemia vera, essential thrombocytosis (ET), idiopathic osteomyelofibrosis, chronic myelomonocytic leukemia (proliferative subtype), idiopathic hypereosinophilic syndrome (HES), myeloproliferative syndrome (unclassifiable)] and on 102 patients with suspected CMPD. Cytogenetic abnormalities occurred in different frequencies ranging from 3 to 40% depending on the subtype, and showed some specific differences with respect to their type. The highest frequency and the most complex pattern of clonal aberrations were observed in idiopathic osteomyelofibrosis. However, clonal aberrations were also found in 10% of patients with suspected CMPD establishing the diagnosis of a malignant disease. In conclusion, cytogenetics are essential in the routine diagnostic setting of CMPD or cases suspicious for CMPD. In ET and in HES the aberration rate was only 3 and 7%, respectively. Thus, cytogenetics can be omitted. However, in some of these cases molecular procedures should be integrated into the routine diagnostic process.