Torsten Haferlach

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.

Patients with de novo acute myeloid leukaemia and complex karyotype aberrations show a poor prognosis despite intensive treatment: a study of 90 patients

The clinical significance of complex chromosome aberrations for adults with acute myeloid leukaemia (AML) was assessed in 920 patients with de novo AML who were karyotyped and treated within the German AML Cooperative Group (AMLCG) trials. Complex chromosome aberrations were defined as three or more numerical and/or structural chromosome aberrations excluding translocations t(8;21)(q22;q22), t(15;17)(q22;q11–q12) and inv(16)(p13q22). Complex chromosome anomalies were detected in 10% of all cases with a significantly higher incidence in patients u200a60u2003years of age (17·8% vs. 7·8%, Pu2003<u20030·0001). Clinical follow‐up data were available for 90 patients. Forty‐five patients were <u200a60u2003years of age and were randomly assigned to double induction therapy with either TAD‐TAD [thioguanine, daunorubicin, cytosine arabinoside (AraC)] or TAD‐HAM (high‐dose AraC, mitoxantrone). Twenty‐one patients achieved complete remission (CR) (47%), 20 patients (44%) were non‐responders and 9% of patients died during aplasia (early death). The median overall survival (OS) was 7u2003months and the OS rate at 3u2003years was 12%. Patients receiving TAD‐HAM showed a significantly higher CR rate than patients receiving TAD‐TAD (56% vs. 23%, Pu2003=u20030·04). Median event‐free survival was less than 1u2003month in the TAD‐TAD group and 2u2003months in the TAD‐HAM group, respectively (Pu2003=u20030·04), with a median OS of 4·5u2003months vs. 7·6u2003months (Pu2003=u20030·13) and an OS after 3u2003years of 7·6% vs. 19·6%. Forty‐five patients were u200a60u2003years of age: 28 of these patient were treated for induction using one or two TAD courses and 17 cases received TAD‐HAM with an age‐adjusted reduction of the AraC dose. The CR rate was 44%, 38% were non‐responders and 18% experienced early death. The median OS was 8u2003months and the OS rate at 3u2003years was 6%. In conclusion, complex chromosome aberrations in de novo AML predicted a dismal outcome, even when patients were treated with intensive chemotherapy. Patients under the age of 60u2003years with complex aberrant karyotypes may benefit from HAM treatment during induction. However, long‐term survival rates are low and alternative treatment strategies for remission induction and consolidation are urgently needed.

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.

Clinical significance of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics

Resistance to chemotherapy-induced apoptosis and a multidrug-resistance (MDR) phenotype, mainly mediated by P-glycoprotein (P-gp), contribute to chemotherapy failure in hematologic malignancies. To study apoptosis-regulating factors in acute myeloid leukemia (AML), we investigated cell samples of adults with de novo AML by flow cytometry for constitutive expression levels of the apoptosis-related molecules CD95 (n = 135), Bcl-2 (n = 131), and Bax (n = 66), as well as spontaneous apoptosis in vitro (n = 104) and susceptibility to anti-CD95-induced apoptosis (CD95 sensitivity) (n = 93). We correlated these findings with P-gp function as detected by the rhodamine123-efflux test (n = 121), immunophenotype, FAB morphology, cytogenetics, and clinical data of the examined patients. Immature FAB M0/1 AML cells expressed significantly more Bcl-2 (P < 0.0002) and less cd95 (P < 0.0003) compared with aml cells of the more mature fab m2–5 subtypes. no maturation-dependent difference in bax expression was observed. fab m2–5 aml cells were more susceptible to anti-cd95-induced apoptosis (P < 0.008) and showed a lower p-gp function (P < 0.002) than fab m0/1 aml cells. leukemic cells of aml patients who achieved a complete remission (cr) after induction chemotherapy expressed less bcl-2 than non-responder (nr) (69 cr, 23 nr; P = 0.05). CR was associated with a higher extent of spontaneous apoptosis in vitro (58 CR, 17 NR; P=0.05) and a tendency towards a higher CD95 expression (73 CR, 23 NR; P = 0.08) compared to NR. CR also correlated with a low P-gp function (70 CR, 21 NR; P = 0.008) and a tendency towards CD34 negativity (73 CR, 23 NR; P = 0.08). No correlation between Bax expression and response to induction chemotherapy (49 CR, 12 NR) was observed. In stepwise logistic regression analyses, P-gp function and the extent of spontaneous apoptosis in vitro as well as CD95 sensitivity but not Bcl-2, CD95, Bax, and CD34 expression levels emerged as significant markers for response to induction chemotherapy. We conclude that the constitutive expression of CD95 and Bcl-2, as well as CD95 sensitivity and P-gp function but not constitutive Bax expression depend on the maturation stage of leukemic cells in adult de novo AML. P-gp function, the extent of spontaneous apoptosis in vitro and CD95 sensitivity are more predictive for response to induction chemotherapy in adult de novoAML than the constitutive expression levels of the apoptosis-related molecules CD95, Bcl-2 and Bax.

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.