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Gerichtsreporter verstehen ihre Aufgabe mitunter nicht allein in der Berichterstattung, sie wollen bisweilen eigene Recherchen anstellen. Manche versuchen gar, der Wahrheitsfindung des Gerichts vorzugreifen, denn Gerichtsberichte müssen heute, wie andere Texte auch, Aufmerksamkeit erwecken. Begibt sich der Gerichtsberichterstatter auf juristisches Glatteis, sollte er seine Grenzen kennen. Wenn Journalisten ohne genügendes Wissen sich anmassen, der Leserschaft zu sagen, wie das Gericht zu entscheiden hat, ist das schlechter Journalismus. Die exakte Analyse wird im heutigen Journalismus allerdings zunehmend verdrängt. Aber für Demokratie und Rechtsstaat ist eine kompetente Gerichtsberichterstattung essenziell. Die juristischen Überlegungen eines Gerichts dem breiten Publikum attraktiv darstellen kann jedoch nur ein Journalist mit guten Rechtskenntnissen. Man mag die Meinung des Gerichts teilen oder nicht. Zunächst sind in jedem Fall dessen Beweggründe, die rechtlichen Grundlagen und deren Auslegung darzutun. Danach ist es dem Berichterstatter unbenommen, Kritik am Gericht zu üben.

Compared with fluorescence in situ hybridization (FISH), conventional metaphase cytogenetics play only a minor prognostic role in chronic lymphocytic leukemia (CLL) so far, due to technical problems resulting from limited proliferation of CLL cells in vitro. Here, we present a simple method for in vitro stimulation of CLL cells that overcomes this limitation. In our unselected patient population, 125 of 132 cases could be successfully stimulated for metaphase generation by culture with the immunostimulatory CpG-oligonucleotide DSP30 plus interleukin 2. Of 125 cases, 101 showed chromosomal aberrations. The aberration rate is comparable to the rate detected by parallel interphase FISH. In 47 patients, conventional cytogenetics detected additional aberrations not detected by FISH analysis. A complex aberrant karyotype, defined as one having at least 3 aberrations, was detected in 30 of 125 patients, compared with only one such case as defined by FISH. Conventional cytogenetics frequently detected balanced and unbalanced translocations. A significant correlation of the poor-prognosis unmutated IgVH status with unbalanced translocations and of the likewise poor-prognosis CD38 expression to balanced translocations and complex aberrant karyotype was found. We demonstrate that FISH analysis underestimates the complexity of chromosomal aberrations in CLL. Therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.

We analyzed 2502 patients with acute myeloid leukemia at diagnosis for NRAS mutations around the hot spots at codons 12, 13, and 61 and correlated the results to cytomorphology, cytogenetics, other molecular markers, and prognostic relevance of these mutations. Two hundred fifty-seven (10.3%) of 2502 patients had NRAS mutations (NRASmut). Most mutations (112 of 257; 43.6%) were found at codon 12, mostly resulting in changes from glycine to asparagine. The history of AML did not differ significantly in association with NRAS mutations. The subgroups with inv(16)/t(16;16) and inv(3)/t(3;3) showed a significantly higher frequency of NRASmut (50 of 133, 37.6% [P < .001], and 11 of 41, 26.8% [P = .004], respectively) than the total cohort. In addition, in these 2 subgroups, mutations of codon 61 were significantly overrepresented (both P < .001). In contrast, NRAS mutations were significantly underrepresented in t(15;17) (2 of 102; 2%; P = .005) in the subgroup with MLL/11q23 rearrangements (3 of 77; 3.9%; P = .061) and in the complex aberrant karyotype (4 of 258; 1.6%; P < .001). Overall, we did not find a significant prognostic impact of NRASmut for overall survival, event-free survival, and disease-free survival. However, there was a trend to better survival in most subgroups, especially when other molecular markers (FLT3-LM, MLL-PTD, and NPM) were taken into account.

Double minutes (dmin)-circular, extra-chromosomal amplifications of specific acentric DNA fragments-are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in similar to 1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of similar to 500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the 'episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplifications. (Less)

Medulloblastoma is a highly malignant embryonal tumor of the cerebellum that accounts for 20%-25% of all intracranial pediatric tumors. The most frequent chromosomal rearrangement in medulloblastoma is isochromosome 17, or i(17q). Its frequency suggests that it serves an important role in tumor pathogenesis, possibly mediated by the disruption or permanent activation of a gene at the breakpoint. To address this question, we performed a detailed analysis of chromosome 17 DNA copy number from 18 medulloblastomas previously shown to carry an apparent i(17q). We identified two breakpoint regions, one well within band 17p11.2 (n = 16) and a second within the pericentromeric region (n = 2). To map the breakpoints more precisely, we constructed a tiling-path matrix-CGH array covering chromosomal band 17p11.2 to the centromere and utilized it to delineate two small breakpoint intervals mapping at Mb 19.0 and 21.7 in seven of the medulloblastomas and in nine hematological neoplasias with i(17q). The former interval contains two breakpoint clusters that each colocalize with a pair of head-to-head inverted DNA sequence repeats, and the latter maps close to a region of alpha-satellite repeats. No consensus coding sequence localizes in these regions. Together, these data strongly suggest that the effects of i(17q) in medulloblastoma are mediated by gene-dosage effects of genes on 17p or 17q rather than by the disruption or deregulation of a "breakpoint" gene. Furthermore, we identified artifacts introduced in DNA copy number data by cross-hybridization of low-copy repeat sequences and discuss the challenge these can pose in the interpretation of diagnostic microarrays.

BACKGROUND. Microarray analysis is considered a future diagnostic tool in leukemias. Whereas data accumulate on specific gene expression patterns in biologically defined leukemia entities, data on the correlation between flow cytometrically determined protein expression, which are essential in the diagnostic setting today, and microarray results are limited. METHODS. The results obtained by microarray analysis were compared using the Affymetrix GeneChip HG-U133 system in parallel with flow cytometric findings of 36 relevant targets in 814 patients with newly diagnosed acute and chronic leukemias as well as in normal bone marrow samples. RESULTS. In a total of 21,581 individual comparisons between signal intensities obtained by microarray analysis and percentages of positive cell as determined by flow cytometry, coefficients of correlation in the range of 0.171 to 0.807 were obtained. In particular, the degree of correlation was high in the following genes critical in the diagnostic setting: CD4, CD8, CD13 (ANPEP), CD33, CD23 (FCER2), CD64 (FCGR1A), CD117 (KIT), CD34, MPO, CD20 (MS4A1), CD7 (range of r, 0.589–0.807). CONCLUSIONS. The present data prove the high degree of correlation between findings obtained by microarray analysis and flow cytometry. They are in favor of a future application of the microarray technology as a robust diagnostic tool in leukemias. Cancer 2006. © 2006 American Cancer Society.

BACKGROUND Different diagnostic methods add information to define complete remission (CR) in patients with acute myeloid leukemia (AML). The detection of minimal residual disease (MRD) for predicting prognosis and for therapeutic planning still are under discussion. METHODS The authors studied 216 patients with AML at the time of initial diagnosis and during follow-up and correlated cytomorphology, interphase fluorescence in situ hybridization (FISH), and flow cytometry results to evaluate response status. They further tested the prognostic impact of those results, especially in patients who achieved a morphologic CR. RESULTS Interphase FISH was found to be correlated significantly with the clinical course at the time of complete cytomorphologic remission and was more reliable than morphology for defining CR. Furthermore, interphase FISH was correlated with immunophenotyping results at all times during follow-up. CONCLUSIONS The current results indicated that interphase FISH may be used as a valid MRD parameter in patients with AML. Multiparameter immunophenotyping for MRD also was correlated strongly with the clinical course, and the authors suggest integrating such immunophenotyping into the routine diagnostic panel at the time of diagnosis and during the clinical course in patients with AML. Cancer 2006. © 2006 American Cancer Society.

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3–length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1–/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM– mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

Mutations within the FLT3 gene are of growing importance for classification, risk assessment, and therapeutic targeting of acute myeloid leukemia (AML). We analyzed 656 AML patients for a recently described single-nucleotide polymorphism (SNP) in the third immunoglobulin-like domain of the extracellular region of FLT3. The FLT3 D324N variant was present in 42 cases (6.4%), but it was not associated with a specific AML subtype and did not show an elevated leukocyte count, as do other FLT3 mutations. In remission samples, a 50% ratio of the normal to the D324N variant was detectable. Stably expressed in IL-3 dependent Ba/F3 cells, the D324N variant did not confer receptor autophosphorylation, factor independent growth, or increased resistance to apoptotic cell death in response to varying doses of FLT3 ligand. In 400 healthy donors, the FLT3 D324N variant was detected in 6 cases (1.5%) and segregated in a family. Thus, it was shown to be a polymorphism with a lower frequency in healthy controls than in patients with AML (P < 0.001). In addition, 21 of 234 CML (9.0%) and 7 of 155 ALL (4.5%) cases carried the FLT3 D324N. Our data suggest that the FLT3 D324N variant might be associated with a predisposition to different subtypes of leukemia. © 2005 Wiley-Liss, Inc.

BACKGROUND Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induced in vitro differentiation of primary acute myeloid leukemia (AML) blasts, an effect enhanced by all-trans retinoic acid (ATRA). Clinical responses to VPA were recently observed in patients with myelodysplastic syndrome (MDS). Herein, the authors have described results of a clinical trial with VPA plus ATRA in 26 patients with poor-risk AML. METHODS VPA (5–10 mg/kg starting dose) and ATRA (45 mg/m2) were administered orally. Low-dose AraC or hydroxyurea were permitted to control leukocytosis. Biologic activity of VPA was confirmed by serial analysis of HDAC2 protein levels in peripheral blood (PB) mononuclear cells. RESULTS Nineteen of 26 patients completed at least 4 weeks of VPA/ATRA treatment; 7 patients were withdrawn prematurely because of rapidly progressive disease (n = 3) or unacceptable neurologic and cardiovascular toxicity (n = 4). Additional cytoreductive treatment was required in 58% of patients enrolled. Median treatment duration was 3 months. No patient achieved complete remission, one with de novo AML had a minor response, and two patients with secondary AML arising from myeloproliferative disorder (MPD) achieved a partial remission and clearance of PB blasts, respectively. The latter responses were accompanied by profound granulocytosis and erythrocytosis in both patients, reminiscent of the response pattern known from ATRA treatment of acute promyelocytic leukemia. However, cytogenetic analysis of isolated CD34+ cells and granulocytes did not reveal terminal differentiation of leukemic blasts. CONCLUSIONS Treatment with VPA/ATRA results in transient disease control in a subset of patients with AML that has evolved from a myeloproliferative disorder but not in patients with a primary or MDS-related AML. Cancer 2005. © 2005 American Cancer Society.

Monitoring minimal residual disease (MRD) becomes increasingly important in the risk-adapted management of patients with acute myeloid leukemia (AML). The two most sensitive and quantitative methods for MRD detection are multiparameter flow cytometry (MFC) and real-time polymerase chain reaction (QRT-PCR). Fusion gene-specific PCR in AML is based on the RNA level, and thus in contrast to MFC expression levels rather than cell counts are assessed. For both methods independent prognostic values have been shown. The strong power of MFC has been shown mainly in the assessment of early clearance of the malignant clone. MRD levels in AML with fusion genes have the strongest prognostic power after the end of consolidation therapy. In addition, with QRT-PCR highly predictive initial expression levels can be assessed. With both methods early detection of relapse is possible. So far, validated PCR-based MRD was done with fusion genes that are detectable in only 20-25% of all AML MFC is superior since it is applicable for most AML. However, QRT-PCR is still more sensitive in most cases. Thus, it is desirable to establish new molecular markers for PCR-based studies. Large clinical trials will determine the role and place of immunologic and PCR-based monitoring in the prognostic stratification of patients with AML.

Genetic and molecular techniques have provided increasing insights into the biology of acute myeloid leukemia (AML). These investigations showed that AML is not a homogeneous disease but a heterogeneous group of biologically different subentities. These subentities are currently primarily defined by cytogenetics by which three main subgroups can be discriminated: AML with balanced translocations, AML with unbalanced aberrations and AML without cytogenetically detectable aberrations. Within the latter group molecular alterations are identified in more than half of cases such as NPM mutations, FLT3 mutations, MLL duplications and mutations of CEBP-alpha. The clinical meaning of these findings is illustrated by substantial differences in response to therapy and long-term outcome. As demonstrated by the recent multicenter trial of the German AML Cooperative Group (AMLCG) and other studies intensification of induction therapy may improve the results in distinct subtypes but fails to do so in others. Therefore, new strategies need to be explored which incorporate the knowledge about the biology of AML to develop biology adapted treatment strategies. This process has just begun and is predominantly determined by the availability of new agents and their evaluation in clinical phase I and II studies. A variety of targets are currently explored and some trials have yielded promising results already. The step towards a biology adapted treatment of AML is long and requires the combined efforts of researchers, clinicians and the pharmaceutical industry. The first steps towards this goal have been taken and give rise to the hope for more effective and more specific therapies of AML.

Undertreatment of the older patients with AML can explain, in part, their inferior outcome when compared with that in younger patients. In analogy to the benefit of patients under the age of 60 years from high-dose AraC there are dosage related therapeutic effects in the patients over 60 years in particular for daunorubicin in the induction treatment, and for maintenance versus no maintenance in the post-remission treatment. Utilizing these effects can partly overcome the mostly unfavorable disease biology in older age AML, whereas the role of risk factors involved is not completely understood and the concept of dose–response needs to be requestioned. We recommend an adequate dosage of 60mg/(m 2 day) daunorubicin for 3 days in a combination with standard dose AraC and 6-thioguanine given for induction and consolidation and followed by a prolonged monthly maintenance chemotherapy. Further improvements in supportive care may help delivering additional anti-leukemic cytotoxicity. As a novel approach, reduced toxicity preparative regimens may open up allogeneic transplantation for older patients with AML. Other new options like MDR modulators, antibody targeted therapies and tyrosine kinase inhibitors are under clinical investigation. A questionnaire study in patients with AML showed that according to patients' self-assessment intensive and prolonged treatment did not result in decreasing quality of life. This finding did not vary by age under or above 60 years. Given the actual median age in this disease being more than 60 years the adequate management of older age AML remains as the major challenge.

Zusammenfassung Die Technik der Fluoreszenz-in-situ-Hybridisierung (FISH) ist eine wichtige Methode, mit welcher Nukleinsäuresequenzen im Zellkern nachgewiesen werden können. Sie hat inzwischen in der Diagnostik und Klassifikation von hämatologischen Neoplasien einen festen Stellenwert. Die Durchführung eines diagnostischen FISH-Assays setzt voraus, dass die Sequenz der zu untersuchenden genetischen Region bekannt ist. Durch das zunehmende Wissen über die genetischen Veränderungen bei Tumoren konnten FISH-Assays für viele klinisch relevante Fragestellungen entwickelt werden. Grundsätzlich können für diverse Fragestellungen Sonden für eine FISH-Untersuchung hergestellt werden, sofern die zu untersuchende genetische Veränderung molekular aufgeklärt und prinzipiell mit FISH erfassbar ist, wie dies bei Deletionen, Translokationen, Duplikationen, Inversionen sowie Zugewinnen und Verlusten ganzer Chromosomen der Fall ist. Kleine Mutationen innerhalb eines einzelnen Gens sind nicht erfassbar. Viele für die Diagnostik wichtige Sonden sind bereits kommerziell erhältlich. Eine Empfehlung zum Einsatz der FISH-Diagnostik bei Leukämien wird zur Zeit im Rahmen des Europäischen Leukämie-Netzes erarbeitet (www.leukemia-net.org).

We performed microarray analyses in AML with trisomies 8 (n=12), 11 (n=7), 13 (n=7), monosomy 7 (n=9), and deletion 5q (n=7) as sole changes to investigate whether genomic gains and losses translate into altered expression levels of genes located in the affected chromosomal regions. Controls were 104 AML with normal karyotype. In subgroups with trisomy, the median expression of genes located on gained chromosomes was higher, while in AML with monosomy 7 and deletion 5q the median expression of genes located in deleted regions was lower. The 50 most differentially expressed genes, as compared to all other subtypes, were equally distributed over the genome in AML subgroups with trisomies. In contrast, 30 and 86% of the most differentially expressed genes characteristic for AML with 5q deletion and monosomy 7 are located on chromosomes 5 or 7. In conclusion, gain of whole chromosomes leads to overexpression of genes located on the respective chromosomes. Losses of larger regions of the genome translate into lower expression of the majority of genes represented by only one allele. The reduced expression of these genes is the most characteristic difference in gene expression profiles between AML with monosomy 7 and AML with deletion 5q, respectively, and other AML subtypes. Therefore, these data provide evidence that gene dosage effects gene expression in AML with unbalanced karyotype abnormalities. Losses of specific regions of the genome determine the gene expression profile more strongly than the gain of whole chromosomes.

We have identified a t(8;9)(p21-23;p23-24) in seven male patients (mean age 50, range 32-74) with diverse hematologic malignancies and clinical outcomes: atypical chronic myeloid leukemia/chronic eosinophilic leukemia (n = 5), secondary acute myeloid leukemia (n = 1), and pre-B-cell acute lymphoblastic leukemia (n = 1). Initial fluorescence in situ hybridization studies of one patient indicated that the nonreceptor tyrosine kinase Janus-activated kinase 2 (JAK2) at 9p24 was disrupted. Rapid amplification of cDNA ends-PCR identified the 8p22 partner gene as human autoantigen pericentriolar material (PCM1), a gene encoding a large centrosomal protein with multiple coiled-coil domains. Reverse transcription-PCR and fluorescence in situ hybridization confirmed the fusion in this case and also identified PCM1–JAK2 in the six other t(8;9) patients. The breakpoints were variable in both genes, but in all cases the chimeric mRNA is predicted to encode a protein that retains several of the predicted coiled-coil domains from PCM1 and the entire tyrosine kinase domain of JAK2. Reciprocal JAK2–PCM1 mRNA was not detected in any patient. We conclude that human autoantigen pericentriolar material (PCM1)–JAK2 is a novel, recurrent fusion gene in hematologic malignancies. Patients with PCM1–JAK2 disease are attractive candidates for targeted signal transduction therapy.

BACKGROUND The advent of imatinib has considerably changed the treatment of chronic myeloid leukemia (CML). Early studies demonstrated high rates of hematologic and cytogenetic responses in all phases of the disease after limited observation periods. METHODS The authors evaluated long-term outcome, rates of response, and resistance in 300 patients with BCR-ABL–positive leukemias (CML in chronic phase after failure to respond to interferon-alpha [CP], n = 139; accelerated phase [AP], n = 80; myeloid blast crisis [BC], n = 76; lymphoid BC and Philadelphia chromosome-positive acute lymphoblastic leukemia, n = 5) who entered clinical trials with imatinib in a single center after an observation time of 4.5 years. RESULTS In CP, hematologic remission was achieved in 97% and major (MCR) and complete cytogenetic remission (CCR) in 61% and 49% of patients, respectively. The chance to achieve MCR was higher in patients commencing imatinib earlier in the course of CML. In AP, the median survival period after the start of imatinib was 44 months, and MCR and CCR were observed in 31% and 26% of patients, respectively. In myeloid BC, the median survival period after the start of imatinib and after diagnosis of BC was 6 and 9 months, respectively. Hematologic resistance occurred in 25%, 41%, and 92% of patients in CP, AP, and myeloid BC, respectively, and was associated with BCR-ABL mutations in 45% of patients and with clonal evolution in 58% of patients. CONCLUSIONS The data emphasized the need for a prolonged follow-up of patients treated with imatinib to define the clinical potential of the drug and to establish methods to optimize therapy. Cancer 2005. © 2005 American Cancer Society.

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.

The cytogenetic patterns in addition to the Philadelphia translocation in Philadelphia-positive (Ph+) ALL and chronic myeloid leukemia (CML) is heterogenous. We investigated 154 patients with Ph+ ALL at diagnosis or at relapse and 174 patients with different phases of CML. Ph+ ALL at diagnosis demonstrated a heterogenous pattern with a high frequency of numerical and structural chromosomal aberrations. CML at diagnosis presented with rare additional chromosomal changes. In Ph+ ALL, the pathway from diagnosis to relapse was characterized by the acquisition of a higher number of chromosomal aberrations, but the pattern of frequent aberrations at relapse did not differ from that observed at diagnosis. In contrast, in CML the pathway from chronic to the advanced phases was characterized by the acquisition of new chromosomal changes and by the development of karyotype complexity. In addition, the investigation of 10 cases of lymphoid blast crisis of CML showed significant differences in the karyotype in comparison to Ph+ ALL at diagnosis. Therefore, the karyotype can be helpful in discriminate de novo lymphoid blast crisis of CML from de novo Ph+ ALL. In conclusion, we were able to demonstrate that the cytogenetic patterns of Ph+ ALL and of CML are different at diagnosis and furthermore follow different pathways during progression or relapse.

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.

Acute promyelocytic leukemia (APL) with t(15;17) appears in two phenotypes: AML M3, with abnormal promyelocytes showing heavy granulation and bundles of Auer rods, and AML M3 variant (M3v), with non- or hypogranular cytoplasm and a bilobed nucleus. We investigated the global gene expression profiles of 35 APL patients (19 AML M3, 16 AML M3v) by using high-density DNA-oligonucleotide microarrays. First, an unsupervised approach clearly separated APL samples from other AMLs characterized genetically as t(8;21) (n ! 35), inv(16) (n ! 35), or t(11q23)/MLL (n ! 35) or as having a normal karyotype (n ! 50). Second, we found genes with functional relevance for blood coagulation that were differentially expressed between APL and other AMLs. Furthermore, a supervised pairwise comparison between M3 and M3v revealed differential expression of genes that encode for biological functions and pathways such as granulation and maturation of hematologic cells, explaining morphologic and clinical differences. Discrimination between M3 and M3v based on gene signatures showed a median classification accuracy of 90% by use of 10-fold CV and support vector machines. Additional molecular mutations such as FLT3-LM, which were significantly more frequent in M3v than in M3 (P < 0.0001), may partly contribute to the different phenotypes. However, linear regression analysis demonstrated that genes differentially expressed between M3 and M3v did not correlate with FLT3-LM. ' 2005 Wiley-Liss, Inc.

Approximately 50 different chromosomal translocations of the human MLL gene are currently known and associated with high-risk acute leukemia. The large number of different MLL translocation partner genes makes a precise diagnosis a demanding task. After their cytogenetic identification, only the most common MLL translocations are investigated by RT-PCR analyses, whereas infrequent or unknown MLL translocations are excluded from further analyses. Therefore, we aimed at establishing a method that enables the detection of any MLL rearrangement by using genomic DNA isolated from patient biopsy material. This goal was achieved by establishing a universal long-distance inverse-PCR approach that allows the identification of any kind of MLL rearrangement if located within the breakpoint cluster region. This method was applied to biopsy material derived from 40 leukemia patients known to carry MLL abnormalities. Thirty-six patients carried known MLL fusions (34 with der(11) and 2 with reciprocal alleles), whereas 3 patients were found to carry novel MLL fusions to ACACA, SELB , and SMAP1 , respectively. One patient carried a genomic fusion between MLL and TIRAP , resulting from an interstitial deletion. Because of this interstitial deletion, portions of the MLL and TIRAP genes were deleted, together with 123 genes located within the 13-Mbp interval between both chromosomal loci. Therefore, this previously undescribed diagnostic tool has been proven successful for analyzing any MLL rearrangement including previously unrecognized partner genes. Furthermore, the determined patient-specific fusion sequences are useful for minimal residual disease monitoring of MLL associated acute leukemias.

In about 55% of acute myeloid leukemia (AML) cases, chromosome aberrations are detectable by cytogenetics. Close correlations between cytomorphology and cytogenetics have been reported. To determine a pattern of cytogenetic abnormalities within the French-American-British (FAB) subtypes AML M0, M1, and M2, we analyzed 48 AML M0, 179 AML M1, and 425 AML M2 and compared cytogenetic data to a cohort of 1,062 AML M3/3v, M4, M4eo, M5a/5b, M6, and M7. Cytogenetic abnormalities were significantly more frequent in AML M0 (71%) compared to M1 (49%), M2 (53%), and the total cohort (56%; P < 0.02). While +8 was the most common numeric abnormality in all FAB subtypes, +13, +14, and +11 were associated with AML M0-M2. The only recurring balanced translocation that was associated with one of these FAB subtypes was t(8;21) in M2 (12.5%) and, rarely, M1 (1.7%) (M0, 0% and M3-7, 0.09%; P=0.001). To evaluate the frequency of cytogenetically undetectable abnormalities, we performed fluorescence in situ hybridization (FISH) analyses in 273 AML M0-M2 with normal karyotype using probes for ETO, ABL, MLL, TEL, RB, P53, AML1, and BCR. In two cases we identified numerical aberrations of RB only in interphases nuclei. In seven additional cases, TEL and MLL abnormalities were found. In conclusion, t(8;21), +11, +13, and +14 are strongly associated with AML M0, M1, and M2. The FISH screening analyses identified abnormalities in an additional 3% in normal karyotypes.

Summary Deficiencies or structural defects of the apoptotic machinery have been postulated as a potential mechanism for a broad resistance of acute myeloid leukaemia (AML) blasts towards cytotoxic therapy comprising chemotherapeutic agents with diverse pharmacodynamic principles but also cell-mediated cytotoxicity of the graft-versus-leukaemia effect, for example, in the setting of allogeneic transplantation. This hypothesis was systematically tested by functionally analysing the early, intermediate and late events of the apoptotic process in primary AML (n = 31) blasts following activation of the intrinsic and extrinsic pathway of apoptosis (etoposide and cytarabine as DNA damaging agents, FAS-ligand as an activator of the death receptor pathway). Activation of the extrinsic pathway by FAS-ligand did not induce apoptosis in primary AML, instead the proapoptotic signal was shown to ‘fade’, even in the early phase of the apoptotic sequence. However, activation of the intrinsic pathway induced severe cytotoxicity in all samples that showed the characteristic features of typical apoptosis, with a prominent apoptotic volume decrease (blebbing) in the early phase, significant increases in caspase 3 activity (intermediate or effector phase) and breakdown of cellular energy production in the late phase of apoptosis. These characteristics did not differ between prognostically favourable versus unfavourable AML karyotypes or between clinically responding versus refractory AML – indicating that a functional apoptotic apparatus is present even in the unfavourable AML subgroups . Our data indicate that the mechanism for a broad clinical resistance is not a dysfunctional apparatus per se but rather the consequence of anti-apoptotic regulation impeding otherwise functional apoptotic machinery.

We report on a 21-year-old man with a mediastinal germ cell tumor (MGCT) who developed a myelodysplastic syndrome (MDS) (refractory anemia with ringed sideroblasts, RARS) 10 months after the start of successful treatment with cisplatin, etoposide, ifosfamide, and paclitaxel. A very rare early occurrence of a therapy-related MDS was suspected. Cytogenetic analysis of the bone marrow revealed an aberrant karyotype, showing a deletion in 12p, an isochromosome 5p, as well as gain of an isochromosome 12p. Isochromosome 12p is a specific aberration frequently observed in MGCT. It also was described in patients who developed hematological transformation of a mediastinal germ cell tumor. In this report the association between mediastinal germ cell tumors and hematological malignancies including the possibility of a common genetic origin is discussed.

Amplification within chromosome arm 11q involving the mixed-lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix-based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700-kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23–24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes. © 2004 Wiley-Liss, Inc.

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.

The standard methods for establishing the diagnosis of acute leukemias are cytomorphology and cytochemistry in combination with multiparameter immunophenotyping. Cytogenetics, fluorescence in situ hybridization, and PCR-based assays add important information regarding biologically defined and prognostically relevant subgroups, and allow a comprehensive diagnosis of well-defined subentities. In the clinical setting, a better understanding of the clinical course of distinct, biologically defined disease subtypes is the basis for a selection of disease-specific therapeutic approaches. As knowledge of deregulated pathways in leukemia increases and accelerates the development of new therapeutics, a detailed and comprehensive diagnostic tool is required. Microarray technology, which quantifies gene expression intensities of thousands of genes in a single analysis, has the potential to become an essential tool for the molecular classification of leukemias. It may, therefore, be used as a routine method for diagnostic purposes in the near future. Furthermore, gene expression profiling may also lead to the detection of new biologically defined and clinically relevant subtypes in leukemia and guide therapeutic decision-making in the future.

Purpose: To examine the efficacy of prolonged maintenance chemotherapy versus intensified consolidation therapy for patients with acute myeloid leukemia (AML). Materials and Methods: Eight hundred thirty-two patients (median age, 54 years; range, 16 to 82 years) with de novo AML were randomly assigned to receive 6-thioguanine, cytarabine, and daunorubicin (TAD) plus cytarabine and mitoxantrone (HAM; cytarabine 3 g/m2 [age < 60 years] or 1 g/m2 [age ≥ 60 years] × 6) induction, TAD consolidation, and monthly modified TAD maintenance for 3 years, or TAD-HAM-TAD and one course of intensive consolidation with sequential HAM (S-HAM) with cytarabine 1 g/m2 (age < 60 years) or 0.5 g/m2 (age ≥ 60 years) × 8 instead of maintenance. Results: A total of 69.2% patients went into complete remission (CR). Median relapse-free survival (RFS) was 19 months for patients on the maintenance arm, with 31.4% of patients relapse-free at 5 years, versus 12 months for patients on the S-HAM arm, with 24.7% of patients relapse-free at 5 years (P = .0118). RFS from maintenance was superior in patients with poor risk by unfavorable karyotype, age ≥ 60 years, lactate dehydrogenase level greater than 700 U/L, or day 16 bone marrow blasts greater than 40% (P = .0061) but not in patients with good risk by complete absence of any poor risk factors. Although a survival benefit in the CR patients is not significant (P = .085), more surviving patients in the maintenance than in the S-HAM arm remain in first CR (P = .026). Conclusion: We conclude that TAD-HAM-TAD-maintenance first-line treatment has a higher curative potential than TAD-HAM-TAD-S-HAM and improves prognosis even among patients with poor prognosis.

We analyzed data of 76 consecutive patients with myelodysplastic syndrome (MDS) and isolated del(5q) (n=66) or del(5q) plus one additional chromosomal abnormality (n=10) included in our MDS database over the last 26 years. The median age of our patient population was 66.8 years. The male to female ratio was 1:1.7. In all, 14 patients (18%) had advanced MDS with an increased medullary blast count. A total of 17 patients (22%) had significant dysplasia in the nonmegakaryocytic cell lines. Nearly half of the study population showed erythroid hypoplasia in the bone marrow. The projected median survival of patients with isolated del(5q) is 146 months for a median follow-up of 67 months. Patients with an increased medullary blast count and those with an additional chromosomal abnormality have a significantly shorter overall survival (24 and 45 months, respectively) than patients with isolated del(5q). We did not find survival differences for different cytogenetic breakpoints, nor did the amount of dysplasia have an impact on survival in our population. In total, 29 patients have died. Deaths occurred primarily due to transformation into acute leukemia, infection, or cardiac failure. Our data support the current definition of a separate entity of MDS with del(5q) that has been suggested by the World Health Organization.

The aim of this study was to compare the pattern of karyotype abnormalities of therapy-related acute myeloid leukemia (t-AML) (n=93) with de novo AML (n=1091), and to evaluate their impact on prognosis. Favorable, intermediate, and unfavorable cytogenetics were observed in 25.8, 28.0, and 46.2% of t-AML, and in 22.2, 57.3, and 20.4% of de novo AML. The median overall survival (OS) was shorter in t-AML than in de novo AML (10 vs 15 months, P=0.0007). Favorable and unfavorable cytogenetics had a prognostic impact with respect to OS in both t-AML (P=0.001 and 0.0001) and de novo AML (P

Overexpression of proto-oncogene c-jun and constitutive activation of the Jun N-terminal kinase (JNK) signaling pathway have been implicated in the leukemic transformation process. However, c-jun expression and the role of the JNK signaling pathway have not been investigated in primary acute myeloid leukemia (AML) cells with frequently observed balanced rearrangements such as t(8;21). In the present study, we report elevated c-jun mRNA expression in AML patient bone marrow cells with t(8;21), t(15;17) or inv(16), and a high correlation in mRNA expression levels of AML1-ETO and c-jun within t(8;21)-positive AML patient cells. In myeloid U937 cells, c-jun mRNA and protein expression increase upon inducible expression of AML1-ETO. AML1-ETO transactivates the human c-jun promoter through the proximal activator protein (AP-1) site by activating the JNK pathway. Overexpression of JNK-inhibitor JIP-1 and chemical JNK inhibitors reduce the transactivation capacity of AML1-ETO on the c-jun promoter and the proapoptotic function of AML1-ETO in U937 cells. An autocrine mechanism involving granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSF-R) might participate in AML1-ETO mediated JNK-signaling, because AML1-ETO induces G-CSF and G-CSF-R expression, and G-CSF-R-neutralizing antibodies reduce AML1-ETO-induced JNK phosphorylation. These data suggest a model in which AML1-ETO induces proto-oncogene c-jun expression via the proximal AP-1 site of the c-jun promoter in a JNK-dependent manner.

Background Flow cytometry (FC) is a standard method for diagnosing and subclassifying acute myeloid (AML) and acute lymphoblastic (ALL) leukemias and allows the analysis of cell surface and intracellular proteins. In the future, diagnostic procedures may include oligonucleotide microarray analysis (MA) to detect expression patterns of large numbers of specific genes. Methods For comparison between methods, we performed FC and MA by using the Affymetrix GeneChip HG-U133A microarray in parallel and correlated protein expression levels and mRNA abundance of 39 relevant genes in 113 patients with newly diagnosed AML and ALL and four normal bone marrow samples. Results In 1,512 of 2,187 (69.1%) comparisons between methods, congruent results were obtained with regard to positivity or negativity of expression, respectively. Specifically, there was a significant correlation between protein expression and mRNA abundance for genes essential for diagnosing and subclassifying AML and ALL with regard to positivity and expression. Conclusions These data suggest that protein expression is highly correlated to mRNA abundance in AML and ALL. Further, expression patterns of specific genes provide important information at diagnosis for patients with AML and ALL that may be used for the discrimination from other leukemias. Cytometry Part B (Clin. Cytometry) 55B:29–36, 2003. © 2003 Wiley-Liss, Inc.

Complex cytogenetic abnormalities confer dismal prognoses in myeloid malignancies. Even bone marrow transplantation from siblings or matched unrelated donors offer minimal chances for cure, suggesting that these cases are not only refractory to chemotherapy but also resist the graft-vs.-leukemia effect. We herein describe the first permanent, factor-independent c-kithi+ cell line CS-1 derived from an unrelated donor stem cell transplanted patient with relapsed acute myeloid leukemia (AML)-M5a of high-risk karyotype [monosomy 7, t(2;11)(q31;p13), t(10;12)(q24;q24)]. Having the same karyotype, CS-1 exhibits an autonomous growth pattern and responds to stem cell factor (SCF). CS-1 did not induce T cell activation in mixed-lymphocyte-tumor-cultures (MLTCs) and, when used as third party stimulators, decreased T cell proliferation in mixed-lymphocyte reactions (MLRs). Cytokines added exogenously or secreted from bystander T cells caused CS-1 to differentiate into dendritic cells (DCs). CS-1-derived DCs, in contrast to DCs originating from non-malignant CD34+ progenitor cells, had virtually no T cell stimulatory effect, indicating that CS-1 is both immunosuppressive and poorly immunogenic. These properties may partially be due to the detected downregulation of costimulatory molecules and appear to involve a soluble factor. CS-1 cells injected subcutaneously (s.c.) to non-obese diabetes/severe combined immunodeficient (NOD/SCID) mice produced solid tumors, disseminating into bone marrow and spleen. The data show that transforming AML blasts with high-risk karyotype into DCs is insufficient to restore their immunogenicity and that the CS-1 cell line is useful to identify tumor-related immunosuppressive mechanisms in vitro and in vivo. © 2003 Wiley-Liss, Inc.

Risk assessment in acute myeloid leukemia (AML) using pretreatment characteristics may be improved by incorporating parameters of early response to therapy. In the 1992 trial of the German AML Cooperative Group (AMLCG), the amount of residual leukemic blasts in bone marrow was assessed one week after the first induction course (day 16 blasts). A total of 449 patients 16 to 76 years of age (median, 53 years) with de novo AML entered the trial and were evaluable. Treatment included TAD/HAM (thioguanine, cytosine arabinoside, and daunorubicin/high-dose cytosine arabinoside and mitoxantrone) double induction, TAD consolidation, and randomly either maintenance therapy or S-HAM consolidation. Cytogenetics were favorable, intermediate, unfavorable and not available in 10.0%, 48.3%, 13.1%, and 28.5%, respectively. Day 16 blasts ranged from 0% to 100% (median, 5%, mean ± SD, 18.6 ± 28.5%). Complete remission (CR) rate was 72.6%, 17.6% had persistent leukemia (PL), and 9.8% succumbed to hypoplastic death. Median overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were 18, 9, and 15 months with 28.4%, 21.6%, and 30.1% at 5 years, respectively. As a continuous variable, day 16 blasts were related to CR rate (P

Etiology of acute myeloid leukemia following intensive therapy for AML – relapse, secondary disease or bad luck?

Background Recombinant adeno-associated virus (rAAV) has many advantages for gene therapeutic applications in comparison with other vector systems. One of the most promising features is the ability of wild-type (wt) AAV to integrate site-specifically into human chromosome 19. However, this feature is lost in rAAV vectors due to the removal of the rep-coding sequences. Methods HeLa cells were transfected with a rep expression plasmid, infected by rAAV and grown with or without selection pressure. Single cell clones were generated and genomic DNA was analyzed for site-specific integration by Southern blotting analysis and fluorescence in situ hybridization (FISH). Results Transfection of HeLa cells with a rep expression plasmid followed by transduction with a rAAV vector resulted in site-specific integration of the transgene at AAVS1 on human chromosome 19 in 7 of 10 cell clones analyzed. In marked contrast, transduction of cells with rAAV alone did not result in any site-specific integration of the transgene. Conclusions The high frequency with which the site-specific integration took place in the presence of Rep protein is comparable with the results observed with wtAAV. These results offer opportunities for the development of specifically integrating rAAV vectors. Copyright © 2002 John Wiley & Sons, Ltd.

Cytogenetic analysis is the most important diagnostic tool for determining prognosis in acute myeloid leukemia (AML). In the majority of patients with AML, acquired clonal chromosome aberrations can be observed. Numerous recurrent karyotype abnormalities have been discovered in AML. These findings on the chromosomal level have paved the way for molecular studies that have identified genes involved in the process of leukemogenesis. The identification of specific chromosomal abnormalities and their correlation with cytomorphologic features, immunophenotype, and clinical outcome have led to a new understanding of AML as a heterogeneous group of distinct biologic entities. The importance of cytogenetic findings in AML for classification and for the understanding of pathogenetic mechanisms is increasingly appreciated in the clinical context, and the new World Health Organization classification of AML uses cytogenetic abnormalities as a major criterion.

The instability of the karyotype may play a role in the development of refractoriness of acute myeloid leukemia (AML) to anti-leukemic therapy. Therefore, in the current study cytogenetic analyses were performed in 117 patients with AML both at diagnosis and at relapse. Changes in karyotype were observed in 38% (36% of initially normal karyotypes, 39% of initially aberrant karyotypes). An evolution of karyotype, ie the acquisition of further aberrations in addition to those present at diagnosis, occurred more frequently in patients with unfavorable karyotypes at diagnosis as compared to all others (60% vs 32%, P = 0.0095). The duration from initial diagnosis to relapse was significantly shorter in cases with an evolution of the aberrant karyotype as compared to cases with no changes in the aberrant karyotype between diagnosis and relapse or with solely regression of aberrations at relapse (9.2 +/- 4.4 vs14.0 +/- 8.5 months, P = 0.0081). In an additional analysis, another cohort of 120 patients with refractory and relapsed AML who were treated uniformly within the respective trial of the German AML Cooperative Group was analyzed cytogenetically at diagnosis and at relapse to further prove the prognostic impact of karyotype aberrations at relapse. Karyotypes were prognostically favorable, intermediate, unfavorable and not available in 8%, 50%, 17% and 25% at diagnosis and in 8%, 49%, 21% and 22% at relapse, respectively. Karyotype aberrations at diagnosis had no impact on response to therapy (P = 0.32) but influenced survival and event-free survival significantly (P = 0.03 and P = 0.02). In contrast, karyotype aberrations at relapse strongly influenced response to therapy (P = 0.05), survival (P = 0.01), and event-free survival (P = 0.002). These data suggest that the instability of the karyotype between diagnosis and relapse and thus karyotype aberrations at relapse in particular contribute to the refractoriness of AML to anti-leukemic therapy.

Conventional metaphase cytogenetics underestimates the frequency of specific chromosome aberrations in B-cell chronic lymphocytic leukaemia (B-CLL) as a result of the very low proliferative activity of these cells in vitro. New molecular approaches, such as fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH), may circumvent this problem, at least in part, but these techniques are either strongly dependent on the knowledge of candidate regions or detect only unbalanced aberrations. In the present study, we analysed 27 B-CLL peripheral blood samples by metaphase cytogenetics after CD40 ligand (CD40L)-induced cell cycle stimulation. In comparison with the simultaneous use of B-cell mitogens such as 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), CD40L stimulation of B-CLL cells induced a threefold increase in metaphases amenable to analysis by conventional cytogenetics. The analysis of these metaphases confirmed all genetic abnormalities detected by FISH. Moreover, CD40L-enhanced cytogenetics revealed complex karyotypic aberrations in 11 out of 27 patients (41%). In one case, a balanced translocation t(11;16)(p15;p13.1), so far unreported in B-CLL, was detected. Taken together, the results of our study show the potential of CD40L-enhanced metaphase cytogenetics to detect more and new chromosome aberrations in B-CLL.

Summary. The French–American–British (FAB) classification and the new World Health Organization (WHO) classification distinguish acute monoblastic leukaemia (AML M5a) from acute monocytic leukaemia (AML M5b). Not much is known about the underlying genetic differences leading to these clearly different phenotypes. We analysed 58 patients with de novo AML M5a and 66 patients with denovo AML M5b in comparison with a whole group of 1603 de novo AML. An aberrant karyotype was found in 75·9% of AML M5a but in only 28·8% of M5b (P

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.