Complex Aberrant Karyotype Research Articles

Further Insights into the Pathogenesis of AML with Complex Aberrant Karyotype by Performing a Combination of Conventional CGH, Gene Expression and Genomic Array Analysis.

Acute myeloid leukemia (AML) with complex aberrant karyotype is a distinct biological entity. It is characterized by: 1) a sharp increase of incidence above age 50, 2) a characteristic pattern of chromosomal gains and especially losses, i.e. losses of 5q14q33, 7q32q35, and 17p13 translating into a reduced expression of genes located in these regions, 3) a unique gene expression pattern including an upregulation of genes involved in DNA repair, 4) a high incidence of TP53 deletions and/or mutations and 5) an overall unfavorable prognosis. So far, the pathogenetic role of the lost and gained chromosomal regions in AML with complex aberrant karyotype is unclear. In a first step we tested whether a correlation between genomic imbalances and changes in gene expression exists. Therefore, gene expression analysis was performed in 24 cases of AML with complex aberrant karyotype and in 57 AML with normal karyotype for comparison. Overall, genes located on 5q, which is deleted in the majority of cases, showed a significantly lower expression in AML with complex aberrant karyotype as compared to AML with normal karyotype. Furthermore, for each chromosomal band on chromosome 5 ratios were calculated between AML with complex aberrant karyotype and AML with normal karyotype. Ratios lower than 0.90 were found for all chromosome bands between 5q14 and 5q34 (range 0.64–0.88) with lowest values for 5q15 and 5q22 (0.64 and 0.68). Between 61% and 90% of genes located in one of the chromosome bands 5q14 to 5q34 showed a lower expression in AML with complex aberrant karyotype compared with AML with normal karyotype. An overall reduced expression of genes was also observed in other frequently lost regions such as 7q and 17p, while an overall higher expression of genes located in gained regions such as 1p, 8q and 11q was detected. However, not all genes located within a deleted or gained region showed an altered expression. In order to perform more precise correlations between the copy number of individual genes and their expression 33 cases with AML and complex aberrant karyotype were analyzed in parallel with conventional comparative hybridization, genomic arrays (Affymetrix 10K arrays) and gene expression arrays (Affymetrix U133A+B or 2.0 plus). The lost and gained regions detected in conventional CGH were confirmed by data obtained from the genomic arrays. In addition, gained and lost regions could be mapped more precisely. Interestingly, the genomic arrays revealed that even large deletions are truly continuous, although not all genes from the respective regions showed a lower expression. In contrast at the borders of amplifications amplified genes alternated with non-amplified genes. In conclusion, a detailed analysis on the genomic as well as on the gene expression level might lead to further insights into the pathogenesis of AML with complex aberrant karyotype and may also serve as a new diagnostic tool in the near future.

NRAS Mutations in AML: Biology, Cytogenetics, and Prognostic Impact in 2502 Patients.

In AML NRAS mutations were reported in 11–30%. The prognostic impact is not yet clarified. We studied 2502 pts. between 18.3 and 91.8 years of age (median 63.4 years). 2037 pts. had de novo AML, 281 secondary AML following MDS, and 181 pts. suffered from therapy-related AML. All samples were evaluated by cytomorphology, cytochemistry, multiparameter flow cytometry, cytogenetics, FISH, and molecular genetics in parallel. The screening for NRAS mutations was performed around the mutational hotspots at codons 12, 13, and 61 using a melting curve based LightCycler assay. All cases tested positive for NRAS mutations were confirmed by sequence analysis. 257/2502 pts. (10.3%) showed NRAS mutations (NRASmut), whereas 2245 cases had wildtype NRAS (NRASwt). In 3 of the 257 mutated cases (1.2%) two different mutations were detected. None of these cases was homozygous for one mutation. The majority of mutations was found at codon 12 (n=112/257; 43.6%). Mutations at codon 13 were found in 64/257 pts. (24.9%). In both cohorts the changes were most frequently from glycin to asparagin (codon 12: n=76; 29.6%; codon 13: n=41; 16%). Mutations at codon 61 were detected in 54/257 pts. (21.0%), mostly from glycin to arginin. For 105 cases paired samples from diagnosis and relapse were analysed. The status was stable in 101 cases (96.2%). The history of AML did not differ significantly in association to NRAS mutations. The median leukocyte count was 28.0 G/l (range 0.4–514.0 G/l) in pts. with NRASmut and 63.4 G/l (range 18.3–91.8G/l) in the pts. with NRASwt (p<0.0001). Cytogenetic analyses were available in all cases. Pts. were grouped as followed: normal karyotype, t(15;17), t(8;21), inv(3)/t(3;3), inv(16)/t(16;16), 11q23/MLL rearrangements, complex aberrant karyotype, +8, 5q- or loss of chromosome 5, 7q- or loss of chromosome 7, and other aberrations. In the subgroups with inv(16)/t(16;16) and with inv(3)/t(3;3) NRASmut showed a high frequency of 37.6% (50/133) (p<0.0001), and 11/41 (26.8%) (p=0.0004), respectively. In contrast, NRASmut were significantly underrepresented in t(15;17) (2/102; 2.0%) (p=0.0048), in the subgroup with MLL/11q23 rearrangements (3/77; 3.9%) (p=0.0616), and in cases with complex aberrant karyotype (4/258; 1.6%)(p<0.0001). With 80% and 55% mutations of codon 61 were significantly overrepresented in pts. with inv(16) and with inv(3) compared to the total group where mutations at codon 12/13 were the most common (both p<0.0001). We did not find a significant prognostic impact of NRASmut for OS, EFS and DFS. This was also true in detail for subgroups with inv(16), inv(3), t(8;21), normal karyotype and other aberrations. In AML with normal karyotype we further took the FLT3 mutation status into account. For pts. without FLT3 mutations the EFS was slightly better if NRAS mutations were detected (426+/−73 days) in comparison to pts. negative for NRAS mutations (378+/−21 days) (p=0.0603). In conclusion, we found a clear pattern of NRAS mutations in defined subgroups of AML supporting the hypothesis of a two hit mutation model in AML. However, a prognostic impact was not found.

Complex Aberrant Karyotypes and Unbalanced Translocations in CLL Correlate with an Unmutated IgVH Status: A Study on 133 Patients Studied with Chromosome Banding Analysis, Interphase FISH, IgVH Mutation Status, ZAP-70 RNA Expression and Immunophenotyping.

CLL is a heterogenous disease from a clinical as well as from a genetic point of view. In order to characterize CLL patients in detail we performed chromosome banding analysis and interphase FISH on 133 cases and analyzed the IgVH status, CD38 and ZAP-70 mRNA expression in parallel. Of the 133 samples that were included in this study, 126 (95%) could be successfully analyzed by cytogenetic banding techniques. 102 (81%) of the 126 samples showed chromosomal aberrations. In comparison, all 133 cases could be successfully analyzed by FISH with a rate of 79% aberrations like deletions of chromosomes 13q (57%, in 38% as sole abnormality), 11q (17%), trisomy 12 (13%), 6q (7%) 17p (6%) and translocations involving 14q32 (4%). 9 cases with a normal karyotype in conventional cytogenetics revealed genetic aberrations by FISH. Additional aberrations not included in the FISH panel of probes, were detected in 47 samples (37%) by conventional cytogenetics. The assessment of additional risk factors in our cohort of CLL patients revealed an unmutated IgVH status in 55%, a positive ZAP-70 expression in 57% and a CD38 expression ≥ 30% in 29% of the samples. ZAP-70 mRNA expression resulted in concordance with the IgVH status in 75% of the cases with the concurrence of ZAP-70 positive/unmutated IgVH or ZAP-70 negative/ mutated IgVH. Genetic poor risk factors like del(11q) and del(17p )correlated well with an unmutated IgVH status with 89% and 75% concordant cases, respectively and also CD38 expression resulted in a significant correlation with the IgVH status (p = 0.028, Fischer's exact test). When analyzing the group of 47 samples with cytogenetic aberrations beyond the FISH panel in more detail, it was remarkable that a large number of 22 cases (47%) was characterized by a complex aberrant karyotype (≥ 3 unbalanced aberrations). The status of somatic mutations of the IgVH gene was available for 39 cases in that group with 74% having an unmutated IgVH-status. This value was significantly increased (p = 0.013) compared to the 44% of unmutated samples calculated from the panel of samples that were concordant in FISH and chromosome analysis (IgVH status was available in 57 cases). No significant correlations were obtained for ZAP-70 and CD38 expression with the different cytogenetic groups. Remarkable was also the incidence of unbalanced translocations in 22 cases, not picked up by the FISH probes, which were strongly associated with an unmutated IgVH-status (83%). In conclusion, a comprehensive laboratory work-up is necessary in order to obtain more insights into the pathophysiology of CLL and for individualized treatment approaches. Interestingly, CLL cases with a more complex karyotype (≥ 3 unbalanced aberrations) or with unbalanced translocations correlated with an unmutated IgVH status and might therefore represent a high risk group of patients.

Nucleophosmin Gene Mutations Are Predictors of Favourable Prognosis in Acute Myeloid Leukemia with a Normal Kayotype and Can Be Used as a New Marker for Quantitative PCR To Detect Minimal Residual Disease.

Nucleoplasmin (NPM) mutations have recently been described as a new kind of mutation in AML. In the presented work we have analyzed 977 AML cases from different prognostic subgroups. Fifteen different mutations (type A:n=245 (78.3%), B:n=21 (6.7%), D: n=28 (11.4%), I:n=3 (1%), J:n=4 (1.3%), K:n=2, G,H, L, M, N, O, P, Q, R: n=1, each (0.3%) were detected in 313 cases. No mutation was found in inv(16) (n=89), t(8;21) (n=98), t(15;17) (n=17), t(11q23)/MLL (n=8), inv(3) (n=8), other reciprocal translocations (n=3), and complex aberrant karyotypes (n=59). In the normal karyotype group the incidence was 299/603 (49.6%) and in all others 13/90 (14.4%). In the latter cohort cases with NPM+ were: − Y (1 case), +4 alone (1 case), +4 and +8 (2 cases), +4 with multiple trisomies (1 case),+8 (1 case), multiple other trisomies (1 case), del(5q) (1 case), − 7 alone (2 cases), del(9q) (2 cases), unbalanced translocation (1 case).Data for evaluation of prognosis were available in 401 AML patients with normal karyotype treated within the AMLCG99 study. Results were calculated in relation to MLL-PTD, FLT3-LM, FLT3-TKD, NRAS, KIT, and CEBPA mutations and correlated to further clinical characteristics. NPM-mutated cases were frequently associated with FLT3 mutations but rarely with other mutations. The NPM-mutated group had a higher CR rate (70.5% vs. 54.7%, p=0.003), a trend to longer OS (median 1012 vs. 549 days, p=0.0762), and significantly longer EFS (median 428 vs. 336 days; p=0.0121). There was a clear favourable impact of NPM+/FLT3-LM- on OS and EFS (264/395 cases; 66.8%). This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM+/FLT3-LM+ double positive pts. was similar to NPM-/FLT3-LM+ cases. We further analysed stability for the NPM mutation at diagnosis and relapse and found stability in 14/15 paired samples. One case with AML M5 lost the NPM mutation at relapse. This case had also a shift from normal karyotype to 46,XX,del(7)(q22),der(7)t(7;21)(p11;q?) and a complete shift of immunophenotype indicating a therapy-related AML. In addition, we have analyzed the applicability of NPM mutations as targets for minimal residual disease (MRD) detection. In total, 82 samples of 22 selected cases were analyzed by use of quantitative real time PCR for the three most common mutation types. Fifteen of these cases had a type A, 3 a type B, and 4 a type D mutation. Samples at diagnosis had an NPM/ABL ratio of 267.6 (range: 54.9 to 3174.7) while each 10 NPM negative cases had a median ratio of 0.03 (assay for A), 0.0001 (assay for B), and 0.002 (assay for D), showing that these assays were nearly specific for the respective mutation. Using limited dilution series of NPM+ in NPM- cDNA a median sensitivity of 1:100.000 could be shown. In both analyzed cases with relapse these were early detectable by increasing NPM levels. Thus, NPM mutations can be used for highly sensitive PCR-based MRD detection in AML, especially if other markers are lacking, i.e. in normal karyotype.In conclusion, our data showa nearly exclusive association of NPM+ to the normal karyotype,a good prognosis of NPM+/FLT3-LM- in normal karyotype.an intermediate prognosis of NPM+/FLT3-LM+ in normal karyotypeNPM as a new marker for PCR-based MRD-detection.

JAK2 Mutation Screening and Chromosome Analysis Are Necessary for a Comprehensive Diagnostic Work up in CMPD: A Study on 469 Cases.

The diagnosis of BCR-ABL negative chronic myeloproliferative disorders (CMPD) is still a challenge for the morphologist and clinician, mainly because of overlapping phenotypes of essential thrombocythemia (ET), polycythemia vera (PV), idiopathic myelofibrosis (IMF) and non-malignant reactive phenotypes. Recently, a new mutation in JAK2 leading to V617F exchange in exon 12 was described in these entities. Therefore, we developed a rapid and easy LightCycler based melting curve assay and screened 469 patients with various malignancies for the respective JAK2 mutation using cDNA prepared from mononucleated cells. All cases tested positive were confirmed by sequencing and without any exception all mutations were G to T exchanges at nucleotide 1849. In total, 61 cases with PV were analysed. 56 (91.8%) were mutated (see table). A karyotype was available in 36 cases. Of the JAK2- cases 4 had normal karyotypes and one 20q-. Of the JAK2+ cases 21 had normal karyotype; four (7.1%) had +9, one +8, one +22, one 20q-, and three showed a complex aberrant karyotype. Of the 47 analyzed ET 26 (55%) were mutated. Cytogenetics was available in 20 cases (9 JAK2-, 11 JAK2+). All JAK2- cases all had normal karyotypes. Of the JAK2+ 10 had a normal karyotype and one a t(9;13)(p24;q22) involving the JAK2 locus. Of the 25 IMF cases 14 (56%) were mutated. Karyotype was available in 17 cases (7 JAK2-, 10 JAK2+). All 7 JAK2- had normal karyotype. Of the 10 JAK2+ 6 had normal karyotype and four were aberrant (+9:n=2; 20q-: n=2). In addition, 2/7 cases (28.6%) with CMML were JAK2+. Furthermore, we analysed 89 BCR-ABL negative MPS that were not further specified. V617F was detectable in 43 patients (48.3%). At next 128 AML were analyzed (84 novo AML, 11 t-AML after a previous malignancy, 15 secondary to MDS, and 16 secondary to MPS (PV:n=7, ET:n=5, OMF:n=2). No V617F was detected in all cases with t-AML and s-AML after MDS. In contrast, in de novo AML 6/84 (7.1%) were JAK2+, of which 4 were proven homozygous. Surprisingly, 4 of these six cases had a +9. In AML secondary to a previous CMPD 11/16 (68.8 %) were JAK2+. Of these 2 had +9, one a normal karyotype and 12 a complex aberrant karyotype. These data suggest that acquisition of +9 may lead to progress or blast crisis in JAK2 mutated MPS and supports the gain of function mechanism of the mutation.In conclusion, more than half of all BCR-ABL-negative MPS harbour a V617F mutation. This helps in the differential diagnosis of MPS versus reactive disorders. V617F is more frequently associated with aberrant karyotype than wildtype JAK2. In addition, there is an increasing level of aberrant cytogenetics and mutation rate with respect to incidence and status from ET<IMF<PV indicating that these diseases might be overlapping or even a continuum. It demonstrates that the present classification is artificial and a new classification of “JAK2” positive diseases may be more adequate. In addition, the same mutation was observed in most cases of AML secondary to CMPD. It was also found in few cases with de novo AML and correlated with +9. JAK2V617F is a new and promising target for therapy as well as for molecular monitoring of therapy response in CMPD.Distribution of JAK2 mutation in various malignanciesETIMFPVMPS*CMMLde novo AMLAML after MPStotal4725618978416mutated (n=)261456432611mutated (% of all)55.0 %56.0 %91.8 %48.3 %28.6 %7.1 %68.8 %homozygous (n=)393319147homozygous (% of all)6.4 %36.0 %54.1 %21.3 %14.3 %4.8 %43.8 %*not further specified

Concept, Feasibility and Results of the Randomized Comparison of Imatinib Combination Therapies for Chronic Myeloid Leukemia: The German CML-Study IV.

Targeted therapy with the BCR-ABL tyrosine kinase inhibitor imatinib induces high response rates in chronic myeloid leukemia (CML) patients (pts). Nevertheless, residual disease remains in virtually all pts on imatinib monotherapy as a potential cause of relapse. In July 2002, the German CML-Study Group activated the four-armed randomized controlled trial comparing imatinib 400mg/d with imatinib+IFN, imatinib+Ara-C, and imatinib after IFN failure in newly diagnosed pts with chronic phase CML. Randomization is stratified according to prognostic risk groups and not biased by consecutive allogeneic stem cell transplantation (SCT). High-risk pts are randomly assigned to primary imatinib-based therapies including a treatment arm with 800mg/d imatinib. By 7/05, 632 pts were randomized: imatinib 400mg/d (n=129), imatinib+IFN (n=179), imatinib+Ara-C (n=156), imatinib after IFN failure (n=157), and imatinib 800mg/d (n=11). According to the Hasford score, 35% of pts were low risk, 54% intermediate risk, and 11% high risk. At baseline, median WBC count was 67/nl (3–529), median platelet count 391/nl (34–2,799) and median hemoglobin 12.6 g/dl (6.1–16.6). We sought to evaluate results of pts with a >12 months follow-up (n=416), recruited between 7/02 and 6/04 (imatinib 400mg/d, n=102; imatinib+IFN, n=126; imatinib+Ara-C, n=104; imatinib after IFN failure, n=81; imatinib 800mg/d, n=3) and of pts with a >24 months follow-up (n=232), recruited between 7/02 and 6/03 (imatinib 400mg/d, n=55; imatinib+IFN, n=74; imatinib+Ara-C, n=54; imatinib after IFN failure, n=49) with respect to response, resistance, and progression. After 12 months of treatment cytogenetic data are available from 238/335 pts (71%) randomized to primary imatinib based therapies. 209 pts (63%) achieved a major cytogenetic remission (MCR; Ph+<35%), being complete in 53%. Q-PCR data were available in 270 pts (81%). 89 pts (27%) achieved a major molecular response (MMR; ratio BCR-ABL/ABL <0.12%). After 24 months cytogenetic data are available from 141/183 pts (77%). 126 pts (69%) achieved a MCR, being complete in 60%. Q-PCR data were available in 149 pts (81%). 73 pts (40%) achieved a MMR. 12/177 pts lost CCR (7%) during the 1st year and 6/110 pts (5%) during the 2nd year of treatment. Within the 1st year 13/335 pts (6 low, 3 intermediate, 4 high risk; 4%) progressed to blast crisis, 4 of them revealed clonal evolution (complex aberrant karyotype, n=3; +8, n=1), two others BCR-ABL mutations (E355G and M244V). Within the 2nd year 3/232 pts (1 each low, intermediate, and high risk; 1%) progressed to blast crisis. During the 1st year of treatment imatinib therapy was stopped due to side effects or resistance in 6% of pts in the imatinib 400mg arm, in 2% of pts in the imatinib+IFN, and in 2% of pts in the imatinib+Ara-C arm. IFN was stopped in 21%, Ara-C in 18% of pts. This interim analysis of a prospective randomized trial with imatinib and imatinib in combination for newly diagnosed pts with CML has proven feasibility of imatinib combinations in addition to high response and low progression rates. Long-term observation will demonstrate whether the promising results will be maintained and will improve survival.

Immunostimulatory CpG-Oligonucleotide Activated Metaphase Cytogenetics Is Feasible in Routine Diagnostics of Chronic Lymphocytic Leukaemia and Reveals More Abnormalities Than Interphase FISH.

Genetic characterization of chronic lymphocytic leukemia (CLL) cells by fluorescence in situ hybridization (FISH) has identified genetic aberrations in 80% of CLL patients. In contrast, conventional metaphase cytogenetics detected chromosomal aberrations in only 40–50% of all cases. Immunostimulatory CpG-oligonucleotides (CpG-ODN) in combination with interleukin 2 (IL-2) have recently been reported to induce proliferation in CLL B cells. Therefore, we evaluated this stimulus for its efficacy in metaphase generation for the detection of chromosomal aberrations by cytogenetic banding techniques. In addition these results were compared with data obtained by interphase FISH. For metaphase induction 107 cells were cultured in growth medium with the CpG-ODN DSP30 and IL-2. After 2 days colchicine was added for another 24h before preparation. Of the 133 samples that were included in this study, all cases could be successfully analyzed by interphase FISH with a rate of 79% aberrations like deletions of chromosomes 13q (57%, in 38% as sole abnormality), 11q (17%), trisomy 12 (13%), 6q (7%), 17p (6%) and translocations involving 14q32 (4%). By FISH 55% of all samples showed a single aberration, 22% two aberrations and 2% 3 aberrations. In comparison, 126 cases (95%) could be successfully analyzed by cytogenetic banding techniques. 102 (81%) of the 126 samples showed chromosomal aberrations, which involved all different chromosomes. 9 cases with a normal karyotype in conventional cytogenetics revealed genetic aberrations by FISH. In all but 1 of these cases the aberrant clone represented < 30% of the cell population. In addition to the aberrations that were detected by the FISH probes, a substantial amount of other aberrations was revealed by chromosome banding analysis. Interestingly, 10 cases of a total of 27 cases without abnormalities detected by FISH displayed aberrations in chromosome analysis indicating that the true amount of CLL patients with aberrations exceeds 79%. Overall, 47 samples (37%) showed chromosomal aberrations in chromosome banding analysis in addition to FISH analysis. Compared to FISH analysis, which detected 2 cases with 3 aberrations, metaphase cytogenetics detected 22 cases with 3 or more unbalanced aberrations, which can be considered as a complex aberrant karyotype. Loss of p53 referred to as del(17p13) in FISH has attracted considerable attention, because of the poor clinical outcome of affected patients. In our study, all del(17p13) cases (n=7) displayed either a loss of the short arm of chromosome 17 (n=6) or a complete loss of chromosome 17 (n=1) indicating that chromosomal regions in addition to the p53 locus might be relevant for this phenotype. Furthermore, numerous recurrent aberrations have been identified in this study beyond the aberrations that are detected by FISH. Of note are gains of 2p and 3q, losses in 11q13 and gains in 11q23–25, gains in 13q31–34, gains of 17q21–25 and cases with trisomy 18 and 19, which occurred in parallel to trisomy 12. The results of the present study show that immunostimulatory CpG-oligonucleotides plus interleukin 2 are a simple and cheap stimulus for efficient metaphase induction in CLL. The rate of aberrations detected by conventional banding techniques was even slighty increased compared to FISH analysis, however, the complexity of cytogenetic aberrations is underestimated by FISH analysis in a large portion of CLL cases (37%).

Proteomics of Acute Myeloid Leukemia: Cytogenetic Risk Groups Differ Specifically in Their Proteome, Interactome and Posttranslational Protein Modifications.

Acute Myeloid Leukemia (AML) is characterized by specific cytogenetic aberrations which are strong determinants of prognostic outcome and therapeutic response. Because the clinical outcome in AML cytogenetic groups differs considerably, we hypothesized that cytogenetic risk groups of AML might differ specifically in their proteome, protein interaction pathways and posttranslational modifications (PTMs). Thus, we determined the proteome of 30 AML patients belonging to various cytogenetic groups based on two-dimensional gel electrophoresis and Nano LC coupled MALDI-TOF-TOF tandem mass spectrometry. We could identify substantial differences in the proteome, protein expression and peak pattern between cytogenetic risk groups of AML. The interactome analysis based on computational bioinformatics using Ingenuity analysis revealed major regulating networks: MAPK8 and MYC for complex aberrant karyotype AML, TP53 for t(8;21)-AML, TP53- MYC- PRKAC for 11q23-AML, JUN and MYC for inv(16)-AML. Most interestingly, peak explorer analysis revealed a modification of O-linked acetyl glucosamine of hnRNPH1 in AML patients with a 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation AML, an increased intensity of dimethylated peptide of hnRNPA2/B1 in AML patients with translocations of t(8;21) and inv(16) in comparison to healthy bone marrow. We show for the first time that cytogenetic risk groups of AML differ specifically both in their proteome, interactome and PTMs. These findings lead to a new thinking about the pathogenesis of AML and has major therapeutic implications because PTMs are the primary drug targets.

Further correlations of morphology according to FAB and WHO classification to cytogenetics in de novo acute myeloid leukemia: a study on 2,235 patients

In routine diagnostic procedures of acute myeloid leukemia (AML), the French-American-British (FAB) and World Health Organization (WHO) classifications both play a central role. Some morphologic subtypes are specifically associated to distinct cytogenetic and molecular aberrations; however, such close correlations do not exist for the majority of entities. We evaluated cytogenetics in 2,235 patients at diagnosis of AML with the FAB subtypes M0-2, M4, and M5-7. The cytogenetic patterns of these subtypes showed differences with respect to the clonal aberration rate and the incidence of complex aberrant karyotypes. The frequency of numerical gains and losses and of structural losses and the incidence of 11q23/MLL rearrangements differed. Thus, cytomorphology of AML may be helpful to support or even initiate other diagnostic procedures, e.g., interphase fluorescence in situ hybridization and polymerase chain reaction. In conclusion, the central role of morphology as defined by the FAB and WHO classification in AML at diagnosis is still justified in combination with other techniques.

Concept and feasibility of the randomized comparison of imatinib with imatinib combination therapies for chronic myeloid leukemia: The German CML Study IV - Pilot Phase

6574 Background: Chronic myeloid leukemia (CML) is considered a clinical model for other neoplastic diseases. Targeted therapy with the BCR-ABL tyrosine kinase inhibitor imatinib (IM) induces high response rates in CML. Nevertheless, residual disease remains in virtually all patients (pts) on IM monotherapy as a potential cause of relapse. Methods: In July 2002, a randomized trial has been activated comparing IM 400 mg/d with IM+IFN, IM+Ara-C, and IM after IFN failure (or 800 mg IM monotherapy in high risk pts) in newly diagnosed CML. The study is accompanied by a comprehensive cytogenetic and molecular surveillance program to monitor response and to search for early signs of relapse. Results: By 12/2004, 505 pts were randomized. 35% were low, 54% intermediate, and 11% high risk according to the Euro score. A cohort of pts (n=279) recruited by 9/03 with a >12 months follow up was evaluated. Cytogenetic data were available from 161/222 pts (73%) randomized to IM based therapies. At 12 months, 142 pts (64%) achieved a major cytogenetic response (Ph+<35%), being complete in 53%. Quantitative PCR data were available in 182 pts (82%). 58 pts (26%) achieved a major molecular response (ratio BCR-ABL/ABL <0.12%). In one patient (0.5%) BCR-ABL became undetectable. 5/116 pts lost CCR (4%) during the first year. 9/222 pts (4 low, 2 intermediate, 3 high risk; 4%) progressed to blast crisis, 4 of them showed clonal evolution (complex aberrant karyotype, n=3; +8, n=1), 2 others BCR-ABL mutations (K247R and E355G). IM therapy was stopped due to side effects or resistance in 6% of pts in the IM 400 mg arm, 2% in the IM+IFN arm, and 4% in the IM+Ara-C arm. IFN was stopped in 21%, Ara-C in 13% of pts. Conclusions: The pilot phase of CML IV has proven feasibility of IM combinations in addition to high response rates. Long-term observation will demonstrate whether the promising results will be maintained and will improve survival. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis, Roche

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.

Myelodysplastic syndrome of donor origin subsequent to successful treatment of myeloid/NK-cell precursor leukaemia with allogeneic PBSCT: two very rare conditions in one patient

We report a 36-year-old male with myeloid/natural killer (NK)-cell precursor acute leukaemia with a complex aberrant karyotype, who was treated according to an acute-myeloid-leukaemia (AML) treatment protocol (idarubicine, cytarabine, and etoposide) followed by high-dose cytarabine consolidation and achieved complete remission. He underwent allogeneic matched unrelated donor (MUD) peripheral blood stem-cell transplantation (PBSCT) and remained in remission throughout his remaining life. Seven months posttransplantation, a myelodysplastic syndrome (MDS) with (20q-) of donor origin was diagnosed causing severe thrombocytopenia and finally leading to infection and death. This patient represents one of the few cases published achieving remission for a significant period of time after being diagnosed with myeloid/NK-cell precursor acute leukaemia, a very rare malignant disease. We conclude, despite the fatal outcome due to infection, that allogeneic PBSCT is a therapeutic option for patients with this entity. In addition, the development of a myelodysplastic syndrome of donor origin is extremely rare and only very few cases are published worldwide.

FISHing — Current Status and Future Prospects for Improved Management of Leukemia

It is now mandatory to perform cytogenetics (CG) study in all new cases of leukemia owing to its usefulness in disease diagnosis, classification and prognostication. The technique of fluorescence in-situ hybridization (FISH), applicable to both interphase and metaphase cells on a variety of samples has proved to be useful in supplementing convention CG, especially in the detection of clinically significant changes when CG is unsuccessful or when genetic abnormalities are cryptic. With improvements in the test sensitivity, FISH can be applied in monitoring molecular response to treatment and detection of residual disease, especially in the setting of monitoring for BCR-ABL in chronic myeloid leukemia patients following imatinib therapy. Newer molecular CG tests such as multicolor karyotyping and comparative genomic hybridization have a clinical potential as they enable resolution of complex karyotypic aberrations and global scanning of genomic imbalances, respectively. In the horizon, important information gained from gene expression profile studies will be useful for the design of tests, molecular based or otherwise, that are suited for routine diagnostic service. However, CG and molecular data must also be integrated with clinical and pathological findings before a definitive diagnosis is made.

Mutations of the TP53 Gene Play an Important Role in the Pathogenesis of Acute Myeloid Leukemia with Complex Aberrant Karyotype.

Acute myeloid leukemia with complex aberrant karyotype is associated with the most unfavorable prognosis of all AML subtypes. Clinical behavior, complexity of karyotype abnormalities and the exponential increase of incidence with age shows similarities to the most common solid tumors. In the latter ones TP53 gene is the most frequently mutated gene identified so far. Therefore, we addressed the role of TP53 in AML with complex aberrant karyotype. In a first step we analyzed 360 AML cases with complex aberrant karyotype using conventional cytogenetics and 24 color FISH to resolve the chromosomal rearrangements in detail. In combination with data from comparative genomic hybridization for a subset of these patients (n=49) we identified 10 genomic regions frequently lost (5q14q33, 7q32q35, 12p13, 13q14, 16q22q24, 17p13, 18q21q22) or gained (11q23q25, 1p33p36, 8q22q24). In all 350 cases interphase FISH with a TP53 probe was performed. In 210 patients (60%) a loss of one TP53 allele was observed. 34 cases (22 with and 12 without TP53 loss) were further evaluated for p53 mutations using the Affymetrix p53 GeneChip assay. All mutations detected were verified by direct sequencing. TP53 mutations were detected in 21 of the 22 cases showing a loss of one TP53 allele (95%), while 9 of 12 cases (75%) without a TP53 loss showed a TP53 mutation on the microarray. Two mutations occurred in introns 4 and 7, while all others were located in exons (e4: n=2, e5: n=10, e6: n=3, e7: n=5, e8: n=8, e9: n=1). 22 mutations were missense mutations resulting in the substitution of a single amino-acid, while 4 were nonsense mutations. One small deletion and one insertion were detected. Four cases without mutations detected by microarray screening and 15 additional cases (9 with and 11 without TP53 loss detected with FISH) were further analyzed by DHPLC (WAVE, Transgenomics). In two of the four cases in which no mutation was detectable on the microarray mutations were found with DHPLC, which are most likely larger deletions not detectable with the p53 GeneChip assay. Furthermore, in 11 of the 15 additional cases a mutation was detected by DHPLC. Thus, in total 43 of 49 cases (88%) showed a TP53 mutation (27/30 (90%) with loss of one TP53 allele and 16/19 (84%) without loss of one TP53 allele). Three of the 6 cases in which no TP53 mutation was detected showed loss of one TP53 allele in FISH analysis. Taken together, only in 3 of 49 cases (6%) no alteration of TP53 was detected. In one of these cases an increased MDM2 expression was found using gene expression microarrays (U133A), another mechanism of inactiving TP53 function. In conclusion, the loss of normal TP53 function by loss of one allele and/or point mutation plays an important role in the pathogenesis of AML with complex aberrant karyotype and may be a major reason for chemoresistance in this prognostically most unfavorable AML subtype. TP53 alterations are detectable in more than 90% of cases with complex aberrant karyotype and are very rare in other AML subtypes. Therefore, we suggest to include the TP53 status in the definition of AML with complex aberrant karyotype.

Gene Expression Profiling Demonstrates Similar Biological Patterns in t-AML vs. de novo AML: A Study Including 106 Patients.

About 10–15% of acute myeloid leukemia (AML) arise secondary after chemotherapeutic treatment and/or radiation for a primary malignant disease and are therefore called therapy-related AML (t-AML). Overall, t-AMLs respond less well to treatment than their de novo counterparts. It has also been shown that the karyotype of the leukemic blasts is an independent prognostic parameter in t-AML. Here we assessed the gene expression profiles of 53 t-AML cases compared to a matched patient cohort of 53 de novo AML patients using Affymetrix DNA-oligonucleotide microarrays (HG-U133 chip design). The following cohort was analyzed (de novo and t-AML each): 2 t(15;17), 3 inv(16), 4 t(8;21), 4 normal karyotypes, 5 other intermediate karyotypes, 2 t(8;16), 5 inv(3), 20 t(11q23)/MLL, and 8 complex aberrant karyotypes. First, we aimed at mining for specific patterns separating de novo from t-AML. Both unsupervised, i.e. hierarchical clustering and principal component analysis, and supervised data analysis algorithms could not identify a signature that robustly separated de novo from therapy-related cases. Then the algorithm was changed to analyze both patient cohorts stratified according to favorable cytogenetics, i.e. t(15;17), t(8;21), inv(16), intermediate cytogenetics, i.e. inv(3), and unfavorable cytogenetics, i.e. t(11q23)/MLL and complex aberrant karyotypes. This resulted in gene expression signatures where de novo and t-AML patients were closely related to each other but separated within genetic subgroups. Particularly in t(11q23)/MLL, complex aberrant karyotypes and t(8;21) samples, de novo cases intercalated with therapy-related cases. When a supervised classification algorithm (Support Vector Machine) was trained to predict the subtype for both de novo and t-AML samples misclassifications were observed. However, these misclassifications mainly occurred within cytogenetic subgroups, e.g. therapy-related AML with complex aberrant karyotype was classified as de novo AML with complex aberrant karyotype, or de novo t(11q23)/MLL as therapy-related t(11q23)/MLL and vice versa. In contrast, an analysis only focusing on cytogenetic subtypes and grouping de novo and t-AML cases together clearly identified signatures related to the biologically distinct AML subtypes. Taken together, we were not able to define a specific expression profile globally associated with therapy-related AML. Instead, t-AML cases of distinct cytogenetic subgroups were demonstrated to be similar to their de novo counterpart. Despite known differences in the genetic events leading to the malignant transformation, i.e. leukemogenic exposures in t-AML through alkylating agents, topoisomerase II inhibitors, or radiation both de novo and t-AML can be considered as biologically identical diseases. Thus, given the underlying biology in AML one should separate patients in clinical trials according to cytogenetics and their history as t-AML or de novo as suggested by the WHO classification. However, differences in prognosis may not be explainable by the respective gene expression profiles in both groups.

Gene Expression Profiling in AML and MDS Identifies Genes Located on 5q Which Are Consistently Lower Expressed in Cases with 5q Deletion as Compared to Cases with Normal Karyotype.

Deletions of the long arm of chromosome 5 occur either as the sole karyotype abnormality in MDS and AML or as part of a complex aberrant karyotype. It was the aim of this study to analyze the impact of the 5q deletion on the expression levels of genes located on chromosome 5q in AML and MDS. Therefore, gene expression analysis was performed in 344 AML and MDS cases using Affymetrix U133A+B oligonucleotide microarrays. The following subgroups were analyzed: AML with sole 5q deletion (n=7), AML with complex aberrant karyotype (n=83), MDS with sole 5q deletion (n=9), and MDS with complex aberrant karyotype (n=9). These were compared to 200 AML and 36 MDS with normal karyotype. In total, 1313 probe sets representing 603 genes cover sequences located on the long arm of chromosome 5. Overall a significant lower mean expression of all genes located on the long arm of chromosome 5 was observed in subgroups with 5q deletion in comparison to their respective control groups (for all comparisons, p<0.05). 36 genes showed a significantly lower expression in all comparisons. These genes are involved in a variety of different biological processes such as signal transduction (CSNK1A1, DAMS), cell cycle regulation (HDAC3, PFDN1) and regulation of transcription (CNOT8). In addition we performed class prediction using support vector machines (SVM). In one approach all 6 different subgroups were analyzed as one class each. While AML and MDS with normal karyotype as well as AML with complex aberrant karyotype were correctly predicted with high accuracies (97%, 81%, and 92%, respectively) AML and MDS with 5q- sole and MDS with complex aberrant karyotype were frequently misclassified as AML with complex aberrant karyotype. In a second approach only two classes were defined: all cases with 5q deletion combined vs. all cases without 5q deletion. 102 out of 108 cases (94%) with 5q deletion were identified correctly supporting the fact that a distinct gene expression pattern is associated with 5q deletion in general. Performing SVM only with genes located on the long arm of chromosome 5 also resulted in a correct prediction of 92 of 108 (85%) stressing the importance of the expression of genes located on chromosome 5 for these AML and MDS subtypes. The top 100 differentially expressed probe sets between cases with and without 5q deletion represented 74 different annotated genes of which 23 are located on the long arm of chromosome 5. They are involved in a variety of different biological functions such as DNA repair (POLE, RAD21, RAD23B), regulation of transcription (ZNF75A, AF020591, MLLT3, HOXB6), protein biosynthesis (UPF2, TINP1, RPL12, RPL14, RPL15) cell cycle control (GMNN, CSPG6, PFDN1) and signal transduction (HINT1, STK24, APP, CAMLG). 10 of the top 74 genes associated with 5q deletion were involved in the CMYC-pathway with upregulation of RAD21, RAD23B, GMMN, CSPG6, APP, POLE STK24 and STAG2, and downregulation of ACTA2, and RPL12. Ten other genes out of the 74 top differentially expressed genes were involved in the TP53 pathway with upregulation of H1F0, PTPN11 and TAF2 and downregulation of DF, UBE2D2, EEF1A1, IGBP1, PPP2CA, EIF2S3, and NACA. In conclusion, loss of parts of the long arm of chromosome 5 leads to a lower expression of genes located on the long arm of chromosome 5. A specific pattern of functionally related genes was identified which shows a lower expression in AML and MDS subtypes with 5q deletion.

Gene Expression Profiling Identifies a Prognostically Favorable Subgroup in AML Independent of Cytogenetic Stratification.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with varying clinical outcome. So far the karyotype of the leukemic blasts as well as molecular genetic abnormalities - both abnormalities on the genomic level - have been proven to be strong prognostic markers. However, even in genetically well defined subgroups clinical outcome is not uniform and a large proportion of AML shows genetic abnormalities of yet unknown prognostic significance. Here we addressed the question whether gene expression profiles are associated with clinical outcome independent of the known genomic abnormalities. Therefore, gene expression analyses were performed using Affymetrix U133A+B oligonucleotide microarrays in a total of 403 AML treated uniformly in the AMLCG studies. This cohort was divided randomly into a training set (n=269) and a test set (n=134). The training set included 18 cases with t(15;17), 22 cases with t(8;21), 29 cases with inv(16), 14 cases with 11q23/MLL-rearrangement, 19 with complex aberrant karyotype and 167 cases with normal karyotype or “other” chromosome aberrations. The respective data for the test set were: 10 t(15;17), 8 t(8;21), 11 inv(16), 8 11q23/MLL, 19 cases with complex aberrant karyotype and 78 with normal karyotype or “other” chromosome aberrations. Based on the clinical outcome the training cohort was divided into 4 equally large subgroups. We trained support vector machines (SVM) with the training set and classified the cases of the test set with the respective most discriminating genes. Next a Kaplan-Meier analysis was performed with the test set cases assigned to prognostic groups 1 to 4 according to SVM classification. Based on the expression level of 100 genes group 1 showed an overall survival rate of 57% at 3 years. 31 of 134 (23%) patients were assigned to this favorable subgroup. They belonged to the following cytogenetic subgroups: t(15;17) n=6, t(8;21) n=4, inv(16) n=3, 11q23/MLL n=4, complex aberrant karyotype n=1 and normal karyotype or “other” chromosome aberration n=13. The overall survival rate of groups 2, 3, and 4 did not differ significantly (17%, 21%, and 19% at 3 years). Among the genes highly expressed in the favorable group were MPO and the transcription factor ATBF1, which regulates CCND1. The unfavorable groups were characterized by a higher expression of the transcription factors ETS2, RUNX1, TCF4, and FOXC1. Interestingly, 10 of the top 40 differentially expressed genes are involved in the TP53-CMYC-pathway with a higher expression of 9 of these in the unfavorable groups (SFRS1, TPD52, NRIP1, TFPI, UBL1, REC8L1, HSF2, ETS2 and RUNX1). In conclusion, gene expression profiling leads to the identification of prognostically important alterations of molecular pathways which have not yet been accounted for by use of cytogenetics. This approach is anticipated to help optimizing therapy for patients with AML.

Impact of Trisomy 8 on Expression of Genes Located on Chromosome 8 in Different AML Subgroups.

Trisomy 8 is the most frequently observed trisomy in acute myeloid leukemia (AML). It occurs as a sole karyotype abnormality or in addition to other chromosome aberrations. It was the aim of this study to analyze the impact of trisomy 8 on the expression of genes located on chromosome 8 in different AML subgroups. Therefore, gene expression analyses were performed in a total of 567 AML cases using Affymetrix U133A+B oligonucleotide microarrays. The following 14 subgroups were analyzed: +8 sole (n=19), +8 within a complex aberrant karyotype (n=11), +8 with t(15;17) (n=7), +8 and inv(16) (n=3), +8 with t(8;21) (n=3), +8 and 11q23/MLL (n=8), and +8 with other abnormalities (n=10). These were compared to 200 AML with normal karyotype and the following subgroups without trisomy 8: complex aberrant karyotype (n=73), t(15;17) (n=36), inv(16) (n=46), t(8;21) (n=37), 11q23/MLL (n=37), and other abnormalities (n=77). In total 1188 probe sets cover sequences located on chromosome 8 representing 580 genes. A significant higher mean expression of all genes located on chromosome 8 was observed in subgroups with +8 in comparison to their respective control groups (for all comparisons, p<0.05). Significantly higher expressed genes in groups with +8 in comparison to the respective groups without +8 were identified in all comparisons. The number of identified genes ranged from 40 in 11q23/MLL to 326 in trisomy 8 sole vs. normal. There was no common gene significantly overexpressed in all comparisons. Three genes (TRAM1, CHPPR, MGC40214) showed a significantly higher expression in 5 out of 7 comparisons. Between 19 and 107 genes with an exclusive overexpression in trisomy 8 cases in only one subtype comparison were identified. In addition, we performed class prediction using support vector machines (SVM) including all probe sets on the arrays. In one approach all 14 different subgroups were analyzed as one class each. Only 3 out of 61 cases with trisomy 8 were assigned into their correct subclass, while 40 cases were assigned to their corresponding genetic subclass without trisomy 8. In a second approach only two classes were defined: all cases with trisomy 8 combined vs. all cases without trisomy 8. Only 26 out of 61 (42.6%) with trisomy 8 were identified correctly underlining the fact that no distinct gene expression pattern is associated with trisomy 8 in general. Performing SVM only with genes located on chromosome 8 did not improve the correct assignment of cases with trisomy 8 overall. Only cases with trisomy 8 sole were correctly predicted in 58% as compared to 11% in SVM using all genes. In conclusion, overall the gain of chromosome 8 leads to a higher expression of genes located on chromosome 8. However, no consistent pattern of genes was identified which shows a higher expression in all AML subtypes with trisomy 8. This data suggest that the higher expression of genes located on chromosome 8 only in part is directly related to a gene dosage effect. Trisomy 8 may rather provide a platform for a higher expression of chromosome 8 genes which are specifically upregulated by accompanying genetic abnormalities in the respective AML subtypes. Therefore, trisomy 8 does not seem to be an abnormality determining specific disease characteristics such as the well known primary aberrations (t(8;21), inv(16), t(15;17), MLL/11q23) but rather a disease modulating secondary event in addition to primary cytogenetic or moleculargenetic aberrations.

MDS and AML Are Closely Related Diseases: Gene Expression Patterns Reveal Clear Similarities with Respect to Karyotypes and Are Less Related to the Bone Marrow Blast Percentages.

MDS and AML are discriminated by percentages of blasts in the bone marrow (BM) according to the FAB as well as to the WHO classification. However, thresholds are arbitrary and demonstrate only a limited reproducibility in interlaboratory testings. Thus, other parameters have been assessed to discriminate these entities with respect to diagnosis and prognosis. In particular, in the majority of cases common karyotype aberrations have been observed between MDS and AML which have a higher prognostic impact than blast percentages. We applied gene expression profiling (U133A+B, Affymetrix) in 70 MDS and 238 AML cases. In accordance with the WHO classification we excluded cases with balanced translocations (i.e. t(8;21), t(15;17), inv(16), or 11q23) which are classified as AML irrespective of BM blast percentage. First we aimed at identifying genes of which the expression correlated to blast count (Spearman correlation). Out of the top 50 genes this analysis revealed only the FLT3 gene which showed a higher expression in cases with high blast count, while 12 genes with a higher expression in cases with lower blast counts were identified (ANXA3, ARG1, CAMP, CD24, CEACAM1, CEACAM6, CEACAM8, CRISP3, KIAA0922, LCN2, MMP9, STOM). Most of the latter genes are expressed in mature granulocytes and are involved in differentiation and apoptosis. In a second step we performed class prediction using support vector machines (SVM) to separate MDS and AML according to blast percentages as defined in the WHO classification (<5%: RA and 5q- syndrome; 5–9%: RAEB-1; 10–19%: RAEB-2; >19% AML). Using 10-fold cross validation and support vector machines the overall prediction accuracy was only 80%. In detail, 230/238 AML cases were correctly assigned to the AML group while 8 cases were classified as MDS RAEB-2. However, none of the RA, 5q- syndrome and RAEB-1 cases were correctly assigned to their groups, respectively, but were either classified as AML or RAEB-2. Furthermore, only 16 of 38 RAEB-2 cases were correctly predicted, while the 20 remaining cases were assigned to the AML group. Thus, no clear gene expression patterns were identified which correlated with AML and MDS subtypes according to WHO classification. Taking the common genetic background observed in MDS and AML into account, both entities were categorized in a third step according to cytogenetics and classified based on their gene expression profiles. In order to assess the impact of the common genetic background, the largest cytogenetically defined subgroups were compared to each other, i.e. AML and MDS with normal karyotype and with complex aberrant karyotype. Intriguingly, while correct classification of AML or MDS was found in 91%, classification into the correct cytogenetic groups was achieved in 95%. Consequently, all cases were devided into the two groups, complex aberrant karyotype (n=60) and other or no aberrations (n=248) irrespective of AML or MDS. A classification into these groups also yielded an accuracy of 93%. Our data suggests that gene expression profiling reveales the biology of MDS or AML to highly correlate with cytogenetics and less with the percentages of BM blasts. These results strengthen the need for a revision of the current MDS and AML classification centering now genetic abnormalities, which may also be used for clinical decisions.

All Clinically Relevant Leukemia Subtypes Can Be Diagnosed and Classified Based Solely on Gene Expression Profiling with an Accuracy of 95.1%: A Study on 1337 Adult Patients.

So far, comprehensive diagnosis of leukemia requires a combination of cytomorphology, immunophenotyping, and genetic methods. We aimed at developing a new diagnostic tool based solely on gene expression profiling to accurately predict all clinically relevant subtypes of leukemia in adults and to distinguish these from normal bone marrow. Therefore, we analyzed samples from 1337 untreated patients at diagnosis and healthy donors using oligonucleotide microarrays. The first series of 937 cases was hybridized to HG-U133A+B microarrays (Affymetrix). The following 13 subgroups were included: 620 AML (42 t(15;17); 38 t(8;21); 49 inv(16); 47 t(11q23); 75 complex aberrant karyotype; 193 normal karyotype; 176 other cytogenetic abn.); 152 ALL (26 Pro-B-ALL/t(11q23); 12 ALL-t(8;14); 32 T-ALL; 82 c-ALL/Pre-B-ALL); 75 CML, 45 CLL, and 45 bone marrows from healthy volunteers or non-leukemia pts. (nBM). For each disease entity the top 100 differentially expressed genes were calculated in a one-versus-all (OVA) approach. Class prediction was performed using support vector machines (SVM). Prediction accuracy was estimated by 10-fold cross validation (CV) and assessed for robustness in a resampling approach. 891 of the 937 samples (95.1%) were correctly classified (10-fold CV). A resampling approach with 2/3 training and 1/3 test cohort (100 runs of SVM) confirmed this high accuracy (median, 93.8%). In particular, a median of 100% sensitivity and specificity was achieved for AML with t(15;17), t(8;21), and inv(16), as well as for Pro-B-ALL/t(11q23), and CLL. The median specificity was at least 99.7% in all subgroups except for AML normal/other (median specificity, 93.7%). In a second step T-ALL cases were separated into cortical and immature ones (accuracy, 84.4%) and c-ALL/Pre-B-ALL into cases with and without t(9;22) (accuracy, 82.9%). The second prospective series comprized 400 unselected cases which were hybridized to the new generation HG-U133 Plus 2.0 microarrays (Affymetrix). To validate the diagnostic accuracy of our approach these cases were processed blinded in parallel to routine diagnostic work-up and classified based on the gene expression signatures discovered in the first series described above. Applying a first classification step as described above the 13 different diagnoses were predicted with an accuracy of 94.5%. Failures were mostly due to misclassification into biologically related subgroups, e.g. AML with del(5q) aberrations classified as AML with complex aberrant karyotype. In the second step (separation of the two T-ALL subtypes, and c-ALL/Pre-B-ALL with or without t(9;22)) accuracies of 100% and 70.6% respectively were achieved. In conclusion, we were able to identify within a routine diagnostic workflow distinct expression profiles for all clinically and prognostically relevant adult leukemia subtypes and their discrimination from nBM based only on gene expression data. Accuracy, sensitivity, and specificity were higher than achieved with each of the gold standard techniques alone used today. Thus, gene expression patterns analyzed by microarrays qualify as a diagnostic tool in a routine setting for leukemia diagnosis and classification and may guide relevant therapeutic decisions.