ALBERT

Description: Gene expression profiling has the potential to offer consistent objective diagnostic test results once a standardized protocol is established. We investigated the robustness, precision, and reproducibility of this technology and present data that complements the Microarray Innovations in LEukemia study (MILE study). In four laboratories, located in Germany (D), Austria (A), and Switzerland (CH) (DACH study), replicates of 112 patient samples were analyzed using the AmpliChip Leukemia research test. Patient samples were centrally collected and diagnosed in daily routine at the Munich Leukemia Laboratory and represented 8 distinct classes of acute and chronic leukemias, with non-leukemia as control group. After purification of the mononuclear cells by Ficoll density centrifugation, 4 × 5 million cells were frozen in lysis buffer and stored at −80°C. Equipped with identical instruments, software, and reagents, study operators were trained on the microarray sample preparation protocol using total RNA from commercially available cell lines. Upon receipt of the frozen lysates each of the four laboratories purified the total RNA from the 112 technical quadruplicates. 99.3% (445/448) of the sample preparations were successfully performed. On average, 8.4 μg, 7.2 μg, 7.4 μg, or 7.5 μg of total RNA, respectively, were isolated from the mononuclear cells from the four laboratories. In three samples less than 1.0 μg of total RNA was obtained and thus the preparation failed. Bland-Altman plots of agreement showed that any two centers were unlikely to have more than an 8.3 μg difference in yield of total RNA from the same sample. On average there was between 0.1 μg to 1.2 μg difference in total RNA yield from the same sample. Further processing of the 445 samples resulted in 437 (98.2%) successfully performed in vitro transcription reactions, i.e. obtained cRNA yield of 〉8.0 μg. On average there was between 0.4 μg to 7.4 μg difference in cRNA yield from the same sample. After hybridization to microarrays on average, 46.1%, 48.6%, 46.5%, and 47.3% of probe sets were detected as present with mean scaling factors of 4.3, 2.9, 3.9, and 3.7, respectively. The mean values and standard deviations of distributions of the coefficient of variation (CV) within each site over all the probe sets of the quantile normalized signals on the chip were 27.2% (StdDev: 12.3%), 27.0% (StdDev: 12.3%), 27.3% (StdDev: 12.3%), 26.9% (StdDev: 12.4%), respectively. Furthermore, in unsupervised hierarchical cluster and principal component analyses replicates from the same patient always clustered closely together, with no indications of association between gene expression profiles due to different operators or laboratories. In conclusion, we demonstrated that microarray analysis can be performed with remarkably high inter-laboratory reproducibility and with comparable quality and high technical precision across laboratories.

Description: CLL is a genetically heterogeneous disease. Genetic aberrations allow to distinguish different biological subgroups within CLL. Based on chromosome banding analysis we identified complete and partial gain of the short arm of chromosome 2 always including 2p23 to 2p25 in 28/1051 CLL cases (2.7%) as a new recurring chromosome aberration. Recurring aberrations accompanying gain of 2p were: loss of 1p (n=3), 1q (n=3), 2q (n=3), 6q (n=3), 8p (n=7), 11q (n=10), 12q (n=4), 13q (n=21), 17p (n=7), 17q (n=3), 18p (n=5), and gain of 2q (n=3), 3q (n=3), 13q (n=3), 21q (n=3). In 24/28 cases the mutational status of the immunoglobulin variable heavy chain gene (IgVH) was available. 20 cases showed an unmutated and only 4 a mutated IgVH status. Thus, 2p gain is significantly associated with an unmutated IgVH status as compared to the non 2p group (83% vs 51%, p=0.002). In 8 cases an ATM deletion (29%) and in 5 cases a TP53 deletion (18%) (1 case showed both) were observed (frequency in non 2p+ cohort: 12%; p=0.036 and 7%; p=0.031). A median number of 4 chromosome aberrations per case was observed in 2p+ CLL (range: 1–16, mean=5.2) as compared to only 1 abnormality per case in the non 2p+ cohort (range: 0–10, mean 1.7) (p

Description: Deletions of the long arm of chromosome 5 are typical aberrations in AML and MDS. They occur either as the sole abnormality or within a complex aberrant karyotype. As the precise determination of the breakpoints and the size of the deletion is not possible with chromosome banding analysis alone we performed genomic arrays (Affymetrix 10K arrays) in 32 AML with 5q-deletion within a complex aberrant karyotype and in 17 cases with a 5q-deletion as the sole abnormality (AML: n=3, MDS as typical 5q- syndrome: n=6, other MDS subtypes: n=8). Gene expression analysis using Affymetrix U133A+B or 2.0 plus was performed in addition in all 32 AML cases with complex aberrant karyotype and in 3 cases with 5q deletion sole. The deleted region could be determined based on the genomic array data in 30 of 32 cases with AML and complex aberrant karyotype and in 9 of 17 cases with 5q deletion sole. All evaluable cases showed the 5q deletion in more than 40% of cells (interphase FISH with a 5q31-probe, range 40 to 98%), while less than 45% of interphase nuclei with 5q31-deletion in not evaluable cases (range 8% to 45%). Genomic array analysis mapped the variable proximal breakpoint in the 5q deletion sole group between 5q14.1 and 5q31.3 and the distal breakpoint between 5q32 and 5q35.1. The size of the deletion varied between 27.14 and 81.20 MB (median 73.13 MB). In cases with complex aberrant karyotype the proximal breakpoint was located between 5q11 and 5q23.1 while the distal was located between 5q32 and 5q35.3. The size of the deletion varied between 34.72 and 132.30 MB (median 94.77 MB). Two approaches were tried to determine the size of the deletion based on gene expression data. As a control groups 40 AML and 40 MDS both with normal karyotype were used. Each probe set expressed in at least 1 case of the control group with a precise localiasation on chromosome 5 available was included in the analysis. For each probe set a median expression within the control groups was calculated. First for each probe set a ratio between the individual patient with 5q deletion and the median expression of the respective control group was calculated. For each chromosomal band on chromosome 5 the proportion of genes showing a ratio

Description: Effects of gene dosage on gene expression have been demonstrated in several leukemia entities. We asked the question whether unbalanced chromosome aberrations can be reliably identified based on gene expression data. Therefore, we performed genomic arrays (Affymetrix 10K arrays) and gene expression arrays (Affymetrix U133A+B or 2.0 plus) in 33 AML with a complex aberrant karyotype. For comparison gene expression data of 100 AML with normal karyotype were used. Based on this data set an algorithm was developed for the detection of higher and lower expression of groups of genes located in chromosomal bands (CB) in individual patients. Therefore, a “normal” expression status was defined for each CB based on gene expression data of AML with normal karyotype. Next for each individual case the gene expression status of each CB in relation to the earlier defined “normal” expression status was calculated and if a significant deviation was observed called “+” or “-”. First the new algorithm was tested for prediction of 5q deletions and their localization as well as for −7 and 17p deletion comprising 64 CB. Thus, overall 2112 pairs of genomic (GD) and gene expression data (GED) were compared in these 33 cases with AML. Based on GD 1011 CB were identified as deleted, 648 of these showed a significantly reduced gene expression. In addition gene expression was not reduced in 898 not deleted CB resulting in 1546/2112 (73.2%) concordant results between GD and GED. Thus, the sensitivity for prediction of deletion based on gene expression was 64% and the specificity 82%. In the next step gene expression data was correlated to karyotype data (KD) obtained by chromosome analysis. Therefore, 48 AML cases with available GED and a karyotype showing unbalanced chromosome aberrations such as trisomies of chromosomes 4 (n=2), 8 (n=14), 9 (n=1), 10 (n=1), 11 (n=3), 13 (n=8), 14 (n=3), 19 (n=1), 21 (n=3), and 22 (n=2) and monosomies of chromosomes 7 (n=7) and 13 (n=1) as well as gain of 1q (n=2), loss of 3q (n=1), 5q (n=11), 7q (n=4), 9q (n=3), 12p (n=1), and 10q (n=1) were evaluated. First we performed in this second cohort an analysis focusing again on loss of 5q, 7 and 17p. Based on KD 607 CB were identified as deleted, 390 of these showed a significantly reduced gene expression, in addition gene expression was not reduced in 2232 not deleted CB resulting in 2622/3408 (76.9%) concordant results between KD and GED. Thus, the sensitivity for prediction of deletion based on gene expression was 64% and the specificity 80%. Next an overall analysis on the whole genome broken down into 653 CB (excluding X and Y chromosome) was performed in the second cohort. Based on KD 1002 CB were gained, 820 lost and 29522 not affected by unbalanced rearrangements. A significantly higher gene expression was found in 570/1002 gained CB, a lower expression in 501/820 lost CB and an unchanged expression in 17366/29522 CB not affected, resulting in 18437/31344 (58.8%) concordant results between KD and GED. Thus, the sensitivity for prediction of deletion based on gene expression was 61% and the specificity 82% and for gain 57% and 81%. In conclusion, we present an approach for predicting unbalanced karyotype changes based on gene expression. It could be demonstrated genome wide that an association between gene dosage and gene expression exists.

Description: It has previously been shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human Organic Cation Transporter 1 (hOCT1; SLC22A1). In more recent work on clinical samples it was further shown that low hOCT1 expression of this influx transporter may be an important mechanism of imatinib resistance. To further evaluate this issue we have retrospectively quantified pretreatment hOCT1 mRNA expression in 92 CML patients (pts) that responded with major molecular remission within the first year of treatment and compared these results to 19 pts with primary resistance to imatinib. We found that all 19 resistant pts had low hOCT1 expression (median: 2.032 (expressed as %hOCT1/ABL); range 0.18–4.24). Although the median hOCT1 expression at diagnosis in the responders was higher (median 8.417) the range was very heterogeneous (0.45–188.2) with only 30% of all responders having a significantly higher expression than the resistant pts. As in vitro studies have shown that genetic variants of the SLC22A1 gene that codes for hOCT1 can have a negative effect on the transport of some substrates we hypothesized that not only certain hOTC1 expression levels but also different genetic variants within the SLC22A1 gene may be associated with different efficiencies of imatinib uptake. Using high resoluting melting and subsequent sequencing we have genotyped exons 1, 2, 5, 6, 7, 9, 10, and 11 in 109 responders as well as in 55 resistant pts, thus each 326 alleles were evaluated. We detected 12 different exonic polymorphisms. Two of these, a G38D and a Y404C were so far undescribed variants. Both nonsynonymous variants were detected in heterozygeous forms, the G38D in one responder and the Y404C variant in one resistant pt. All other variants were detected in frequencies similar to those that have already been described (R61C: 0.07, L160F: 0.76, P341L: 0.01, G401S:

Description: Leukemia specific fusion genes such as CBFB-MYH11 play a major role in the pathogenesis of distinct AML entities. However, additional genetic aberrations seem necessary for the development of full blown leukemia. This study was performed to decipher CBFB-MYH11 rearrangements and their accompanying genetic lesions at the molecular level. Therefore, Affymetrix SNP Array 6.0 analyses, featuring 〉1.8 million markers for genetic variation (〉906,600 SNPs and 〉946,000 probes for the detection of copy number variations), were performed in 35 newly diagnosed AML with inv(16) (p13q22) or t(16;16)(p13;q22) and CBFB-MYH11-rearrangement. First, as a proof of principle, additional gains and losses of chromosomal material as observed by cytogenetics were also detected by the SNP technology. This included gains of whole chromosome 8 (n=7) and 22 (n=8). In addition, a partial trisomy 13 and a partial trisomy 6 resulting from an unbalanced translocation were confirmed. In two cases a 7q deletion was observed by chromosome banding analysis. One of these was missed by SNP array as the 7q deletion occurred in a subclone only (11% of cells with 7q deletion as determined by interphase FISH). However, SNP array analyses detected loss of 7q in two additional cases which was missed by cytogenetics. Based on SNP array data the commonly deleted region was identified to range from 7q36.1 to 7q36.3 (size: 8.5 MB; physical map position 147,549,804–156,038,680). In addition to a gain of the whole chromosome 8, frequently observed as an additional aberration, in one case SNP array analyses revealed only a partial gain on 8q ranging from 8q24.13 to 8q24.3 (size: 25.3 MB; physical map position 120,986,982–146,268,936). Furthermore, a recurrent deletion (n=2) on chromosome 18 was detected by SNP array but not detected by cytogenetics. The commonly deleted region was localized in 18q23 (size: 3.1 MB; physical map position 72,481,657–75,604,994). In two cases the CBFB-MYH11 rearrangement was cryptic and could not be detected by chromosome banding analysis or FISH using two probes flanking the breakpoints within the CBFB gene, however, a CBFB-MYH11 transcript was amplified by RT-PCR. In one of these cases SNP array data revealed a small gain on 16p13 including 3′ part of the MYH11 gene (size: 71 kb; physical map position 15,654,558–15,725,636) suggesting the insertion of additional 3′ MYH11 sequences into the CBFB rearrangement leading to a CBFB-MYH11 fusion gene. Interestingly, four cases showed a deletion on 16p13 (sizes: 176 kb, 461 kb, 464 kb, 468 kb; physical map positions 15,729,932–15,906,308, 15,726,920–16,188,116, 15,725,663–16,189,984, 15,721,133–16,189,807). All included the 5′ part of the MYH11 gene, and in 3 cases, the ABCC1 gene (multidrug resistance-associated protein 1) was included in the deleted region, which could have an impact on prognosis. The patient with the smallest deletion in 16p13 also showed a deletion on 16q22 including the ′ part of CBFB (size: 35 kb, physical map position 65,672,864–65,707,954). This would be in line with findings in chronic myeloid leukemia where comparable small deletions in the breakpoint region of BCR and ABL have been described. Furthermore, large regions of copy-neutral loss of heterozygosity were observed for the whole short arm of chromosome 1 in two cases, for 17q12 to 17qter and 19q in one case each. In conclusion, a novel mechanism leading to a CBFB-MYH11 fusion gene was identified: A cytogenetically cryptic insertion of additional MYH11 sequences into the CBFB locus. A distinct pattern of additional aberrations was confirmed showing gains of whole chromosomes 8 and 22. Small copy number changes not observable in chromosome banding analysis were detected on 7q, 8q and 18q. A recurrent region of loss of heterozygosity without copy number change was found for the whole short arm of chromosome 1 suggesting that candidate genes in this region are mutated and potentially play a pathogenetic role in AML with CBFB-MYH11-rearrangement.

Description: Chromosomal rearrangements involving the MLL gene occur in 3–5% of adult AML. More than 50 different partner genes have been described in acute leukemia with 11q23-abnormalities. Although MLL-rearrangements per se have a high leukemic potential, additional genetic aberrations occur. This study was intended to decipher MLLrearrangements and their accompanying genetic lesions at the molecular level. Therefore, Affymetrix SNP 6.0 microarray analyses were performed in 47 newly diagnosed AML with 11q23 aberrations. First, as a proof of principle, all gains and losses of chromosomal material as observed by cytogenetics were also detected by the SNP technology. This included recurring gains of whole chromosomes; 4 (n=3), 8 (n=7), and 19 (n=2). In addition, the following unbalanced abnormalities were detected: gain of 1q31.3 to 1q43 (n=5) and a gain of 3q (n=2). In 40/47 cases the following partner genes had been identified based on the translocation observed in chromosome banding analysis and RTPCR: AF9 (n=27), AF6 (n=4), AF10 (n=3), ELL (n=2), AF4 (n=1), AF17 (n=1), ENL (n=1), SEPT5 (n=1). In 4/47 cases results from chromosome banding analysis suggested partner genes to be located at 11q13 (n=1), 10p11 (n=1), and 19p13 (n=2). In 3/47 cases the MLL rearrangement was cryptic and only suspected by FISH analysis. Two of those (#1, #2) showed a del(11)(q23q25) in chromosome banding analyses and FISH analyses demonstrated a loss of the 3′ flanking MLL probe. In the remaining case (#3) cytogenetics showed an i(21)(q10). FISH analysis on metaphase spreads identified an additional copy of the 5′ flanking MLL probe which localized on 6q27. SNP analyses were able to resolve all three cases: #1) The deletion was fine-mapped by SNP microarray data and ranged from physical map position 117,859,541 to 11qter including exons 10 to 28 of the MLL gene. In addition, SNP microarray data revealed a gained segment on 6q ranging from physical map position 167,977,103 to 6qter including exons 2 to 28 of AF6. #2) In this case the 11q deletion spans from physical map position 117,859,541 to 121,033,713 including exons 10 to 28 of the MLL gene. SNP microarray data revealed a gained segment on 6q ranging from physical map position 168,036,784 to 168,457,799 including exons 9 to 28 of AF6. #3) Corresponding to FISH analysis SNP microarray data revealed a gained segment on 11q ranging from physical map position 117,760,488 to 117,859,673, including exons 1 to 9 of MLL. Moreover, on chromosome 6 a small deletion of 177 kb was detected, starting at physical map position 167,804,673 towards 167,982,457. This deletion included exon 1 of AF6 and a small adjacent centromeric region. In all 3 cases, subsequent RT-PCR analyses confirmed the predicted MLL-AF6 fusion. Analyzing the MLL gene further in the remaining cases revealed copy number changes in 2 cases showing gains of 11q starting from exon 12 of the MLL gene to 11qter (physical map position 117,863,291 to 11qter and 117,862,916 to 11qter). These were due to an extra copy of der(4)t(4;11)(q21;q23) and der(19)t(11;19)(q23;p13.3), respectively. In two additional cases very small deletions within MLL with a size of 4.831 kb including exons 10 and 11 (physical map position 117,859,541 to 117,864,372) and 1.699 kb including exons 10 and 11 (physical map position 117,859,541 to 117,861,240) were observed (MLL-AF6- and MLL-AF4-rearrangement). With respect to the various MLL partner genes, deletions starting in the partner genes were observed in 2 cases with MLL-AF9 rearrangement (size: 8 MB and 6.1 MB, physical map position 20,334,335 to 28,350,412 and 20,342,604 to 26,451,390). The region deleted in both cases spanned 37 genes, including several genes of the interferon alpha family and the tumor suppressor candidate TUSC1. Copy number gains were observed in the region of the partner genes in both cases with a doubling of der(4)t(4;11)(q21;q23) and der(19)t(11;19)(q23;p13.3). In conclusion, using high resolution SNP arrays we identified three novel mechanisms leading to MLL-AF6 fusions which are cytogenetically cryptic and associated with atypical FISH signal constellations. Furthermore, a distinct pattern of additional aberrations was observed showing trisomies of chromosomes 4, 8 and 19. SNP microarray data also revealed a small deletion on the short arm of chromosome 9 as a recurrent additional genetic change in AML with MLL-AF9-rearrangements.

Description: Chronic lymphocytic leukemia (CLL) is a genetically heterogeneous disease. Recently, several genetic aberrations have been identified that allow to distinguish different biological subgroups within CLL. Translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 is a rare but recurrent abnormality in CLL and still poorly described. Based on karyotype data we identified 12 cases with t(14;19)(q32;q13) in a cohort of 1051 CLL (1.1%). In all these cases 1 to 10 chromosomal aberrations were observed in addition to t(14;19) (median: 3). Recurring accompanying aberrations were: +12 (n=8), loss of 18p (n=2), and gain of 10q (n=2). Interestingly, trisomy 12 is also the most frequent additional abnormality in CLL with t(14;18)(q32;q21). Remarkably, neither 13q deletions nor 11q deletions which are frequently observed in CLL overall, were found in addition to t(14;19). A TP53-deletion and a 6q21 deletion were observed in one case each. In 8/12 cases the mutation status of the immunoglobulin variable heavy chain gene (IgVH) was available. All 8 cases showed an unmutated IgVH status. Gene expression analysis (Affymetrix, HG U133 Plus 2.0) was performed in 9 cases with t(14;19) and compared to 44 cases with CLL comprising various chromosome aberrations excluding t(14;19). Using 10fold cross validation resulted in an assignment of 7 out of 9 cases with t(14;19) into the correct class, none of the cases without t(14;19) was classified into the t(14;19) group (accuracy 96%, sensitivity 78%, specificity 100%). Classification based on an independent test set led to comparable results (median accuracy 94%, sensitivity 67%, specificity 100%). The 10 most differentially expressed genes showing a higher expression in t(14;19)+ CLL were: TUBB6, CPSF6, RFC5, MAP3K8, CUGBP2, BCAT1, BCAT1, LOC647135, TSPAN13, SIGLEC6 and are involved in transition of mitotic cell cycle, DNA replication and RNA processing. The 10 most differentially expressed genes showing a lower expression in t(14;19)+ CLL were: LSR, APLP2, C2orf10, HS3ST1, LRRC32, PALM2-AKAP2, DFNB31, PDE4A, CTLA4, PDCD4 and are involved in signal transduction, apoptosis and immune response. In conclusion: t(14;19)(q32;q13) is a rare, recurrent chromosome abnormality in CLL. It is very frequently accompanied by additional chromosomal aberrations. The most frequent additional aberration is trisomy 12. t(14;19) is associated with an unmutated IgVH status. Comparable to other translocations leading to fusion genes it is associated with a distinct gene expression profile.

Description: 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.

Description: 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.