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Leukemia (2005) 19, 1224–1228 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Genomic gains and losses influence expression levels of located within the affected regions: a study on acute myeloid leukemias with 8, 11, or 13, 7, or 5q

C Schoch1, A Kohlmann1, M Dugas1, W Kern1, W Hiddemann1, S Schnittger1 and T Haferlach1

1Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich, Germany

We performed microarray analyses in AML with 8 aim of this study to investigate whether gains and losses on the (n ¼ 12), 11 (n ¼ 7), 13 (n ¼ 7), monosomy 7 (n ¼ 9), and deletion genomic level translate into altered genes expression also in 5q (n ¼ 7) as sole changes to investigate whether genomic gains and losses translate into altered expression levels of other areas of the in AML. genes located in the affected chromosomal regions. Controls were 104 AML with normal . In subgroups with trisomy, the median expression of genes located on gained Materials and methods was higher, while in AML with monosomy 7 and deletion 5q the median expression of genes located in deleted Samples regions was lower. The 50 most differentially expressed genes, as compared to all other subtypes, were equally distributed Bone marrow samples of AML patients at diagnosis were over the genome in AML subgroups with trisomies. In contrast, 30 and 86% of the most differentially expressed genes analyzed: 12 cases with (AML-TRI8), seven with characteristic for AML with 5q deletion and monosomy 7 are trisomy 11 (AML-TRI11), seven with trisomy 13 (AML-TRI13), located on chromosomes 5 or 7. In conclusion, gain of whole and nine cases with monosomy 7 (AML-MO7), and seven cases chromosomes leads to overexpression of genes located on the with a deletion of the long arm of 5 (5q) (AML- respective chromosomes. Losses of larger regions of the DEL5q), as well as 104 AML with normal karyotype (AML-NK). genome translate into lower expression of the majority of All karyotype abnormalities were the sole aberrations. All cases genes represented by only one allele. The reduced expression of these genes is the most characteristic difference in were analyzed by cytomorphology, chromosome-banding expression profiles between AML with monosomy 7 and AML analysis and fluorescence in situ hybridization (FISH) on with deletion 5q, respectively, and other AML subtypes. There- interphase nuclei using loci-specific probes for EGR1 (5q31), fore, these data provide evidence that gene dosage effects gene RB (13q14), the centromeric regions of chromosomes 7, 8, and expression in AML with unbalanced karyotype abnormalities. 11.3 The proportion of cells carrying the genetic abnormality Losses of specific regions of the genome determine the gene was above 60% in all cases as determined by FISH analyses. The expression profile more strongly than the gain of whole chromosomes. studies abide by the rules of the local Internal Review Board and Leukemia (2005) 19, 1224–1228. doi:10.1038/sj.leu.2403810 the tenets of the revised Helsinki protocol. Published online 19 May 2005 Keywords: ; ; gene dosage Gene expression and statistical analysis

We performed gene expression analysis using oligonucleotide Introduction microarrays covering 33 000 transcripts (Affymetrix U133 set) according to the protocol of the manufacturer applying quality Acute myeloid leukemia (AML) is a heterogeneous group of controls as described elsewhere.4,5 For further analysis, only diseases. From a genetic point of view, three subgroups can be probe sets with a known chromosomal localization (www.affy distinguished: (1) AML with normal karyotype, (2) AML with a metrix.com/analysis/) and those expressed at least in one sample primary balanced chromosome aberration, and (3) AML with were used. All probe sets used in this study together with the unbalanced karyotype abnormalities characterized by gains gene they represent and their chromosomal localization are and/or losses of usually larger regions of the genome and no 1 shown in Tables S1a–S1e of the Supplementary Information. For known primary balanced abnormality. The important pathoge- each probe set, the mean expression value was calculated for netic role of leukemia-specific fusion transcripts such as PML- each AML subgroup. Then for each probe set, a ratio ‘mean RARA, AML1-ETO, and CBFB-MYH11 has been proven. The expression level AML unbalanced/mean expression level AML role of genomic gains and losses in AML is less clear. It has been normal’ was determined. In addition, differentially expressed assumed that gene dosage effects may play a role in the genes were identified by means of the two-sample t-statistic pathogenesis of this AML subgroup. Virtaneva et al supported score with correction for unequal variances.6 The analysis was this hypothesis showing that AML with trisomy 8 as the sole programmed using R (version 1.7.1; www.r-project.org/). karyotype abnormality overexpressed genes located on chromo- some 8 compared to AML with normal karyotype.2 It was the Results and discussion Correspondence: Dr C Schoch, Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, University Hospital Grosshadern, Gene copy number abnormalities are characteristic for malig- Ludwig-Maximilians-University of Munich, Marchioninistr. 15, nant diseases and are also found in association with develop- Munich 81377, Germany; Fax: þ 49 89 7095 4971; E-mail: [email protected] mental abnormalities and/or mental retardation. It is reasonable Received 30 November 2004; accepted 12 April 2005; Published to propose that the transcription of genes mapping to online 19 May 2005 chromosomal regions gained or deleted is altered due to a Gene dosage effects in AML C Schoch et al 1225 dosage effect. Differences in the transcriptional regulation of However, DEFA1 was not confirmed (ratio: 0.74). In our series genes may explain why a change in copy number does not the 3 genes most significantly upregulated in AML-TRI8 were: always result in a higher or lower expression of the affected PTK2B, CNOT7, and LYPLA1. PTK2B was identified also by gene. It was the aim of this study to determine the effect of gene Virtaneva et al as upregulated in AML-TRI8 in a comparison of dosage in AML with trisomies 8, 11, 13, monosomy 7, or AML-TRI8 vs AML-NK vs CD34 þ normal stem cells, while the deletion 5q as a sole abnormality. other two genes were not mentioned.

Trisomy 11 and 13 Trisomy 8 In all, 1263 of 1693 and 440 of 530 probe sets covering A total of 1052 probe sets cover sequences localized on sequences localized on chromosomes 11 and 13, respectively, (Table S1a, supplemental material). In all, 835 of were expressed in at least one sample (Table S1b and S1c, them were expressed in at least one sample and were thus used Supplementary Information). The mean expression of 828 out of for further analyses. The mean expression level of 607 of these 1263 (65.6%) probe sets was higher in cases with trisomy 11, 835 (72.6%) probe sets was higher in cases with trisomy 8 as and of 275 out of 440 (62.5%) higher in cases with trisomy 13 as compared to AML with normal karyotype (Figure 1a). The compared to AML with normal karyotype, respectively median expression level of probe sets on chromosome 8 was (Figure 1b). The median expression of probe sets on chromo- 1.27-fold higher in AML-TRI8 cases than in AML-NK (Figure 2a some 11 was 1.25-fold higher in AML-TRI11 cases than in AML- and b) confirming a gene dosage effect as described by NK and the expression of probe sets on was 2 Virtaneva et al. In that series, seven out of 29 genes most 1.14-fold higher in AML-TRI13 cases than in AML-NK, significantly upregulated in AML-TRI8 for which a chromosomal demonstrating a gene dosage effect also in AML with trisomy assignment could be made, map to chromosome 8: FABP5, 11 and 13 (Figure 2b). PTDSS1, LY6E, COX6C, LYN, SIAHBP1, and DEFA1.In contrast, none of the 25 genes upregulated in AML-NK map to chromosome 8. The following genes identified by Virtaneva et al Monosomy 7 as up-regulated in AML-TRI8 were confirmed in our series: FABP5 (ratio: 1.73), PTDSS1 (ratio: 1.65), LY6E (ratio: 2.0), A total of 1441 probe sets cover sequences localized on COX6C (ratio: 1.34), LYN (ratio: 1.4), and SIAHBP1 (ratio: 1.37). (Table S1d, Supplementary Information). In all,

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Figure 1 Ratios of mean expression of probe sets affected by change of gene dosage between the respective subtype with gain or loss of one chromosome/chromosomal region in comparison to AML with normal karyotype. (a) In all, 73% of the 835 probe sets located on chromosome 8 show a higher mean expression (ratio41) in cases with trisomy 8 than in AML with normal karyotype. (b) In all, 65% of the 1263 probe sets located on show a higher mean expression (ratio41) in cases with trisomy 11 than in AML with normal karyotype. (c) In all, 87% of the 1088 probe sets located on chromosome 7 show a lower mean expression (ratioo1) in cases with monosomy 7 than in AML with normal karyotype. (d) In all, 67% of the 502 probe sets located on the long arm of within the chromosomal bands 5q13–5q31 show a lower mean expression (ratioo1) in cases with deletion 5q than in AML with normal karyotype.

Leukemia Gene dosage effects in AML C Schoch et al 1226 a 1.8

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0.2 Chr. 8 Chr. 11 Chr. 13 Chr. 7 Chr. 5q13q31 AML-TRI8 AML-TRI11 AML-TRI13 AML-MO7 AML-DEL5q vs. AML-NK vs. AML-NK vs. AML-NK vs. AML-NK vs. AML-NK Figure 2 Gene dosage influences levels of gene expression. (a) AML samples with trisomy 8 (n ¼ 12) exhibit higher expression for genes located on chromosome 8 than AML samples with normal karyotype (n ¼ 104). The median expression levels and the second and third quartile for probe sets by chromosome in AML with þ 8 relative to AML with normal karyotype are shown. (b) The median expression levels and 25 and 75% interval for probe sets on chromosomes 8, 11, 13, 7, and on chromosomal bands 5q13–5q31 in AML with the respective gain or loss of the chromosome/chromosomal region relative to AML with normal karyotype are shown.

1088 were expressed in at least one sample. The mean 5q31 was 0.82-fold in AML-DEL5q cases compared to AML-NK expression of 948 out of 1088 (87.1%) probe sets was lower demonstrating a gene dosage effect also in AML with 5q deletion in cases with monosomy 7 than in AML with normal karyotype (Figure 2b). In Figure 3b, the median expression ratios are (Figure 1c). The median expression of probe sets on chromo- separately shown for genes located on 5p and the chromosomal some 7 was 0.57-fold in AML-MO7 cases compared to AML- bands 5q11, 5q12, 5q13, 5q14, 5q15, 5q21, 5q22, 5q23, 5q31, NK, demonstrating a strong effect of gene dosage on gene 5q32, 5q33, 5q34, and 5q35. The expression ratios are only expression also in AML with monosomy 7 (Figure 2b). In Figure clearly below 1 for those chromosomal bands located within the 3a, the median expression ratios are separately shown for genes most commonly deleted region (5q14–5q33), showing the located on 7p and the chromosomal bands 7q11, 7q21, 7q22, lowest ratio for genes located within the chromosomal band 7q31, 7q32, 7q33, 7q34, 7q35, and 7q36. The expression ratios 5q15 (median ratio 0.61). Genes located on the short arm of are clearly below 1 in all chromosomal regions ranging from chromosome 5 or in chromosomal bands 5q11, 5q12, and 0.48 to 0.63. 5q13, showed on average a slightly higher expression ratio in cases with 5q deletion than in AML with normal karyotype.

5q deletion Differences in the global gene expression profile In all, 1374 probe sets cover sequences localized on chromo- some 5 (Table S1e, Supplementary Information), 644 of these In order to evaluate the effect of gene dosage on the subtype- are located in chromosome bands 5q13–5q31, the region most specific gene expression signature, the top 50 differentially commonly deleted in AML with 5q deletion. In all, 502 of them expressed probe sets for each subtype vs all other subtypes were were expressed in at least one sample. The mean expression of determined using ANOVA and t-test statistics (Welch t-test). The 337 out of 502 (67.1%) probe sets was lower in cases with 5q 50 most differentially expressed probe sets were equally deletion than in AML with normal karyotype (Figure 1d). The distributed over the genome for each of the comparisons AML- median expression of probe sets located in the region 5q13– TRI8, AML-TRI11, AML-TRI13 vs all other subtypes. However,

Leukemia Gene dosage effects in AML C Schoch et al 1227 a 1

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Figure 3 (a) Median expression levels for probe sets located on chromosome 7 separately shown for 7p and the chromosomal bands 7q11, 7q21, 7q22, 7q31, 7q32, 7q33, 7q34, 7q35, and 7q36. Ratios have been calculated between cases with monosomy 7 and AML with normal karyotype. (b) Median expression levels for probe sets located on chromosome 5 separately shown for 5p and the chromosomal bands 5q11, 5q12, 5q13, 5q14, 5q15, 5q21, 5q22, 5q23, 5q31, 5q32, 5q33, 5q34, and 5q35. Ratios have been calculated between cases with deletion 5q and AML with normal karyotype. comparing AML-DEL-5q with all other subtypes revealed that 10 location is known. Of these 43 probe sets, 39 representing 36 of the 34 probe sets for which chromosomal location was different genes are localized on chromosome 7. They are available are located on chromosome 5 within the region involved in mismatch repair (PMS2L1, PMS2L3, PMS2L5, affected by the deletion. These probe sets represent eight genes PMS2L8, PMS2L9), apoptosis (TAX1BP1, CASP2, CARD4), involved in (HINT1, PDE8B, SNX2, DNA replication (RIP60, SSBP1), and signal transduction CSNK1A1, ANXA6), radioadaptive response (HSPA4), and (AKAP9, CARD4). Also HOXA3 and HOXA9 were significantly suppression of invasion (CTNNA1), respectively. Recently, lower expressed in AML-MO7 than in all other subtypes. CTNNA1 was reported to show a lower expression in In inborn diseases as well as in several malignant diseases, a leukemia-initiating cells of cases with del(5q) MDS/AML than pathogenetic role of haploinsufficiency has been demon- in normal hematopoietic stem cells and leukemia-initiating cells strated.7,8 Especially for several tumor suppressor genes, of AML/MDS without 5q deletion (Liu et al. Blood 2004; 104: including TP53, data suggest that some genes, when expressed 62a; abstract). In Figure 4, the CTNNA1 expression levels in at half normal levels, that is from only one functional allele, the different AML subgroups of our cohort are shown demon- cannot fully suppress tumor growth.9,10 Therefore, genes located strating the significantly lower CTNNA1 expression in cases in deleted regions showing a reduced expression may be new with 5q deletion. candidate genes playing an important role in the pathogenesis of For 43 of the top 50 probe sets differentially expressed malignancies with unbalanced . The concept of between AML-MO7 and all other subtypes, the chromosomal halpoinsufficiency, especially in AML with 5q deletion is

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Figure 4 The median expression levels and the 25 and 75% interval of CTNNA1 for the different AML subgroups are shown.

strengthened also by the fact that despite a lot of effort no tumor acute myeloid leukemia with complex aberrant karyotype: a suppressor gene was identified within the deleted region, being detailed analysis of 125 cases using conventional chromosome critical for the pathogenesis of this AML subtype. Therefore, the analysis and fluorescence in situ hybridization including 24-color deletion of large regions of the long arm of chromosome 5 may FISH. Genes, Chromosomes 2002; 35: 20–29. 2 Virtaneva K, Wright FA, Tanner SM, Yuan B, Lemon WJ, Caligiuri not initiate tumor formation in itself but could foster transforma- MA et al. Expression profiling reveals fundamental biological tion by rendering the genetically unstable and therefore differences in acute myeloid leukemia with isolated trisomy 8 and more likely to sustain other and chromosomal normal . Proc Natl Acad Sci USA 2001; 98: rearrangements as is frequently observed, in addition to 5q 1124–1129. deletions in cases with complex aberrant karyotypes.1 3 Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger In conclusion, gain of whole chromosomes leads to over- U et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-FISH, qualitative and quantitative PCR for expression of genes located on the respective chromosomes. diagnosis and for follow-up in chronic myeloid leukemia: a study However, the altered expression of genes located on the gained on 350 cases. Leukemia 2002; 16: 53–59. chromosome does not dominate the specific gene expression 4 Schoch C, Kohlmann A, Schnittger S, Brors B, Dugas M, signature of the specific subtype. These data suggest that trisomy Mergenthaler S et al. Acute myeloid leukemias with reciprocal 8, 11, or 13 may not be the primary disease determining rearrangements can be distinguished by specific gene expression aberration but rather secondary events associated with progres- profiles. Proc Natl Acad Sci USA 2002; 99: 10008–10013. 5 Kohlmann A, Schoch C, Schnittger S, Dugas M, Hiddemann W, sion. This is supported by the fact that especially trisomy 8 Kern W et al. Pediatric acute lymphoblastic leukemia (ALL) gene occurs in addition to a large variety of primary aberrations in AML expression signatures classify an independent cohort of adult ALL such as t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q22;q12), patients. Leukemia 2004; 18: 63–71. 11q23/MLL rearrangements.11,12 6 Altmann DG. Practical statistics for medical research. London: In contrast, losses of larger regions of the genome translate not CRC Press, 2004. 7 Seidman JG, Seidman C. Transcription factor haploinsufficiency: only into lower expression of the majority of genes represented when half a loaf is not enough. J Clin Invest 2002; 109: 451–455. by only one allele, but also is the most characteristic difference 8 Santarosa M, Ashworth A. Haploinsufficiency for tumour suppres- in gene expression between AML with monosomy 7 or AML sor genes: when you don’t need to go all the way. Biochim Biophys with 5q deletion and other AML subtypes. Therefore, these data Acta 2004; 1654: 105–122. provide evidence that gene dosage effects play an important role 9 Largaespada DA. Haploinsufficiency for tumor suppression: the in AML with unbalanced karyotype abnormalities, leading to hazards of being single and living a long time. J Exp Med 2001; losses of genetic material. Genes located in the lost regions 193: F15–F18. 10 Venkatachalam S, Shi Y-P, Jones SN, Vogel H, Bradley A, Pinkel D showing also a reduced expression are candidate genes and et al. Retentio of wild-type p53 in tumore from p53 heterozygous their role in pathogenesis of AML needs to be clarified. mice: reduction of p53 dosage can promote cancer formation. EMBO J 1998; 17: 4657–4667. 11 Schoch C, Haase D, Fonatsch C, Haferlach T, Lo¨ffler H, Supplementary Information Schlegelberger B et al. The significance of trisomy 8 in de novo acute myeloid leukaemia: the accompanying chromo- Supplementary Information accompanies the paper on the some aberrations determine the prognosis. Br J Haematol 1997; 99: 605–611. Leukemia website (http://www.nature.com/leu). 12 Schoch C, Schnittger S, Klaus M, Kern W, Hiddemann W, Haferlach T. AML with 11q23/MLL abnormalities as defined by References the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected 1 Schoch C, Haferlach T, Bursch S, Gerstner D, Schnittger S, Dugas series of 1897 cytogenetically analyzed AML cases. Blood 2003; M et al. Loss of genetic material is more common than gain in 102: 2395–2402.

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