(2010) 24, 1139–1145 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu ORIGINAL ARTICLE

High-density single nucleotide array analysis and ASXL1 mutation screening in chronic myeloid leukemia during disease progression

J Boultwood1, J Perry1, R Zaman1, C Fernandez-Santamaria1, T Littlewood1, R Kusec2, A Pellagatti1, L Wang3, RE Clark3 and JS Wainscoat1

1LRF Molecular Haematology Unit, NDCLS, University of Oxford, John Radcliffe Hospital, Oxford, UK; 2Department of Clinical Sciences, Department of Haematology, University Hospital Merkur, Zagreb, Croatia and 3University Department of Haematology, Royal Liverpool University Hospital, Liverpool, UK

We have undertaken a -wide single nucleotide poly- patients not treated with imatinib and also in those who have morphism (SNP) array analysis of 41 chronic myeloid leukemia become resistant to the drug.6,7 (CML) patients. In total, 44 regions of uniparental disomy (UPD) Although it is recognized that cytogenetic and molecular 43 Mb were identified in 24 of 32 patients in chronic phase (CP), and 21 regions of UPD 43 Mb were identified in 13 of 21 patients changes occur in the majority of CML patients during evolution in blast crisis (BC). 8 had the highest frequency of to BC, the molecular events responsible for leukemic transfor- UPD regions in both CP and BC samples. Eight recurrent regions mation remain unclear. It has been suggested that BCR/ABL may of UPD were observed among the 41 patients, with chromosome have a direct or an indirect role in promoting genomic instability 8 showing the highest frequency. Ten regions of copy number and 60–80% of patients with CML develop additional non- change (CNC) 43 Mb were observed in 4 of 21 patients in BC, random chromosomal abnormalities involving 8, whereas none were observed in CP. We have identified several 8 recurrent regions of UPD and CNC in CML that may be of 17, 19 and 22. In addition, marker chromosomes arising from B pathogenetic importance. Overrepresentation of genomic aberra- complex chromosome rearrangements are present in 20% of tions (UPD and copy number gain) mapping to chromosome 8 the BC CML cases and may remain unresolved by banding was observed. Selected candidate mapping within the analysis. Some combinations, such as trisomy 8, double Ph aberrant genomic regions were sequenced and mutation of the chromosome and i(17q), are more frequent than others; TP53 gene was observed in one case in BC and of the ASXL1 however, neither the presumed order of appearance nor the gene in 6 of 41 cases in CP or BC. Mutation of ASXL1 represents combination itself seems to have a clear impact on the prognosis an important new molecular abnormality in CML. 8 Leukemia (2010) 24, 1139–1145; doi:10.1038/leu.2010.65; of BC CML. published online 22 April 2010 We have shown that telomere shortening and diminishing Keywords: CML; SNP array analysis; UPD; ASXL1 gene levels of hTERT are associated with disease evolution in CML.9,10 Mutation of the tumor suppressor gene is detected in B25–30% of CML-myeloid BC,11 whereas B50% of the patients with lymphoid BC present a homozygous deletion of the INK4A/ARF gene.12 Most recently, it has been shown that Introduction the loss of the IKAROS (IKZF1) gene is an important event in the development of lymphoid BC in CML and IKAROS is deleted in Chronic myeloid leukemia (CML) is a clonal disorder of the B80% of such patients.13 hematopoietic stem cell that typically evolves from an initial At the molecular level, BC CML is a heterogeneous disease.12 chronic phase (CP) to a terminal acute phase termed blast crisis It is essential to the management and treatment of CML to (BC) that resembles acute leukemia.1 In some patients, a understand the biology of CML transformation.12 We have transitional accelerated phase precedes BC.1 The Philadelphia applied single nucleotide polymorphism (SNP) array analysis to chromosome (Ph1), or t(9;22) encoding the BCR/ABL fusion the study of CML to achieve a better understanding of the oncoprotein, is the hallmark of CML.2,3 Until recently, transition pathogenic mechanisms central to disease heterogeneity and to BC was the inevitable outcome of CML except in a cohort of progression. To study disease progression without the complicat- patients receiving allogeneic bone marrow transplants early in ing factor of imatinib treatment, we have performed this study the CP CML.1 The development of the BCR/ABL tyrosine kinase exclusively on CML samples obtained in the immediate ‘pre- inhibitor imatinib mesylate (Gleevec, Novartis, East Hanover, imatinib era’. The identification of recurrent regions of unipar- NJ, USA) has revolutionized the treatment of CML.4 Imatinib is ental disomy (UPD), genomic deletions, amplifications or other now established as the standard first-line therapy for patients cryptic rearrangements in CML during disease evolution would with newly diagnosed CP CML.5 Virtually all patients treated clearly be of great value not only as potential new prognostic with this drug in the early stages of CML respond, but in indicators, but also as clues to the location of oncogenes or tumor advanced phases the leukemia usually becomes resistant within suppressor genes central to leukemic transformation. months.6,7 Therefore, leukemic transformation remains a serious clinical problem in CML and is seen both in the small group of Patients and methods Correspondence: Dr J Boultwood, LRF Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK. Patients and samples E-mail: [email protected] Forty-one patients with CML were included in this study. A total Received 10 November 2009; revised 28 January 2010; accepted 22 of 53 bone marrow or peripheral blood samples were obtained February 2010; published online 22 April 2010 from them, of which 32 were from patients in CP and 21 from Copy number changes and LOH in CML J Boultwood et al 1140 patients in BC at the time of investigation. Paired (sequential) (patients 12 and 40) identified with a deletion at 17p13.3–p11 samples obtained in CP and also at BC phase were available for by SNP analysis. The primers and amplifications conditions 12 of these 41 patients. None of the CML patients included in this were as previously published.17 Sequences spanning 1–7 study were being treated with imatinib at the time the samples of IKAROS (IKZF1) were amplified from CP CML DNA samples were taken. The samples from CML patients were obtained from by PCR in two cases (patients 20a and 41) identified with a two sources: Oxford and Liverpool (UK). The study was approved UPD or deletion at 7p13-q11.2, respectively, by SNP analysis. by the ethics committees of the institutes involved (Oxford Primers and amplifications conditions were as previously C00.196, Liverpool 02/11/230/A) and informed consent was published.13 Sequences spanning 12 of the ASXL1 (addi- obtained. The patient are shown in Supplementary Table tional sex combs 1) gene were amplified from all patient DNA 1. The BCR-ABL mRNA transcript level was measured in some samples using PCR. All previously reported mutations of ASXL1 patients by quantitative real-time PCR as previously described,14 in hematological malignancies were found exclusively in exon using ABL as the control gene, in line with recent recommenda- 12 of the gene.18,19 Primers and amplifications conditions tions.15 All BCR-ABL/ABL ratios were obtained from RNA extracted were as previously published.19 PCR products were purified from the same population of cells used for SNP analysis. and directly sequenced using BigDye Terminator v1.1 cycle sequencing (Applied Biosystems, Foster City, CA, USA) and an ABI 3100 Genetic analyzer (Applied Biosystems). Sequence DNA preparation data was analyzed using Sequence Analysis 1.6.0 (Informagen, Bone marrow aspirates were collected in PBS supplemented Greenland, NH, USA) and Mutation Surveyor v3.25 (Soft- with 2 mM EDTA and peripheral blood samples were collected genetics, State College, PA, USA). in EDTA tubes. Mononuclear cell fractions were obtained from bone marrow samples by Histopaque density gradient centrifu- gation (Sigma-Aldrich, Dorset, UK). For patients in BC, the Results leukemic blast fraction (mononuclear fraction) was separated by Histopaque density gradient centrifugation from peripheral Quality control of SNP genotype arrays and regions of blood samples. Genomic DNA was isolated by phenol/chloro- UPD form extraction using standard methods and evaluated for The accuracy of whole-genome sampling analysis was deter- quality and concentration using an ND-1000 spectrophotometer mined using GeneChip Operating Software. The mean call rate (Nanodrop, Wilmington, DE, USA). of all the CML patient samples was 95.76%. Regions of UPD were defined as continuous stretches of 4 20 SNP mapping assay homozygous SNP calls 3 Mb without copy number loss. We observed a total of 32 regions of UPD in 23 of 45 healthy control The SNP mapping assay was carried out according to the subjects dispersed across the genome on 15/22 chromosomes manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). (Supplementary Table 2). For the patients, we report only those Briefly, 250 ng DNA were digested with HindIII, ligated to the regions of UPD not observed in any of our group of 45 normal adaptor and amplified by PCR using a single primer. PCR controls or in normal healthy control subjects reported in the products were purified with the DNA amplification clean-up literature.20,21 A total of 65 regions of UPD 43 Mb were kit (Clontech, Mountain View, CA, USA) and the amplicons detected in 29 of 41 patients, dispersed on 17/22 chromosomes were quantified. Purified amplicons (40 mg) were fragmented, (Supplementary Table 3, Figure 1). Differences in the number of end-labeled and hybridized to a Genechip Mapping 50K HindIII UPDs on each chromosome was significant for chromosome 8 array at 48 1C for 16–18 h in a Hybridization Oven 640 (CP versus controls; P ¼ 0.019). In the CP samples, 44 regions of (Affymetrix). After washing and staining in a Fluidics Station UPD 43 Mb were identified in 24 of 32 patients. In the BC 450 (Affymetrix), the arrays were scanned with a GeneChip samples, 21 regions of UPD 43Mb were identified in 13 of 21 Scanner 3000 (Affymetrix).16 patients. In both CP and BC samples, chromosome 8 had the highest frequency of UPD regions, with a total of 14. SNP array data analysis Chromosomes 2 and 4 also showed a high frequency of UPDs, Cell intensity calculations and scaling were carried out with a total of 10 and 8, respectively. using GeneChip Operating Software. Data were analyzed using GeneChip Genotyping Analysis Software Version 4.0 (Affymetrix) and CNAG software version 2.0. Quality control Recurrent regions of UPD was performed within the genotyping software after scaling Eight regions of UPD 43 Mb were seen in two or more cases in the signal intensities of all arrays to a target of 100. DNA the group of CML patient samples (including CP and BC phase): copy number was analyzed with both the chromosome copy 2q21.3-q22.1 (three patients), 2q32.3-q33.1 (two patients), number tool (CNAT) version 3.0 and CNAG version 2.0. CNAT 4p15.1 (three patients), 7q22.1-q22.3 (two patients), 8p23.1 compares obtained SNP hybridization signal intensities with (two patients), 8q11.21-q11.22 (two patients), 8q23.2-q23.3 SNP intensity distributions of a reference set from more than (five patients) and 12p11.21-q12 (two patients). The sizes of 100 healthy individuals of different ethnicity. For analysis with these recurrent regions of UPD and the genes that map within CNAG, we used a pool of 45 healthy controls included in the them are shown in Table 1. study as a reference set.16 Paired sample analysis Mutational analysis Paired (sequential) samples obtained in CP and also at BC phase Mutational analysis was carried out on selected candidate genes were available for 12 of the CML patients. Altogether, 27 regions mapping within the aberrant genomic regions identified in this of UPD 43 Mb were identified in these paired samples study. Sequences spanning exons 5–8 of TP53 were amplified (Supplementary Table 4). Paired samples were also analyzed from CP and BC CML DNA samples by PCR in two cases according to whether regions of UPD were only seen

Leukemia Copy number changes and LOH in CML J Boultwood et al 1141

Figure 1 Percentage of UPD (43 Mb) on each chromosome. Control: control group, CP: chronic phase group, BC: blast crisis group.

Table 1 Recurrent regions of UPD identified in patients with CML

ID Status Chromosome Length of recurrent Genes mapping to recurrent region region (Mb)

31 CP 2q21.3-q22.1 2.53 RAB3GAP1, ZRANB3, R3HDM1, UBXD2, LCT, MCM6, DARS, CXCR4, THSD7B 15 CP 1a CP 1b BC 41 CP 2q32.3-q33.1 4.23 SLC39A10, DNAH7, STK17B, HECW2, CCDC150, GTF3C3, C2orf66, PGAP1 7b BC 31 CP 4p15.1 2.85 37 CP 28 CP 5a CP 7q22.1-q22.3 2.31 AP4M1, CUX1, SH2B2, AC005088.3, AC073517.6, PRKRIP1, ORAI2, ALKBH4, LRWD1, AC093668.4, POLR2J, AC004084.4, POLR2J3, AC004084.7, RASA4, POLR2J2, AC105052.3, FBXL13, LRRC17, ARMC10, PSMC2, NAPEPLD, RELN, DPY19L2P2, AC004668.1, PMPCB, ZRF1, SLC26A5, ORC5L, LHFPL3 5b BC 28 CP 7a CP 8p23.1 3.00 AC068353.34, MFHAS1, ERI1, PPP1R3B, TNKS, MSRA, UNQ9391, RP1L1, C8orf74, SOX7, AC105001.3, XKR6, C8orf16 7b BC 28 CP 9b BC 8p11.21-q11.22 2.36 SNTG1, PXDNL 20 CP 11a CP 8q23.2-q23.3 1.22 11b BC 13 CP 16 CP 23 CP 15 CP 25 CP 12p11.21-q12 4.62 DNM1L, YARS2, PKP2, SYT10, ALG10, ALG10B 32 BC Abbreviations: BC, blast crisis; CML, chronic myeloid leukemia; CP, chronic phase; UPD, uniparental disomy. in BC phase. Two regions of UPD 43 Mb were identified samples and no CNC were identified in CP samples. Five regions only in the BC phase (Supplementary Table 5). Following on of copy number gain (Table 2), including one patient (5b) with from the recent publication of common mutations of the ASXL1 an apparent trisomy 8 were identified. The remaining four gene in other myeloproliferative disorders (MPDs),18 we regions of copy number gain were all seen in Patient 40 and looked for UPDs smaller than our standard cutoff of 43Mb were observed on chromosomes 9, 17 and 22 and ranged from at the 20q11 region. We found a 0.5 Mb UPD in one patient 7.93 to 56.36 Mb. (both in CP and BC phases) at 20q11, which encompasses the A total of five regions of copy number loss (Table 3) were ASXL1 gene. observed in a total of three patients on chromosomes 12, 16 and 17 and ranged from 20.12 to 33.88 Mb. All the copy number losses were identified in BC samples and were not observed in Copy number changes the paired CP samples. Common regions of copy number loss Regions of copy number gain and loss 43 Mb were recorded. were identified in two patients in the BC phase at 17p13.3-p11.2 The copy number changes (CNCs) were identified in 4 of 21 BC and at 12p13.3-p12.2.

Leukemia Copy number changes and LOH in CML J Boultwood et al 1142 In addition, some smaller regions of CNC were recorded polymorphisms (Table 5). The mutations were identified in (o3 Mb). One patient (patient 41) showed a small deletion patient samples from both CP and BC. However, in one positive (B1 Mb) encompassing the IKAROS gene. We also observed case for which we had paired samples available (2a and b), we two recurrent regions of copy number gain o3 Mb. These observed a nonsense mutation of ASXL1 in the BC sample, but regions were identified at 3q25.33 and 8q23.1, and were not in the CP sample. None of the ASXL1 mutations identified in relatively frequent in the CML patient group (both being the patients with CML were identified in the DNA extracted identified in at least five CML patients). Table 4 shows two from the peripheral blood samples of 111 normal individuals regions of recurrent copy number gain and the genes that map to that were also sequenced, or are recorded in the NCBI that area. SNP database.

Mutational analysis Discussion Two cases (patients 12 and 40) were identified with a deletion at 17p13.3-p11.2 encompassing the TP53 gene. We carried out We have analyzed a group of 41 patients with CML by SNP mutational analysis of the TP53 gene in the CP and BC samples array analysis for the presence of CNCs and loss of hetero- of these cases (with the exception of patient 40 from whom only zygosity. All the CML samples used in this study were obtained a BC sample was available). A known A4G mutation in exon 5 22 before the widespread adoption of imatinib as treatment. (K132R) of the TP53 gene was observed in patient 12 in the Patients were studied at CP and BC to identify disease-related BC, but not CP sample. The one other case (patient 40) did not genomic abnormalities in CML and those related to disease show mutation of TP53. Two cases (20a and 41) were identified, progression. one with a region of UPD and one with a deletion at 7p13- We observed a total of 32 regions of UPD 43Mb in 23 q11.21, a region containing the IKAROS gene. We carried out of 45 control subjects dispersed across the genome on 15/22 mutational analysis of the IKAROS gene in these cases, but no chromosomes. The frequency of regions of UPD in normal mutations were detected. In addition, we carried out mutational individuals observed in this and our previous study is compar- analysis of exon 12 of the ASXL1 gene in all the CML cases and able with that observed by other groups, including the study by we found ASXL1 mutations in 6 of 41 cases (Table 5). Five Gibson et al.21 These studies show that homozygous tracts are patients had a frameshift mutation and one patient had a common in normal individuals and that a control pool is nonsense mutation. In addition, three cases had substitutions essential for the analysis of SNP array data. In this study, we that we did not take into account as they could represent report only those regions of UPD not observed in any of our group of 45 normal controls or in normal healthy control subjects reported in the literature by others.20,21 Table 2 Regions of copy number gain (43 Mb) identified in SNP analysis that enables the detection of UPD has already patients with CML proven very informative in the study of myeloid malignan- cies.20,23,24 For example, up to 20% of cases of acute myeloid Status ID Chromosome Length (Mb) leukemia (AML) have been found to have large-scale cryptic 24 BC 5b 8p23.3-q24.3 145.81 regions of acquired homozygosity in the form of UPD. BC 40 9p24.3-p13.3 35.77 Subsequently, an impressive association between UPD and BC 40 9q34.11-q34.3 7.93 homozygous gene mutation in AML was determined.23 By BC 40 17p11.2-q25.3 56.36 BC 40 22q11.1-q11.23 8.76

Abbreviations: BC, blast crisis; CML, chronic myeloid leukemia. Table 5 Mutations of the ASXL1 gene identified in patients with CML

Sample Phase Mutation Table 3 Regions of copy number loss (43 Mb) identified in patients with CML 2b BC C3202T R1068X 14 CP 1934dupG G646WfsX12 Status Patient Chromosome Length (Mb) 15 CP 1934dupG G646WfsX12 H1008Q C3204Ga 19 CP G4016A G1339Ea BC 12b 12p13.33-p11.1 33.88 31 CP 1934dupG G646WfsX12 BC 10b 12p13.33-p12.2 20.76 36 BC 1934dupG G646WfsX12 A3745G M1249Va BC 10b 16q12.2-q24.3 34.41 40 BC del1888–1910 E635RfsX15 BC 12b 17p13.3-p11.2 22.87 BC 40 17p13.3-p11.2 20.12 Abbreviations: BC, blast crisis; CML, chronic myeloid leukemia; CP, chronic phase. Abbreviations: BC, blast crisis; CML, chronic myeloid leukemia. aSubstitutions, possibly representing polymorphisms.

Table 4 Recurrent regions of copy number gain (o3 Mb) identified in patients with CML

Patient Chromosome Length (Mb) Genes mapping to recurrent region

1a, 3b, 15, 7a, 41 3q25.33 0.41 KPNA4, ARL14, PPM1L 3a, 3b, 7a, 15, 23, 36 8p23.1 0.90 CTSB, AC107918.5, AC130366.5, ZNF705C, USP17, FAM90A2P, AC145124.2, DEFB130, ZNF705F, AC087203.12, AC068587.16, LONRF1 Abbreviation: CML, chronic myeloid leukemia.

Leukemia Copy number changes and LOH in CML J Boultwood et al 1143 analogy with AML and myelodysplasia, SNP analysis may BC phase in two patients and are thus associated with disease illuminate the location of disease genes in CML. evolution in these individual CML patients. For the patient group as a whole, we have determined In addition, a small UPD at 20q11, which encompasses the disease-related UPD on the basis of size (43 Mb) and also the ASXL1 gene, a putative tumor suppressor, was found in one absence of the corresponding UPD in control groups. We find patient in both the CP and BC phases. Most recently, that regions of UPD are very common in both CP and BC patient heterozygous frameshift mutations of exon 12 of the ASXL1 groups. A total of 65 regions of UPD 43 Mb were detected in 30 have been reported in 11% of patients with myelodysplasia19 of 41 CML patients dispersed on 17/22 chromosomes. Thus the and in B7% patients with the MPD essential thrombocythemia frequency of UPD 43 Mb in CML is considerably greater (by and primary myelofibrosis.18 Thus, we chose to sequence exon approximately twofold) than that observed in normal controls. In 12 of the ASXL1 gene in our group of patients with the MPD the CP samples, 44 regions of UPD 43 Mb were identified in 24 CML. We identified mutations of ASXL1 in 6 of 41 patients. Five of 32 patients. In the BC samples, 21 regions of UPD 43Mb of these mutations were frameshift mutations and the other was were identified in 13 of 21 patients. The regions of UPD are not a nonsense mutation. The mutations were identified in patient evenly distributed across the various chromosomes. In both CP samples from both CP and BC. Thus, this gene mutation can and BC patients, chromosome 8 had the highest frequency of represent an early event in CML, which is intriguing given the UPD regions, with chromosomes 2 and 4 also showing a high rarity of gene mutations (other than the BCR/ABL) in CP. The frequency of UPD. function of the human ASXL1 is poorly understood, but Eight regions of UPD 43 Mb were identified in two or more there is some evidence to suggest that it may represent a cases in CP and BC patients and are thus considered recurrent component of DNA- and/or histone-modifying complexes.39 abnormalities: two different regions on 2q, one region on 4p, ASXL1 belongs to a family of three members that encode one on 7q, one region on 8p and two different regions on 8q, belonging to polycomb and mixed lineage leukemia/ and one region on 12p. trithorax chromatin modifier complexes.39 ASXL1 is a fusion Three recurrent regions of UPD were observed on chromo- partner of PAX5 in acute lymphoblastic leukemia.40 In this some 8 and this chromosome showed the highest frequency of study, we have shown, for the first time, that mutation of ASXL1 recurrent UPD regions. The UPD at 8p23.1 was found in two is a frequent event in CML, adding further weight to the proposal CML patients and contains several genes of interest including that mutation of ASXL1 may have an important role in the MFHAS1 (MASL1),25,26 TNKS and SOX7.27,28 We have pre- pathogenesis of MPD. viously shown that TNKS (tankyrase), a TRF1-interacting factor The BC stage of CML is commonly associated with non- that has an important role in the control of telomere dynamics, is random secondary chromosomal changes that, in addition to upregulated in CP and accelerated phase CML.10 The UPD at the t(9;22), include þ Ph, þ 8, i(17q), þ 19, t(3;21)(q26;q22) 8q11 was found in two CML patients. The most frequent UPD and t(7;11)(p15;p15).8 These karyotypic changes involve the on chromosome 8 at 8q23, occurring in five patients, contained loss or gain of large chromosomal regions together with the no known genes. Our demonstration of frequent regions of UPD concomitant loss of tumor suppressor genes and/or gain of on chromosome 8 in CML is intriguing given the strong oncogenes. However, the molecular mechanisms responsible association of abnormalities of chromosome 8 with disease for disease progression in CML are not fully understood.3,30 progression in this disorder.29,30 Indeed, trisomy 8 is the most Ten regions of copy number gain or loss 43 Mb were common secondary chromosomal aberration in CML, occurring observed in 4 of 21 BC samples and none were observed in the in 34% of cases with additional changes.8 The importance of CP samples. Thus, the CNC observed in this study are clearly trisomy 8 in the pathogenesis of CML has been long recognized, associated with disease evolution and most probably reflect but the causative gene(s) mapping to chromosome 8 has increased genomic instability in BC CML. Regions of copy remained elusive.12 It is possible that the regions of UPD on number loss were identified on chromosomes 12, 16 and 17, chromosome 8 identified by our study give the location for two of which were recurrent. One region of copy number loss at genes of importance in the pathogenesis of CML. 17p13.3-p11.2, encompassing the TP53 gene, was identified in Two recurrent regions of UPD were observed on the long arm two patients. The loss of 17p, due to i(17q), –17, or other of . The UPD at 2q21-q22 was found in three changes resulting in 17p– is frequently reported in BC CML and CML patients and contains several genes of interest including is often associated with mutation of the TP53 gene.11,12 We MCM-631 and CXCR4. CXCR4 is a chemokine receptor known therefore carried out mutational analysis of the TP53 gene in the to be downregulated in CML by BCR-ABL and this is associated two cases showing loss of 17p13.3-p11.2 and one patient had a with cell-migration defects in CML.32,33 The UPD at 2q32-q33 TP53 mutation in the BC but not CP phase of the disease. A was found in two CML patients and contains the zinc transporter region of copy number loss at 12p13.33-p12.2 was found in two ZIP10 (SLC39A10).34 patients in BC. This recurrent region contains many genes A recurrent region of UPD was observed on . known to have a role in carcinogenesis including JARID1A, Interestingly, the UPD at 7q22 encompasses a commonly WNK1, FOXM1, ECR1 and NOL1. Intriguingly, 12p13 is a deleted region previously identified in a range of malignant chromosomal region often involved in structural rearrangements myeloid disorders by Le Beau and colleagues.35 The 7q22 in BC CML.8 In addition, one patient showed a small deletion commonly deleted region contains several candidate genes (B1 Mb) encompassing the IKAROS gene. We carried out including FBXL13 and LRRC17.35 mutational analysis of the IKAROS gene in this case, but no A UPD at 12p11.21-q12 was found in two CML patients and mutations were detected. The regions of CNC identified in this contains the DNM1L and PKP2 genes. DNM1L (DRP1) mediates study were found only in BC CML and therefore may give the caspase-independent in CLL36 and PKP2 is frequently location for novel genes of importance in disease evolution in overexpressed in malignancy.37,38 CML. Paired samples obtained in both CP and BC phase were In addition, we observed two recurrent regions of copy available for a proportion of CML patients (12 patients) included number gain that were smaller than 3 Mb and occurred in this study. In these cases, two regions of UPD 43Mb on relatively frequently in the patient group under investigation chromosomes 2 and 8, respectively, were identified only in the and were found in both CP and BC phases. One of these

Leukemia Copy number changes and LOH in CML J Boultwood et al 1144 amplified regions mapped to chromosome 8 at 8q23, and was the first-line treatment of chronic myeloid leukemia. Leukemia found in five patients. The amplified region at 8p23 overlaps 2009; 23: 1054–1061. with a recurrent amplicon found in esophageal adenocarcinoma 6 Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM and contains several genes known to have a role in carcinogen- et al. Activity of a specific inhibitor of the BCR-ABL tyrosine 41,42 29 kinase in the blast crisis of chronic myeloid leukemia and acute esis including the CTSB gene. Interestingly, Gribble et al. , lymphoblastic leukemia with the Philadelphia chromosome. using metaphase CGH, have demonstrated the amplification of N Engl J Med 2001; 344: 1038–1042. 29 the 8q23/q24 region in six of eight CML-derived cell lines. 7 Volpe G, Panuzzo C, Ulisciani S, Cilloni D. Imatinib resistance in More recently, Hosoya et al.,30 revealed a number of novel CML. Cancer Lett 2009; 274: 1–9. CNCs involving chromosome 8 in some patients with CML using 8 Johansson B, Fioretos T, Mitelman F. Cytogenetic and molecular array CGH. These alterations included a gain at 8p23.2. genetic evolution of chronic myeloid leukemia. Acta Haematol It is desirable for SNP studies such as those reported 2002; 107: 76–94. 9 Boultwood J, Peniket A, Watkins F, Shepherd P, McGale P, here to include constitutional controls. However, this study Richards S et al. Telomere length shortening in chronic myelo- was conducted with archival DNA samples collected in the genous leukemia is associated with reduced time to accelerated ‘pre-imatinib’ era and so this did not prove to be feasible. phase. Blood 2000; 96: 358–361. Nevertheless, it is clear that the differences in the frequency 10 Campbell LJ, Fidler C, Eagleton H, Peniket A, Kusec R, Gal S et al. of UPDs between patients and controls are significant. For hTERT, the catalytic component of telomerase, is downregulated in example, the chromosome 8 UPDs are far more common in the the haematopoietic stem cells of patients with chronic myeloid patient group than in controls. It should also be noted that this leukaemia. Leukemia 2006; 20: 671–679. 11 Ahuja H, Bar-Eli M, Advani SH, Benchimol S, Cline MJ. Alterations particular study more closely approximates to the natural history in the p53 gene and the clonal evolution of the blast crisis of of disease progression in CML than a contemporaneous study chronic myelocytic leukemia. Proc Natl Acad Sci USA 1989; 86: would. The evolution of CML in the present era includes 6783–6787. elements possibly not relevant in the pre-imatinib era such as 12 Calabretta B, Perrotti D. The biology of CML blast crisis. Blood kinase domain mutations of BCR-ABL, but nonetheless it is 2004; 103: 4010–4022. always important to try and establish the natural pattern of 13 Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J et al. disease progression. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 2008; 453: 110–114. To our knowledge this study represents the first SNP array 14 Wang L, Pearson K, Pillitteri L, Ferguson JE, Clark RE. Serial analysis of CML yet reported. We have identified several monitoring of BCR-ABL by peripheral blood real-time polymerase recurrent regions of UPD in patients with CML, which may be chain reaction predicts the marrow cytogenetic response to of pathogenetic importance and may underlie the clinical imatinib mesylate in chronic myeloid leukaemia. Br J Haematol heterogeneity observed in this disorder. Our demonstration of 2002; 118: 771–777. frequent mutation of ASXL1, a putative tumor suppressor gene, 15 Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, likely represents an important new molecular abnormality in Kaeda J et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations CML. We have also shown that BC CML is associated with an for harmonizing current methodology for detecting BCR-ABL increase in CNC. In particular, this study has demonstrated transcripts and kinase domain mutations and for expressing results. overrepresentation of genomic aberrations (recurrent UPD and Blood 2006; 108: 28–37. copy number gain) mapping to chromosome 8 in CML. The role 16 Wang L, Fidler C, Nadig N, Giagounidis A, Della Porta MG, of chromosome 8 in CML deserves further study, and our study Malcovati L et al. Genome-wide analysis of copy number changes provides a starting point for a range of future investigations. and in with del(5q) using high-density single nucleotide polymorphism arrays. Haematologica 2008; 93: 994–1000. Conflict of interest 17 Fidler C, Watkins F, Bowen DT, Littlewood TJ, Wainscoat JS, Boultwood J. NRAS, FLT3 and TP53 mutations in patients with myelodysplastic syndrome and a del(5q). Haematologica 2004; The authors declare no conflict of interest. 89: 865–866. 18 Carbuccia N, Murati A, Trouplin V, Brecqueville M, Adelaide J, Rey J et al. Mutations of ASXL1 gene in myeloproliferative Acknowledgements neoplasms. Leukemia 2009; 23: 2183–2186. 19 Gelsi-Boyer V, Trouplin V, Adelaide J, Bonansea J, Cervera N, This work was supported by the Kay Kendall Leukaemia Fund Carbuccia N et al. Mutations of polycomb-associated gene ASXL1 and by Leukaemia Research UK. in myelodysplastic syndromes and chronic myelomonocytic leukaemia. 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