Leukemia (2009) 23, 85–94 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Genome profiling of acute myelomonocytic leukemia: alteration of the MYB locus in MYST3-linked cases A Murati1,13, C Gervais2,13, N Carbuccia1,13, P Finetti1, N Cervera1, J Ade´laı¨de1, S Struski2, E Lippert3, F Mugneret4, I Tigaud5, D Penther6, C Bastard6, B Poppe7, F Speleman7, L Baranger8, I Luquet9, P Cornillet-Lefebvre9, N Nadal10, F Nguyen-Khac11, CPe´rot12, S Olschwang1, F Bertucci1, M Chaffanet1, M Lessard2, M-J Mozziconacci1 and D Birnbaum1 on behalf of the Groupe Francophone de Cytoge´ne´tique He´matologique (GFCH) 1De´partement d’Oncologie Mole´culaire, Centre de Recherche en Cance´rologie de Marseille, Institut Paoli-Calmettes, Marseille, France; 2Laboratoire d’He´matologie, CHU de Hautepierre, Strasbourg, France; 3Laboratoire d’He´matologie, Hoˆpital Cardiologique-Haut Le´veˆque, Bordeaux, France; 4Laboratoire de Cytoge´ne´tique, CHU du Bocage, Dijon, France; 5Laboratoire d’He´matologie, CHU Lyon Sud, Lyon, France; 6De´partement de Ge´ne´tique, Centre Henri Becquerel, Rouen, France; 7Centre de Ge´ne´tique Me´dicale, Ghent, Belgique; 8Laboratoire de Ge´ne´tique, CHU d’Angers, Angers, France; 9Service de Ge´ne´tique, Hoˆpital Maison Blanche, Reims, France; 10Laboratoire d’He´matologie, Hoˆpital Nord, Saint-Etienne, France; 11Laboratoire de Cytoge´ne´tique He´matologique, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France and 12Laboratoire de Cytoge´ne´tique Onco-He´matologique, Hoˆpital Saint-Antoine, Paris, France The t(8;16)(p11;p13) is a rare translocation involved in de novo NCOA3 at 20q12.6 MYST3-linked acute myeloid leukemias and therapy-related myelomonocytic and monocytic acute (AMLs) share specific features, such as frequent extramedullary leukemia. It fuses two genes encoding histone acetyltrans- involvement, disseminated intravascular coagulation, erythro- ferases (HATs), MYST3 located at 8p11 to CREBBP located at 7 16p13. Variant translocations involve other HAT-encoding phagocytosis and a poor prognosis. They are often secondary, 8,9 genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked therapy-related AMLs. Because of its rarity, the molecular acute myeloid leukemias (AMLs) share specific clinical and biology of the MYST3-linked AMLs remains poorly understood. biological features and a poor prognosis. Because of its rarity, A study of three cases using DNA microarrays has shown that the molecular biology of MYST3-linked AMLs remains poorly they have a distinct gene expression profile compared with other understood. We have established the genome and gene 10 expression profiles of a multicentric series of 61 M4/M5 AMLs AMLs. including 18 MYST3-linked AMLs by using array comparative Here, we have established the genome and/or gene expres- genome hybridization (aCGH) (n ¼ 52) and DNA microarrays sion profiles of a series of 61 M4/M5 AMLs including MYST3- (n ¼ 44), respectively. We show that M4/5 AMLs have a variety of linked AMLs and variants by using array comparative genome rare genomic alterations. One alteration, a gain of the MYB hybridization (aCGH) and DNA microarrays, respectively. We locus, was found recurrently and only in the MYST3-linked show that MYST3-linked AMLs have specific gene alterations AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and gene expression profiles within the M4/M5 group. and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor. Leukemia (2009) 23, 85–94; doi:10.1038/leu.2008.257; Patients and methods published online 25 September 2008 Keywords: array-CGH; acute myeloid leukemia; gene expression Patients profiling; MYB; MYST3; t(8;16) We collected a series of 53 bone marrow (BM) and eight peripheral blood samples from 61 patients at the time of diagnosis for primary or secondary AML. The characteristics of the patients and samples are summarized in Supplementary Table 1. According to FAB classification11 and World Health Introduction Organization12 criteria, the panel comprised 16 M4, 44 M5, and one case of AML with no more data available. Some cases have The t(8;16)(p11;p13) is a rare translocation involved in de novo been previously reported in detail, namely M45-1, M45-3, M45- and therapy-related myelomonocytic and monocytic acute 4, M45-5, M45-7, M45-11, M45-13, M45-16, M45-19, M45-20, leukemia (French-American-British classification (FAB) AML- M45-21, M45-23, M45-26,7 M45-052,13 M45-031,14 M45-0326 M4, M5a and M5b). It fuses two genes encoding histone M45-062 and M45-063.4 Three BM samples were used as acetyltransferases (HAT), MYST3 (also called MOZ) located at 1–3 normal controls (normal bone marrow (NBM)). They were 8p11 to CREBBP (also called CBP) located at 16p13. Variant collected from breast cancer patients without micrometastases. translocations involve other HAT-encoding genes, such as 4 5 The aCGH study was carried out on 52 of the 61 samples, EP300 at 22q13, MYST4 at 10q22, NCOA2 at 8q13 or including 18 MYST3-linked AMLs, 16 AMLs with normal karyotype, nine AMLs with 11q23 (mixed lineage leukemia Correspondence: Dr D Birnbaum, Centre de Recherche en Cance´rologie (MLL)) abnormalities, four AMLs with inv(16; CBF-b-MYH11 de Marseille, UMR891 Inserm, 27 Bd. Leı¨ Roure, 13009 Marseille, fusion), one AML with t(8;21) (RUNX1-RUNX1T1 aka AML1- France. ETO fusion) and four AMLs with trisomy 8. E-mail: [email protected] 13These authors contributed equally to this work. A total of 44 M4/M5 cases with good quality RNA were Received 6 May 2008; revised 1 August 2008; accepted 21 August selected for global gene expression profile analysis, including 2008; published online 25 September 2008 eight MYST3-linked AML and 36 other samples comprising 18 MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 86 cases with normal karyotype, six with 11q23 abnormalities, four Before hierarchical clustering, filtered data were log2 trans- with inv(16), two with trisomy 8 and six with various karyotypes. formed and submitted to the cluster program using the median- Of the 61 cases analyzed by aCGH and/or gene expression centered data on genes, Pearson correlation as similarity metric profile, mutations of nucleophosmin 1 (NPM1) were found in 11 and centroid linkage clustering. Results were displayed using cases (mostly in AMLs with normal karyotype, as expected), and TreeView program. To identify and rank genes discriminating an internal tandem duplication and mutation of the FLT3 gene in eight cases of MYST3-linked and 36 M4/M5 AMLs, a supervised four and six cases, respectively (Table 1). analysis was applied to the 26 376 genes/ESTs. A discriminating score (DS) was calculated for each gene. DS ¼ (M1ÀM2)/ (S1ÀS2) where M1 and S1, respectively, represent mean and Nucleic acids extraction s.d. of expression levels of the gene in MYST3-linked subgroup, Bone marrow aspirates or peripheral blood samples were and M2 and S2 in M4/M5 AMLs subgroup. Confidence levels collected from 61 patients at the time of diagnosis. Blasts and were estimated by 100 random permutations of samples. mononuclear cells were purified after density gradient centrifu- Functional processes and pathways were identified by using gation of BM aspiration or whole blood (cases M45-3, M45-4, Ingenuity software (Ingenuity Systems, Redwood City, CA, USA). M45-11, M45-16, M45-010, M45-035, M45-036 and M45- 037), and processed immediately or cryoconserved at À80 1C. High-quality total RNA and DNA was extracted by the Allprep Results DNA/RNA isolation kit (Qiagen, Germany) from blood or BM blast cells. RNA quality and purity were assessed with Agilent aCGH profiling Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Using genome-wide, high-density arrays, we established the NBM samples were processed similarly. aCGH profiles of 52 samples. Examples of profiles are shown in Figure 1 and results are summarized in Table 1. Three main types of profiles were observed. In 60% of the cases (33/52), the Array comparative genomic hybridization profile was said ‘normal-like’ because no alteration was Genomic imbalances were analyzed by aCGH using 244K CGH detected. A second type of profiles showed gains or losses Microarrays (Hu-244A, Agilent Technologies, Massy, France) visible on the karyotype and affecting large regions of the following a previously described protocol.15 Scanning was genome, such as trisomy 8, gains of 11q23-qter (Figure 1a), performed with Agilent Autofocus Dynamic Scanner (G2565BA, deletions of 12p (including the CDKN1B locus; Figure 1b), Agilent Technologies). Data analysis was performed as des- rearrangement of chromosome 13 (Figure 1c), gain of 3q26-qter cribed previously15 and visualized with CGH analytics 3.4 (Figure 1d) or 6q22–25 (case 4). Finally, few profiles showed software (Agilent Technologies). Extraction data (log2 ratio) was rare and limited gains or losses that affected few or single genes, performed from CGH analytics, whereas normalized and filtered such as deletions encompassing CTNNA1 and CXXC5 at 5q31 log2 ratio were obtained from ‘feature extraction’ software (case 011, normal karyotype, Figure 1e), reminiscent of a (Agilent Technologies). Data generated by probes mapped to 5q-syndrome,18 CXXC4 at 4q24 (case 23, MYST3, Figure 1f), X and Y chromosomes were eliminated. The final data set IKZF1/Ikaros at 7p12 (case 055) or a cluster of chemokines at contained 225 388 unique probes covering 22 509 genes and 17q12 (case 010). Among these alterations, a gain of MYB at intergenic part following the hg17 human genome mapping. 6q23 was detected in seven MYST3-linked AML samples Copy number changes were characterized as reported pre- (Figure 2). In six cases, only MYB and its neighbor gene AHI1 viously.15 Results were displayed using TreeView program.