Genomic Gain at 6P21: a New Cryptic Molecular Rearrangement in Secondary Myelodysplastic Syndrome and Acute Myeloid Leukemia
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Leukemia (2006) 20, 958–964 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE Genomic gain at 6p21: a new cryptic molecular rearrangement in secondary myelodysplastic syndrome and acute myeloid leukemia R La Starza1, A Aventin2, C Matteucci1, B Crescenzi1, S Romoli1, N Testoni3, V Pierini1, S Ciolli4, C Sambani5, A Locasciulli6, E Di Bona7, M Lafage-Pochitaloff8, MF Martelli1, P Marynen9 and C Mecucci1 1Hematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy; 2Servei de Hematologia, Hospital De La Santa Creu I Sant Pau, Barcelona, Spain; 3‘Istituto Seragnoli’, Hospital S Orsola, Bologna, Italy; 4Hematology Unit, University of Firenze, Firenze, Italy; 5Laboratory of Health Physics and Environmental Hygiene, NCSR ‘Demokritos’, Athens, Greece; 6Hematology, Hospital ‘S Camillo’ of Rome, Rome, Italy; 7Hematology, Hospital ‘S Bortolo’ Vicenza, Vicenza, Italy; 8Laboratoire de Biopathologie, Institut Paoli-Calmettes, INSERM U119, Marseille, France and 9Center for Human Genetics and Flanders Interuniversity Institute for Biotechnology (VIB), University of Leuven, Campus Gasthuisberg, Leuven, Belgium Fluorescence in situ hybridization and comparative genomic ter aberrantly activates the cyclin D3, which is overexpressed;5,6 hybridization characterized 6p rearrangements in eight primary in MM with t(6;14)(p25;q32), the IgH gene juxtaposes to MUM/ and in 10 secondary myeloid disorders (including one patient IRF4.7 with Fanconi anemia) and found different molecular lesions in In secondary myelodysplastic syndrome (MDS) and AML, 6p each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different break- rearrangements account for less than 2% of cytogenetic 8 points along the 6p arm. Reciprocal translocations were found abnormalities. Although mainly found in complex karyotypes, in seven. In the 10 patients with secondary acute myeloid which also bear À5/del(5)(q) and/or À7/del(7)(q), rare cases of leukemia/myelodysplastic syndrome (AML/MDS), the short arm isolated 6p rearrangements have been observed.9–11 Deletions, of chromosome 6 was involved in unbalanced translocations in balanced and unbalanced translocations, insertions and dupli- 7. The other three patients showed full or partial trisomy of the cations have been described with a der(1)t(1;6) change as the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two 12–14 patients). In 5/7 patients with unbalanced translocations, DNA only recurrent abnormality. No specific correlations have sequences were overrepresented at band 6p21 as either cryptic been found with previous exposure because 6p rearrangements duplications (three patients) or cryptic low-copy gains (two have been linked not only to chemo- and/or radiotherapy, but patients). In the eight patients with cytogenetic or cryptic 6p also to different environmental agents.9,10 gains, we identified a common overrepresented region extend- We used a molecular cytogenetic approach to characterize 6p ing for 5–6 megabases from the TNF gene to the ETV-7 gene. 6p rearrangements in primary and secondary myeloid disorders and abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p found different molecular lesions in each clinical subgroup. gains are major pathogenetic events arising from acquired and/ or congenital genomic instability. Leukemia (2006) 20, 958–964. doi:10.1038/sj.leu.2404208; Patients, materials and methods published online 13 April 2006 Keywords: primary and secondary myeloid malignancies; 6p Patients rearrangements; genomic gain Inclusion criteria were diagnosis of a myeloid disorder, a 6p rearrangement and availability of fixed cells for fluorescence in situ hybridization (FISH) studies. Patients were retrieved from the Departments of Hematology, Universities of Florence, Bologna, Perugia, the ‘S Bortolo’ Hospital in Vicenza, and the Introduction ‘San Camillo’ Hospital in Rome, Italy; the Servei of Hemato- logia, Hospital Sant Pau, Barcelona, Spain; the Health Physics In hematological malignancies, rearrangements at the short arm and Environmental Hygiene Lab, NCSR Demokritos, Athens, of chromosome 6 are characterized by heterogeneous chromo- Greece; the Laboratoire de Biopathologie, Institut Paoli-Calm- some abnormalities, breakpoints and partner chromosomes. In ettes, INSERM U119, Marseille, France. primary disorders, specific clinical–hematological and genetic entities have been identified. In acute myeloid leukemia (AML), Cytogenetics the t(6;9)(p23;q34)/DEK-CAN is associated with bone marrow Bone marrow cells were cultured for 24–48 h. Metaphases were dysplasia and basophilia, a high incidence of FLT3 mutations G-banded with Wright stain, and karyotypes were described and unfavorable prognosis;1,2 in B-cell non-Hodgkin’s lympho- according to the International System for Human Cytogenetic ma (B-NHL), the H4 or SRP20 gene deregulates the BCL-6 Nomenclature.15 oncogene as a result of t(3;6)(q26;p21);3,4 in B-NHL and multiple myeloma (MM) with t(6;14)(p21;q32), the IgH promo- Fluorescence in situ hybridization Correspondence: Professor C Mecucci, Hematology and Bone Marrow Metaphase FISH was performed as described16 with a panel of Transplantation Unit, Policlinico Monteluce, via Brunamonti 51, 6p DNA clones ordered from band p25 to band p12 as shown in 06122 Perugia, Italy. E-mail: [email protected] Table 1. To fully characterize 6p unbalanced translocations, Received 11 October 2005; revised 13 February 2006; accepted 27 whole chromosome painting (WCP) for chromosomes 1, 6, 8, February 2006; published online 13 April 2006 16, 18, 19 and for the short arm of 20, probes for the alpha 6p changes in myeloid disorders R La Starza et al 959 Table 1 Breakpoint restriction in three primaryK and in seven trisomy/splitting (3p14/FHIT 1.5%, 5q33/CSF1R 0.5%, 7q31/ secondary* AML/MDS D7S486 0.5%, 12p13/ETV6 2.5%, 13q14/D13S25 1%, 17p13/ TP53 2%, 21q22/AML1 2.5%) were set at the upper limits Band DNA clones Loci/genes for false positive results as established by our laboratory standard.16,17 Telomere p25 RP3-409kg CHLC.GGAT14C06 p24 RP1-233G22 AFMa272zb5 p23 RP3-511E16 AFMa296zb1 KK Comparative genomic hybridization RP3-366E2 AFM337wg9 KK Comparative genomic hybridization (CGH) was performed as 18 p22 RP1-135L22 WI-5592 already reported. Chromosomal regions were considered RP1-73M23 SSADH * overrepresented if the corresponding green/red ratio exceeded RP3-501N12 WI-4509 1.17 and underrepresented if the ratio was below 0.83. Negative RP1-45P21 StSG2643 control thresholds were established on profiles from hybridiza- p21 RP1-153G14 ZNF184 RP1-15D7 WI-2632 * tion of two differently labeled DNA samples from healthy RP1-97D16 TRMI1 donors. FISH and CGH analyses were carried out using a RP3-408B2O OR2W6P, OR2B6, 0R2W4P * fluorescence microscope (Provis, Olympus, Milan, Italy) RP1-313I6 ZNF165, OR2W2P, equipped with a cooled CCD camera (Sensys, Photometrics) OR2B7P, 0R2B8P run by PathVysion softwares (Vysis, Stuttgart, Germany). RP1-265C24 ZNF435, ZNF192 RP11-424I5 C60RF194 * RP5-874C20 KIAA0426 Results RP5- GPX5 1186N24 RP11-60E24 D6S248 Patients Chromosome6 RP1-111M5 RFP Tables 2 and 3 report, respectively, clinical, hematological and RP1-88J8 HS6M1-15, 0R2W1 * cytogenetic data in 10 patients with secondary and eight with RP5-974I11 HS6M1-6, 0R2U1P primary AML/MDS. cosmid Cah5 TNF RP1-22011 HSET K** RP1-160J11 KNLS2 Cytogenetics RP1-28OE11 StSG28858 K RP1-99J17 HMGA1 In secondary AML/MDS, 6p rearrangements were isolated in RP3-329A5 ZNF76 four cases and included in complex karyotypes in six. The 6p RP1-109F14 FANCE changes included i(6)(p10) (patient 1), 6p duplication (patients 2 RP1-124L9 WI-10190, WI-73111 and 3) and unbalanced translocations (patients 4–10) with RP1-162J16 WI-6620, R12494 diverse chromosome partners or unidentified material (Table 2). RP1-50J22 ETV7 In primary AML/MDS 6p abnormalities were isolated in six RP5-1043E3 StSG22477 RP5-904F15 CBF-A cases, associated with one numerical change in 1 and included RP5-1106P1 CCND3 in complex karyotype in 1. Balanced translocations with diverse RP5-895C5 WI-8795 K chromosome partners were present in patients 11–17. Patient 18 RP3-374I13 RUNX2 had add(6)(p) (Table 3). p12 RP3-381E2 AFM123xe1 K RP4-753D5 TFAP2B Centromere Metaphase fluorescence in situ hybridization and Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplastic multi-color FISH syndrome; TNF, tomor necrosis factor. Secondary acute myeloid leukemia/myelodysplastic syndrome. In patient 1, all 6p probes were present in three copies as expected: one on normal 6 and two on each arm of i(6)(p10) (Figure 1a). In patients 2 and 3 with dup(6)(p), both satellite region of chromosomes 1/5/19 (D1Z7/D5Z2/D19Z3) duplications fell within band p21 and extended, respectively, to and chromosome 6 (D6Z1) were used (Oncor, Appligene include clones RP1-153G14 to RP3-381E2 and cosmid CAH5 to Gaithersburg, MD, USA; Vysis, Stuttgart, Germany; ListarFish, RP4-753D5 (Figure 1b). Breakpoints of 6/7 unbalanced trans- Milan, Italy). Multi-color FISH (M-FISH) experiments with the locations (patients 4–8 and 10) clustered at band p21; the other 24XCyte human multi-color FISH probe kit (MetaSystem, Zeiss, breakpoint in patient 9 was at band p23 (Table 1). Altlussheim, Germany) were performed in patient 8 (Table 2). In patients 4, 6 and 7, with unbalanced translocations, FISH Five to ten abnormal metaphases were evaluated by FISH