417-423 31/12/2008 08:43 Ì ™ÂÏ›‰·417 INTERNATIONAL JOURNAL OF ONCOLOGY 34: 417-423, 2009 417 Delineation of yet unknown cryptic subtelomere aberrations in 50% of acute myeloid leukemia with normal GTG-banding karyotype MADELEINE GROSS1, HASMIK MKRTCHYAN1, MELANIE GLASER1, HANS JÖRG FRICKE2, KLAUS HÖFFKEN2, ANITA HELLER1, ANJA WEISE1 and THOMAS LIEHR1 1Institut für Humangenetik und Anthropologie, Kollegiengasse 10, D-07743 Jena; 2Klinik fuer Innere Medizin II, Erlanger Allee 101, D-07747 Jena, Germany Received August 29, 2008; Accepted November 3, 2008 DOI: 10.3892/ijo_00000165 Abstract. Acute myeloid leukemia (AML) is a heterogeneous hematopoietic progenitor cells. At the time of diagnosis ~55% disease with respect to clinical prognosis and acquired of the patients show at least one chromosomal aberration in chromosomal aberrations. After routine banding cytogenetic their bone marrow blasts where >200 different aberrations are analysis 45% of AML patients show a normal karyotype described until now (1). However, 45% of the patients with (NK-AML). For a better understanding of development and de novo AML still show a cytogenetic normal karyotype progression in AML, it is important to find markers which (NK-AML) (2,3). Recent studies gave evidence that in could be primary genetic aberrations. Therefore, in this addition to chromosomal aberrations genetic changes can study 31 patients with NK-AML were analyzed by new occur. These include e.g. mutations in the FLT3 (4), the high resolution molecular cytogenetic approaches. A NPM1 (5,6) or the MLL gene (7,8). Also acquired isodisomy combination of multitude multicolor banding and metaphase were described (9-11). The latter are regions of homo- microdissection-based comparative genomic hybridization zygosity of which the importance for the development of revealed deletions of the subtelomeric regions in 6% of the AML has not been sufficiently clarified yet. studied cases. According to these results, locus-specific Patients with NK-AML are classified in the intermediate probes for the subtelomeric regions of chromosomes 5, 9, 11, prognostic group, which allows no precise prediction of the 12 and 13 were applied on 22 of the studied 31 NK-AML outcome or an individual therapy for each patient (12,13). cases. Surprisingly, 50% of them showed deletions or Besides the age of the patients, cytogenetic aberrations are duplications. These aberrations occurred in the in vitro important for therapy planning and prognosis. However, proliferating as well as in the non-proliferating cells. Meta- bone marrow chromosomes show a poor resolution and analysis of the aberrant regions revealed that they often morphology and therefore banding cytogenetic analysis of include genes known to be associated with tumors, e.g. RASA3 such chromosomes is performed on band levels between on chromosome 13. These results implicate that aberrations in 200-300 bands per haploid karyotype. Therefore, it is a well the subtelomeric regions of NK-AML occur quite often and known problem in leukemia cytogenetics that cryptic may be considered as primary genetic changes, and should chromosomal aberrations can easily be disregarded. not be neglected in future diagnostic approaches. The introduction of molecular cytogenetics, especially of multicolor fluorescence in situ hybridization (FISH) leads to Introduction better results and identification of previously cryptic chromosomal aberrations in leukemia cytogenetics and The occurrence of acute myeloid leukemia (AML) is based on furthermore make interphase nuclei accessible (1,2). Compared acquired genetic alterations, resulting in an accumulation of to whole genome approaches like array-based comparative genomic hybridization (array-CGH), a further advantage of FISH is the analysis of single cells. The latter allow also _________________________________________ the study of rare cell lines which are typically found in leukemia patients. Previous FISH studies (14-18) including metaphase comparative genomic hybridization (CGH) on Correspondence to: Dr Thomas Liehr, Institut für Humangenetik NK-AML (19,20) showed that these approaches are suited to und Anthropologie, Postfach, D-07740 Jena, Germany pick up cryptic chromosomal rearrangements in 5-8% of the E-mail: [email protected] cases. In the present study, we analyzed 31 cases with NK-AML Key words: acute myeloid leukemia, microdissection-based with a new, previously not applied combination of FISH comparative genomic hybridization, multitude multicolor banding, subtelomere methods. We used two genome-wide FISH approaches, one interphase, i.e. microdissection-based comparative genomic hybridization (micro-CGH) (21) and one metaphase cell 417-423 31/12/2008 08:43 Ì ™ÂÏ›‰·418 418 GROSS et al: ACUTE MYELOID LEUKEMIA WITH NORMAL GTG-BANDING KARYOTYPE Figure 1. mMCB result of case 28. In (a) the staining of the chromosomes in three of the five fluorochromomes (TexasRed, cyanine 5 and diethylamino- coumarine) and (b) the results are depicted in pseudo-colors (ISIS, Metasystems, Altlussheim, Germany). (c) Inverted DAPI banding of the same karyogram. In this case no aberrations were detected by mMCB. directed approach, i.e. multitude multicolor banding (mMCB) was performed according to the modifications, as previously (22). Furthermore, the results were confirmed by locus-specific described (25). For mMCB and for micro-CGH 15-20 probes, which were used to analyze the proliferating and metaphases were evaluated. the non-proliferating cells of the studied cases. According to Locus-specific DNA probes from the subtelomeric region the mMCB and micro-CGH results, subtelomeric probe of chromosomes 5, 9, 11, 12 and 13 were purchased as BAC sets were subsequently applied on 22 of the 31 NK-AML clones from the Children's Hospital Oakland Research Institute cases. (CHORI), Oakland, CA, USA. Plasmid DNA from BAC clones was isolated, amplified and labeled by PCR and Materials and methods subsequently used for FISH, as described (26). In order to have a probe set that is also reliable in interphase FISH at least Patients. Thirty-one patients with de novo AML or AML three BAC clones from the same chromosomal region were derived from MDS (myelodysplastic syndromes) presenting combined to a probe-set and labeled in the same color at diagnosis a normal karyotype after banding cytogenetics, (Table II). For chromosome 5 only one BAC clone from the were included in the present study (for details see Table I). subtelomeric region was available. For each FISH probe The age range was 27 to 78 with a median of 55 years and a 15-20 metaphases and 150 interphase nuclei were analyzed. male-to-female ratio of 2:4. The number of blasts varied The cut-off level for the analyses of interphase nuclei was from 10-100%. According to the French-American-British determined as 5%. Classification, cases from all AML-subtypes except AML-M7 were present. Meta-analysis. The regions identified to be deleted in the aforementioned FISH-experiments were analyzed for their Banding cytogenetics. Cytogenetic studies applying GTG- genetic content using database of NCBI (36.2) (http://www. banding was performed according to standard protocols (23). ncbi.nlm.nih.gov) and database of genetic and cytogenetic in Two hundred to 350 bands per haploid karyotype and at least haematology and oncology (http://atlasgeneticsoncology. 15 metaphases were analyzed per patient. As aforementioned org//index.html). GTG-banding revealed a karyotype of 46,XX or 46,XY, respectively. Results Fluorescence in situ hybridization (FISH). FISH was done Micro-CGH. In 17 of 31 NK-AML cases, sufficient amount of according to standard protocols described in Liehr et al (26). suspension for analysis with micro-CGH was available. In two The mMCB probe set was used as published in (22). These cases gain of copy numbers for chromosome 19 and in another probes were further specified later (24). Microdissection- case loss of copy number for parts of chromosome 17 and based comparative genomic hybridization (micro-CGH) (21) whole chromosome 19 were observed. Application of further 417-423 31/12/2008 08:43 Ì 417-423 31/12/200808:43Ì Table I. List of studied cases including the number of the case, the AML-subtype according to French-American-British Classification, gender, age, percentage of blasts and the applied molecular cytogenetic approaches (miro-CGH, mMCB). ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Subtelomeric FISH-Regions ––––––––––––––––––––––––––––––––––––––––––––––– Case AML-Subtype Gender Age % Blasts micro-CGH mMCB Metaphase FISH Interphase FISH Results FISH ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 1 AML-M6 Male 57 54 + + del(13)(q34)(11%) del(13)(q34)(8,18%) del(13)(q34) 2 AML-M2 Male 62 63.2 + + del(5)(q35.3)(10%) del(12)(p13.33)(6%) del(12)(p13.33) (no data for 5q) ™ÂÏ›‰·419 3 AML-M2 Male 71 84 + + del(9)(p24.3)(33%) del(9)(p24.3)( 6,75%) del(9)(p24.3) INTERNATIONAL JOURNALOFONCOLOGY34:417-423,2009 4 AML-M1 Male 36 88.5 + + - no aberr. (no data for 9p) no aberr. 5 AML-M6 Male 62 80-90 + + del(5)(p15.5)(39%), - del(5)(p15.5), del(5)(q35)(14%) del(5)(q35) 6 AML-M1 Male 68 20-25 + + del(5)(q35.3)(17%) no aberr. (no data for 13q) del(5)(q35.3) 7 AML-M4 Female 53 100 + + no aberr. no aberr. no aberr. 8 AML-M2 Female 74 39 + + del(12)(p13.33)(14%), del(9)(p24.3)(7%), del(9)(p24.3), dup(12)(p13.33)(24%) dup(12)(p13.33)(13%) del(12)(p13.33), dup(12)(p13.33) 9 AML-M2 Male 43 28 + + del(5)(q35.3)(9%), no aberr. del(5)(q35.3), del(12)(p13.33)(7%) del(12)(p13.33) 10 AML from MDS Male 55 16 + - - - - 11 AML-M4 Male 64 91 + + del(13)(q34)(63%) del(11)(q25)(16,37%), (no data for 5q, 11p, 11q) del(13)(q34) (17,12%) del(11)(q25), (no data for 5q) del(13)(q34) 12 AML from MDS Male 34 13.6 + + no aberr. no aberr. no aberr.