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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 JRG FRICKE2, KLAUS HFFKEN2, ANITA HELLER1, ANJA WEISE1 and THOMAS LIEHR1 1Institut fr 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 disease with respect to clinical prognosis and acquired chromosomal aberrations. After routine banding cytogenetic analysis 45% of AML patients show a normal karyotype (NK-AML). For a better understanding of development and progression in AML, it is important to find markers which could be primary genetic aberrations. Therefore, in this study 31 patients with NK-AML were analyzed by new high resolution molecular cytogenetic approaches. A combination of multitude multicolor banding and metaphase microdissection-based comparative genomic hybridization revealed deletions of the subtelomeric regions in 6% of the studied cases. According to these results, locus-specific probes for the subtelomeric regions of chromosomes 5, 9, 11, 12 and 13 were applied on 22 of the studied 31 NK-AML cases. Surprisingly, 50% of them showed deletions or duplications. These aberrations occurred in the in vitro proliferating as well as in the non-proliferating cells. Metaanalysis of the aberrant regions revealed that they often include genes known to be associated with tumors, e.g. RASA3 on chromosome 13. These results implicate that aberrations in the subtelomeric regions of NK-AML occur quite often and may be considered as primary genetic changes, and should not be neglected in future diagnostic approaches. Introduction The occurrence of acute myeloid leukemia (AML) is based on acquired genetic alterations, resulting in an accumulation of _________________________________________ Correspondence to: Dr Thomas Liehr, Institut fr Humangenetik und Anthropologie, Postfach, D-07740 Jena, Germany E-mail: i8lith@mti.uni-jena.de Key words: acute myeloid leukemia, microdissection-based comparative genomic hybridization, multitude multicolor banding, subtelomere hematopoietic progenitor cells. At the time of diagnosis ~55% of the patients show at least one chromosomal aberration in their bone marrow blasts where >200 different aberrations are described until now (1). However, 45% of the patients with de novo AML still show a cytogenetic normal karyotype (NK-AML) (2,3). Recent studies gave evidence that in addition to chromosomal aberrations genetic changes can occur. These include e.g. mutations in the FLT3 (4), the NPM1 (5,6) or the MLL gene (7,8). Also acquired isodisomy were described (9-11). The latter are regions of homozygosity of which the importance for the development of AML has not been sufficiently clarified yet. Patients with NK-AML are classified in the intermediate prognostic group, which allows no precise prediction of the outcome or an individual therapy for each patient (12,13). Besides the age of the patients, cytogenetic aberrations are important for therapy planning and prognosis. However, bone marrow chromosomes show a poor resolution and morphology and therefore banding cytogenetic analysis of such chromosomes is performed on band levels between 200-300 bands per haploid karyotype. Therefore, it is a well known problem in leukemia cytogenetics that cryptic chromosomal aberrations can easily be disregarded. The introduction of molecular cytogenetics, especially of multicolor fluorescence in situ hybridization (FISH) leads to better results and identification of previously cryptic chromosomal aberrations in leukemia cytogenetics and furthermore make interphase nuclei accessible (1,2). Compared 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 NK-AML (19,20) showed that these approaches are suited to pick up cryptic chromosomal rearrangements in 5-8% of the cases. In the present study, we analyzed 31 cases with NK-AML with a new, previously not applied combination of FISH methods. We used two genome-wide FISH approaches, one interphase, i.e. microdissection-based comparative genomic hybridization (micro-CGH) (21) and one metaphase cell 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 diethylaminocoumarine) 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) (22). Furthermore, the results were confirmed by locus-specific probes, which were used to analyze the proliferating and the non-proliferating cells of the studied cases. According to the mMCB and micro-CGH results, subtelomeric probe sets were subsequently applied on 22 of the 31 NK-AML cases. Materials and methods Patients. Thirty-one patients with de novo AML or AML derived from MDS (myelodysplastic syndromes) presenting at diagnosis a normal karyotype after banding cytogenetics, were included in the present study (for details see Table I). The age range was 27 to 78 with a median of 55 years and a male-to-female ratio of 2:4. The number of blasts varied from 10-100%. According to the French-American-British Classification, cases from all AML-subtypes except AML-M7 were present. Banding cytogenetics. Cytogenetic studies applying GTGbanding was performed according to standard protocols (23). Two hundred to 350 bands per haploid karyotype and at least 15 metaphases were analyzed per patient. As aforementioned GTG-banding revealed a karyotype of 46,XX or 46,XY, respectively. was performed according to the modifications, as previously described (25). For mMCB and for micro-CGH 15-20 metaphases were evaluated. Locus-specific DNA probes from the subtelomeric region of chromosomes 5, 9, 11, 12 and 13 were purchased as BAC clones from the Children's Hospital Oakland Research Institute (CHORI), Oakland, CA, USA. Plasmid DNA from BAC clones was isolated, amplified and labeled by PCR and subsequently used for FISH, as described (26). In order to have a probe set that is also reliable in interphase FISH at least three BAC clones from the same chromosomal region were combined to a probe-set and labeled in the same color (Table II). For chromosome 5 only one BAC clone from the subtelomeric region was available. For each FISH probe 15-20 metaphases and 150 interphase nuclei were analyzed. The cut-off level for the analyses of interphase nuclei was determined as 5%. Meta-analysis. The regions identified to be deleted in the aforementioned FISH-experiments were analyzed for their genetic content using database of NCBI (36.2) (http://www. ncbi.nlm.nih.gov) and database of genetic and cytogenetic in haematology and oncology (http://atlasgeneticsoncology. org//index.html). Results Fluorescence in situ hybridization (FISH). FISH was done according to standard protocols described in Liehr et al (26). The mMCB probe set was used as published in (22). These probes were further specified later (24). Microdissectionbased comparative genomic hybridization (micro-CGH) (21) Micro-CGH. In 17 of 31 NK-AML cases, sufficient amount of suspension for analysis with micro-CGH was available. In two cases gain of copy numbers for chromosome 19 and in another case loss of copy number for parts of chromosome 17 and whole chromosome 19 were observed. Application of further 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) 3 AML-M2 Male 71 84 + + del(9)(p24.3)(33%) del(9)(p24.3)( 6,75%) del(9)(p24.3) INTERNATIONAL JOURNAL OF ONCOLOGY 34: 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. (no data for 11p, 13q) 13 AML-M5b Female 48 10 + + del(9)(p24.3)(25%), no aberr. del(9)(p24.3), del(13)(q34)(33%) del(13)(q34) 14 AML-M2 Male 78 82 + + del(11)(p15.5)(12%) del(12)(p13.33)(10,47%) del(12)(p13.33) 15 AML from MDS Male 65 20 + + del(5)(q35.3)(15%), del(5)(q35.3)(5%), del(5)(q35.3), del(11)(p15.5)(21%) del(11)(p15.5)(13%) del(11)(p15.5) 16 AML-M2 Male 48 30-35 + + del(11)(p15.5)(15%), del(11)(p15.5)(13%) del(11)(p15.5) del(11)(q25)(9%) 17 AML-M1 Male 54 81.3 + - - del(12)(p13.33)(15,13%) del(12)(p13.33) 18 AML-M1 Female 66 100 - + - -- 19 AML-M1 Male 70 60 -+ - del(12)(p13.33)(5,03%) no aberr. 20 AML-M0 Female 62 - -+ - -- 21 AML-M1 Male 38 - -+ - del(13)(q34)(5,06%) no aberr. 419 22 AML-M4 Male 65 - -+ - -- 23 AML-M4 Male 58 90 -+ - no aberr. no aberr. 420 GROSS et al: ACUTE MYELOID LEUKEMIA WITH NORMAL GTG-BANDING KARYOTYPE For the subtelomeric probes only those results detecting aberrations in interphase and/or metaphase are shown and summarized. No abber, no aberrations; `+', analysis was done and `-', no analysis. -- -- -- dup(12)(p13.33) -- del(2)(q37) -- dup(11)(q25) dup(12)(p13.33)(7,10%) del(13)(q34) (5,13%) dup(11)(q25)(13,45%) del(11)(q25)(5,26%), - - - (no data for 5q) no aberr. - (no data for 5q, 9q) del(2)(q37)(8%) - - -+ -+ - - - + -+ -+ Results FISH Interphase FISH Metaphase FISH - - - - Gender Age % Blasts micro-CGH mMCB Figure 2. Hybridization result of a subtelomeric probe contigue (subtel 13q) with a whole chromosome paint for chromosome 13 (wcp 13) in case 11. The BAC contigue were labeled in TexasRed (TR), the wcp probe in Spectrum Green (SG). Due to the deletion the signal of the BAC clones is visible only on one of the homologue chromosomes. On the left hand of the FISH figures the inverted DAPI-banding (inv. DAPI) result is shown. FISH probes (mMCB and subtelomere 19 specific probes) could not confirm these results. Thus, micro-CGH did not pick up any aberrations in the 17 studied cases. mMCB. Metaphases suited for analysis by mMCB were obtained in 26 of 31 NK-AML cases. The mMCB probe-set revealed aberrations in 2 of the 26 studied cases which were confirmed by additional FISH probes. In case 5, three different cell clones were detectable: 46,XY,del(5)(p15.5)[11]/46,XY, del(5)(q35)[4]/46,XY[13]. In case 26, a deletion in the subtelomeric region in 2q could be described. This deletion occurred in 8% of the metaphases. In the remaining analyzed NK-AML cases no aberrations were detectable by mMCB (Fig. 1). Subtelomeric regions. In 22 of 31 cases, sufficient amount of suspension was available for the analysis of subtelomeric regions by interphase and metaphase FISH. In 50% of the analyzed 22 NK-AML cases aberrations in the subtelomeric regions were detectable, including deletions and duplications of the appropriate regions (Table I). For each case the number of detected cell clones varied from one to three. The aberrations were found in the proliferating cells, in the nonproliferating cells or in both cell types. Fig. 2 shows a deletion on chromosome 13 detected in case number 11. Discussion In the present study, 17 NK-AML cases were analyzed by micro-CGH and no aberrations could be described. These results are comparable to others using metaphase-based CGH for analyzing NK-AML (19-20,27). However, as already described (25) micro-CGH enables the direct analysis of tumor cells which is more informative than conventional metaphase-based CGH. Thus, no major copy number changes can be expected in NK-AML detectable by metaphase CGH. The false-positive/false negative copy number changes affected especially chromosome 19 - a region known to be not reliably informative in CGH-studies (25,28). - Female 37 AML-M6 Female 69 36 Male 43 100 Male 44 80-90 50 Female 41 AML-M0 Male 49 - AML-M2 Male 27 - 88 AML from MDS Female 61 AML-M3 AML-M4 AML-M1 AML-M4 AML-Subtype Table I. Continued. 31 30 29 28 27 26 25 24 Case INTERNATIONAL JOURNAL OF ONCOLOGY 34: 417-423, 2009 421 Table II. BAC clones from the subtelomeric regions of the chromosomes 5, 9, 11, 12 and 13 used for interphase and metaphase FISH, based on NCBI build 36.2. Chromosome Band BAC clone Accession code Start kb End kb 5 q35.3 RP11-240G13 - 180.512 180.626 9 p34.3 RP11-393D13 BH141105.1 236 427 9 p34.3 RP11-1021N5 AQ593522.1 150 333 9 p34.3 2241C11 (Cosmid) - -- 11 p15.5 RP11-1021K7 AQ697676.1 668 668 11 p15.5 RP11-51L17 AQ052363.1 764 942 11 p15.5 RP11-496F2 AZ695077 668 872 11 p15.5 2209A2 (Cosmid) - -- 11 q25 RP11-186N3 AQ422456.1 134.184 134.356 11 q25 RP11-1077G24 AQ744064.1 133.998 134.194 11 q25 RP11-265F9 AQ484019.1 134.272 134.411 11 q25 RP11-267D5 AQ487857.1 133.964 134.130 12 p13.3 RP11-519B13 AZ916624.1 28 214 12 p13.3 RP11-12H17 B75812.1 345 484 12 p13.3 RP11-367L11 AQ529919.1 366 537 13 q34 RP11-63L17 AQ200393.1 113.782 113.954 13 q34 RP11-960N24 AQ739689.1 113.904 114.103 13 q34 RP11-450H16 AZ773446.1 113.958 114.117 BAC, bacterial artificial chromosome; Band, chromosomal subband; kb, kilobasepairs and `-', not available. The application of mMCB could identify deletions in 6% of the 31 NK-AML cases. The proof of principle for mMCB to detect cryptic aberrations was done on cases with MDS (22) and acute lymphoblastic leukemia (29). The FISH method mMCB is also useful to analyze mixtures of cells such as in AML cases where the studied cells comprise healthy and leukemic cells (unpublished data). In previous multicolor FISH studies whole chromosome paints as probes were applied on NK-AML cases (14-18). Surprisingly, the results from the present study are similar to those in the published studies, even though the FISH-banding approach (for review see ref. 30) mMCB allows the detection of aberrations which are not detectable by other cytogenetic methods. This means, that in NK-AML intrachromosomal changes are sparse. A limitation of all multicolor FISH approaches using whole or partial chromosome paints as well as for CGH is that they do not provide reliable information on changes within the subtelomeric regions of the chromosomes. Therefore, in the present study locus-specific probes for the subtelomeric regions were applied. The analyzed regions were chosen according to chromosomes which are often involved in aberrations in AML (http://atlasgeneticsoncology. org//index.html). Cryptic aberrations in 50% of the 22 NKAML cases were found. These aberrations included deletions and duplications and occurred in the in vitro proliferating as well as the non-proliferating cells. The size of the deleted regions was at least 200 to 600 kb according to the BAC clones used for FISH. Database analysis of these regions showed that they include genes known to be associated with tumors. The deleted region on chromosome 13 has a size of at least 300 kb and contains the RASA3 gene. The product of this gene is a GTPase activating protein which stimulates the GTPase activity of RAS p21 (31). It is further known that RAS genes have a function as proto-oncogenes and are involved in a variety of tumors (32,33). The deleted regions are, excluding those on chromosomes 5 and 9, regions that contain published copy number variations (34,35). For the chromosomes 9 and 11 segmental duplications of the corresponding regions are reported, which are in general known to be predisposed for non-allelic homologues recombination (36). The deleted region on chromosome 12 is known to be often involved in rearrangements in AML (29,37-39). Our data have shown a correlation between deletion 12p and AML-subtype M2 which was also described before (40,41). The remaining aberrations occurred independently from the AML-subtype, age or percentage of blasts. Furthermore, aberrations were observed in the proliferating as well as in the non-proliferating cells with no correlation to the aberration. In conclusion, this is the first study using genome-wide FISH methods as well as locus-specific FISH probes to detect aberrations in NK-AML. In ~50% of the cases submicroscopic deletions could be described. 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