acute myeloid leukemia
Gomes e Silva, F.P.
Citation
Gomes e Silva, F. P. (2009, March 3). Genome wide characterization of minimally differentiated acute myeloid leukemia. Retrieved from https://hdl.handle.net/1887/13569
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13569
Note: To cite this publication please use the final published version (if applicable).
5
Fernando PG Silva, Bruno Morolli, Clelia T Storlazzi, Antonella Zagaria, Luciana Impera, Binie Klein, Harry Vrieling, Hanneke C Kluin-Nelemans &
Micheline Giphart-Gassler
Published as a letter in:
Leukemia 2008;22:1639-1643
differentiated acute myeloid leukemia
Abstract
Chromosome 12 short arm abnormalities are frequent occurrences in AML and take mainly two forms: deletions and translocations. The high frequency of deletions in this region suggests the presence of a tumor suppressor gene and often includes the ETV6 and CDKN1B locus. Translocations are less common but t(4;12)(q12;p13) is recurrent, specially in AML- M0. We have studied in detail 52 AML-M0 patients and observed abnormalities in the short arm of chromosome 12 in five cases, and ETV6 involvement in eight. Three patients presented deletions ranging from 3,2 to 14,3 Mb. One of the deletions was associated with a t(10;12) (q11;p11). The minimal overlap region of deletion between these patients included ETV6 but excluded CDKN1B. Two others patients had a t(4;12)(q12;p13). In both patients we detected an out of frame CHIC2-ETV6 fusion transcript. The reciprocal transcript was not detected supporting the view that t(4;12) does not result in a oncogenic fusion protein. In addition, we detected ETV6 inactivating biallelic mutations in one patient and mutations leading to ETV6 truncated proteins in another two patients. Here we discuss the involvement of ETV6 and the chromosome 12 short arm in AML.
Keywords: AML-M0; ETV6; t(4;12); chromosome 12; mutation; deletion
Introduction
Abnormalities of the short arm of chromosome 12 (12p) are found in about 5 per cent of acute myeloid leukemias (AML) and myelodysplastic syndrome (MDS). Karyotypes associated with 12p aberrations are mostly complex and the patients have poor remission and survival rates.1 Most abnormalities are a total or partial loss of chromosome 12p arm usually affecting ETV6 and CDKN1B.1 The loss of material at 12p is often associated with unbalanced translocations.1-9 Loss of heterozygosity (LOH) of 12p region containing ETV6, CDKN1B or both is also frequent in acute lymphoblastic leukemia (ALL) and T-cell prolymphocytic leukemia.10-12
ETV6 (ets translocation variant gene 6), also known as TEL, encodes a transcription repressor belonging to the ETS family of DNA-binding proteins. In mice, ETV6 is essential for the establishment of hematopoiesis of all lineages in the bone marrow and for adult hematopoietic stem cell survival.13;14 ETV6 is known as a proto-oncogene involved in translocation with over 40 partners.15 In ALL the most frequent translocation is the t(12;21) involving ETV6 and RUNX1.16 The residual non-rearranged ETV6 allele in t(12;21) is frequently deleted suggesting that ETV6 can also be a tumor suppressor gene (TSG).17 The idea that ETV6 may have a tumor suppressor function is supported by functional experiments. 18;19 In AML only a few rare translocations result in transforming fusion proteins,16 suggesting that ETV6 oncogenic role does not play a major part in AML. Recently, heterozygous mutations of ETV6 resulting in loss of repressor activity were found in AML adding to the view that ETV6 might have tumor suppressor characteristics.20
Here we investigate the ETV6 status in 52 AML-M0 patients. We report for the first time two t(4;12)(q11;p13) with breakpoint within ETV6 and CHIC2 (alias BTL) resulting in an out of frame fusion transcript and an overlapping deleted region in the short arm of chromosome 12 that includes ETV6 but not CDKN1B. Most importantly, we found ETV6 heterozygous mutations in two patients and biallelic insertions in another.
Results
Single nucleotide polymorphism (SNP) analysis
DNA from leukemic cells isolated from samples of 52 AML-M0 patients was analyzed and compared to a T cell control DNA using a GeneChip 10k array (Affymetrix). Deletions in the short arm of chromosome 12 were detected as loss of heterozygosity and copy number variation in patients 1, 9 and 45 (Figure 1, Table 1). The deletion in patient 9 was accompanied by a t(10;12)(q11;p11) detected by karyotyping (Table 1). The deletions in patients 1, 9 and 45 spanned at maximum over a region of 6,8 Mb (rs252028 to rs1074716), 3,2 Mb (rs252028 to rs747726) and 14,3 Mb (rs917859 to rs2417848), respectively. While deletions in patients 1 and 45 included both ETV6 and CDKN1B, the proximal deletion breakpoint in patient 9 excluded CDKN1B (Figure 1). The minimal deleted overlapping region extended from rs252028 (position 9,814 Kb) to rs747726 (position 12,561 Kb). In patients 2 and 43, both with a t(4;12)(q12;p13), loss of heterozygosity (LOH) was not detected at 12p (Table 1;
Figure 1). All other samples were cytogenetically normal for chromosome 12 (confirmed by SNP analysis) with exception of patient 33, showing the loss of one chromosome 12 and an
unbalanced t(8;12)(q21;q13).
Figure 1. Single nucleotide polymorphism analyses of patients with chromosome 12 abnormalities.
A) LOH values based on haplotype using the paired normal. Each box represents the combined call between tumor sample and respective control (T-cells). Black boxes represent LOH, dark grey boxes no loss and light grey boxes non-informative markers. Boxes are displayed proportionally to the position of the SNP that they represent in relation to the cytogenetic band to the left of the panel. LOH was detected in patients 1, 9 and 45 at bands 12p13.31 to 12p12.3. In addition patient 45 shows LOH from position 119,661 Kbp to 122, 430 Kbp. B) Chromosome copy number calculated for the common region showing LOH in panel a (amplified). Copy number for the tumor samples was inferred using the paired normal as reference and a median smoothing. Grey boxes represent 2 copies and black boxes 1 copy (deletion) for each chromosome locus. The minimal deleted overlapping region is localized between SNP rs252028 (position 9,814 Kbp) to SNP rs747726 (position 12,561 Kbp) (underlined SNPs). C) Schematic representation of the genes present in the minimal deleted overlapping region defined in panel B based on the UCSC genome browser.48 Solid black boxes do not represent genes but clusters of related genes. The minimum overlap region defined by Baens et al.7 and La Starza et al.8 extends from marker d12s358 to gene PRB3 also represented in this panel.
Fluorescent in situ hybridization
Fluorescence in situ hybridization (FISH) showed that the t(4;12) breakpoint in patient 2 was within the ETV6 and CHIC2 regions (Table 2). Splitting signals on derivative chromosomes 4 and 12 [der(4) and der(12)] were observed with BAC probes RP11-367N1, encompassing
Table 1. Clinical, hematological, cytogenetic features and mutational status of ETV6.
Pat Age Diagnoses Karyotype ETV6
Allele 1 Allele 2 1I 65 AML-M0 46,XX,del(16)(q22?),i(17)(q10),
del(20)(q?) Deleted wt
2I 67 sAML-M0 47,XX,t(4;12)(q12;p13),-21,
+der21del(q?)x2 Translocated wt
6I 37 AML-M0 46,XY S107DfsX21 V345_Y346insR
9I 47 sAML-M0
52,XX,t(1;4) (p13;p12),+6,+8, t(10;12) (q11;p11),+18,+19,+20,+21
Deleted wt
21 59 AML-M0 46,XY R360X wt
43 64 AML-M0 46,XY,t(4;12)(q12;p13) Translocated wt
45 - AML-M0 n.d. Deleted wt
58 50 AML-M0 46,XY,idic(21)(p11.2) F103LfsX11 wt
I – patients used in Silva et al., 200346; s – secondary; n.d.- not done; wt – wild type
the whole CHIC2, and with pooled RP11-96B19 and RP11-418C2, covering the ETV6 gene (Figure 2b). RP11-651C2 was shown to be retained on chromosome der(4). FISH analysis using BAC probes confirmed ETV6 hemizygous deletion in patient 9 and showed that the deletion breakpoints coincided with the translocation breakpoint linking these two events (Figure 2a; Supplementary figure). Metaphases for patient 43 were not available for FISH analysis.
Table 2. FISH results obtained on patient 2.
BAC RP11-651C2 RP11-367N1 RP11-586A2 RP11-96B19
RP11-418C2
Includes 4q11 (cen) CHIC2-PDGFRA KIT ETV6
Chromosome
4 + + + -
12 - - - +
der(4) + + - +
der(12) - + + +
Symbols +/- indicate presence/absence of signal on target chromosome Detection of fusion transcripts
We screened patients 2 and 43 for CHIC2-ETV6 transcripts using the same approach described by Cools et al.21 In both patients, we could detect an ETV6-CHIC2 fusion transcript that consisted of the first exon of ETV6 and exons 2 to 6 of CHIC2 (Figure 2c). The fusion transcript, mainly consisting of exon 1 of ETV6, encodes a very short out of frame protein (Figure 2c,d). We repeatedly failed to detect the reciprocal CHIC2-ETV6 transcript, which, if present, would consist of the first exon of CHIC2 and exons 2 to 8 of ETV6 producing again an out of frame fusion transcript.
Figure 2. Schematic representation of FISH analysis of the breakpoints in t(10;12) in patient 9 and t(4;12) in patient 2 and fusion transcripts in patients 2 and 43. A) Determination of the deletion and translocation breakpoints in patient 9. The deleted region in chromosome 12 is localized between SNP rs252027 to SNP rs747726 (single copy SNPs are not underlined) as determined using the GeneChip 10K array. By FISH analysis, we determined that the distal translocation breakpoint occurred between BACs RP11-685G3 and RP11-959H9, whereas the proximal breakpoint occurred between BACs RP11-165F6 and RP11-1150D7. BACs represented by white boxes are hemizygously deleted, by black boxes are retained in the original chromosome, by dark gray boxes are translocated to the partner chromosome whereas light gray boxes show an intrachromosomal cross-hybridization signal on der(12) due to the occurrence of repeats and a split signal and split signal with der(10.) Only known genes are represented and white boxes represents clusters of related genes. B) BACs used in chromosome 4 and 12 to determine the t(4;12) breakpoint in patient 2. The gap in chromosome 4 is approximately 0.75 Mb.
Color code for the BAC probes as in panel A with the exception of striped boxes which represent BACs with a split signal. C) Genomic structure of CHIC2 (4q12) and ETV6 (12p13) according to UCSC Genome Browser48 and positions of primers used for PCR and sequencing (genes are not represented in the same scale). Dotted crossed lines represent the probable area where the t(4;12) occurred in patients 2 and 43. D) ETV6-CHIC2 transcripts detected in both patients 2 and 43. Sequence of ETV6-CHIC2 cDNA showed an out of frame fusion between exon 1 of ETV6 and exon 2 of CHIC2. The arrow indicates the boundary between the ETV6 and CHIC2 exons.
ETV6 mutations screening
We amplified and sequenced all 8 exons of ETV6 using genomic DNA of the 52 patients. We found insertions in patients 6 and 58 and a point mutation in patient 21 (Figure 3, Table 1).
Patient 6 had two mutations, an insertion of 13 base pairs (589_590insGATACGTCTCTCG) in exon 3, resulting in a truncated protein S107DfsX21, and a second 3 base pair in-frame insertion (1305_1306insTCG) in exon 6 leading to Y345_V346insR. We determined by cloning that Y345_V346insR was independent of S107DfsX21, that is, the mutations were in different alleles (biallelic). The result was confirmed by amplification and sequencing of the cDNA.
Patient 58 had a heterozygous frame-shift mutation in exon 3 (589_590 insGGGC) leading to the truncated protein F103LfsX11. Patient 21 nonsense mutation in exon 6 (1349C>T) changed the coding sequence to a stop codon at R360. In all cases the mutations were absent in the control cells (T cells). All mutations occurred in either the pointed domain (patients 6 and 58) or in the ETS DNA-binding domain (patients 6 and 21) (Figure 3).
In patients 1, 2, 9, 43 and 45, in which one ETV6 allele was either rearranged by translocation or lost by deletion, we did not find mutations in the remaining allele.
Figure 3. Schematic representation of the predicted ETV6 mutant products. Amino acid positions are shown under the wild type ETV6 protein. The pointed domains (PNT) and ETS DNA-binding domain are represented by a black and a grey box, respectively. New ORFs resulting from frame-shifts are drawn as a stripped box. Patient 6 has two predicted proteins corresponding to each one of the mutated alleles.
Discussion
We studied a cohort of 52 patients diagnosed with AML-M0. From these, 5 presented abnormalities in the short arm of chromosome 12. Patients 1 and 45 showed deletions, patients 2 and 43 translocations and patient 9 both types of abnormalities.
Deletions at the short arm of chromosome 12 in AML predict the presence of a tumor suppressor gene. Because of their role in hematopoiesis, ETV6 and CDNK1B (a cyclin-
dependent kinase inhibitor) are considered as potential candidates. Several articles point for a minimal deletion overlap including ETV6 and CDKN1B.2;4-6 Others studies excluded either ETV6 7;22;23 or CDKN1B 7;8;23, but a deletion excluding both genes simultaneously was never reported. Our minimal region of overlap set the proximal breakpoint in chromosome 12 short arm at SNP rs747726 (patient 9) and excluded CDKN1B (Figure 1). Together with the studies of Baens et al.7 and La Starza et al.8, with proximal breakpoint at d12s358 (a marker close to rs747726), these are the most telomeric break points including ETV6 ever reported. Both studies set the distal breakpoint at the PRB3 gene, delimiting a region of 1200 Kb (Figure 1c).7;8 Because inactivation of the second allele of ETV6, or another gene, in the deleted region was never found, haploinsufficiency of ETV6 has been suggested.5;6 The conflicting data regarding deletions in the short arm of chromosome 12 in AML, including or excluding ETV6 and CDKN1B, opens the possibility that there is more than one target in the deleted region or even a synergistic effect between targets, as argued for ALL.11;24
The t(4;12)(q12;p13), although rare, is a recurrent event in AML, particularly in subtype M0.15;21;23;25-29 Previous studies have mapped the breakpoints of this translocation to ETV6 in chromosome 12 and three regions in chromosome 4: CHIC2, HSG2 and an area between these two genes.21;28;30 Cools et al.21 reported four cases where the t(4;12)(q12;p13) fused the first 3 exons of CHIC2 on 4q12 to exons 2-8 of the ETV6 gene on 12p13, resulting in the expression of a hybrid CHIC2-ETV6 transcript in all cases. In contrast, we only detected an out of frame ETV6-CHIC2 transcript in patients 2 and 43 and no CHIC2-ETV6 transcript (which would also be out of frame) (Figure 2). This finding is in line with the absence of transforming ability of the CHIC2-ETV6 protein described by Cools et al.28 and further supports the idea that the fusion protein is not the element of pathogenesis. The heterogeneity of the t(4;12) adds to this view.28;31 Several AML studies reported translocations involving ETV6 but lacking functionally fusion proteins. In t(12;13) and t(3;12) it has been shown that ectopic expression of protooncogenes at the partner chromosomes might be the malignant event.32-34 In t(4;12) and t(7;12) (HLXB9-ETV6) , ectopic expression of genes neighboring the translocation breakpoints was reported, but its malignant potential was not determined.28;35-38 Still in t(1;12), t(5;12), t(7;12) (AF7P14-ETV6), t(12;17) and t(5;12;22) neither a functional fusion protein nor an alternative malignant mechanism was found.39-43 This led to the suggestion that heterozygous disruption of ETV6 by the translocation resulting in ETV6 haploinsufficiency is part of the process resulting in AML.37;40;43 It is worth to observe that translocations in AML involving breakpoints outside the ETV6 locus are sometimes accompanied by cryptic deletions that include ETV6,8 like in our patient 9, indicating targeting of this region.
Supporting the idea that ETV6 is a tumor suppressor gene we found one case with biallelic mutations of ETV6. Patient 6 showed a frame-shift mutation in one allele resulting in a truncated protein and a second insertion in the remaining allele. Mutations of ETV6 have only been previously reported in 5 AML patients (M1 and M2) and in prostate cancer.20 ETV6 mutant proteins with truncation comparable to ours (patients 6, 21 and 58; Table 1, Figure 3) were shown to have impaired transcriptional repression activity.20 Furthermore, patient 6 insertion leading to V345_Y346insR is similar to a published mutation at position Y344_V345insG that also affected the transcription repression ability of the ETV6 mutant.20
Interestingly, all our mutants resulted in loss of the ETS domain or its binding activity, but not of the PTN domain (Figure 3). The mutants reported by Barjesteh van Waalwijk et al.20 lacking the ETS domain showed a dominant-negative effect when ETV6 wild type constructs were co-transfected with seven-fold higher amounts of mutant construct. Similar results were obtained by another group, but when constructs were co-transfected in nearly similar amounts the dominant-negative effect was negligible.44 It is probable that the dominant-negative effect is due to over expression of mutant ETV6,44 something less likely to occur in vivo. In fact, expression of mutated ETV6 protein was not detected in the Barjesteh van Waalwijk et al.20 study, arguing against a dominant-negative effect. The infrequency of homozygous mutations of ETV6 is contradictory to a potential role for this gene as a tumor suppressor. However, it is possible that the need of ETV6 for adult hematopoietic stem cell survival makes its total loss not viable in most cases, specially in early stages of leukemogenesis.13
In conclusion, the number and variety of ETV6 translocations not resulting in a fusion protein together with the loss of ETV6 by deletion and heterozygous or homozygous mutations makes a mutually supportive and compelling case for loss and haploinsufficiency of ETV6 as a leukemogenic step in AML-M0.
Materials and methods
Patient material
Cryo-preserved mononuclear cells isolated from 52 patients, classified morphologically and immunophenotypically as AML-M0 were used in this study.45 Pure tumor cell populations were sorted by flow cytometry from mononuclear cells isolated from bone marrow or peripheral blood at the time of diagnosis.46 T-cells were expanded for each sample, to be used as a control, using previously described conditions.46 DNA and RNA were isolated using QIAamp DNA Blood Mini Kit and QIAamp RNA Blood Mini Kit following manufacturer instructions (Qiagen, Hilden, Germany).
Karyotyping and fluorescence in situ hybridization analyses (FISH)
Cytogenetic analysis was performed on GTG-banded chromosomes and the karyotype was assigned at the time of diagnosis according to the International System for Human Cytogenetic Nomenclature (ISCN) criteria.47
Metaphase preparations from cultured cells were obtained according to standard cytogenetic techniques. FISH experiments were done as described using BAC probes RP11-651C2 (Accession No. AC093880), RP11-367N1 and RP11-586A2 for chromosome 4 and RP11- 96B19 (Accession No. AC084430), RP11-639O1, RP11-1136K1, RP11-685G3, RP11- 1053G20, RP11-159B4, RP11-58C14, RP11-418C2 (Accession No. AC084358), RP11- 100M3, RP11-346G18, RP11-959H9, RP11-311P12, RP11-144O23, RP11-958J24, RP11- 297N18, RP11-165F6 and RP11-1150D7 for chromosome 12 (Figure 2a).46 These clones were chosen accordingly to the latest release (March 2006-hg18 assembly) of the University of California Santa Cruz (UCSC) Human Genome Browser.48
Detection of fusion transcripts
cDNA was prepared from 500 ng of RNA using an oligo-dT adapter primer (5’ AGC TGG TCA GTC GTC AGC TGA (T)16 3’) and SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Breda, The Netherlands). Detection of the CHIC2-ETV6 transcript was done using primers BTLF1, ETV6R2, ETV6R3B, ETV6R (5’ CGT CAC ACT TCC AGC AAT CC 3’) or an adapter primer (5’ AGC TGG TCA GTC GTC AGC TGA 3’) and of the ETV6-CHIC2 transcript using primers ETV6F1A, BTLR4 or the adapter primer. Sequences for non-described primers and PCR conditions can be obtain elsewhere.21 Sequencing of the forward and reverse strands was performed as described previously using the PCR primers.46
ETV6 mutation analysis and cloning
ETV6 mutation screening was performed on genomic DNA using primers and conditions described elsewhere with the exception of primers for exon 5 (Forward: 5’ GAG TTT CCT GTC CTG 3’; Reverse: 5’ GGG GAG AGT GGG ACT TTG TC 3’) and for exon 8 (Forward:
5’ TTC AGA GTG AAG ACA GCT TTA GG 3’; Reverse: 5’ CGT CAC ACT TCC AGC AAT CC 3’).49 Sequencing of the forward and reverse strands was performed as described previously using the PCR primers.46
To determine heterozygosity of mutations in patient 6, ETV6 cDNA was amplified using ETV6F1 and ETV6R primers and cloned using GeneJET PCR cloning kit (Fermentas, St.Leon-Rot, Germany) following manufacturers instructions. The inserted fragments were amplified by colony PCR using the primers provided in the GeneJET PCR cloning kit (Fermentas) and sequenced.
Sequence numbering is according to the GenBank database: BC043399.
Single nucleotide polymorphism analyses
SNP analysis was performed using the GeneChip Mapping 10k 2.0 array (Affymetrix, Santa Clara, CA). Briefly, 250 ng of total genomic DNA was digested with XbaI restriction enzyme and ligated to an universal adapter. The ligated fragments were then amplified by PCR using primers complementary to the universal adapters. PCR products were purified, fragmented (DNase I), labeled with biotinylated ddATP and hybridized to the array. The arrays were scanned using a GeneChip Scanner (Affymetrix). Affymetrix GeneChip 5.0 genotyping software was used to examine the SNP hybridization patterns and to make SNP calls. The resulting data were analyzed with the dChip software package.50
Supplementary information available on the Leukemia web site.
Acknowledgements
We would like to thank H Wessels for support with karyotyping. Patient material was kindly provided by PJ Valk, Erasmus University Medical Center, W-D Ludwig, Medical University of Berlin, WAF Marijt, Leiden University Medical Center and WR Sperr, Medical University of Vienna. This work was supported by grants from the Calouste Gulbenkian Foundation and the Foundation for Science and Technology (Portugal) to FPG Silva.
Reference list
Streubel B, Sauerland C, Heil G et al. Correlation of cytogenetic, molecular cytogenetic, and clinical 1.
findings in 59 patients with ANLL or MDS and abnormalities of the short arm of chromosome 12.
Br.J.Haematol. 1998;100:521-533.
Hoglund M, Johansson B, Pedersen-Bjergaard J, Marynen P, Mitelman F. Molecular characterization 2.
of 12p abnormalities in hematologic malignancies: deletion of KIP1, rearrangement of TEL, and amplification of CCND2. Blood 1996;87:324-330.
Kobayashi H, Rowley JD. Identification of cytogenetically undetected 12p13 translocations 3.
and associated deletions with fluorescence in situ hybridization. Genes Chromosomes.Cancer 1995;12:66-69.
Kobayashi H, Montgomery KT, Bohlander SK et al. Fluorescence in situ hybridization mapping 4.
of translocations and deletions involving the short arm of human chromosome 12 in malignant hematologic diseases. Blood 1994;84:3473-3482.
Sato Y, Suto Y, Pietenpol J et al. TEL and KIP1 define the smallest region of deletions on 12p13 in 5.
hematopoietic malignancies. Blood 1995;86:1525-1533.
Wlodarska I, Marynen P, La Starza R, Mecucci C, Van den BH. The ETV6, CDKN1B and 6.
D12S178 loci are involved in a segment commonly deleted in various 12p aberration in different hematological malignancies. Cytogenet.Cell Genet. 1996;72:229-235.
Baens M, Wlodarska I, Corveleyn A et al. A physical, transcript, and deletion map of chromosome 7.
region 12p12.3 flanked by ETV6 and CDKN1B: hypermethylation of the LRP6 CpG island in two leukemia patients with hemizygous del(12p). Genomics 1999;56:40-50.
La Starza R, Stella M, Testoni N et al. Characterization of 12p molecular events outside ETV6 in 8.
complex karyotypes of acute myeloid malignancies. Br.J.Haematol. 1999;107:340-346.
Hernandez JM, Gonzalez MB, Garcia JL et al. Two cases of myeloid disorders and a t(8;12) 9.
(q12;p13). Haematologica 2000;85:31-34.
Stegmaier K, Pendse S, Barker GF et al. Frequent loss of heterozygosity at the TEL gene locus in 10.
acute lymphoblastic leukemia of childhood. Blood 1995;86:38-44.
Takeuchi S, Bartram CR, Miller CW et al. Acute lymphoblastic leukemia of childhood: identification 11.
of two distinct regions of deletion on the short arm of chromosome 12 in the region of TEL and KIP1. Blood 1996;87:3368-3374.
Hetet G, Dastot H, Baens M et al. Recurrent molecular deletion of the 12p13 region, centromeric 12.
to ETV6/TEL, in T-cell prolymphocytic leukemia. Hematol.J. 2000;1:42-47.
Hock H, Meade E, Medeiros S et al. Tel/Etv6 is an essential and selective regulator of adult 13.
hematopoietic stem cell survival. Genes Dev. 2004;18:2336-2341.
Wang LC, Swat W, Fujiwara Y et al. The TEL/ETV6 gene is required specifically for hematopoiesis 14.
in the bone marrow. Genes Dev. 1998;12:2392-2402.
Odero MD, Carlson K, Calasanz MJ et al. Identification of new translocations involving ETV6 in 15.
hematologic malignancies by fluorescence in situ hybridization and spectral karyotyping. Genes Chromosomes.Cancer 2001;31:134-142.
Bohlander SK. ETV6: a versatile player in leukemogenesis. Semin.Cancer Biol. 2005;15:162- 16.
174.
Cave H, Cacheux V, Raynaud S et al. ETV6 is the target of chromosome 12p deletions in t(12;21) 17.
childhood acute lymphocytic leukemia. Leukemia 1997;11:1459-1464.
Fenrick R, Wang L, Nip J et al. TEL, a putative tumor suppressor, modulates cell growth and cell 18.
morphology of ras-transformed cells while repressing the transcription of stromelysin-1. Mol.Cell Biol. 2000;20:5828-5839.
Rompaey LV, Potter M, Adams C, Grosveld G. Tel induces a G1 arrest and suppresses Ras-induced 19.
transformation. Oncogene 2000;19:5244-5250.
Barjesteh van Waalwijk van Doorn-Khosrovani, Spensberger D, de Knegt Y et al. Somatic 20.
heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid
leukemia. Oncogene 2005;24:4129-4137.
Cools J, Bilhou-Nabera C, Wlodarska I et al. Fusion of a novel gene, BTL, to ETV6 in acute 21.
myeloid leukemias with a t(4;12)(q11-q12;p13). Blood 1999;94:1820-1824.
Andreasson P, Johansson B, Arheden K et al. Deletions of CDKN1B and ETV6 in acute myeloid 22.
leukemia and myelodysplastic syndromes without cytogenetic evidence of 12p abnormalities.
Genes Chromosomes.Cancer 1997;19:77-83.
Andreasson P, Johansson B, Billstrom R et al. Fluorescence in situ hybridization analyses of 23.
hematologic malignancies reveal frequent cytogenetically unrecognized 12p rearrangements.
Leukemia 1998;12:390-400.
Wlodarska I, Baens M, Peeters P et al. Biallelic alterations of both ETV6 and CDKN1B genes in a 24.
t(12;21) childhood acute lymphoblastic leukemia case. Cancer Res. 1996;56:2655-2661.
Harada H, Harada Y, Eguchi M, Dohy H, Kamada N. Characterization of acute leukemia with 25.
t(4;12). Leuk.Lymphoma 1997;25:47-53.
Nathan PC, Chun K, Abdelhaleem M, Malkin D. Isochromosome (17)(q10) and translocation 26.
(4;12)(q12;p13) in a child with acute myeloid leukemia. Cancer Genet.Cytogenet. 2001;131:82- 85.
Chauffaille ML, Fermino FA, Pelloso LA et al. t(4;12)(q11;p13): a rare chromosomal translocation 27.
in acute myeloid leukemia. Leuk.Res. 2003;27:363-366.
Cools J, Mentens N, Odero MD et al. Evidence for position effects as a variant ETV6-mediated 28.
leukemogenic mechanism in myeloid leukemias with a t(4;12)(q11-q12;p13) or t(5;12)(q31;p13).
Blood 2002;99:1776-1784.
Kuchenbauer F, Schoch C, Holler E et al. A rare case of acute myeloid leukemia with a CHIC2- 29.
ETV6 fusiongen and multiple other molecular aberrations. Leukemia 2005;19:2366-2368.
Burtey S, De Roux C, Carbuccia N. The t(4;12)(q11;p13) translocation associated with AML is a 30.
complex molecular entity [abstract]. Blood 2000;96:693a.
Berger R, Le Coniat M, Lacronique V et al. Chromosome abnormalities of the short arm of 31.
chromosome 12 in hematopoietic malignancies: a report including three novel translocations involving the TEL/ETV6 gene. Leukemia 1997;11:1400-1403.
Chase A, Reiter A, Burci L et al. Fusion of ETV6 to the caudal-related homeobox gene CDX2 in 32.
acute myeloid leukemia with the t(12;13)(p13;q12). Blood 1999;93:1025-1031.
Rawat VP, Cusan M, Deshpande A et al. Ectopic expression of the homeobox gene Cdx2 is the 33.
transforming event in a mouse model of t(12;13)(p13;q12) acute myeloid leukemia. Proc.Natl.
Acad.Sci.U S.A 2004;101:817-822.
Peeters P, Wlodarska I, Baens M et al. Fusion of ETV6 to MDS1/EVI1 as a result of t(3;12) 34.
(q26;p13) in myeloproliferative disorders. Cancer Res. 1997;57:564-569.
Tosi S, Giudici G, Mosna G et al. Identification of new partner chromosomes involved in 35.
fusions with the ETV6 (TEL) gene in hematologic malignancies. Genes Chromosomes.Cancer 1998;21:223-229.
Slater RM, von Drunen E, Kroes WG et al. t(7;12)(q36;p13) and t(7;12)(q32;p13)--translocations 36.
involving ETV6 in children 18 months of age or younger with myeloid disorders. Leukemia 2001;15:915-920.
Beverloo HB, Panagopoulos I, Isaksson M et al. Fusion of the homeobox gene HLXB9 and 37.
the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13). Cancer Res.
2001;61:5374-5377.
von Bergh AR, van Drunen E, van Wering ER et al. High incidence of t(7;12)(q36;p13) in infant 38.
AML but not in infant ALL, with a dismal outcome and ectopic expression of HLXB9. Genes Chromosomes.Cancer 2006;45:731-739.
Odero MD, Vizmanos JL, Roman JP et al. A novel gene, MDS2, is fused to ETV6/TEL in a 39.
t(1;12)(p36.1;p13) in a patient with myelodysplastic syndrome. Genes Chromosomes.Cancer 2002;35:11-19.
Yagasaki F, Jinnai I, Yoshida S et al. Fusion of TEL/ETV6 to a novel ACS2 in myelodysplastic 40.
syndrome and acute myelogenous leukemia with t(5;12)(q31;p13). Genes Chromosomes.Cancer 1999;26:192-202.
Yuji K, Mitani K, Ueno H et al. A new partner gene of the
41. TEL/ETV6, AF7p15, cloned in acute
myeloid leukemia with t(7;12)(p15;p13). [abstract]. Blood 2000;96:693a.
Penas EM, Cools J, Algenstaedt P et al. A novel cryptic translocation t(12;17)(p13;p12-p13) in a 42.
secondary acute myeloid leukemia results in a fusion of the ETV6 gene and the antisense strand of the PER1 gene. Genes Chromosomes.Cancer 2003;37:79-83.
Belloni E, Trubia M, Mancini M et al. A new complex rearrangement involving the ETV6, 43.
LOC115548, and MN1 genes in a case of acute myeloid leukemia. Genes Chromosomes.Cancer 2004;41:272-277.
Sasaki K, Nakamura Y, Maki K et al. Functional analysis of a dominant-negative DeltaETS TEL/
44.
ETV6 isoform. Biochem.Biophys.Res.Commun. 2004;317:1128-1137.
Silva FP, Lind A, Brouwer-Mandema G, Valk PJ, Giphart-Gassler M. Trisomy 13 correlates 45.
with RUNX1 mutation and increased FLT3 expression in AML-M0 patients. Haematologica 2007;92:1123-1126.
Silva FP, Morolli B, Storlazzi CT et al. Identification of RUNX1/AML1 as a classical tumor 46.
suppressor gene. Oncogene 2003;22:538-547.
ISCN 2005. An International System for Human Cytogenetics Nomenclature. Basel: Karger;
47.
2004.
Karolchik D, Baertsch R, Diekhans M et al. The UCSC Genome Browser Database. Nucleic Acids 48.
Res. 2003;31:51-54.
Kibel AS, Faith DA, Bova GS, Isaacs WB. Mutational analysis of ETV6 in prostate carcinoma.
49.
Prostate 2002;52:305-310.
Lin M, Wei LJ, Sellers WR et al. dChipSNP: significance curve and clustering of SNP-array-based 50.
loss-of-heterozygosity data. Bioinformatics. 2004;20:1233-1240.
