2120

ETV6

TEL|TEL/ABL;ets variant 6;ETV6;ets variant 6

ETS translocation variant 6|ETS-related protein Tel1|TEL1 oncogene|ets variant gene 6 (TEL oncogene)|transcription factor ETV6

12p13

protein-coding

Cytogenetic deletions on the short arm of chromosome 12 are common, recurring alterations found in a wide range of hematological neoplasias, including childhood acute lymphoblastic leukemia (ALL), the most frequent pediatric malignancy. This loss of genetic material suggests the presence of a tumor suppressor gene playing an important role in growth regulation or in the differentiation of hematopoietic stem cells. In order to substantiate this hypothesis and determine the chromosomal location of this putative gene, we and others have applied a deletion mapping strategy based on the detection of loss of heterozygosity (LOH) at specific genomic loci in leukemic cells. Hemizygous deletions at chromosome 12p12.3 were observed in childhood B cell precursor ALL,and proved to be one of the most frequent genetic alterations seen in this disease. The shortest region of overlapping deletions (SRO) was delimited by themarkers D12S89 (distal) and D12S358 (proximal), separated by a genetic interval of approximately 3 cM. LOH in the same region in other hematological diseases, as well as in a variety of solid tumors, suggests either the presence of several tumor suppressor genes or the existence of a single gene with a wide range of activity. This genetic interval contains two known genes: TEL/ETV6, an ets-like transcription factor, and the cyclin-dependant kinase inhibitor, p27/kip1. Accumulating evidence suggests that an as yet unidentified tumor suppressor geneis closely linked to these two genes. Long-range restriction mapping of the SRO region allowed the construction of a approximately 750 kb physical map containing 4 known genes, 7 STSs, 3 chromosome 12 ESTs and 8 CpG islands. The construction of a 12p12.3 framework map is a crucial step towards the identification of candidate genes and should provide a valuable tool for the characterization of transcriptional units.

Rearrangements of the short arm of chromosome 12 are frequently observed in hematological malignancies and in certain solid neoplasias. Loss of heterozygosity studies identified a small genetic interval on chromosome 12p12.3that is frequently deleted in childhood acute lymphoblastic leukemia (ALL). Two genes, ETV6 and CDKN1B, are located within this interval although evidence has accumulated that an as yet unidentified tumor suppressor gene is closely linked.Here we report the physical mapping of the G-protein coupled receptor 19 (GPR19)at approximately 40kb from CDKN1B. The delineation of the gene order tel-ETV6-CDKN1B-GPR19-cen excluded GPR19 from the region commonly deleted in childhood ALL, but it could still be the target of genetic alterations found in other cancers.

Rearrangements of the short arm of chromosome 12 are frequently observed in hematological disorders. Previous studies of loss of heterozygosity identified asmall genetic interval on chromosome 12p12.3 that is frequently deleted in childhood acute lymphoblastic leukemia (ALL). Two genes, ETV6/TEL and p27/KIP1, are located in this interval. Evidence has accumulated that an as-yet unidentified tumor suppressor gene is closely linked to these. To facilitate theidentification of candidate genes, a long-range high-resolution restriction map of the ALL locus was constructed using a contig of YAC clones. Several marker loci, including 11 STS, three newly developed YAC end-based STS, six EST, and seven genes were unambiguously positioned in the new map. The map covers 1.8Mb and extends from the distal salivary proline-rich protein gene cluster to the proximal p27/KIP1 gene. The data confirmed the order tel-D12S358-p27/KIP1-cen and excluded p27/KIP1 as a candidate tumor suppressor gene. The critical region delimited by D12S89 and D12S358 is a 750kb CpG-island rich region that includes the 240kb TEL/ETV6 gene as well as CLAPS3 (clathrin-adaptor small chain 3). The new map provides a molecular framework for the identification of novel genes andtranscriptional units in the ALL interval.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood. The improvements of therapies have increased the number of long-term survivors. However, an increased incidence of secondary neoplasias has been observed in this cohort. Our purpose was to evaluate the late effects of cancer therapy in cured patients previously treated for ALL, considering previous reports on the occurrence of gene fusions as putative markers of chromosomal instability. Twelve ALL patients (aged 5 to 16 years) and twelve healthy subjects (aged 18 to 22 years) were studied for the presence of ETV6/RUNX1 (TEL/AML1) translocations, which were detected by FISH (fluorescence in situ hybridization). The blood samples were collected months or years after completion of the therapy, and the frequencies of gene fusions in lymphocytes were compared with those obtained retrospectively for bone marrow samples at the time of diagnosis, and also for the control group. It was demonstrated that ETV6/RUNX1 gene fusion was a frequent event (0.59-1.84/100 cells) in peripheral blood lymphocytes from normal individuals and the ALL patients who underwent chemotherapy showed significantly(P = 0.0043) increased frequencies (0.62-3.96/100 cells) of the rearrangement when compared with the control groups (patients at diagnosis and healthy subjects). However, a significant difference was not found between the groups ofpatients at diagnosis and healthy subjects, when the two patients who were positive for the rearrangement were excluded. Therefore, increased frequencies of ETV6/RUNX1 fusions in ALL cured patients indicate the influence of previous exposure to anti-cancer drugs, and they may represent an important genetic marker for estimating the risk of relapse, or development of secondary neoplasias.#CI- Copyright 2003 S. Karger AG, Basel

Allelic losses on chromosome 12p12-13 are associated with childhood acute lymphoblastic leukemia (ALL) and several solid neoplasias, suggesting the presence of a tumor suppressor locus. The recent construction of a transcriptionmap of this locus has enabled the identification of eight genes, of which five were previously known: ETV6, BCL-G, LRP6, MKP-7, and CDKN1B. The three other candidate genes, LOH12CR1, LOH12CR2, and LOH12CR3, have no known functions. To evaluate whether one (or more) of the candidate genes is the actual target of the 12p12-13 deletions, we examined the genomics and the expression status of these genes in ALL patients. Although we found nine DNA variants in these genes, no inactivating mutations were found in the leukemia cells of patients with 12p hemizygous deletions. expression analysis revealed that most 12p hemizygously deleted samples also carried a t(12;21) translocation, of which none expressed ETV6 from the nontranslocated allele. Furthermore, we observed one case of t(12;21) without deletion of ETV6, in which the expression of this gene was greatly reduced, indicating a different mechanism of inactivation. None of the other genes showed a significant decrease in expression, suggesting that ETV6 isindeed the target of deletions in ALL patients.

childhood cancer is rare comprising less than 1% of ALL malignancies diagnosed each year in developed countries. Leukaemia is the commonest form of cancer in children accounting for around a third of ALL childhood cancer, with acute lymphoblastic leukaemia (ALL) being the most prevalent. Biologically specific subtypes of ALL and acute myeloblastic leukaemia (AML), the other major morphological type of childhood leukaemia, are characterised by chromosomal changes. Whilst over 200 genes have been associated with chromosomal translocations, to date, only MLL, TEL, and AML1 have been linked with childhoodleukaemia. Interestingly, there is increasing evidence to support the theory that gene rearrangements such as these may originate in utero. As with many other human diseases, both genetic and environmental factors have been implicated in the aetiology of the disease. Although much has been documented with regard to diet, smoking, alcohol consumption and recreational and prescription drug use during pregnancy, there is no consistent evidence to support a link with any of these factors and childhood leukaemia. However, findings from studies investigating prenatal and early life exposures are often based on small numbersof cases as both the type of cancer and exposure are rare. Furthermore, accurateinformation relating to past exposures can be difficult to obtain and is often reliant on self-reporting. To further our understanding of the aetiology of childhood leukaemia and lymphoma, there are areas which clearly warrant investigation. These include collection of parental dietary folate data combinedwith genetic analysis of the folate related genes, in utero exposure to DNA topoisomerase II inhibitors, and the possible effects of assisted reproduction technology on disease susceptibility.

High-resolution single nucleotide polymorphism genomic microarray (SNP-chip) is a useful tool to define gene dosage levels over the whole genome, allowing precisedetection of deletions and duplications/amplifications of chromosomes in cancer cells. We found that this new technology can also identify breakpoints of chromosomes involved in unbalanced translocations, leading to identification of fusion genes. Using this technique, we found that the PAX5 gene was rearranged to a variety of partner genes including ETV6, FOXP1, AUTS2, and C20orf112 in pediatric acute lymphoblastic leukemia (ALL). The 3' end of the PAX5 gene was replaced by the partner gene. The PAX5 fusion products bound to PAX5 recognitionsequences as strongly as wild-type PAX5 and suppressed its transcriptional activity in a dominant-negative fashion. In human B cell leukemia cells, bindingof wild-type PAX5 to a regulatory region of BLK, one of the direct downstream target genes of PAX5, was diminished by expression of the PAX5-fusion protein, leading to repression of BLK. expression of PAX5-fusion genes in murine bone marrow cells blocked development of mature B cells. PAX5-fusion proteins may contribute to leukemogenesis by blocking differentiation of hematopoietic cells into mature B cells. SNP-chip is a powerful tool to identify fusion genes in human cancers.

We sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed ahigh frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.

Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ~5-10% of pediatric T-ALLs and are associated with poor prognosis. However, the geneticdefects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors.

TEL is a new member of the ETS family of transcription factors which is rearranged in a number of hematologic malignancies with translocations involvingchromosome band 12p13. In some cases, both TEL alleles are affected, resulting in loss of wild-type TEL function in the leukemic cells. In addition, 5% of children with acute lymphoblastic leukemia (ALL) have 12p12-p13 deletions, suggesting that a tumor suppressor gene resides on 12p. These observations led us to consider whether TEL loss of function may contribute to the pathogenesis of ALL. In this report we show that the TEL gene maps between the polymorphic markers D12S89 andD12S98, and we use these flanking markers to screen paired diagnosis and remission samples from 81 children with ALL for loss of heterozygosity (LOH) at the TEL gene locus. Fifteen percent of informative patients showed TEL LOH whichwas not evident on cytogenetic analysis. Detailed examination of patients with LOH at this locus showed that the critically deleted region included two candidate tumor suppressor genes: TEL and KIP1, the gene encoding the cyclin-dependent kinase inhibitor p27. These studies show that LOH at the TEL locus is a frequent finding in childhood ALL.

The presence of ETV6 deletions was investigated in 215 children with acute lymphoblastic leukemia (ALL) using the loss of heterozygosity (LOH) approach. Weused four intragenic or juxtagenic microsatellite markers to detect allelic deletions. In this series of unselected patients, LOH of ETV6 markers was found in 23% of cases (6% of T-ALL and 26% of B lineage ALL) confirming that chromosome 12p12-13 deletions represent a major genetic alteration in childhood ALL, frequently missed by cytogenetic analysis. The presence of a t(12;21)(p13;q22) was studied by RT-PCR and/or FISH in a total of 134 patients (125 B lineage ALL,nine T-ALL) including 42 cases with LOH. Thirty-four out of 44 patients (77%) for whom a t(12;21) was observed displayed LOH of the ETV6 markers. When associated with a t(12;21), ETV6 is very likely to be the target of deletions as indicated by the detection of intragenic deletions in three patients. Although deletion ofETV6 and t(12;21) were associated in most patients, in eight cases (six B lineage and two T-ALL) LOH was detected at the ETV6 locus without ETV6-AML1 hybrid RNA. FISH studies conducted in five of these eight patients confirmed the absence of translocation involving ETV6. In such patients, the other allele of ETV6 could be disrupted by either a small deletion, a point mutation, or an epigenetic modification and it will be of interest to study the structure and expression ofthe remaining allele of ETV6 in these cases. Alternatively, a tumor suppressor gene located close to ETV6 and CDKN1B could be the target of deletions.