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Therapy-related myelodysplastic syndrome is a long-term complication of cancer treatment in patients receiving cytotoxic therapy, characterized by high-risk genetics and poor outcomes. Allogeneic hematopoietic cell transplantation is the only potential cure for this disease, but the prognostic impact of pre-transplant genetics and clinical features has not yet been fully characterized. We report here the genetic and clinical characteristics and outcomes of a relatively large cohort of patients with therapy-related myelodysplastic syndrome (n=67) who underwent allogeneic transplantation, comparing these patients to similarly treated patients with de novo disease (n=199). The 5-year overall survival was not different between patients with therapy-related and de novo disease (49.9% versus 53.9%; P=0.61) despite a higher proportion of individuals with an Intermediate-2/High International Prognostic Scoring System classification (59.7% versus 43.7%; P=0.003) and high-risk karyotypes (61.2% versus 30.7%; P

Though rare, inherited forms of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are being recognized with increasing frequency. Germline mutations in well-characterized genes such as RUNX1, CEBPA, GATA2 and ETV6 have been found to predispose to MDS/AML.[1][1]–[4][2] Families with

Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages.

Chromosome 7 abnormalities are common in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Often, there is partial loss of 7q as the result of deletion (del(7q)) or loss of one entire copy of chromosome 7 (monosomy 7 or -7). Del(7q) and -7 may occur in isolation, in simple or in

The ETV6 gene (previously known as TEL) belongs to the ETS (E26 transformation specific) family of transcription factors characterized by 2 important domains: the C-terminal Ets domain responsible for specific DNA-binding activities and the N-terminal helix–loop–helix (HLH) oligomerization

Background Acute myeloid leukemia is a clonal hematopoietic malignant disease; about 45–50% of cases do not have detectable chromosomal abnormalities. Here, we identified hidden genomic alterations and novel disease-related regions in normal karyotype acute myeloid leukemia/myelodysplastic syndrome samples.Design and Methods Thirty-eight normal karyotype acute myeloid leukemia/myelodysplastic syndrome samples were analyzed with high-density single-nucleotide polymorphism microarray using a new algorithm: allele-specific copy-number analysis using anonymous references (AsCNAR). Expression of mRNA in these samples was determined by mRNA microarray analysis.Results Eighteen samples (49%) showed either one or more genomic abnormalities including duplication, deletion and copy-number neutral loss of heterozygosity. Importantly, 12 patients (32%) had copy-number neutral loss of heterozygosity, causing duplication of either mutant FLT3 (2 cases), JAK2 (1 case) or AML1/RUNX1 (1 case); and each had loss of the normal allele. Nine patients (24%) had small copy-number changes (< 10 Mb) including deletions of NF1, ETV6/TEL, CDKN2A and CDKN2B. Interestingly, mRNA microarray analysis showed a relationship between chromosomal changes and mRNA expression levels: loss or gain of chromosomes led, respectively, to either a decrease or increase of mRNA expression of genes in the region.Conclusions This study suggests that at least one half of cases of normal karyotype acute myeloid leukemia/myelodysplastic syndrome have readily identifiable genomic abnormalities, as found by our analysis; the high frequency of copy-number neutral loss of heterozygosity is especially notable.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the presence of different collaborating cytogenetic and molecular aberrations that are associated with treatment response.1–3 Approximately 20% of pediatric AML patients are classified as cytogenetically normal AML (CN-AML).4 In the past decade, new prognostic relevant aberrations have been identified in CN-AML, such as NPM1, WT1, FLT3-ITD and CEBPA double mutations (CEBPAdm), which may improve future risk-group stratification.5–8 In addition to these mutations, we recently described recurrent cryptic NUP98/NSD1 and NUP98/KDM5A translocations in pediatric CN-AML.9,10 Still, some CN-AML samples have not been fully characterized. Recently, Grossmann et al. discovered a somatic mutation in the BCL6 co-repressor (BCOR) gene in an adult CN-AML patient using whole-exome sequencing.11 BCOR mutations were subsequently identified in 10 of 262 (approx. 4%) adult CN-AML patients, unselected for cytogenetic abnormalities, and for whom a poor outcome had been predicted. In addition, Li et al. discovered mutations in the BCL6 corepressor-like 1 (BCORL1) gene in 2 of 8 patients with secondary adult AML.12 Further exploration identified somatic BCORL1 mutations in 10 of 173 (6%) of adult AML patients, of which 5 cases were diagnosed with secondary or treatment-related AML. In 8 of 10 patients these included nonsense, splice site, and frame-shift mutations that were predicted to result in truncation of the protein, suggesting that BCORL1 is a tumor suppressor gene that may be inactivated by mutations.13 In pediatric AML, the role of BCOR and BCORL1 is unknown. Therefore, we explored the frequency and impact of BCOR and BCORL1 mutations in pediatric AML in a molecularly well documented cohort of 230 pediatric AML patients. Genomic DNA was PCR amplified using the primers described in Online Supplementary Table S1. Purified PCR products were directly sequenced. Sequence data were analyzed using CLCWorkbench (CLC Bio, Aarhus, Denmark). BCOR variations were determined in comparison to the coding DNA sequence ENST00000342274, and BCORL1 variations to ENST00000540052. A mutation was defined as a nucleotide change not reported in the dbSNP database. SNPs were defined as a nucleotide change as described in the dbSNP database. Characteristics of the 230 pediatric AML patients included in this mutational screening are listed in Table 1. Table 1. Clinical characteristics of pediatric acute myeloid leukemia patients included in mutation screening. The complete coding sequence of BCOR and BCORL1 was screened in a nested cohort of 83 de novo pediatric AML patients, and 17 AML cell lines. This cohort represented all relevant cytogenetic subgroups in pediatric AML, with an enrichment for CN-AML (n=48). All CN-AML patients were screened for NPM1, CEBPAdm, FLT3-ITD, IDH1/2, WT1, cKIT, N/K-RAS, DNMT3A, ASXL1 and RUNX1 mutations, and MLL-PTD, NUP98/NSD1, NUP98/KDM5A, and MLL-rearrangements (Online Supplementary Table S2). None of the cell lines showed a mutation. In 4 of 83 patients a BCOR mutation (detailed in Table 2) was identified; 3 in exon 4 and 1 in exon 12. Three of these cases were CN-AML (3 of 48, 6.3%), and the fourth mutation was seen in an MNX1/ETV6 translocated AML. In only one of 83 patients a BCORL1 mutation was identified which resulted in a stop codon (Table 2). This CN-AML patient had an underlying xeroderma pigmentosum. Table 2. Characteristics of BCOR and BCORL1 aberrations detected in childhood cytogenetically normal-acute myeloid leukemia patients. An additional 147 pediatric AML patients were screened for mutations in BCOR and BCORL1 exon 4, based on the occurrence of mutations in this exon; 136 cases were de novo AML, 11 cases were secondary AML, of which 6 cases were MDS which progressed to AML and 5 were secondary to earlier therapy because of another malignancy. None of the additional cases were CN-AML. No additional mutations were found. In 2 of 230 cases a missense SNP was found in BCOR (p.V679I, rs144722432) and in 35 of 230 (15%) a missense SNP in BCORL1 (p.G209S, rs5932715). Both SNPs are predicted as tolerated by SIFT analysis, and in line with the predicted prevalence in the normal population.14 In one case missense SNP rs139887979 (p.D94N) was identified in BCORL1, predicted as damaging by SIFT analysis.14 We were unable to confirm this in germline or remission because such material was not available for these patients. Altogether we identified a BCOR mutation in 4 of 230 (1.7%) cases. The frequency of mutations in pediatric CN-AML patients (6.3%; 95%CI: 2.1–16.8) was comparable to that in adult CN-AML (3.8%; 95%CI: 2.1–6.9; P=ns). The frequency of BCORL1 mutations (1 of 83, 1.2%; 95%CI: 0.2–6.5) was significantly lower than in adults (10 of 173, 5.8%; 95%CI: 3.2–10.3; P

In 1994 the ETV6-PDGFRB ( TEL-PDGFRB ) fusion gene was identified by Todd Golub and Gary Gilliland in patients with chronic myelomonocytic leukemia (CMML) and t(5;12)(q33;p13).[1][1] The implications of the molecular characterization of this translocation go far beyond the identification of the ETV6

Background Chromosomal translocations resulting in alternative fusions of the human TEL ( ETV6 ) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated. Design and Methods TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro . Results Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis. Conclusions This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.

Among 4,780 consecutive adult acute lymphoblastic/myeloblastic leukemia patients, we identified 117 (2.4%) patients with mixed-phenotype acute leukemia fulfilling WHO 2008 criteria; these were classified as: Blymphoid+ myeloid (n=64), T-lymphoid+myeloid (n=38), B+T-lymphoid (n=14) and trilineage (n=1). Of 92 patients karyotyped, 59 were abnormal and were classified as: complex (22 of 92), t(9;22)(q34;q11) (14 of 92), monosomy 7 (7 of 92), polysomy 21 (7 of 92), t(v;11q23) (4 of 92), t(10;11)(p15;q21) (3 of 92), while STIL-TAL1 fusion was detected in one (T+My) patient. After investigating common acute leukemia-related mutations in 17 genes, 12 of 31 (39%) patients were found to have at least one mutation, classified with: IKZF1 deletion (4 of 31), and EZH2 (3 of 31), ASXL1 (3 of 31), ETV6 (2 of 31), NOTCH1 (1 of 31), and TET2 (1 of 31) mutations. Array-CGH revealed genomic deletions of CDKN2A (4 of 12), IKZF1 (3 of 12), MEF2C (2 of 12), BTG1 (2 of 12), together with BCOR, EBF1, K-RAS, LEF1, MBNL1, PBX3, and RUNX1 (one of 12 each). Our results indicate that mixed-phenotype acute leukemia is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular genetic features.

We evaluated CD123 expression in 95 pediatric and 24 adult ALL patients and compared the results with the CD123 expression in normal B-cell precursors. Early B-cell precursors were negative while intermediate precursors and mature B cells showed weak CD123 expression. Leukemic blasts in 31% of precursor-B ALL samples exhibited strong expression of CD123, 61% had moderate CD123 expression and 8% were negative; 81.5% of ALL with hyperdiploid karyotype (/= 52 chromosomes) showed strong CD123 overexpression. In contrast, cases with ETV6/RUNX1 rearrangement had weak CD123 expression. Our study suggests that overexpression of CD123 is an aberrant phenotype present in a subset of precursor-B ALL with hyperdiploid genotype, and represents an additional marker of good prognosis in pediatric precursor-B ALL. Moreover, aberrant CD123 expression in ALL is a good marker for monitoring of minimal residual disease.

While great progress has been achieved in the clinical management and molecular understanding of Ph+ chronic myeloid leukemia (CML), little is known about the optimal approach to monitor and treat patients with ETV6-ABL1+ myeloproliferative neoplasms. Nine BCR-ABL1-negative CML patients with a

The ETV6/GOT1 fusion, resulting from t(10;12) (q24;p13), has been recently described in a myelodysplastic syndrome. We reported a second case of t(10;12)-positive myelodysplastic syndrome in whom fluorescent in situ hybridization confirmed the non-random translocation but molecular biology analyses revealed a ETV6/GOT1 chimera varying from the first case described.

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.