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1.  Spectra of Chromosomal Aberrations in 325 Leukemia Patients and Implications for the Development of New Molecular Detection Systems 
Journal of Korean Medical Science  2011;26(7):886-892.
This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.
doi:10.3346/jkms.2011.26.7.886
PMCID: PMC3124718  PMID: 21738341
Leukemia; Chromosomal Abnormalities; Molecular Detection System
2.  Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene 
The Journal of Experimental Medicine  2009;206(13):3131-3141.
MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4+ B-ALL. Unlike leukemic blasts, MLL-AF4+ BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4+ BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors.
doi:10.1084/jem.20091050
PMCID: PMC2806455  PMID: 19995953
3.  ETV6-RUNX1 Rearrangement in Tunisian Pediatric B-Lineage Acute Lymphoblastic Leukemia 
Advances in Hematology  2009;2009:924301.
In this study, Forty-one out of fifty-seven Tunisian children with B-lineage acute lymphoblastic leukemia (B-ALL), and without cytogenetically detectable recurrent abnormalities at the time of the diagnosis, were evaluated by fluorescence in situ hybridization (FISH) for the t(12;21). This translocation leads ETV6-RUNX1 (previously TEL-AML1) fusion gene. 16 patients (28%) had ETV6-RUNX1 rearrangement. In addition to this rearrangement, two cases showed a loss of the normal ETV6 allele, and three others showed an extra signal of the RUNX1 gene. Seven patients without ETV6-RUNX1 rearrangement showed extra signals of the RUNX1 gene. One out of the 7 patients was also associated with a t(3;12) identified by FISH. This is the first Tunisian study in which we report the incidence of t(12;21) among childhood B-lineage ALL and in which we have found multiple copies of RUNX1. Finally, our findings confirm that additional or secondary genetic changes are commonly encountered in pediatric B-lineage ALL with ETV6-RUNX1 gene fusion which is envisaged to play a pivotal role in disease progression.
doi:10.1155/2009/924301
PMCID: PMC2799269  PMID: 20049174
4.  Prenatal origin of childhood AML occurs less frequently than in childhood ALL 
BMC Cancer  2006;6:100.
Background
While there is enough convincing evidence in childhood acute lymphoblastic leukemia (ALL), the data on the pre-natal origin in childhood acute myeloid leukemia (AML) are less comprehensive. Our study aimed to screen Guthrie cards (neonatal blood spots) of non-infant childhood AML and ALL patients for the presence of their respective leukemic markers.
Methods
We analysed Guthrie cards of 12 ALL patients aged 2–6 years using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements (n = 15) and/or intronic breakpoints of TEL/AML1 fusion gene (n = 3). In AML patients (n = 13, age 1–14 years) PML/RARalpha (n = 4), CBFbeta/MYH11 (n = 3), AML1/ETO (n = 2), MLL/AF6 (n = 1), MLL/AF9 (n = 1) and MLL/AF10 (n = 1) fusion genes and/or internal tandem duplication of FLT3 gene (FLT3/ITD) (n = 2) were used as clonotypic markers. Assay sensitivity determined using serial dilutions of patient DNA into the DNA of a healthy donor allowed us to detect the pre-leukemic clone in Guthrie card providing 1–3 positive cells were present in the neonatal blood spot.
Results
In 3 patients with ALL (25%) we reproducibly detected their leukemic markers (Ig/TCR n = 2; TEL/AML1 n = 1) in the Guthrie card. We did not find patient-specific molecular markers in any patient with AML.
Conclusion
In the largest cohort examined so far we used identical approach for the backtracking of non-infant childhood ALL and AML. Our data suggest that either the prenatal origin of AML is less frequent or the load of pre-leukemic cells is significantly lower at birth in AML compared to ALL cases.
doi:10.1186/1471-2407-6-100
PMCID: PMC1463004  PMID: 16630339
5.  Clinical utility of FISH analysis in addition to G-banded karyotype in hematologic malignancies and proposal of a practical approach 
The Korean Journal of Hematology  2010;45(3):171-176.
Background
Fluorescence in situ hybridization (FISH) analysis can provide important information in the management of patients with hematologic malignancies. However, FISH performed in addition to G-banded karyotype can be labor-intensive and expensive. The aim of this study was to evaluate whether FISH gives additional information in the setting of adequate conventional cytogenetics in cases of hematologic malignancies.
Methods
Bone marrow aspirates were obtained from 135 patients at diagnosis (56 AML, 32 MDS, 20 ALL, and 27 MM) between 2005 and 2010. Interphase FISH was performed using the following probes: BCR/ABL1, AML1/ETO, PML/RARA, CBFB, MLL, EGR1, CEP8, and D7S486 for AML; CEP8, D20S108, EGR1, and D7S486 for MDS; BCR/ABL1, MLL, CDKN2A (p16), ETV6, and 6q21/c-myc for ALL; IgH, TP53, D13S25, IgH/CCND1, IgH/MAF, IgH/FGFR3, and 1q21/8p21 for MM. We compared the results of FISH with the corresponding aberrations identified by G-banded karyotype.
Results
Additional genetic aberrations detected by FISH (which were not identified by G-banded karyotype) were 4%, 9%, 50%, and 67% in AML, MDS, ALL, and MM, respectively. In ALL, CDKN2A and ETV6 FISH revealed additional genetic aberrations in 33% and 28% of cases, respectively. In MM, FISH was of benefit in detecting IgH, D13S25, TP53, and 1q21 rearrangements, not detected by G-banded karyotype (31%, 36%, 20%, and 40%, respectively).
Conclusion
These results suggest that performing FISH in addition to G-banded karyotype may contribute little additional genetic information in AML and MDS, whereas routine FISH analysis appears to be an efficient screening method in ALL and MM.
doi:10.5045/kjh.2010.45.3.171
PMCID: PMC2983032  PMID: 21120205
FISH; Karyotype; Acute myeloid leukemia; Myelodysplastic syndrome; Acute lymphoblastic leukemia; Multiple myeloma
6.  Genetic Variants Modify Susceptibility to Leukemia in Infants: A Children’s Oncology Group Report 
Pediatric blood & cancer  2012;60(1):31-34.
Background
The mixed lineage leukemia (MLL) gene is commonly rearranged in infant leukemia (IL). Genetic determinants of susceptibility to IL are unknown. Recent genome wide association studies for childhood acute lymphoblastic leukemia (ALL) have identified susceptibility loci at IKZF1, ARID5B, and CEBPE.
Procedure
We genotyped these loci in 171 infants with leukemia and 384 controls and evaluated associations overall, by subtype (ALL, acute myeloid leukemia (AML)), and by presence (+) or absence (−) of MLL rearrangements.
Results
Homozygosity for a variant IKZF1 allele (rs11978267) increased risk of infant AML (Odds Ratio (OR)=3.9, 95% Confidence Interval (CI)=1.8–8.4); the increased risk was similar for AML/MLL+ and MLL− cases. In contrast, risk of ALL/MLL− was increased in infants homozygous for the IKZF1 variant (OR=5.1, 95%CI=1.8–14.5) but the variant did not modify risk of ALL/MLL+. For ARID5B (rs10821936), homozygosity for the variant allele increased risk for the ALL/MLL− subgroup only (OR=7.2, 95%CI=2.5–20.6). There was little evidence of an association with the CEBP variant (rs2239633).
Conclusion
IKZF1 is expressed in early hematopoiesis, including precursor myeloid cells. Our data provide the first evidence that IKZF1 modifies susceptibility to infant AML, irrespective of MLL rearrangements, and could provide important new etiologic insights into this rare and heterogeneous hematopoietic malignancy.
doi:10.1002/pbc.24131
PMCID: PMC3381932  PMID: 22422485
leukemia; genetic susceptibility; infants
7.  Incidence of Common Preleukemic Gene Fusions in Umbilical Cord Blood in Slovak Population 
PLoS ONE  2014;9(3):e91116.
The first event in origination of many childhood leukemias is likely the presence of preleukemic clone (transformed hematopoietic stem/progenitor cells with preleukemic gene fusions (PGF)) in newborn. Thus, the screening of umbilical cord blood (UCB) for PGF may be of high importance for developing strategies for childhood leukemia prevention and treatment. However, the data on incidence of PGF in UCB are contradictive. We have compared multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (RT qPCR) in neonates from Slovak National Birth Cohort. According to multiplex PCR, all 135 screened samples were negative for the most frequent PGF of B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To explore the prevalence of prognostically important TEL-AML1, MLL-AF4 and BCR-ABL (p190), 200 UCB were screened using RT qPCR. The initial screening showed an unexpectedly high incidence of studied PGF. The validation of selected samples in two laboratories confirmed approximately ¼ of UCB positive, resulting in ∼4% incidence of TEL-AML1, ∼6.25% incidence of BCR-ABL1 p190, and ∼0.75% frequency of MLL-AF4. In most cases, the PGF presented at very low level, about 1–5 copies per 105 cells. We hypothesize that low PGF numbers reflect their relatively late origin and are likely to be eliminated in further development while higher number of PGF reflects earlier origination and may represent higher risk for leukemia.
doi:10.1371/journal.pone.0091116
PMCID: PMC3951330  PMID: 24621554
8.  Additional Genomic Aberrations Identified by Single Nucleotide Polymorphism Array-Based Karyotyping in an Acute Myeloid Leukemia Case with Isolated del(20q) Abnormality 
Annals of Laboratory Medicine  2012;32(6):445-449.
Prognosis is known to be better in cases with isolated chromosomal abnormalities than in those with complex karyotypes. Accordingly, del(20q) as an isolated abnormality must be distinguished from cases in which it is associated with other chromosomal rearrangements for a better stratification of prognosis. We report a case of an isolated del(20q) abnormality with additional genomic aberrations identified using whole-genome single nucleotide polymorphism array (SNP-A)-based karyotyping. A 39-yr-old man was diagnosed with AML without maturation. Metaphase cytogenetic analysis (MC) revealed del(20)(q11.2) as the isolated abnormality in 100% of metaphase cells analyzed, and FISH analysis using D20S108 confirmed the 20q deletion in 99% of interphase cells. Using FISH, other rearrangements such as BCR/ABL1, RUNX1/RUNX1T1, PML/RARA, CBFB/MYH11, and MLL were found to be negative. SNP-A identified an additional copy neutral loss of heterozygosity (CN-LOH) in the 11q13.1-q25 region. Furthermore, SNP-A allowed for a more precise definition of the breakpoints of the 20q deletion (20q11.22-q13.31). Unexpectedly, the terminal regions showed gain on chromosome 20q. The patient did not achieve complete remission; 8 months later, he died from complications of leukemic cell infiltrations into the central nervous system. This study suggests that a presumably isolated chromosomal abnormality by MC may have additional genomic aberrations, including CN-LOH, which could be associated with a poor prognosis. SNP-A-based karyotyping may be helpful for distinguishing true isolated cases from cases in combination with additional genomic aberrations not detected by MC.
doi:10.3343/alm.2012.32.6.445
PMCID: PMC3486942  PMID: 23130347
Deletion; Chromosome 20; Isolated; AML; Cytogenetics; Single nucleotide polymorphism; Array
9.  Molecular rearrangements of the MLL gene are present in most cases of infant acute myeloid leukemia and are strongly correlated with monocytic or myelomonocytic phenotypes. 
Journal of Clinical Investigation  1994;93(1):429-437.
Cytogenetic studies have previously identified abnormalities of chromosome band 11q23 in many cases of infant acute leukemia. Recent studies by ourselves and others have demonstrated breakpoint clustering in acute leukemias bearing translocations involving 11q23, and a Drosophila trithorax gene homologue (called MLL, HRX, or ALL-1) has been shown to span the 11q23 breakpoints of these translocations. To determine if this gene is affected in infant acute myeloid leukemia (AML), we have analyzed 26 infant AML cases for molecular alterations of this 11q23 gene. 15 out of 26 cases studied (58%) showed rearrangement of the MLL gene at the molecular level, and these rearrangements were clustered within an approximately 11-kb region containing nine exons of this gene. Moreover, 14 of the 15 cases with 11q23 rearrangements (93%) had myelomonocytic or monocytic phenotypes (M4 or M5 FAB subtypes, respectively), both of which are associated with a poor prognosis in childhood AML. In contrast, only 1 of 11 nonrearranged cases had an M4 or M5 phenotype (P = 0.00002). Rearrangement also correlated significantly with hyperleukocytosis (P = 0.02), another clinical parameter associated with poor outcome in this disease. Our results demonstrate that molecular rearrangements of MLL are common in M4 or M5 infant AML, and suggest that alteration of this gene may result in abnormal control of proliferation and differentiation in monocytic progenitor cells.
Images
PMCID: PMC293805  PMID: 8282816
10.  MLL rearrangements in pediatric acute lymphoblastic and myeloblastic leukemias: MLL specific and lineage specific signatures 
BMC Medical Genomics  2009;2:36.
Background
The presence of MLL rearrangements in acute leukemia results in a complex number of biological modifications that still remain largely unexplained. Armstrong et al. proposed MLL rearrangement positive ALL as a distinct subgroup, separated from acute lymphoblastic (ALL) and myeloblastic leukemia (AML), with a specific gene expression profile. Here we show that MLL, from both ALL and AML origin, share a signature identified by a small set of genes suggesting a common genetic disregulation that could be at the basis of mixed lineage leukemia in both phenotypes.
Methods
Using Affymetrix® HG-U133 Plus 2.0 platform, gene expression data from 140 (training set) + 78 (test set) ALL and AML patients with (24+13) and without (116+65) MLL rearrangements have been investigated performing class comparison (SAM) and class prediction (PAM) analyses.
Results
We identified a MLL translocation-specific (379 probes) signature and a phenotype-specific (622 probes) signature which have been tested using unsupervised methods. A final subset of 14 genes grants the characterization of acute leukemia patients with and without MLL rearrangements.
Conclusion
Our study demonstrated that a small subset of genes identifies MLL-specific rearrangements and clearly separates acute leukemia samples according to lineage origin. The subset included well-known genes and newly discovered markers that identified ALL and AML subgroups, with and without MLL rearrangements.
doi:10.1186/1755-8794-2-36
PMCID: PMC2709660  PMID: 19549311
11.  Zinc Finger Nuclease Induced DNA Double Stranded Breaks and Rearrangements in MLL 
Mutation research  2013;740(1-2):34-42.
Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the breakpoint cluster region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.
doi:10.1016/j.mrfmmm.2012.12.006
PMCID: PMC3578303  PMID: 23291497
12.  A novel spliced fusion of MLL with CT45A2 in a pediatric biphenotypic acute leukemia 
BMC Cancer  2010;10:518.
Background
Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia.
Methods
Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia.
Results
Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences.
Conclusion
We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first Cancer/Testis antigen family gene found fused with MLL in acute leukemia, future studies addressing its biologic relevance for leukemogenesis are warranted.
doi:10.1186/1471-2407-10-518
PMCID: PMC2956734  PMID: 20920256
13.  Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1-positive leukemia 
BMC Genomics  2007;8:385.
Background
The t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR.
Results
We compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI – highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings.
Conclusion
Gene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.
doi:10.1186/1471-2164-8-385
PMCID: PMC2211320  PMID: 17956600
14.  MLL-Rearranged B Lymphoblastic Leukemias Selectively Express the Immunoregulatory Carbohydrate-Binding Protein Galectin-1 
Purpose
Patients with mixed lineage leukemia (MLL)–rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL.
Experimental Design
Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation.
Results
Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells.
Conclusion
In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.
doi:10.1158/1078-0432.CCR-09-2765
PMCID: PMC2920144  PMID: 20332322
15.  Mll partial tandem duplication induces aberrant Hox expression in vivo via specific epigenetic alterations  
Journal of Clinical Investigation  2006;116(10):2707-2716.
We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%–7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. MllPTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. MllPTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.
doi:10.1172/JCI25546
PMCID: PMC1564428  PMID: 16981007
16.  Refinement of IKZF1 recombination hotspots in pediatric BCP-ALL patients 
Chromosomal translocations resulting in chimeric fusion genes are prototypic for pediatric leukemia patients. The most known fusions are ETV6-RUNX1 or BCR-ABL1 in B-cell progenitor (BCP)-ALL, and rearrangements of MLL in pediatric ALL and AML. Genome-wide sequencing projects have revealed additional, recurrent gene mutations in B cell malignancies. One of these mutations comprises the IKZF1 gene, encoding the IKAROS transcription factor which is one of the essential transcription factors driving lymphoid development. IKZF1 deletions were first identified by SNP arrays in ALL patients, and later identified with a high prevalence in BCR-ABL1+ patients. IKZF1 deletions turned out to be an independent prognostic marker associated with a poor outcome. Here, we characterized IKZF1 deletions in pediatric BCP-ALL patients by combining MLPA mapping experiments with long distance inverse PCR. The aim of our study was also to compare existing methods with our approach. Our attempt confirmed many of the existing data but revealed a more complex pattern of recombination sites, including a total of 4 recombination hotspots. This extended knowledge was translated into a novel, multiplex PCR assay that allows to perform IKZF1 deletion analyses by using a 2-tube PCR approach.
PMCID: PMC3649816  PMID: 23675566
Childhood leukemia; cancer genetics; gene deletion; IKAROS; IKZF1; leukemia markers
17.  The prognostic value of FISH as an adjunct to conventional cytogenetics for the detection of cryptic gene rearrangements on chromosome 16. A retrospective investigation of 13 patients from Northern Ireland diagnosed with M4Eo acute myeloid leukaemia. 
The Ulster Medical Journal  2003;72(1):16-21.
M4Eo acute myeloid leukaemia (AML) patients with the typical chromosome 16 abnormalities have a favourable prognosis. These subtle 16q22 gene rearrangements can be difficult to detect by conventional cytogenetic methods and if missed could lead to the incorrect assignment of prognostic group and hence subsequent treatment strategies. We retrospectively studied 13 patients diagnosed with M4Eo AML for such chromosome 16 abnormalities comparing conventional cytogenetic (G-banding) and molecular (FISH) methods. G-banded analysis detected only 2 patients with definite chromosome 16 abnormalities whereas FISH detected 4 patients, one with the typical inversion and three with the typical chromosome 16 translocation. FISH analysis also confirmed a false +ve G-banded result in one patient and a false -ve G-banded result in another patient. Finally, FISH confirmed a deletion of one chromosome 16 homologue in another patient indicating a poor prognosis. The overall survival of patients with the typical 16q22 rearrangements (n=4) was also significantly better (P=0.007) than patients with normal chromosome 16 homologues or having other numerical and/or structural abnormalities (n=9). This set of data shows that FISH is a more accurate method for the detection of cryptic 16q22 gene rearrangements and because of the prognostic implications has become a mandatory test along with conventional cytogenetics for all newly diagnosed M4Eo AML patients in Northern Ireland.
Images
PMCID: PMC2475396  PMID: 12868698
18.  Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia 
BMC Cancer  2009;9:147.
Background
A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature.
Methods
Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient.
Results
When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023)
Conclusion
We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.
doi:10.1186/1471-2407-9-147
PMCID: PMC2689242  PMID: 19445675
19.  Inter-Platform comparability of microarrays in acute lymphoblastic leukemia 
BMC Genomics  2004;5:71.
Background
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and has been the poster-child for improved therapeutics in cancer, with life time disease-free survival (LTDFS) rates improving from <10% in 1970 to >80% today. There are numerous known genetic prognostic variables in ALL, which include T cell ALL, the hyperdiploid karyotype and the translocations: t(12;21)[TEL-AML1], t(4;11)[MLL-AF4], t(9;22)[BCR-ABL], and t(1;19)[E2A-PBX]. ALL has been studied at the molecular level through expression profiling resulting in un-validated expression correlates of these prognostic indices. To date, the great wealth of expression data, which has been generated in disparate institutions, representing an extremely large cohort of samples has not been combined to validate any of these analyses. The majority of this data has been generated on the Affymetrix platform, potentially making data integration and validation on independent sample sets a possibility. Unfortunately, because the array platform has been evolving over the past several years the arrays themselves have different probe sets, making direct comparisons difficult.
To test the comparability between different array platforms, we have accumulated all Affymetrix ALL array data that is available in the public domain, as well as two sets of cDNA array data. In addition, we have supplemented this data pool by profiling additional diagnostic pediatric ALL samples in our lab. Lists of genes that are differentially expressed in the six major subclasses of ALL have previously been reported in the literature as possible predictors of the subclass.
Results
We validated the predictability of these gene lists on all of the independent datasets accumulated from various labs and generated on various array platforms, by blindly distinguishing the prognostic genetic variables of ALL. Cross-generation array validation was used successfully with high sensitivity and high specificity of gene predictors for prognostic variables. We have also been able to validate the gene predictors with high accuracy using an independent dataset generated on cDNA arrays.
Conclusion
Interarray comparisons such as this one will further enhance the ability to integrate data from several generations of microarray experiments and will help to break down barriers to the assimilation of existing datasets into a comprehensive data pool.
doi:10.1186/1471-2164-5-71
PMCID: PMC522810  PMID: 15387886
20.  Modeling the evolution of ETV6-RUNX1-induced B-cell precursor acute lymphoblastic leukemia in mice 
Blood  2011;118(4):1041-1051.
The t(12;21) translocation which generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. SNP array analysis of ETV6-RUNX1 patient samples have identified multiple additional genetic alterations, however the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that co-operate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to co-express the ETV6-RUNX1 fusion and Sleeping Beauty (SB) transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a SB transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insights into the cooperating genetic alterations in ETV6-RUNX1 leukemia.
doi:10.1182/blood-2011-02-338848
PMCID: PMC3622520  PMID: 21628403
ETV6-RUNX1; leukemia; precursor-B cell; insertional mutagenesis
21.  The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells  
Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-β. This facilitates the competitive expansion of TEL-AML1–expressing cells in the presence of TGF-β. Further analysis indicated that TEL-AML1 binds to a principal TGF-β signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-β–mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34+CD38–CD19+) and a marked growth advantage in the presence of TGF-β. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1–expressing preleukemic clones.
doi:10.1172/JCI36428
PMCID: PMC2662549  PMID: 19287094
22.  Clonal analysis of childhood acute lymphoblastic leukemia with "cytogenetically independent" cell populations. 
Journal of Clinical Investigation  1989;83(6):1971-1977.
Acute lymphoblastic leukemia (ALL) is generally regarded as a clonal disease in which a single abnormal progenitor cell gives rise to neoplastic progeny. Five of 463 cases of childhood ALL with adequately banded leukemic cells were found to have two cytogenetically independent cell populations. In addition, two of the four cases tested had more than two rearranged immunoglobulin genes and (or) T cell receptor genes. To investigate the clonality of these unusual leukemias, we examined the neoplastic cells for X-linked markers extrinsic to the disease. Leukemic cells from each of the three patients heterozygous for an X-linked, restriction fragment length polymorphism showed a single active parental allele, suggesting that both apparently independent cell populations developed from a common progenitor. These cases provide evidence that leukemogenesis involves a multistep process of mutation and suggest that karyotypic abnormalities may be a late event of malignant transformation.
Images
PMCID: PMC303920  PMID: 2566623
23.  H3K79 methylation profiles define murine and human MLL-AF4 leukemias 
Cancer cell  2008;14(5):355-368.
Summary
We created a mouse model where conditional expression of an Mll-AF4 fusion oncogene induces B-precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 Lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4 associated gene expression profiles in murine ALLs, and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We have thus demonstrated that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4 driven gene expression.
Significance
The t(4;11) encodes an MLL-AF4 fusion protein, and predicts a particularly poor prognosis when found in acute lymphoblastic leukemias (ALL). Recent studies suggest certain MLL-fusion proteins enhance gene expression by recruitment of the histone H3 lysine79 (H3K79) methyltransferase DOT1L. We demonstrate that H3K79 methylation is enhanced at many loci in leukemia cells from a murine model of Mll-AF4 and in human MLL-AF4 leukemia cells and this elevation is correlated with enhanced gene expression. Furthermore, suppression of H3K79 methylation leads to inhibition of gene expression in MLL-AF4 cells. These data demonstrate that inhibition of DOT1L may be a therapeutic approach in this disease, and that this mouse model should be useful for assessment of therapeutic approaches for MLL-rearranged ALL.
doi:10.1016/j.ccr.2008.10.001
PMCID: PMC2591932  PMID: 18977325
24.  Myeloid antigens in childhood lymphoblastic leukemia:clinical data point to regulation of CD66c distinct from other myeloid antigens 
BMC Cancer  2005;5:38.
Background
Aberrant expression of myeloid antigens (MyAgs) on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon, although its regulating mechanisms are unclear. MyAgs in ALL are interpreted e.g. as hallmarks of early differentiation stage and/or lineage indecisiveness. Granulocytic marker CD66c – Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is aberrantly expressed on ALL with strong correlation to genotype (negative in TEL/AML1 and MLL/AF4, positive in BCR/ABL and hyperdiploid cases).
Methods
In a cohort of 365 consecutively diagnosed Czech B-precursor ALL patients, we analyze distribution of MyAg+ cases and mutual relationship among CD13, CD15, CD33, CD65 and CD66c. The most frequent MyAg (CD66c) is studied further regarding its stability from diagnosis to relapse, prognostic significance and regulation of surface expression. For the latter, flow cytometry, Western blot and quantitative RT-PCR on sorted cells is used.
Results
We show CD66c is expressed in 43% patients, which is more frequent than other MyAgs studied. In addition, CD66c expression negatively correlates with CD13 (p < 0.0001), CD33 (p = 0.002) and/or CD65 (p = 0.029). Our data show that different myeloid antigens often differ in biological importance, which may be obscured by combining them into "MyAg positive ALL". We show that unlike other MyAgs, CD66c expression is not shifted from the onset of ALL to relapse (n = 39, time to relapse 0.3–5.3 years). Although opposite has previously been suggested, we show that CEACAM6 transcription is invariably followed by surface expression (by quantitative RT-PCR on sorted cells) and that malignant cells containing CD66c in cytoplasm without surface expression are not found by flow cytometry nor by Western blot in vivo. We report no prognostic significance of CD66c, globally or separately in genotype subsets of B-precursor ALL, nor an association with known risk factors (n = 254).
Conclusion
In contrast to general notion we show that different MyAgs in lymphoblastic leukemia represent different biological circumstances. We chose the most frequent and tightly genotype-associated MyAg CD66c to show its stabile expression in patients from diagnosis to relapse, which differs from what is known on the other MyAgs. Surface expression of CD66c is regulated at the gene transcription level, in contrast to previous reports.
doi:10.1186/1471-2407-5-38
PMCID: PMC1112585  PMID: 15826304
25.  Etoposide Sensitivity Does Not Predict MLL Rearrangements or Risk of Therapy-related Acute Myeloid Leukemia 
Therapy-related acute myeloid leukemia (t-AML) can arise from topoisomerase II agents (e.g. etoposide and teniposide), via drug-induced MLL gene fusions. However, whether MLL rearrangements (rMLL) and subsequent leukemogenesis are inextricably linked to the cytotoxic effects of the drug remains controversial. To this end, we compared 1) rMLL in blood of children with acute lymphoblastic leukemia (ALL) who did and did not develop t-AML, 2) epipodophyllotoxin-related toxicity in t-AML cases and controls, and 3) the level of rMLL in cells that were sensitive and resistant to etoposide. In children with ALL, rMLL during etoposide treatment appeared independent of the cumulative dose (P = 0.5), although slightly more frequent in children who developed t-AML than those who did not (7 case-control pairs, P = 0.04). Similarly, the frequency of etoposide- or teniposide-related acute toxicities did not differ between t-AML cases and controls (26 case-control pairs, P>0.17). Finally, in 25 human lymphoblastoid cell lines, MLL fusions were common after equitoxic etoposide treatment vs. controls (P < 0.0001) but did not differ in etoposide-sensitive versus resistant cell lines (P = 0.91). Together, these results indicate that epipodophyllotoxin-mediated leukemogenesis is not directly linked to the cytotoxicity of the drug.
doi:10.1038/clpt.2008.86
PMCID: PMC2583166  PMID: 18509329

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