Etienne De Braekeleer

ETV6 fusion genes in hematological malignancies: A review

Translocations involving band 12p13 are one of the most commonly observed chromosomal abnormalities in human leukemia and myelodysplastic syndrome. Their frequently result in rearrangements of the ETV6 gene. At present, 48 chromosomal bands have been identified to be involved in ETV6 translocations, insertions or inversions and 30 ETV6 partner genes have been molecularly characterized. The ETV6 protein contains two major domains, the HLH (helix-loop-helix) domain, encoded by exons 3 and 4, and the ETS domain, encoded by exons 6 through 8, with in between the internal domain encoded by exon 5. ETV6 is a strong transcriptional repressor, acting through its HLH and internal domains. Five potential mechanisms of ETV6-mediated leukemogenesis have been identified: constitutive activation of the kinase activity of the partner protein, modification of the original functions of a transcription factor, loss of function of the fusion gene, affecting ETV6 and the partner gene, activation of a proto-oncogene in the vicinity of a chromosomal translocation and dominant negative effect of the fusion protein over transcriptional repression mediated by wild-type ETV6. It is likely that ETV6 is frequently involved in leukemogenesis because of the large number of partners with which it can rearrange and the several pathogenic mechanisms by which it can lead to cell transformation.

ABL1 fusion genes in hematological malignancies: a review

Chromosomal rearrangements involving the ABL1 gene, leading to a BCR‐ABL1 fusion gene, have been mainly associated with chronic myeloid leukemia and B‐cell acute lymphoblastic leukemia (ALL). At present, six other genes have been shown to fuse to ABL1. The kinase domain of ABL1 is retained in all chimeric proteins that are also composed of the N‐terminal part of the partner protein that often includes a coiled‐coil or a helix‐loop‐helix domain. These latter domains allow oligomerization of the protein that is required for tyrosine kinase activation, cytoskeletal localization, and neoplastic transformation. Fusion genes that have a break in intron 1 or 2 (BCR‐ABL1, ETV6‐ABL1, ZMIZ1‐ABL1, EML1‐ABL1, and NUP214‐ABL1) have transforming activity, although NUP214‐ABL1 requires amplification to be efficient. The NUP214‐ABL1 gene is the second most prevalent fusion gene involving ABL1 in malignant hemopathies, with a frequency of 5% in T‐cell ALL. Both fusion genes (SFPQ‐ABL1 and RCSD1‐ABL1) characterized by a break in intron 4 of ABL1 are associated with B‐cell ALL, as the chimeric proteins lacked the SH2 domain of ABL1. Screening for ABL1 chimeric genes could be performed in patients with ALL, more particularly in those with T‐cell ALL because ABL1 modulates T‐cell development and plays a role in cytoskeletal remodeling processes in T cells.

Identification of MLL partner genes in 27 patients with acute leukemia from a single cytogenetic laboratory.

Chromosomal rearrangements involving the MLL gene have been associated with many different types of hematological malignancies. Fluorescent in situ hybridization with a panel of probes coupled with long distance inverse‐PCR was used to identify chromosomal rearrangements involving the MLL gene. Between 1995 and 2010, 27 patients with an acute leukemia were found to have a fusion gene involving MLL. All seven ALL patients with B cell acute lymphoblastic leukemia were characterized by the MLL/AFF1 fusion gene resulting from a translocation (5 patients) or an insertion (2 patients). In the 19 AML patients with acute myeloblastic leukemia, 31.6% of all characterized MLL fusion genes were MLL/MLLT3, 21.1% MLL/ELL, 10.5% MLL/MLLT6 and 10.5% MLL/EPS15. Two patients had rare or undescribed fusion genes, MLL/KIAA0284 and MLL/FLNA. Seven patients (26%) had a complex chromosomal rearrangement (three‐way translocations, insertions, deletions) involving the MLL gene. Splicing fusion genes were found in three patients, leading to a MLL/EPS15 fusion in two and a MLL/ELL fusion in a third patient. This study showed that fusion involving the MLL gene can be generated through various chromosomal rearrangements such as translocations, insertions and deletions, some being complex or cryptic. A systematic approach should be used in all cases of acute leukemia starting with FISH analyses using a commercially available MLL split signal probe. Then, the analysis has to be completed, if necessary, by further molecular cytogenetic and genomic PCR methods.

Cytogenetics in pre-B and B-cell acute lymphoblastic leukemia: a study of 208 patients diagnosed between 1981 and 2008

The detection of chromosome abnormalities by conventional cytogenetics, now combined with analyses using fluorescence in situ hybridization (FISH), is an important component in assessing the risk stratification of acute lymphoblastic leukemia (ALL). Identification of specific chromosome abnormalities led to the recognition of genetic subgroups based on modal chromosomal number, reciprocal translocations in B-cell ALL, or both. We report here the cytogenetic analysis of 208 patients with pre-B and B-cell ALL referred to a single laboratory between 1981 and 2008. Chromosome abnormalities were observed in 82.9% of L1/L2 ALL patients and in 83.3% of L3 patients with successful analysis at diagnosis. The proportion of diploid karyotypes tended to decrease during the period of study, from 26% to 13%, in association with technical progress and the introduction of FISH techniques. As previously reported, the incidence of high hyperdiploidy (51-67 chromosomes) was higher among children, whereas pseudodiploidy and hypodiploidy were higher among those >15 years of age. Structural chromosome abnormalities were more frequently observed among patients older than 15 years than in children (75.9% vs. 68.5%, respectively). As previously reported, t(9;22)(q34;q11) and t(12;21)(p13;q21) were the most frequent structural rearrangements among adults (26.9%) and children (19.7%), respectively. Almost 17% of the patients studied at diagnosis had further cytogenetic analyses at relapse, the majority showing clonal evolution toward a more complex karyotype. Although the detection of chromosome abnormalities by conventional cytogenetics and FISH techniques is an important tool in assessing risk stratification of ALL, some patients lack abnormalities with clinical relevance. The use of array comparative genomic hybridization (aCGH) offers an alternative for identifying copy number alterations, but cannot detect balanced chromosomal rearrangements.

RUNX1-MTG16 fusion gene in acute myeloblastic leukemia with t(16;21)(q24;q22): case report and review of the literature

We report here a 73-year old female who was admitted for hematomas, dyspnea, and fever. Hematological data showed pancytopenia with 9% blast cells positive for CD13, CD33, CD34, HLAD2, and myeloperoxydase. A diagnosis of acute myeloid leukemia (AML) type 2 (FAB classification) was made. Banding cytogenetic techniques performed on bone marrow cells showed a 48,XX,+8,+9,del(9)(q22q33)x2 ,t(16;21)(q24;q22)[20]/46,XX[2] karyotype. Fluorescence in situ hybridization (FISH) with BACs covering the RUNX1 (alias AML1) (band 21q22) and MTG16 (band 16q24) gene showed a fusion of both genes. The t(16;21)(q24;q22) has been described in 16 AML cases, including ours. Eleven patients had received chemotherapy for a previous cancer, most of them were been treated with DNA-topoisomerase II inhibitors known to be associated with chromosomal translocations involving the RUNX1 gene. The significant homology between MGT16 and MTG8 suggests that the RUNX1-MTG16 fusion gene induced by the t(16;21)(q24;q22) is a variant of the RUNX1-MTG8 that shares similar activity.

FLNA, a new partner gene fused to MLL in a patient with acute myelomonoblastic leukaemia

Translocations involving chromosomal band 11q23 are found in most cases of infant acute lymphoblastic leukaemia and acute myeloblastic leukaemia. The majority of these translocations lead to the rearrangement of the MLL gene and the formation of new chimeric genes (De Braekeleer et al, 2005). A 5 month-old boy was first seen at the paediatric emergency room for left hemiplegia and right hemianopsia. Scan imaging revealed a thrombosis in the right sylvian artery. Haematological data were as follows: haemoglobin 68 g/l, white blood cell (WBC) count 21Æ6 · 10/l with 10Æ5% blast cells, 55Æ5% lymphocytes, 2Æ5% neutrophils, 29% promonocytes and monocytes, and platelet count 9 · 10/l. The bone marrow aspirate showed hypercellularity with 64Æ5% blasts having a high nucleocytoplasmic index and a basophilic cytoplasm without Auer rods. Immunophenotyping showed the blast cells to be CD7, CD11b, CD11c, CD13, CD14, CD18, CD33, CD45 and myeloperoxydase positive. A diagnosis of myelomonoblastic leukaemia (French-American-British classification type 4) with severe diffuse intravascular coagulopathy was made. The boy received aracytidine for 7 d, but given his neurological status, he was only given palliative care thereafter. He died 2 months later (Arnaud et al, 2004). Cytogenetic analysis was performed on bone marrow cells with standard techniques. All 12 metaphases observed after R-banding showed a 46,XY,ins(11;X)(q23;q28q12). The insertion of material of the X chromosome in the long arm of a chromosome 11 was confirmed by fluorescent in situ hybridization (FISH) with WCPX and WCP11 (Qbiogene, Illkirch, France) probes. FISH analysis using the LSI MLL dual colour probe (Abbott, Rungis, France) confirmed the disruption of the MLL gene and showed the insertion of chromosomal material between the green (5¢ region of MLL) and red (3¢ region of MLL) signals in all 20 metaphases observed. Long distance inverse-polymerase chain reaction (LDI-PCR) was then used to identify the MLL fusion partner involved in the chromosomal rearrangement (Meyer et al, 2005). Genomic DNA was isolated from methanol/acetic acid-fixed cells of leukaemia patients stored at )20 C. DNA was extracted using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer recommendations. After DNA digestion, resulting DNA fragments were religated to form DNA circles prior to LDI-PCR studies. LDI-PCR reactions were performed as described (Meyer et al, 2005) and according the recommendations of the manufacturer (Triple Master Kit; Eppendorf, Hamburg, Germany). PCR amplimers were excised from the gel and subjected to sequence analyses (Fig 1A). Sequence analysis revealed that the 5¢ region of MLL (break in intron 10) was fused in-frame with the 3¢ region of Filamin a (FLNA) (break in intron 19), a gene located in chromosomal band Xq24 (Fig 1D) while the 3¢ region of MLL (break in exon 10) was fused out-of-frame with the 5¢ region of FOXO4 (also known as MLLT7) (break in intron 1), a less frequent partner gene of MLL, located in band Xq12. The FOXO4-FLNA fusion has not been verified at the genomic level. The chromosomal insertion was investigated in more detail using Bacterial Artificial Chromosome (BAC) clones, RP1191A14 and RP11-770J1 covering MLL, CTD-2238E23, CTD2565C16 and CTD-2511C7 spanning FLNA, and RP11-753F2 and CTD-3168H8 covering FOXO4. Two fusion signals were observed, one involving MLL and FLNA, the other MLL and FOXO4 (Fig 1C). To verify the MLL-FLNA fusion on a more functional level, total RNA was also isolated from methanol/acetic acid-fixed cells with RNeasy Mini kit (Qiagen), then reverse transcribed with Omniscript Reverse transcription kit (Qiagen). The MLL forward primer was: 5¢-CTGGGACTGATATTACTTCTGTGAC-3¢ and FLNA reverse primer was: 5¢-ATGAGTAGGCAGCACCTTGAC-3¢. The MLL-FLNA transcript fusion was amplified by HotStarTaq polymerase (Qiagen) with the following conditions (Applied Biosystems 9700): 15 min at 95 C; 35 cycles at 94 C for 30 s, 58 C for 45 s and 72 C for 1 min 30 s; extension at 72 C for 5 min. PCR products were analysed on a 1Æ5% agarose gel and visualized by ethidium bromide staining. Thus, the genomic MLL-FLNA fusion gene was readily expressed and the presence of the resulting MLL-FLNA fusion mRNA was confirmed in the patient cells (Fig 1B). Filamins are a family of widely distributed actin-binding proteins implicated in a large variety of cellular activities, including cytoskeletal organization. Three filamin genes have been thus far discovered: FLNA encoding a-filamin (ABP-280) mapped on Xq28, FLNB encoding b-filamin (ABP278) mapped on 3p14Æ3, and FLNC encoding c-filamin (ABP-L) mapped on 7q32-q35 (Gariboldi et al, 1994). FLNA gene mutations have been shown to be responsible for two X-linked disorders. Truncating mutations were reported in periventricular heterotopia, a neuronal migration disorder affecting predominantly females and leading to early prenatal death in males. Missense or small deletions/insertions were associated with a group of skeletal dysplasias known as oto-palato-digital spectrum disorders (Robertson et al, 2003). FLNA consists of 48 exons and 47 introns that span approximately 26kb of genomic DNA (Patrosso et al, 1994). Correspondence

3q26/EVI1 rearrangements in myeloid hemopathies: a cytogenetic review

The EVI1 gene, located in chromosomal band 3q26, is a transcription factor that has stem cell-specific expression pattern and is essential for the regulation of self-renewal of hematopoietic stem cells. It is now recognized as one of the dominant oncogenes associated with myeloid leukemia. EVI1 overexpression is associated with minimal to no response to chemotherapy and poor clinical outcome. Several chromosomal rearrangements involving band 3q26 are known to induce EVI1 overexpression. They are mainly found in acute myeloid leukemia and blastic phase of Philadelphia chromosome-positive chronic myeloid leukemia, more rarely in myelodysplastic syndromes. They include inv(3)(q21q26), t(3;3)(q21;q26), t(3;21)(q26;q22), t(3;12)(q26;p13) and t(2;3)(p15-23;q26). However, many other chromosomal rearrangements involving 3q26/EVI1 have been identified. The precise molecular event has not been elucidated in the majority of these chromosomal abnormalities and most gene partners remain unknown.

A complex 1;19;11 translocation involving the MLL gene in a patient with congenital acute monoblastic leukemia identified by molecular and cytogenetic techniques

Dear Editor, Translocations involving chromosomal band 11q23 are found in most of the infant acute lymphoblastic leukemia and in acute myeloblastic leukemia. The majority of these translocations lead to the rearrangement of the MLL gene and the formation of new chimeric genes [1]. We report here a case of congenital acute monoblastic leukemia and myeloid sarcoma associated with an apparent t(1;11)(p36; q23) which proved to be in fact a complex 1;19;11 translocation. This female child was born after a pregnancy of 33 weeks and 6 days complicated by gravidic toxemia and hydramnios. At birth, the baby was cyanosed and showed no reaction to stimuli. She had disseminated intravascular coagulation with petechiae and hematoma on the neck and the limbs. Cutaneous tumors were observed on the abdomen, the right arm and the left thigh. Her hematological data were as follows: hemoglobin 14 g/dl, white blood cells (WBC) 162×10/L with 0.5% neutrophils, 0.5% eosinophils, 9.5% lymphocytes, 8% monocytes and 80% monoblasts and promonocytes, and platelets 50×10/L. A diagnosis of acute monoblastic leukemia (FAB type M5) was made. The biopsy of one cutaneous tumor showed a massive derma infiltration by monoblasts; therefore, these skin tumors were considered to be myeloid sarcomas. Palliative care solely was applied. The child developed acute renal insufficiency and massive cerebral hemorrhage, of which she died 24 h after birth [2]. Cytogenetic analysis was performed on bone marrow cells with standard techniques. We observed a translocation which appeared to be balanced in R-banding between chromosomes 1 and 11; thus, the karyotype was written as 46,XX,t(1;11)(p36.2;q23). FISH analysis using the LSI MLL dual color probe (Abbott, Rungis, France) confirmed the disruption of the MLL gene. It showed the translocation of the 3′ region of MLL (red signal) to the derivative chromosome 1 while the 5′ region (green signal) remained on the der(11). We then used long-distance inverse PCR (LDI-PCR) to identify the MLL fusion partner involved in the chromosomal translocation of that particular patient [3]. Genomic DNA was isolated from methanol/acetic acid-fixed cells of leukemia patient that were stored at −20°C. DNA was extracted using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer recommendations. After DNA digestion, resulting DNA fragments were re-ligated to form DNA circles prior to LDIPCR studies. LDI-PCR reactions were performed as Ann Hematol (2009) 88:795–797 DOI 10.1007/s00277-008-0656-8

MLL - ELL fusion gene in two infants with acute monoblastic leukemia and myeloid sarcoma

, June 2012; 53(6): 1222–1224© 2012 Informa UK, Ltd.ISSN: 1042-8194 print / 1029-2403 onlineDOI: 10.3109/10428194.2011.648632Cor respondence: Pr. Marc De Braekeleer, Laboratoire de Cytog e n e tique, Facult e de Me decine et des Sciences de la Sant e, Universit e de Bretagne Occidentale, 22, avenue Camille Desmoulins, CS 93837, F-29238 Brest cedex 3, France. Tel: 33(0)2-98-01-64-76. Fax: 33(0)2-98-01-81-89. E-mail: marc.debraekeleer@univ-brest.fr

Identification of a MLL-MLLT4 fusion gene resulting from a t(6;11)(q27;q23) presenting as a del(11q) in a child with T-cell acute lymphoblastic leukemia.

Faculte de Medecine et des Sciences de la Sante, Universite de Brest, Brest, France, Institut National de la Sante et de la Recherche Medicale (INSERM), U613, Brest, France, Service de Cytogenetique, Cytologie et Biologie de la Reproduction, CHU Brest, Hopital Morvan, Brest, France, DCAL/Institute of Pharmaceutical Biology/ZAFES, Goethe-University of Frankfurt, Frankfurt/Main, Germany, Service d’Hematologie clinique, CHU Brest, Hopital Morvan, Brest, France, and Laboratoire de Genetique moleculaire et d’Histocompatibilite, CHU Brest, Hopital Morvan, Brest, France