Breakpoint heterogeneity in t(10;11) translocation in AML-M4/M5 resulting in fusion of AF10 and MLL is resolved by fluorescent in situ hybridization analysis.

Ten AML-M4/M5 patients' samples containing a t(10;11) translocation, but with different cytogenetic breakpoints on chromosome 11q (11q13-23), were studied by G- and R-banding and fluorescent in situ hybridization. Southern blotting analysis, studied in five patients, revealed a rearranged MLL gene. Reverse transcription-PCR analysis carried out in six patients showed a 5' MLL-3' AF-10 fusion transcript. Fluorescent in situ hybridization studies suggested that in 8 of 10 patients, the rearrangement/fusion transcript resulted from an inversion of a part of 11q (q13q23) translocated to 10p12. In the other two patients, it is assumed that an inversion/translocation has occurred of a part of 10p to the der(11). The results suggest that the orientation of the AF-10 gene on 10p is 5' telomeric and 3' centromeric. This is the first example of opposite-oriented genes being involved in translocation to yield fusion transcripts.

Ph1+bcr- acute leukemias: implication of Alu sequences in a chromosomal translocation occurring in the new cluster region within the BCR gene.

Chromosomal breakpoints on chromosome 22 are located in the first intron of the bcr gene in half of the Philadelphia-positive acute leukemias (Ph1+bcr- AL). We have previously shown that, in these cases, the breakpoints are clustered in the 3' portion of the bcr gene first intron, particularly in a region called bcr2 or m-bcr-1. In order to search for mechanisms underlying the reciprocal chromosome translocation, molecular analysis of breakpoints on chromosome 9 and 22 were performed in a Ph1+bcr- acute lymphoblastic leukemia with bcr2 rearrangement. The comparison of rearranged sequences with their normal counterparts showed that human repetitive Alu sequences were physically linked to the translocation on both chromosomes. In addition an inverted Alu repeat was found 5' to each rearranged Alu sequence on chromosome 22 and 9, with an intervening sequence of 210 and 90bp respectively. This allows to propose a new model, still to be confirmed, of recombination after formation of two hairpin structures which could facilitate the genesis of the chromosomal accident.

The TEL gene products: nuclear phosphoproteins with DNA binding properties.

The human TEL gene is involved in several 12p13 chromosomal abnormalities present in various human hematological malignancies, the most frequent being the t(12;21)(p13;q22), specific for childhood acute lymphoblastic leukemia. The predicted product of TEL harbours an amino acid region similar to the ETS DNA binding domain. We now report the isolation of the murine TEL cDNA and the characterization of the human TEL proteins. Human and murine TEL proteins are particularly homologous within their aminoterminal regions and their ETS domains. TEL proteins are nuclear and display specific DNA binding activity toward classical ETS binding sites. In addition, we show that TEL mRNAs initiate translation at either of the two first inframe ATGs (codon 1 and 43) to encode 50 kDa and 57 kDa TEL proteins. In vivo, each of these primary translational products is modified by multiple phosphorylation events.

Analysis of TEL proteins in human leukemias.

Chromosomal translocations involving the human 12p13 band frequently affect the TEL gene, usually resulting in gene fusion between TEL and genes encoding proteins of various types. The most frequent 12p13 translocation is the t(12;21)(p13;q22), which recombines TEL with the AML1 gene on chromosome 21 and is frequently associated with deletion of the untranslocated TEL allele. Using antisera against different parts of TEL and against the AML1 proteins, we undertook Western blot and immunofluorescence analyses of leukemic samples with and without 12p13 abnormalities. In t(12;21) samples, TEL-AML1 was detected as several protein species in the nuclei, whereas the AML1-TEL protein, was inconsistently expressed. AML1 was found to be expressed but no normal TEL proteins were detected. A survey of the TEL proteins in a panel of human leukemic samples without t(12;21) revealed a variation in the ratio of TEL protein isoforms. We also analysed a leukemic cell line bearing a t(12;22)(p13;q11) that was found to affect the 5' untranslated (UT) region of TEL and to be associated with inactivation of the untranslocated TEL allele. No MN1-TEL fusion could be detected upon RT-PCR analysis, in contrast to the previously investigated t(12;22). Strikingly, extremely low levels of apparently normal TEL proteins, expressed from the translocated allele, were detected by Western blot analysis. These results suggest that the level of TEL expression can be important for leukemogenesis.

Centric and pericentric chromosome rearrangements in hematopoietic malignancies.

Cytogenetic and fluorescence in situ hybridization (FISH) analysis of 10 patients with various hematopoietic malignancies revealed the presence of dicentric chromosomes or pericentric chromosome rearrangements. Dicentrics were only ascertained by FISH studies in six patients. Two types of pericentric chromosome rearrangements have been observed: 'classical' dicentrics with two clearly separated centromeric regions, and more unusual rearrangements with a breakpoint within the centromeric or heterochromatic area, but outside the alphoid domain. FISH analysis of partial chromosome 1 q duplications present in three Burkitt lymphoma cell lines confirmed the partial involvement of the non-alphoid centromeric domain in the duplicated chromosome segment. The incidence of centromeric and pericentromeric rearrangements in hematopoietic malignancies may be higher than hitherto admitted. The chromosomal localization of these rearrangements suggests several mechanisms possibly involved in the malignant process and deserves more systematic study.

A new breakpoint, telomeric to TEL/ETV6, on the short arm of chromosome 12 in T cell acute lymphoblastic leukemia.

Abnormalities of the short arm of chromosome 12 frequently involve the TEL/ETV6 gene in acute leukemias. In two cases of T cell acute lymphoblastic leukemia with translocation t(12;14)(p13;q11) and t(7;12)(q35;p13), respectively, the breakpoints were located telomeric to the TEL/ETV6 locus. Further fluorescence in situ hybridization (FISH) studies showed that the breakpoint was located between two markers, FGF6 (centromeric) and D12S983 (telomeric) on 12p in both patients. This result suggests that a new chromosomal breakpoint can nonrandomly involve rearrangements in T cell malignancies. The breakpoint on chromosome 14 was localized centromeric to the TRCA/D locus.

Faconi anemia and bone marrow clonal chromosome abnormalities.

Clonal chromosome abnormalities were detected in bone marrow cells of 20 patients with Fanconi anemia investigated at various stages of the disease. Two presented with acute leukemia, six with myelodysplastic syndrome, and 12 had minor or no morphological abnormalities of hematopoietic cells. Abnormalities of chromosome 7 were detected in nine patients (monosomy, isochromosome, or other structural rearrangement), and chromosome 1 was rearranged in four. The types and the significance of clonal chromosome abnormalities which may be present without apparent evolution toward acute leukemia or myelodysplastic syndrome in Fanconi anemia patients are discussed.

Abnormalities of the short arm of chromosome 12 in T cell prolymphocytic leukemia.

Abnormalities of the short arm of chromosome 12 are nonrandom events in T cell prolymphocytic leukemia (T-PLL). Fluorescence in situ hybridization (FISH) studies were performed in three patients with T-PLL and one patient with T cell peripheral lymphoma and rearrangement of 12p. Whereas the rearrangements of 12p were different in the four patients, a breakpoint centromeric to the ETV6 gene was present in the three T-PLL patients. In addition, loss of heterozygosity for a chromosomal segment telomeric to ETV6 with loss of the RAD52 locus was also shown by FISH studies. In contrast, the breakpoint was telomeric to ETV6 in the patient with peripheral lymphoma.

Chromosomal rearrangement on chromosome 11q14-q21 in T cell acute lymphoblastic leukemia.

Deletion and translocation involving the bands 11q14 and 11q21 have been detected in five patients with T cell acute lymphoblastic leukemia (ALL). The breakpoint on chromosome 11 was on the same band as that previously described in some acute nonlymphocytic leukemias: monocytic or myelomocytic. The existence of a new nonrandom rearrangement involving bands 11q14-q21 is postulated in ALL. An unusual rearrangement on 11q13 in a patient with T cell ALL is also reported.

An interstitial 11q23 deletion proven to be a rearrangement interrupting the MLL gene in an infant with acute myeloblastic leukemia.

Chromosome studies of an infant with acute myeloblastic leukemia (AML), classified as M2 in the FAB nomenclature revealed an unusual karyotype with del(11)(q23) and a marker chromosome resembling a small chromosomal fragment present in all metaphase cells examined. Fluorescence in situ hybridization (FISH) showed the splitting of a YAC probe containing a part of MLL between the del(11) and mar chromosomes. Painting showed that the mar chromosome contained DNA sequences from chromosome 11, but that the centromeric region was not marked by a chromosome 11-specific alphoid probe. The chromosomal breakpoint was located within the MLL gene by Southern blot experiments. The deletion of 11q was thus interstitial. This case illustrates the importance of associating cytogenetics, several FISH techniques, and molecular studies to analyze unusual karyotypes in leukemia.

Interstitial telomere repeats in translocations of hematopoietic disorders.

Cytogenetic and fluorescence in situ hybridization studies have shown the presence of telomeric repeats in translocation present in three patients with hematopoietic malignancies. One had jumping translocations, involving 1q12 and 2q, 16p, and 19q. These sequences were detected by FISH only in derivative chromosomes t(1;16) and t(1;19) in the first patient, and t(1;7) in the second. They were not seen in derivative t(1;2) and t(7;8), respectively. Interstitial telomeric sequences were observed in der(2)t(1;2) in about half of the metaphases in the third patient. The instability of interstitial telomeric DNA repeats in translocations is shown by the present findings. Moreover it supports the hypothesis that the presence of interstitial telomere repeats is not sufficient to make it functional.

Deletion of the short arm of chromosome 12 is a secondary event in acute lymphoblastic leukemia with t(12;21).

Translocation t(12;21) has been described as a nonrandom event in acute lymphoblastic leukemia (ALL) in patients with deletion of the short arm of chromosome 12, using fluorescence in situ hybridization techniques. Extensive FISH experiments were performed in order to re-examine the short arm of chromosome 12 in three children with ALL, previously shown to have t(12;21). It was shown that the t(12;21) is undetectable by routine R-banding technique and that the translocated 12 looks like a cytogenetically normal chromosome 12 in the three patients. Partial 12p deletion involving the TEL locus was shown to be interstitial in one patient with 12p- by using cosmid and YAC probes. In the second patient, the 12p- chromosome was secondary to the translocation since it was observed in about one half of the metaphases analyzed with FISH. In the third patient, the region of TEL usually rearranged in the t(12;21) displayed a germline pattern by Southern blotting, at diagnosis and in relapse. A few metaphases showed associated 12p- by standard cytogenetics, only in relapse. Thus we conclude that the TEL allele not involved in t(12;21) is inconstantly lost in patients with this subtype of ALL and occurs on the 12p- chromosome. These data question the status of tumor suppressor gene hypothesized for TEL.

Translocation t(11;11)(q13;q23) and HRX gene rearrangement associated with therapy-related leukemia in a child previously treated with VP16.

A child with acute myelomonocytic leukemia, bone marrow eosinophilia and inv(16) received first-line therapy including etoposide (VP-16). Cytopenia and monocytosis appeared 7 months after complete remission while the child was treated with maintenance chemotherapy. Blood abnormalities persisted after discontinuation of treatment. Nine months after complete remission, t(11;11)(q13;q23) and HRX rearrangement were detected. Five months later, overt leukemia of monocytic type occurred. The responsibility of VP-16 therapy in this treatment-related acute myelocytic leukemia is discussed.

MLL-AF1q fusion resulting from t(1;11) in acute leukemia.

The MLL gene, located on chromosome band 11q23 is fused to different partner genes as a result of various chromosomal translocations in hematopoietic malignancies. A t(1;11) (q21;q23) resulting in a MLL-AF1q fusion gene has previously been reported. Cytogenetic studies on six cases are reported, including one three-way translocation. FISH analysis using a YAC encompassing the MLL gene and a YAC encompassing the AF1q locus showed splitting in three cases and two patients, respectively. PCR analysis of two cases confirmed that AF1q is specifically associated with t(1;11)(q21;q23). The MLL-AF1q fusion mRNA was similar to that previously described in one case and involved MLL exon 7 in the other. This study confirms the specific involvement of AF1q in t(1;11) (q21;q23)-positive acute leukemia with monocytic involvement.

Chromosome abnormalities of the short arm of chromosome 12 in hematopoietic malignancies: a report including three novel translocations involving the TEL/ETV6 gene.

A wide variety of abnormalities of the short arm of chromosome 12 has been reported in hematologic malignancies. The most frequent rearrangements result from t(12;21)(p13;q22) of childhood acute lymphoblastic leukemia, a translocation cryptic when leukemic cells are analyzed with chromosome banding techniques. This translocation results in a fusion of the TEL/ETV6 and AML1 genes. In this report, examples of rearrangements of 12p are presented. Study of two complex chromosome abnormalities associated with t(12;21) emphasizes the importance of using FISH in detection of such translocations. Three novel translocations involving the TEL/ETV6 gene localized on 12p13 are also reported: t(X;12)(q28;p13), t(1;12)(q21;p13), and t(9;12)(p23-24;p13). Finally, the presentation of two translocations with breakpoints located centromeric to TEL/ETV6 highlights the not uncommon involvement of genes other than TEL/ETV6 on 12p.

Distinct MLL gene rearrangements associated with successive acute monocytic and lymphoblastic leukemias in the same patient.

A patient with acute monocytic leukemia (AMoL) and t(6;11)(q27;q23) developed acute lymphoblastic leukemia (ALL) and t(4;11)(q21;23), 10 months after complete remission of the AMoL. The MLL gene, normally located at band 11q23, appeared differently rearranged in the cells of these two leukemias, showing a different origin for the two malignant clones. The responsibility of etoposide, used in treatment of the AML, in the occurrence of the ALL is probable in this patient.

Molecular analysis of chromosomal breakpoints in three examples of chromosomal translocation involving the TEL gene.

The TEL gene is involved in several chromosomal abnormalities of human hematopoietic malignancies. The chromosome 12 breakpoints frequently lie within the fifth intron of the gene, particularly in the most frequent translocation involving TEL, the t(12;21)(p13;q22). In order to search for a peculiar mechanism involved in the genesis of these translocations, we have established the sequence of two t(12;21) and a t(9;12)(q24;p13) breakpoints. Our data do not reveal the involvement of VDJ recombinase activity or Alu sequences but favor the occurrence of staggered breaks and DNA repair activity in the genesis of these translocations.

Fluorescence in situ hybridization analysis of chromosome 1 abnormalities in hematopoietic disorders: rearrangements of DNA satellite II and new recurrent translocations.

Using fluorescence in situ hybridization analysis, breakpoints involving the long arm of chromosome 1 (1q) were localized in 36 patients with various hematopoietic disorders and rearrangements of the proximal part of 1q, as ascertained with banding techniques. The breakpoint was localized within the satellite II (sat II) domain in 14 patients with various abnormalities, between the sat II domain and the BCL9 locus in eight, between the BCL9 and ARNT loci in two, between sat II and ARNT in two others, and distal to ARNT in seven. A dicentric chromosome 1 was present in two patients. A high incidence of heterochromatin heteromorphism of chromosome 1 was present in this series. Two recurrent translocations were identified, t(1;2)(q12;q37) in three patients suffering from three different acute leukemia subtypes, and t(1;16)(q12;q24) in two patients with different diseases. Two patients had jumping translocations. Most of the rearrangements of 1q were secondary abnormalities, included in complex karyotypes. The roles of methylation, interactions with the proteins interfering with heterochromatin and possible gene silencing due to heterochromatin rearrangements are discussed.

5q- anomaly in acute lymphoblastic leukemia.

We report two new cases of common acute lymphoblastic leukemia (ALL) with 5q-. This abnormality, which is uncommon in ALL, was previously reported in 14 cases of ALL of various subtypes and appears to occur less frequently in common ALL than in other types of ALL.

Chromosome abnormalities in bone marrow of Fanconi anemia patients.

We report the clonal chromosome abnormalities of five patients with Fanconi anemia (FA). In one with myelodysplastic syndrome (MDS), an abnormal clone was present in the bone marrow (BM): 47,XY,trp(1)(q32q44), + mar. Two had acute myeloblastic leukemia (AML), one with monosomy 7 and the other with 46,XY,add(1)(p34),del(7)(p13). In the two others without signs of MDS or AML, pseudodiploidy with 46,XX,-5, +8 and 46,XX, +5, -21 were present, respectively. The significance of these abnormalities is discussed.