Split-signal FISH for detection of chromosome aberrations in acute lymphoblastic leukemia.

Chromosome aberrations are frequently observed in precursor-B-acute lymphoblastic leukemias (ALL) and T-cell acute lymphoblastic leukemias (T-ALL). These translocations can form leukemia-specific chimeric fusion proteins or they can deregulate expression of an (onco)gene, resulting in aberrant expression or overexpression. Detection of chromosome aberrations is an important tool for risk classification. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for six of the most frequent chromosome aberrations in precursor-B-ALL and T-ALL. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity.     

Molecular cytogenetic analysis of 10;11 rearrangements in acute myeloid leukemia.

MLLT10 (previously called AF10) is a moderately common MLL fusion partner predominantly occurring in acute monoblastic leukemia (AML-M5). 10;11 rearrangements require at least three breaks in order to generate an in-frame MLL-MLLT10 fusion as a result of the opposite orientations of both genes on the respective chromosome arms. In this study, we describe a detailed molecular cytogenetic analysis of MLL-MLLT10 positive 10;11 rearrangements in two patients. We observed an as yet unreported chromosomal mechanism with at least four breakpoints, leading to MLL-MLLT10 gene fusion in a 24-year-old male. An inversion of 11q13-q23 with a breakpoint in the MLL gene was followed by an additional break 3' of MLL prior to insertion of the 11q segment into MLLT10. In a second patient, a 37-year-old male with AML-M5b, molecular cytogenetic analysis of an apparent 10;11 reciprocal translocation showed an intrachromosomal inversion of 3'MLLT10followed by a reciprocal translocation between 10p12 and 11q23. Review of the literature showed that all cases were the result of an inversion of either 10p or 11q followed by translocation 10p;11q or insertion of the inverted segment into MLLT10 or MLL.     

Chromosome abnormalities and MLL rearrangements in acute myeloid leukemia of infants.

Of 29 infants with acute myeloid leukemia (AML), 14 (48%) had various 11q23 translocations. MLL rearrangements were examined in 21 of the 29 patients, and 11 (52%) showed the rearrangements. 11q23 translocations and/or MLL rearrangements were found in 17 (58%) of the 29 patients. While all but one of the 17 patients with 11q23/MLL rearrangements had M4 or M5 type of the FAB classification, the 12 patients without such rearrangements had various FAB types, including M2, M4, M4EO, M6 and M7. Of the 12 patients with other chromosome abnormalities or normal karyotypes, two had inv(16) ort(16;16), one had t(1;22)(p13;q13), and two had a novel translocation, t(7;12)(q32;p13). The breakpoint on 12p of the t(7;12) was assigned to intron 1 or the region just upstream of exon 1 of the TEL/ETV6 gene by fluorescence in situ hybridization. The event-free survival at 5 years for the 17 patients with 11q23/MLL rearrangements was 42.2%, and that for the 12 patients without such rearrangements was 31.3% (P = 0.5544). 11q231MLL rearrangements have been frequently reported and a poor prognosis in infant acute lymphoblastic leukemia implied. Our study showed that while 11q23/MLL rearrangements were also common in infant AML, AML infants with such rearrangements had a clinical outcome similar to that of AML infants without such rearrangements.     

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.     

Rearrangement of MLL in a patient with congenital acute monoblastic leukemia and granulocytic sarcoma associated with a t(1;11)(p36;q23) translocation

Band 11q23 is known to be involved in translocations and insertions with a variety of partner chromosomes. In most cases, they lead to MLL rearrangements, resulting in a fusion with numerous genes. We report here a newborn girl who had disseminated intravascular coagulation and cutaneous tumors (granulocytic sarcomata) in whom a diagnosis of acute myeloblastic leukemia (AML) FAB-M5 was made. Conventional cytogenetics using R-banding showed 11 of the 17 metaphases observed to have a 46,XX,t(1;11)(p36.2;q23) karyotype. FISH analysis confirmed the disruption of the MLL gene. Two adult patients solely have been found to have a t(1;11)(p36;q23); however, no FISH analysis with a MLL probe was performed in both cases. Since the diagnosis was made at birth, this implies that the MLL rearrangement and the onset of the disease occurred in utero. Twenty children, including 3 newborns, have been reported to have granulocytic sarcoma associated with 11q23/MLL rearrangement. To the best of our knowledge, this is the first report of a case of congenital AML with GS arising in a patient with proven MLL rearrangement.     

Comparison of cytogenetics, Southern blotting, and fluorescence in situ hybridization as methods for detecting MLL gene rearrangements in children with acute leukemia and with 11q23 abnormalities.

Bone marrow samples from 67 children with acute leukemia and with cytogenetic evidence of chromosome 11 band q23 (11q23) abnormalities were characterized by fluorescence in situ hybridization (FISH) and Southern blot analysis to determine whether FISH could reliably detect MLL gene rearrangements in this population. Among the 42 patients with acute lymphoblastic leukemia (ALL), MLL gene rearrangements were detected in cells from 23 patients (54.8%) by both FISH and Southern blot analysis. FISH identified allelic deletions of MLL gene in five of 12 patients (42%) with ALL and with deletion of 11q23. In 22 of 25 children (88%) with AML, FISH detected MLL gene rearrangements, whereas Southern blot analysis identified rearrangements in 24 of 25 patients (96%). For children with acute leukemia and with 11q23 abnormalities, we recommend that FISH be used for the rapid screening of MLL gene rearrangements and that Southern blot analysis be used for the definitive assessment of the MLL gene status.     

Malignant hematopoietic cell lines: in vitro models for the study of MLL gene alterations.

Human tumor cell lines are powerful tools for investigating basic and applied aspects of cell biology. Leukemia-lymphoma cell lines have been instrumental in the cytogenetic and molecular analysis of recurring chromosome rearrangements, notably translocations and inversions, thus illuminating the pathogenesis of hematological malignancy. Chromosomal translocations targeting the MLL gene at 11q23 have come to represent a paradigm in acute leukemias. These translocations result in the in-frame joining of the MLL gene with a partner gene to generate unique fusion proteins of putatively novel function. More than 30 partner genes that participate with MLL in the more than 60 known 11q23 translocations have been reported. Cell lines provide territory to both explore the detailed structures of 11q23 translocations and investigate the leukemogenic activities of MLL fusion proteins. We review here the leukemia cell lines that have been described to carry 11q23 translocations and MLL fusion genes. Except for the t(10;11)(p12;q23), each of the following relatively frequent 11q23/MLL translocations is represented by one or more cell lines: 16 cell lines with t(4;11)(q21;q23), two cell lines with t(6;11)(q27;q23), seven cell lines with t(9;11)(p22;q23), and eight cell lines with t(11;19)(q23;p13). For each of three rare translocations, one cell line has been reported: t(5;11)(q15;q23), t(11;16)(q23;p13), and t(X;11)(q13;q23). Of these 36 cell lines with 11q23 translocations, 17 have been made available to us; we confirmed the occurrence of the alterations reported in these cell lines at the chromosomal and/or gene level. A second type of MLL gene alteration is the partial tandem duplication (PTD), which occurs in acute myeloid leukemia (AML). We found four AML cell lines with an MLL PTD; one acute lymphoblastic leukemia-derived cell line was reported to show a partial nontandem duplication. Finally, a third rearrangement involves intrachromosomal amplification of the unrearranged MLL gene leading to multiple copies of the gene and (presumably) increased expression. Three cell lines carrying such MLL amplifications have been described. The availability of these cell lines as model systems provides the opportunity to explore the altered expression or functions of MLL genes and their partners in oncogenesis.     

Rapid isolation of chromosomal breakpoints from patients with t(4;11) acute lymphoblastic leukemia: implications for basic and clinical research.

Chromosomal translocations t(4;11)(q21;q23) are associated with a group of acute lymphoblastic leukemias with very poor prognosis. From the complete sequences of the breakpoint cluster regions of the human MLL and AF-4 translocation partner genes, a novel set of 66 oligonucleotides that facilitates the rapid identification of translocation breakpoints by PCR analysis of genomic DNA was designed. For each breakpoint, a pair of optimally snited primers can be assigned, which improves the monitoring of the disease during treatment. Comparison of the breakpoints with the corresponding parental sequences also contributes to our better understanding of the illegitimate recombination events leading to these translocations.     

Novel FISH probes designed to detect IGK-MYC and IGL-MYC rearrangements in B-cell lineage malignancy identify a new breakpoint cluster region designated BVR2.

Detection of translocations involving MYC at 8q24.1 in B-cell lineage malignancies (BCL) is important for diagnostic and prognostic purposes. However, routine detection of MYC translocations is often hampered by the wide variation in breakpoint location within the MYC region, particularly when a gene other than IGH, such as IGK or IGL, is involved. To address this issue, we developed and validated four fluorescence in situ hybridization (FISH) probes: two break apart probes to detect IGK and IGL translocations, and two dual-color, dual-fusion FISH (D-FISH) probes to detect IGK-MYC and IGL-MYC. MYC rearrangements (four IGK-MYC, 12 IGL-MYC and four unknown partner gene-MYC) were correctly identified in 20 of 20 archival BCL specimens known to have MYC rearrangements not involving IGH. Seven specimens, all of which lacked MYC rearrangements using a commercial IGH/MYC D-FISH probe, were found to have 8q24 breakpoints within a cluster region >350-645 kb 3' from MYC, provisionally designated as Burkitt variant rearrangement region 2 (BVR2). FISH is a useful ancillary tool in identifying MYC rearrangements. In light of the discovery of the distally located BVR2 breakpoint cluster region, it is important to use MYC FISH probes that cover a breakpoint region at least 1.0 Mb 3' of MYC.     

Molecular Cloning of 19p13 Breakpoint Region in Infantile Leukemia with t(11;19)(q23;p13) Translocation

We studied the breakpoint regions involved in t(11;19)(q23;p13) translocation associated with infantile leukemias. Southern blot analysis with the partial cDNA clone for the MLL gene at 11q23 which we had isolated previously detected gene rearrangements in all three cell lines and three leukemia samples from the patients with t(11;19) translocation, indicating that these breakpoints were clustered within the 8.5 kb Bam HI germline fragment detected by the probe. To study the breakpoint region, a genomic library of one of the cell lines, KOCL-33, was made. We have isolated the der(19) allele containing the breakpoint as well as the germline alleles at 19p13 and 11q23. Using the genomic probes on chromosome 19 near the breakpoint, Southern blot analysis was performed. The breakpoints at 19p13 of the two other cell lines and the three leukemia samples were not located within 36 kilobases of the KOCL-33 breakpoint, although pulsed-field gel electrophoresis showed that the breakpoints of all three cell lines were on the same Nru I fragment of 230 kilobases. These results showed that the breakpoints at 19p13 were not clustered like those at 11q23 in t(11;19) translocation.     

Acute myeloblastic leukaemia with alternations of MLL proto-oncogene protein (11q23/MLL+ AML)

One of the most common chromosomal breakpoint regions in acute myeloid leukaemia is the chromosome band 11q23. The analysis of this region led to the discovery of the extremely promiscuous MLL gene, in which more than 60 MLL translocation partner genes have been described. Among the most frequent are t(9;11)(p21-22;q23)/MLL-AF9, t(10; 11)(p13; q23)/MLL-AF10, t(11;19)(q23;p13)/MLL-ELL, ENL and t(6;11)(q27;q23)/MLL-AF6. The presented work provides an overview of the molecular mechanisms by means of which MLL proto-oncogene can be converted into oncogene. Genetic alternations of the MLL Proto-Oncogene Protein besides translocation are also represented by complex chromosomal rearrangements, deletions, insertions, partial tandem duplications, amplifications and gains. These genetic alterations are described in the work from the diagnostic and prognostic point of view. Abnormalities of the MLL ProtoOncogene Protein are usually connected with bad prognosis. For that reason, in oncological practice, particular attention is paid to introducing new genetic methods for their identification. The above work gives well arranged information about different types of genetic tests and their outcomes, which can help oncologists in predicting the prognosis, in minimal residual disease monitoring and in modifying oncological patient treatment.     

The RCK gene associated with t(11;14) translocation is distinct from the MLL/ALL-1 gene with t(4;11) and t(11;19) translocations.

We previously demonstrated that the 11q23 breakpoint region, designated the RCK locus, of the RC-K8 B-lymphoma cell line with t(11;14)(q23;q32) is centromeric to PBGD, while breakpoints of infantile leukemia cell lines with t(11;19)(q23;p13) are detectable by pulsed-field gel electrophoresis with the CD3D probe. In the present study, using a probe within 1.0 kilobase of the t(11;14) breakpoint, we isolated a partial complementary DNA clone for the putative RCK gene, which detects a 7.5-kilobase mRNA. Sequence analysis predicted a novel protein of 472 amino acids which demonstrated sequence homology to a translation initiation factor/helicase family. We also isolated a phage clone from the CD3D/G yeast artificial chromosome clone (yB22B2) which detects 11- and 12-kilobase mRNAs, most likely for the MLL/ALL-1 gene associated t(4;11)(q21;q23) and t(11;19)(q23;p13) translocations. By pulsed-field gel electrophoresis after NotI digestion, this recombinant clone is on a 96-kilobase fragment, while RCK and PBGD probes are on a more telomeric 690-kilobase NotI fragment. These results, altogether, suggested that two different genes, RCK and MLL/ALL-1, are associated with 11q23 translocation of hematopoietic tumors.     

Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology?

The human AF4 (ALL-1 fused gene on chromosome 4) gene (4q11) is recurrently involved in reciprocal translocations to the MLL (mixed lineage leukemia) gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Owing to the constant presence of the derivative (11) product, it was hypothesised that only MLL.TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while other MLL fusions failed. Here, we demonstrate growth-transforming potential of AF4 wild-type and the AF4.MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4.MLL fusion protein against proteosomal degradation.