Leading Article

Leukemia (2004) 18, 895–908. doi:10.1038/sj.leu.2403340 Published online 25 March 2004

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

M van der Burg1, T S Poulsen2, S P Hunger3, H B Beverloo4,5, E M E Smit4,5, K Vang-Nielsen2, A W Langerak1 and J J M van Dongen1

  1. 1Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
  2. 2DakoCytomation Denmark A/S, Glostrup, Denmark
  3. 3Department of Pediatric Hematology/Oncology, University of Florida College of Medicine, Gainesville, FL, USA
  4. 4Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
  5. 5Department of Cell Biology and Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Correspondence: Professor JJM van Dongen, Department of Immunology, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. Fax: +31 10 4089456; E-mail: j.j.m.vandongen@erasmusmc.nl

Received 3 November 2003; Accepted 3 February 2004; Published online 25 March 2004.



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.


fluorescence in situ hybridization (FISH), split-signal FISH, acute lymphoblastic leukemia (ALL), chromosome aberrations, fusion gene, peptide nucleic acid (PNA)


Chromosome aberrations in acute lymphoblastic leukemia (ALL)

Chromosome aberrations play an important role in hematological malignancies.1 In ALL, most of these aberrations concern balanced translocations involving genes that play key roles in the development and function of lymphoid cells, such as transcription factors, cell cycle regulators, and signal transduction molecules. Balanced translocations can result in fusion of two genes that encode leukemia-specific chimeric (fusion) proteins. The fusion proteins have functional features that differ from the corresponding wild-type proteins and mostly play a role in leukemogenesis. In addition to the new features of the fusion protein, loss of wild-type activity due to the translocation (in some translocations enhanced by deletion of the second allele) might contribute to oncogenesis. Alternatively, chromosome translocations can result in deregulated expression of (onco)genes as a direct consequence of a translocation to a regulatory element, for example, an immunoglobulin (Ig) or T-cell receptor (TCR) enhancer.2,3

The most frequent translocations in precursor-B-ALL are t(1;19)(q23;p13) t(4;11)(q21;q23), t(12;21)(p13;q22), and t(9;22)(q34;q11), all four of which result in generation of fusion genes. The t(1;19)(q23;p13) fuses the transcription factor-encoding gene TCF3 (E2A) with the transcription factor PBX1. In t(4;11)(q21;q23), the MLL gene at 11q23, which encodes a putative DNA-binding protein, is translocated to the MLLT2 (AF4) gene. The MLL gene is involved in many other translocations in ALL and acute myeloid leukemia (AML). Until now, more than 30 partner genes have been identified.4 The t(12;21)(p13;q22) involves the ETV6 (TEL) gene at 12p13 and the transcription factor-encoding gene RUNX1 (AML1). Finally, t(9;22)(q34;q11) results in fusion of the BCR gene at 22q11 with the cytoplasmic tyrosine kinase gene ABL.

In T-cell acute lymphoblastic leukemias (T-ALL), the most frequent chromosomal aberrations result in transcriptional activation of genes encoding transcription factors. Aberrations involving the TAL1 gene are frequently observed. The majority of TAL1 aberrations concern a submicroscopic deletion resulting in the fusion of the SIL and TAL1 genes: del(1)(p32p32). As a result of this approx90 kb deletion, all coding exons of the SIL gene and the 5' untranslated region of the TAL1 gene are lost, placing the TAL1 coding region under direct control of the SIL promoter. Ectopic TAL1 gene expression can also be induced by translocations involving TAL1.5,6,7,8,9 Recently, a new recurrent, but cryptic translocation t(5;14)(q35;q32) has been described in T-ALL.10,11 In the majority of patients with this translocation, the breakpoint is located within or downstream of the RANBP17 gene at 5q35; the breakpoints at 14q32 are very heterogeneous. This translocation results in overexpression of the TLX3 (HOX11L2) gene, which is located downstream of RANBP17. Finally, the MLL gene can also be involved in T-ALL-associated translocations.2,12,13

Several clinical studies have demonstrated that chromosomal translocations are useful markers contributing to risk group classification. Other risk factors are age, white blood cell count, and early treatment response to remission–induction therapy. With current treatment protocols, t(12;21) is correlated with a moderate to good prognosis, whereas t(9;22) and translocations involving 11q23 such as t(4;11) are especially correlated with a poor prognosis. The t(1;19) is generally associated with more aggressive disease, although this can be overcome with more intensive chemotherapy.14,15,16 The correlation with prognosis is less clear for chromosome aberrations involving TAL1.17,18 HOX11L2 aberrations have a relatively poor prognosis.19,20 Detection of chromosome aberrations at diagnosis, and also at relapse, is therefore an important factor in risk group classification.


Detection of chromosome aberrations

Several techniques can be used for the detection of chromosome aberrations, each having its inherent advantages and disadvantages (Table 1). An advantage of conventional cytogenetics is that it is highly informative as virtually all abnormalities can be detected. This includes not only structural abnormalities, but also numerical abnormalities such as hypo-, or hyperploidy. However, the interpretation may be difficult if the karyotype is complex. Another disadvantage is that for some samples no reliable results can be obtained because of a low mitotic index or poor chromosome morphology. In addition, some chromosome abnormalities are cryptic, that is, they cannot be identified via conventional cytogenetics, because changes in chromosome banding patterns are too marginal to be detected, such as t(12;21), t(5;14), and SIL-TAL1 fusions.21

Chromosome aberrations can also be identified via Southern blot or PCR analysis on genomic DNA. Southern blotting is considered to be technically demanding and laborious and the applicability to the detection of chromosome translocations is limited, because the breakpoints in many translocations are scattered over large regions (>25 kb). Nevertheless, Southern blot analysis has proven to be useful for detection of MLL translocations. As the MLL gene can have many translocation partner genes and the breakpoint region is relatively small (6.5 kb), Southern blot analysis is suitable for the detection of MLL rearrangements, independent of the partner gene.22,23 Southern blotting has also been used for detection of ETV6 gene rearrangements, since the majority of ETV6 breakpoints are located in a breakpoint region of 15 kb.24

PCR analysis on the DNA level is relatively easy for detection of SIL-TAL1 fusion genes.25,26 but much more complex for other translocations, mainly because PCR analysis needs multiple primers, if genomic breakpoint regions are larger than 2–4 kb.27,28,29

An alternative approach, which is suitable for detection of chromosome translocations resulting in formation of fusion genes, is detection of fusion genes or fusion gene transcripts via (nested) PCR or RT-PCR analysis.30 The advantage of this approach is that it reaches sensitivities of one cell in 103 to one cell in 106 cells, enabling detection of minimal residual disease (MRD).30,31,32 A disadvantage of PCR-based methods is that variant translocations can more easily be missed, if these variants are not covered by the used primers.

Detection of chromosome translocations with fusion genes can also be performed at the protein level via the specific detection of the fusion proteins. This technique, however, has not yet been implemented in routine diagnostics, due to lack of appropriate antibodies that specifically detect only the fusion protein and not the two wild-type proteins of which the fusion protein is composed.

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique, which uses fluorescently labeled probes for detection of specific chromosome aberrations. The advantage of this technique is that besides dividing cells (metaphase nuclei), also nondividing cells (interphase nuclei) can be analyzed, which allows a rapid screening of a large number of cells even if the malignant clone did not divide under culture conditions. In addition, also cryptic aberrations can be detected.33,34 A disadvantage of FISH analysis compared to cytogenetics is that this technique is focused on a specific type of aberration, determined by the applied probe set.

Fusion-signal FISH vs split-signal FISH

There are two main approaches of FISH probe design for use on (interphase) nuclei, that is, fusion-signal FISH and split-signal FISH. The classical fusion-signal FISH approach uses two differentially labeled probes, red and green, which flank the breakpoint regions of the two genes, which are involved in the translocation (Figure 1a). In normal karyotypes, that is, without chromosome aberration, two red signals and two green signals are detectable. In case of a translocation, a red and a green signal will be juxtaposed giving rise to a colocalized green/red signal, which will generally appear as a yellow signal. In addition, separate green and red signals of the unaffected chromosomes will be visible.

Figure 1.
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Differences between fusion-signal FISH and split-signal FISH. (a) Fusion-signal FISH with two probes located in the two genes, which are involved in the chromosome translocation. In normal situations, two green and two red signals will be present. In case of a translocation, a green and a red signal colocalize generally appearing as a yellow signal together with the separate green and red signals of the unaffected genes. (b) Spit-signal FISH with two probes positioned at opposite sides of the breakpoint region in one of genes, which are involved in the chromosome translocation. In normal situations, two yellow signals will be present, while in case of a translocation separate green and red signals will be present together with the colocalized signal of the unaffected gene.

Full figure and legend (136K)

The split-signal FISH approach also uses two differentially labeled probes, but these probes are located in only one of the two involved genes, hereafter called the target gene, and are positioned at opposite sides of the breakpoint region of the target gene (Figure 1b).35,36 In normal karyotypes, two colocalized green/red signals usually appearing yellow will be visible. A translocation will result in a split of one of the colocalized signals, resulting in a separate green and red signal together with a fused signal of the unaffected chromosome.35,36

The split-signal FISH approach has several advantages over the more traditional fusion-signal FISH. First, the detection of a translocation is independent of the involved partner gene. This is particularly of great interest for target genes with multiple partner genes such as MLL and ETV6. Although the detection is independent of the involved partner gene or partner chromosome, split-signal FISH in principle allows the identification of the partner chromosome, if metaphase spreads are present on the slide. As a result of the translocation, one of the probes moves to the partner chromosome, that is, der(partner), while the other probe remains on the der(target) chromosome. The split-signal approach therefore also allows the detection of new partner chromosomes or chromosome regions. Further molecular analysis can then be performed to identify the new partner gene, such as panhandle PCR or long distance inverse PCR.37,38

Another advantage of split-signal FISH is absence of the traditionally high levels of false-positivity as observed via the fusion-signal FISH approach, which range between 5 and 10%. False-positivity occurs as a result of coincidental colocalization of two signals, which actually represent two separate signals in a three-dimensional nucleus, but due to the two-dimensional analysis of the nucleus they are visible as a single colocalized signal. On the other hand, one could argue that split-signal FISH can give rise to low frequencies of false-negativity due to the same type of coincidental colocalization of two separate signals making these cells indistinguishable from normal nuclei. However, 5–10% false-negativity (percentage deduced from fusion-signal FISH) within the leukemic cell population will not alter the result in diagnostic material where the percentage of malignant cells is virtually always over 25%. Consequently, 10% reduction from 25 to 22.5% has no diagnostic meaning.


Technical aspects of the new FISH procedure using PNA-based blocking

The successful use of large genomic probes for FISH is dependent on blocking of the undesired background staining derived from repetitive sequences present throughout the human genome. The finishing of the human genome project has shown that a large proportion of the human genome is comprised of tandem repeated sequences (ie arranged in blocks) and interspersed tandem repeated sequences (distributed all around the genome).

Previously, heat denaturation and reannealing studies on DNA of higher organisms have distinguished three populations of genomic DNA: a slowly reannealing component (45% of the total DNA) containing unique sequences of protein-encoding genes, and intermediate and quickly reannealing components (30 and 25% of the total DNA, respectively) representing repetitive sequences.39 The fast component contains small (a few nucleotides long), highly repetitive DNA sequences, while the intermediate component contains the interspersed repetitive DNA that can be classified as either SINEs (short interspersed nuclear elements), LINEs (long interspersed nuclear elements), or LTRs (long terminal repeats).40,41,42,43 The repetitive units of the intermediate reannealing component are the major reason that large genomic nucleic acid probes are not well suited for hybridization analysis without blocking the repetitive elements to prevent undesired staining.

Blocking of repetitive sequences can be achieved using a component of the total DNA, Cot-1 DNA, enriched with repetitive sequences.44 Recently, a novel method has been developed based on selection of specific peptide nucleic acid (PNA) oligos, directed against the Alu sequences, which is the most frequent repetitive element within and around genes. PNA is a DNA analogue in which the deoxyribose phosphodiester backbone is replaced by a pseudopeptide backbone of N-(2-aminoethyl)-glycine units to which the nucleobases are attached through a methylene carbonyl linker (Figure 2).45,46 The charge of the pseudopeptide backbone of PNA is neutral, whereas the charge of the deoxyribose phosphodiester backbone of DNA is negative. Owing to the lower electrostatic repulsion, a PNA–DNA interaction occurs faster and is stronger than a DNA–DNA interaction.47 Different PNA oligos were selected in such a way that they cover both the upper and lower strand of the repetitive sequences and could therefore be used as a blocking reagent.48

Figure 2.
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Chemical structure of a PNA and a DNA backbone molecule. 'Base' indicates a purine (adenine, guanine) or a pyrimidine (cytosine, thymidine).

Full figure and legend (24K)

This novel PNA-based method for suppression of background staining is now included in our FISH procedure (DakoCytomation, Glostrup, DK, EU). A paraformaldehyde pretreatment is used to improve the brightness of the fluorescence signals. The premixed ALL probe sets contain PNA oligos and the fluorescently labeled DNA probes, and are denaturated together with the target DNA before hybridization in a humified environment overnight. Excess of probe and PNA oligos is removed by washing under stringent condition, before embedding and examination of the hybridization area (Figure 3).

Figure 3.
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Protocol for FISH with PNA-based suppression of background staining. Slides with tissue or cytology preparation are pretreated to increase the access of target DNA for the labeled probes. The probe mixture containing PNA oligos and fluorescent-labeled probes is applied to the target DNA and codenaturated, before hybridization. Unspecifically bound probe is removed by washing before the slide is scored with a fluorescent microscope. Normal cells present on the slides serve as control cells.

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Probe design for split-signal FISH

1. Translocations involving the TCF3 (E2A) gene (19p13.2–p13.3)

Translocations involving the TCF3 (E2A) gene (also called ITF1) are found in precursor-B-ALL (Table 2). The E2A protein is a transcription factor, which contains three critical domains, that is, a leucine zipper motif, a helix-loop-helix (HLH) dimerization domain, and a DNA-binding domain, which are encoded by exon 14, 17, and 18, respectively.49 So far, three types of translocations have been described. First, translocation t(1;19)(q23;p13) is found in approximately 25% of childhood pre-B-ALL (Table 2). It can be present both as balanced and as unbalanced form, that is, der(19)t(1;19) with loss of der(1).49,50 The unbalanced type may arise by nondisjunction leading to loss of the der(1) and replacement with a second copy of the unaffected chromosome 1. Loss of der(1) can arise during clonal evolution as both balanced and unbalanced t(1;19) can be detected within one patient sample.50,51,52 In 90–95% of cases, this translocation results in fusion of the TCF3 (E2A) gene to the PBX1 gene, leading to expression of the chimeric E2A-PBX1 protein.53,54,55 In the remaining cases, the TCF3 (E2A) and PBX1 genes are not involved and therefore this translocation is referred to as a E2A-PBX1-negative t(1;19).56 The E2A-PBX1 fusion protein is able to transform cells by constitutive activation of genes, which are normally regulated by PBX1 or other members of the PBX1 protein family. In addition, the leukemogenic effect of t(1;19)(q23;p13) might also result from a reduced level of wild-type E2A protein, which has recently been shown to have antiproliferative capacity in B-cell progenitors.57,58 Translocation t(1;19) is generally correlated with a poor prognosis, which can be overcome with more intensive chemotherapy, except for cases with the balanced t(1;19).14,15,16

In t(17;19)(q22;p13), TCF3 (E2A) is fused to the transcription factor HLF (hepatic leukemia factor) gene, which is found in approx1% of precursor-B-ALL (Table 2), especially in a rare form of high risk pro-B-ALL in adolescents.59,60,61 E2A-HLF influences an evolutionary conserved antiapoptotic pathway (reviewed by Seidel et al).61

A third TCF3 (E2A) gene alteration concerns a rare cryptic inversion inv(19)(p13;q13), which results in the TCF3-FB1 fusion gene (Table 2).62 The function of FB1 is as yet unknown, but analogous to the fusion proteins E2A-PBX1 and E2A-HLF, a role for E2A-FB1 in development and/or progression of leukemogenesis has been suggested.62

Split-signal FISH analysis for translocations involving TCF3 (E2A)

The TCF3 (E2A) gene consists of 18 exons and spans a region of approximately 41 kb (Figure 4a).53 The translocation breakpoints in the TCF3 (E2A) gene occur almost exclusively in a 3.5 kb intron region between exon 15 and 16 (Figure 4a).54,63 Two probes were designed (TCF3-U and TCF3-D), which flank the breakpoint region without overlapping it.56

Figure 4.
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Split-signal FISH for detection of breaks in the TCF3 (E2A) gene. (a) TCF3 (E2A) gene (19p13) with the position of the breakpoint region and the positions of the centromeric TCF3-U probe (286 kb, red) and the telomeric TCF3-D probe (567 kb, green). (b) Metaphase spread of a healthy donor. (c) Precursor-B-ALL without TCF3 (E2A) gene aberration. (d) Precursor-B-ALL with balanced TCF3 (E2A) gene translocation. (e) Precursor-B-ALL with unbalanced TCF3 (E2A) gene translocation with loss of der(1).

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In cases without a translocation, two colocalized signals will be present (Figure 4b and c). A balanced translocation involving the TCF3 (E2A) gene results in split of one of the colocalized signals giving rise to one separate green signal and one separate red signal, together with a fusion signal of the unaffected chromosome (Figure 4d). An unbalanced t(1;19) involving TCF3 (E2A) leads to the presence of a separate red signal of the der(19) and a fusion signal of the unaffected chromosome (Figure 4e). This TCF3 (E2A) probe set has been proven to detect translocations t(1;19) and t(17;19) and should theoretically also detect inv(19), although due to their rarity ALL samples bearing such aberrations have not yet been analyzed.56

2. Translocations involving the MLL gene (11q23)

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene (also called ALL-1, HRX, TRX1) on chromosome band 11q23 are found in 85% of infant ALL and in 60% of infant AML, whereas the frequency in pediatric and adult ALL is only 5–10% (Table 3).64,65,66 MLL translocations in ALL are associated with the pro-B-ALL immunophenotype (CD19+/CD10-) and are characterized by a poor prognosis, particularly in infants.67 MLL gene aberrations are also frequently found in patients with de novo AML and secondary AML following therapy including topoisomerase II inhibitors.68,69

More than 54 different partner chromosome regions have now been identified, of which at least 37 partner genes are known (Table 4).4,70 The t(4;11)(q21;q23) and t(11;19)(q23;p13.3) are the most common translocations in ALL, whereas t(6;11)(q27;q23), t(9;11)(p21-p22;q23), t(10;11)(p12;q23), and t(11;19)(q23;p13.1) are most frequent in AML.71,72,73,74,75,76

The MLL gene is the human homologue of the Drosophila trithorax gene.77 The MLL protein contains a transcription repression domain, a transcription activation domain, and two types of DNA-binding domains (minor groove DNA-binding 'AT-hook' motifs and major groove DNA-binding zinc-fingers).78 Absence of the MLL gene blocks hematopoietic differentiation in vitro and is therefore possibly important in leukemogenesis.79 MLL gene translocations disrupt the gene between the two types of DNA-binding motifs.77,80 The 'AT-hook' motifs and the transcription repression domain remain on the der(11), while the zinc-fingers and the transcription activation domain are either lost or translocated to the der(partner) chromosome.78 In most translocations, the fusion protein of the der(11) chromosome, with the 5' part of the MLL gene fused to the 3' part of the partner gene, contributes to the oncogenic process.81,82

Split-signal FISH analysis for translocations involving MLL

The human MLL gene consists of 37 exons and spans a region of approximately 100 kb (Figure 5a).83 The breaks are clustered within a 6.5 kb breakpoint region, which is positioned between exon 9 and exon 14. A FISH probe set was designed in such a way that the two probes flank both sides of the MLL breakpoint region without overlap (adapted from the probes described by van der Burg et al).35,84

Figure 5.
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Split-signal FISH for detection of breaks in the MLL gene. (a) MLL gene (11q23) with the position of the breakpoint region and the positions of the centromeric MLL-U probe (239 kb, red) and telomeric MLL-D probe (513 kb, green). (b) Metaphase spread of a healthy donor. (c) Precursor-B-ALL without MLL gene aberration. (d) Precursor-B-ALL with MLL gene translocation. (e) Precursor-B-ALL with MLL gene translocation with loss of 3' part of MLL gene.

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In normal situations without an MLL gene translocation, two colocalized signals will be present (Figure 5b and c), whereas the presence of an MLL translocation results in separate green and red signals of the MLL-U and MLL-D probes, respectively (Figure 5d). In approximately 30% of cases with an MLL translocation, a region telomeric to the breakpoint region is lost, which results in loss of signal of the MLL-D probe (Figure 5e).35,85

3. Translocations involving the ETV6 gene (12p13)

The ETV6 (TEL) gene is a member of the ETS family of transcription factors, which are characterized by a helix-turn-helix DNA-binding domain (ETS domain) with a transcriptional regulatory function, which is encoded by exons 6–8. ETV6 also contains a HLH domain that mediates homotypic oligomerization encoded by exons 3 and 4.86,87,88

Translocations involving the ETV6 gene are found in a broad range of leukemias, including ALL, AML, chronic myelomonocytic leukemia (CMML), and myelodysplastic syndrome (MDS).86,89 In the various types of leukemia, different partner genes are involved and it is noteworthy that in these translocations different exons and functional domains of the ETV6 gene are involved (Table 5).

The t(12;21)(p13;q22) involving the RUNX1 (AML1, CBFA2) gene is the most frequent translocation in children with ALL. This translocation is present in approx25% of childhood precursor-B-ALL and in <2% of adult precursor-B-ALL and is correlated with a moderate to favorable prognosis.90,91,92 The t(12;21) concerns a cryptic translocation that cannot be detected via standard cytogenetics and that is frequently accompanied by aberrations in the 12p region of the nontranslocated ETV6 allele (39% as identified by cytogenetics).93 The cytogenetically detectable 12p aberrations represent deletions, dicentric and (unbalanced) translocations.93 In 72% of cases with t(12;21), the wild-type ETV6 gene on the second allele is lost.93 Loss of heterozygosity studies even report higher frequencies of loss of the nontranslocated ETV6 allele, which is assumed to contribute to the oncogenic process as wild-type ETV6 might interfere with the oncogenic properties of the ETV6-containing fusion proteins.94,95 Recently, it was shown that loss of ETV6 expression is a critical secondary event for leukomogenesis in ALL with a t(12;21)(p13;q22).96

Split-signal FISH analysis for translocations involving ETV6

The ETV6 gene consists of 8 exons and spans a region of approximately 240 kb (Figure 6a).88 Probe design focused on detection of translocations in precursor-B-ALL with breakpoints in intron 4 or 5 (Figure 6a). The two probes were designed flanking this breakpoint region.

Figure 6.
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Split-signal FISH for detection of breaks in the ETV6 gene in precursor B-ALL. (a) ETV6 gene (12p13) with the position of the breakpoint region in precursor B-ALL and the positions of the telomeric ETV6-U probe (264 kb, red) and the centromeric ETV6-D probe (483 kb, green). (b) Metaphase spread of a healthy donor. (c) Precursor-B-ALL without ETV6 gene aberration. (d) Precursor-B-ALL with ETV6 gene translocation. (e) Precursor-B-ALL with ETV6 gene translocation with loss of second ETV6 allele.

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In normal karyotypes, two colocalized signals are present in the nucleus (Figure 6b, c). In case of a translocation, a separate red and a separate green signal are visible (Figure 6d). Owing to high frequency of loss of the nontranslocated ETV6 allele, the colocalized signal of the nontranslocated allele will be frequently lost (Figure 6e).

4. Translocations involving the BCR gene (22q11)

The Philadelphia (Ph) chromosome results from the reciprocal translocation t(9;22)(q34;q11) and is the hallmark of chronic myeloid leukemia (CML), being present in >95% of cases (Table 6).97 BCR-ABL-negative CML is associated with a very poor prognosis.98 Translocation t(9;22) is also found in 4–7% of childhood ALL and in 25–45% of adult precursor-B-ALL and correlates with a poor prognosis.30,97,99,100 The Ph translocation results in the joining of the 3' end of the ABL gene (9q34) to the 5' part of the BCR gene (22q11), creating the BCR-ABL fusion gene. The ABL gene encodes a tyrosine kinase, which has an important role in signal transduction and regulation of cell growth. The BCR protein contains a unique serine/threonine kinase activity and at least two SH2-binding sites encoded by the first exon and a C-terminal domain that functions as a GTPase-activating protein for p21rac.101 In the BCR-ABL fusion protein, the N-terminal part of BCR interferes with the ABL regulatory domain making ABL constitutively active.97

The breakpoints in the ABL gene on chromosome 9 almost exclusively occur in a 200 kb breakpoint region upstream of exon a2. Breakpoints in the BCR gene are clustered in three breakpoint cluster regions (bcr) (Table 6, Figure 7a).102 The breakpoints of CML cases occur almost exclusively in the major bcr (M-bcr), which can lead to two types of mRNA molecules, that is, b2-a2 or b3-a2, dependent on occurrence of the breakpoints in intron 13 or intron 14, respectively.97,103,104 Both transcripts encode a BCR-ABL fusion protein of 210 kDa (p210BCR-ABL). The M-bcr is also involved in approx40% of Ph+ ALL. In the remaining cases of Ph+ALL, the breakpoints are found in the minor bcr (m-bcr) between the two alternative exons and exon 2 (Figure 7a).97,105,106 The resulting e1-a2 fusion transcript encodes the p190BCR-ABL fusion protein. Although the p190BCR-ABL fusion protein is mainly found in ALL, it is linked to 3% of atypical CML cases.107,108 A third bcr in the BCR gene located between exons 19 and 20 was identified in a small proportion of CML representing a milder form (Figure 7a).109 Breakpoints in this micro bcr (mu-bcr) result in the generation of the c3-a2 fusion transcript encoding a 230 kDa fusion protein (p230BCR-ABL).109

Figure 7.
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Split-signal FISH for detection of breaks in the BCR gene. (a) BCR gene (22q11) with the position of the minor, major, and micro breakpoint cluster regions (m-bcr, M-bcr, mu-bcr) and the positions of the centromeric BCR-U probe (333 kb, red) and the telomeric BCR-D probe (408 kb, green) (b) Metaphase spread of a healthy donor. (c) Precursor-B-ALL without BCR gene aberration. (d) Precursor-B-ALL with BCR gene translocation involving the m-bcr as determined by RT-PCR.30 (e) CML with BCR gene translocation involving the m-bcr as determined by RT-PCR.30

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It has been suggested that large deletions adjacent to the breakpoint on the der(9) might be associated with a subgroup of CML patients with less favorable prognosis.110 This finding could not be explained by lack of ABL-BCR expression, because absence of this expression did not correlate with deletions on the der(9) or with a shorter survival in CML patients.111,112 So, the molecular basis of the negative effect of large deletions on prognosis has not been identified.111

Split-signal FISH analysis for translocations involving BCR

The BCR gene consists of 23 exons and spans a region of approximately 135 kb (Figure 7a).102 Two large probes were developed located upstream of the m-bcr and downstream of the mu-bcr (Figure 7a). This allows detection of BCR translocations involving any of the three breakpoint cluster regions, all giving rise to the presence of a separate green and red signal and one colocalized signal of the unaffected allele (Figure 7d, e), as compared to two fusion signals if no translocation is present (Figure 7b and c).

5. Aberrations involving the TAL1 gene (1p32)

In all, 10–25% of T-ALL have aberrations in the TAL1 gene (T-cell acute leukemia gene 1), also called SCL or TCL5, in chromosome region 1p32 (Table 4).5,9,113 These aberrations result in ectopic TAL1 protein expression. In another group of T-ALL (approx30%), TAL1 protein is also ectopically expressed in the leukemic blasts, but in these T-ALL no apparent TAL1 gene abnormalities have been found.113,114 TAL1 is required for embryonic and adult hematopoiesis, as well as for terminal erythroid differentiation.115,116,117 TAL1 belongs to the basic helix-loop-helix (bHLH) family of transcription factors and can form heterodimers with more widely expressed bHLH class I proteins referred to as E proteins.118

The majority of TAL1 gene aberrations comprises intrachromosomal submicroscopic deletions of approx90 kb in region 1p32 with an incidence of 10–25% in childhood T-ALL and <10% in adult ALL (Table 7).119,120,121,122 This deletion removes all coding exons of the SIL (SCL interrupting locus) gene and the 5' untranslated region of the TAL1 gene, placing the TAL1 coding region under direct control of the SIL promoter. As the SIL gene is ubiquitously expressed, the SIL-TAL1 fusion gene transcript results in ectopic TAL1 expression in T cells.123 In the SIL gene, three deletion breakpoints (sildb1–3) have been identified, of which sildb1 is most frequently used (95%).119,121,124 The TAL1 gene contains seven deletion breakpoints (taldb1–7), with two being involved in 98% of cases (taldb1 and 2).9,17,30,120,121,124,125

In 3% of T-ALL, ectopic TAL1 expression is caused by t(1;14)(p32;q11) (Table 7).6 This translocation involves the T-cell receptor delta (TCRD) locus at chromosome 14q11, which replaces the noncoding 5' part of the TAL1 gene.126 As a result of t(1;14), the TAL1 gene is controlled by the regulatory elements of the TCRD gene, resulting in ectopic TAL1 expression. Three additional rare TAL1 translocations have been reported: t(1;7)(p32;p35) involving the TCRB locus, with a breakpoint 35 kb downstream of the TAL1 coding sequences;7 t(1;3)(p32;p21) involving the TCTA gene;5,127 and t(1;5)(p32;q31) in which the exact partner gene has not yet been identified.8 Like in t(1;14), the breakpoints in t(1;3) and t(1;5) are located in the 5' untranslated region of the TAL1 gene. Although aberrations in the TAL1 gene are the most common defects in T-ALL, no clear correlation was found between TAL1 gene aberrations and clinical outcome in a large series of 182 children with newly diagnosed T-ALL.17 Kikuchi et al,18 however, suggested that the presence of SIL-TAL1 fusion genes is correlated with a good prognosis.128

Split-signal FISH analysis for TAL1 gene aberrations

The human TAL1 gene consists of six exons spanning 16 kb.129 The SIL gene consists of 18 exons spanning 65 kb and is located just upstream of the TAL1 gene.123 Two probes are required for the detection of both types of TAL1 gene aberrations, that is, the SIL-TAL1 fusion gene (microscopic deletion) and TAL1 translocations, in a single FISH test. The upstream FISH probe is positioned in the approx90 kb region that is deleted during a SIL-TAL1 fusion, whereas the downstream probe is positioned downstream of the TAL1 breakpoints (Figure 8a).34

Figure 8.
Figure 8 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Split-signal FISH for detection of TAL1 gene aberrations. (a) SIL-TAL1 gene region (1p32) with the centromeric SIL probe (67 kb, red) and the telomeric TAL1 probe (566 kb, green). For detailed description of SIL and TAL1 breakpoints, see van der Burg et al.34 (b) Metaphase spread of a healthy donor. (c) T-ALL without TAL1 gene aberration. (d) T-ALL with TAL1 gene translocation. (e) T-ALL with SIL-TAL1 gene fusion.

Full figure and legend (183K)

Two colocalized signals are present in normal situations without a TAL1 aberration (Figure 8b, c). A TAL1 translocation will result in a split-signal (one red and one green signal) together with a colocalized signal (Figure 8d). In cases with SIL-TAL1 fusion genes, the upstream probe will be lost giving rise to one separate green signal of the downstream probe and one colocalized (green/red) signal of the unaffected SIL-TAL1 region (Figure 8e).

6. Translocations involving the RANBP17 and TLX3 (HOX11L2) region (5q35)

The cryptic translocation t(5;14)(q35;q32) is found in both childhood and adolescent T-ALL with frequencies of 16 and 22%, respectively (Table 8).10,11 In the majority of T-ALL patients carrying this translocation, the breakpoints on band 5q35 are clustered within or downstream of the RANBP17 gene. This translocation does not result in deregulation of RANBP17, but in overexpression of the TLX3 (HOX11L2) gene that is located downstream (telomeric) of RANBP17. Ectopic HOX11L2 expression can also be found in T-ALL without a 5q35 translocation by RT-PCR analysis.19,130 HOX11L2 is a member of the homeobox-containing protein family.131 Overexpression of HOX11L2 has been shown to correlate with a poor prognosis.19,20 In another study, patients with HOX11L2 overexpression did not have a significant different clinical outcome than patients without HOX11L2 overexpression.128 The breakpoint region on chromosome 14q32 was mapped centromeric to the IGH gene between the AKT1 gene (centromeric) and TCL1 gene (telomeric) and probably concerns the BCL11B gene.10 In t(5;14)(q35;q11), the region of the RANBP17 and TLX3 (HOX11L2) genes recombined with the TCRD locus.132 This is a rare translocation also found in childhood ALL.132

Split-signal FISH analysis for breaks in the RANBP17 and TLX3 (HOX11L2) gene region

The RANBP17 gene consists of 28 exons spanning 421 kb and the homeobox gene TLX3 (HOX11L2) consists of three exons spanning 2.2 kb.133,134 The breakpoint region on 5q35 spans the 110 kb region between RANBP17 exon 20 and TLX3 (HOX11L2) exon 1. For this purpose, two probes were designed (TLX3-U and TLX3-D) flanking the breakpoint region (Figure 9a) (Van Zutven, manuscript submitted). In normal situations, two colocalized signals are present in the nucleus (Figure 9b, c). A translocation will give rise to two separate signals (Figure 9d).

Figure 9.
Figure 9 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Split-signal FISH for detection of breaks in the RANBP17 and TLX3 (HOX11L2) region. (a) RANBP1 gene and TLX3 (HOX11L2) gene, consisting of only three exons, with the centromeric TLX3-U probe (192 kb, red) and the telomeric TLX3-D probe (368 kb, green). (b) Metaphase spread of a healthy donor. (c) T-ALL without TLX3 (HOX11L2) gene aberration. (d) Metaphase of T-ALL with TLX3 (HOX11L2) gene translocation t(5;14)(q35;q11). (e) Interphase of T-ALL with TLX3 (HOX11L2) gene translocation.

Full figure and legend (176K)


Concluding remarks

In this review, we describe the application of split-signal FISH probe sets for the six most frequent aberrations in ALL, that is, four translocations with fusion genes in precursor B-ALL involving the TCF3 (E2A), MLL, ETV6, and BCR genes, and two aberrations with TAL1 or TLX3 (HOX11L2) overexpression in T-ALL. Each split-signal FISH probe set consists of two differentially labeled probes (generally composed of several BAC/PAC clones), which are located in the target gene at opposite sides of the breakpoint region.34,35,36,56 All six probe sets are directly labeled and work smoothly in combination with the newly developed PNA-blocking system, which allows combined blocking and hybridization in a single step. This single-step hybridization procedure makes split-signal FISH an easy, rapid, and sensitive tool for molecular cytogenetics (Figure 3).

The split-signal FISH approach has three major advantage over fusion-signal FISH. First, translocations involving the target gene can be detected independent of the involved partner gene. Second, split-signal FISH allows identification of the partner gene or partner chromosome region, if metaphases are present. The third advantage is the absence of high levels of false-positivity due to coincidental colocalization, as observed in the traditional fusion-signal FISH approach. One could argue that split-signal FISH can give rise to similar frequencies of false-negativity due to the same type of coincidental colocalization, but 5–10% false-negativity as deduced from fusion-signal FISH within the leukemic cell population will not alter the result in diagnostic material where the blast percentage is virtually always >25%.

Although we focused in this review on chromosome translocations in ALL, additional probe sets can be developed according to the split-signal FISH approach for other translocations, which frequently occur in other disease categories, for example, for translocations in AML and (mature) B-cell and T-cell malignancies.135,136,137,138,139



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We thank Mr T van Os for preparation of the figures and Mrs ILM Wolvers-Tettero, Mrs BH Barendregt and Mr B Brinkhof for technical assistance. We thank DakoCytomation for excellent scientific support. We are grateful to Dr T Szczepanski for critical reviewing of the manuscript and we thank Prof R Benner (head of the department of Immunology, Erasmus MC) for continuous support.



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Backtracking leukemia to birth

Nature Medicine News and Views (01 Feb 1998)