Chromosomal abnormalities are a hallmark of cancer, with numerous examples of non-random translocations found in leukaemia [1,2]. Recurrent chromosomal abnormalities are used in the clinical practice for their diagnostic and prognostic value, their association with specific subtypes and subsequently as parameters on which to base therapy decisions. A number of genes with a pivotal role in leukaemia have been found rearranged with several partners in different cases [3,4,5]. One of these genes is the ets variant 6 gene, ETV6, that has been reported to be rearranged with approximately 48 chromosomal bands in different types of abnormality. Several mechanisms of actions have been proposed for this gene in leukaemogenesis, showing an array of behavioural patterns depending on the partner involved [6].

The t(7;12)(q36;p13) has been identified as a recurrent abnormality in a subset of paediatric AML. However, there are few reports of t(7;12) found in childhood ALL (see Table 1). This rearrangement involves the ETV6 gene in 12p13 and variable breakpoints on 7q36 [7,8,9,10], usually proximal to the homeobox HB9 gene, HLXB9.

Several reports have investigated the incidence of the t(7;12) in infant leukaemia and have shown that this rearrangement occurs in approximately one third of paediatric patients with age between 0–2 years [9,10,11,12]. According to these studies, the t(7;12) has not been associated with any particular AML subtype and constitutes a poor prognostic factor. From the cytogenetic point of view, the t(7;12) has been found as a sole abnormality in 2 out of 44 cases reported to date (see Table 1 for a summary of the literature). In the majority of cases, this rearrangement was accompanied by the presence of other abnormalities, mainly numerical. The presence of an extra chromosome 19 was found in the majority of cases, with an incidence of 33 out of 44 patients. This additional chromosome was also accompanied by the presence of an extra chromosome 8 (in 10 of these cases), in the same (8 cases) or different clones (2 cases), or an extra chromosome 13 (2 cases). The presence of these additional abnormalities in such a consistent fashion has prompted the screening for t(7;12) cases on infant patients selected on the basis of having a +19 and/or +8 in their leukaemic cells [9,10,12].

The mechanism of oncogenesis at the basis of t(7;12) leukaemias remains to be elucidated. Reverse-Transcription PCR experiments have shown that this translocation results in a fusion transcript between exon 1 of the HLXB9 gene, localized in 7q36, and exon 3 of ETV6 [13]. However, studies on larger series of patients have shown that such a fusion transcript is present only in approximately 50% of t(7;12) leukaemias [12,14]. To date, there is no report that confirms the presence of a chimeric protein in the positive cases. Nevertheless, all of the t(7;12) patients have shown an ectopic expression of HLXB9, both at transcript and protein level, suggesting that this might promote leukaemogenesis in these cases [12,14,15].

Overexpression of HLXB9 has been recently reported in other forms of cancers, such as colorectal cancer and hepatocellular carcinoma, suggesting a common pathway of oncogenesis [16,17].

ChIP-on-chip studies have been undertaken to explore specific pathways of HLXB9 involvement in order to identify possible targets for this transcription factor [18]. These have shown that the latter binds to the promoter of the prostaglandin E receptor 2 gene (PTGER2) and decreases its expression. Comparative expression studies using microarrays have shown that t(7;12) leukaemias have a very different expression profile than MLL-positive childhood AML, to indicate that these two subsets of paediatric leukaemia are ontogenically and biologically very different [19].

It is therefore important to be able to discriminate among different types of leukaemia by setting appropriate diagnostic tests for the detection of t(7;12) rearrangements. Considering that conventional methods of chromosome banding often do not reach the resolutive power to identify translocations between chromosome ends, molecular cytogenetics represents the most accurate diagnostic method. The designing of probes with adequate specificity and sensitivity is crucial for the success of fluorescence in situ hybridization assays.

In this study we describe the use of a novel three-colour FISH assay for the detection of the t(7;12). We have here demonstrated that this new approach can identify the rearrangement accurately in straight forward t(7;12) cases and also as cryptic translocation in a complex rearrangement.

A total of eight patients were analysed in this study. Of these, five patients had previously been reported. Patients nos. 1–4 had already been studied for the presence of the t(7;12) [8,10,23], whereas patient no. 5 failed to show a t(7;12) in previous reports [19], although this patient sample showed an ectopic expression of HLXB9 and a fusion transcript HLXB9-ETV6. Patients nos. 6–8 were not reported previously. Morphological and cytogenetic characteristics of all patients are summarized in Table 2. FISH studies were carried out using a new three-colour probe set on all patients. Furthermore, patient no. 5 was investigated using whole chromosome 16 paint (wcp16) and whole chromosome 12 paint (wcp12) simultaneously. In addition to these probes, a revised version of the ETV6 dual colour probe was also applied at a later stage to get a better definition of the breakpoint on 12p13. The new three colour probe set identified the t(7;12)(q36;p13) in seven patients out of eight. Of these, four patients (patients nos. 1–4) were already investigated in previous studies and were used as positive controls in this work. Interestingly, patients nos. 1 and 8 both showed a deletion of 7q in the same chromosome involved in the t(7;12) rearrangement (see Figure 2 and Figure 3), although the deletion breakpoint in patient no. 8 seems more proximal. The t(7;12) rearrangement in patient no. 8 should be then revised as der(7)t(7;12)(q36;p13)del(7)(q11q36). Similarly, the rearrangement in patient no. 1 could be further refined as der(7)t(7;12)(q36;p13)del(7)(q22q36).

The t(7;12) rearrangement in patients nos. 2–4 was successfully confirmed by FISH using the three colour probe set (a representative FISH image from patient 2 only is shown in Figure 4A). In these cases, same as for patients nos. 1 and 8, it is possible to appreciate a clear split of the ETV6 probe, with a green signal localized on the der(7) and an orange signal localized on the der(12), proximal to the blue signal indicating translocation of the HLXB9 region from chromosome 7.

A different situation has been observed for patients nos. 6 and 7, where the three colour probe set has shown a different distribution of hybridization signals, with the orange signals, specific for the probe proximal to ETV6, present on both normal chromosome 12 and der(12), the green signals, specific for the probe distal to (and overlapping with) ETV6, present in three locations [normal 12, der(12) and der(7)], and blue signals also present in three locations [normal 7, der(7) and der(12)]. This hybridization pattern results in the two derivatives carrying signals for all three fluorophores (see Figure 5).

The remaining patient (no. 5) was selected on the basis of its expression profile, indicative for the presence of the t(7;12), although this rearrangement was not seen at the cytogenetic level [19]. Patient no. 5 was reported as having a translocation t(7;16), as shown by G-banding analysis (see Figure 6).

However, this appeared to be a more complex rearrangement involving a cryptic t(7;12) in the form of t(7;12;16) (see Figure 7). The use of WCP16 confirmed that the terminal portion of the der(7) was chromosome 16 material. Chromosome 12 insertion into the der(7) was not shown by WCP12. However, the three-colour probe set showed that signals corresponding to the three fluorophores were present on the der(7) (Figure 7). Only orange signals were present on the der(7) when using the new version of the break-apart ETV6 probe (see Figure 8). In the revised version, the green labelled probe was designed to be distal to the one in the previous version and would not overlap with ETV6 sequences. The blue hybridization signals corresponding to the HLXB9 region were found on the normal chromosome 7 and the der(16) (see Figure 7).

Our study showed the effectiveness of a novel three colour FISH approach for the detection of t(7;12) rearrangements involving the HLXB9 region in 7q36 and the ETV6 region in 12p13. Abnormalities of these regions are often undetected by conventional cytogenetic methods. We foresee the usefulness of such FISH assay in the diagnostic setting, for a more effective screening of specific rearrangements whose incidence might be to date underestimated.