An interphase FISH analysis with BCR ABL DCDF
An interphase FISH analysis with BCR-ABL1-DCDF (dual colour dual fusion) probes (Abbott) showed a normal signal pattern. In addition, an RT-PCR screen with primers specific for the BCR-ABL1 p210 and p190 fusion transcripts and a JAK2V617F mutation analysis were both negative. Chromosomal analysis of the bone marrow cultures revealed a 46, XX,t(10;12)(q24;p13) karyotype in 5 of 10 metaphases. The remaining metaphase had a normal karyotype (46, XX,t(10;12)(q24;p13)/46, XX). Whole chromosome painting probes (WCP) specific for Lonafarnib 10 and 12 were used to confirm the t(10;12) translocation. As band 12p13 was involved in the rearrangement, we assessed the status of the ETV6 locus with an ETV6 break apart probe (Dako). This analysis detected a break in one of the ETV6 loci. Interestingly, instead of on chromosome 10, the probe corresponding to the 5’ genomic portion of ETV6 hybridized, quite unexpectedly, to 9q34 suggesting an involvement of the ABL1 locus. However, when we repeated the BCR/ABL DCDF FISH analysis on metaphase chromosomes there was no alteration of the ABL1 loci visible. Only a combination of the BCR-ABL1-DCDF and an ETV6/AML1-ES (extra signal) dual colour probes (Fig. 1A) revealed a co-localization of the 5’ ETV6 genomic region with the ABL1 locus (Fig. 1B). These findings suggested the presence of an ETV6-ABL1 rearrangement. RT-PCR using ETV6-ABL1 fusion transcript-specific primers indeed detected a ‘type B’ ETV6-ABL1 fusion transcript, which results from the fusion of the first 5 exons of ETV6 to exon 2 of ABL1. Sequence analysis of the RT-PCR product confirmed the presence of the ‘type B’ – ETV6-ABL1 fusion transcript, showing an in-frame fusion between exon 5 of ETV6 and exon 2 of ABL1 (Fig. 1C). These results suggested that an insertion of the 5′ portion of the ETV6 locus up to ETV6 exon 5 into the ABL1 locus had occurred. We also performed a PCR experiment to detect the reciprocal ABL1(exon1a/1b)/ETV6(exon6) fusion and a more 5′ fusion which might result from the insertion (ABL1(exon1a/1b)/ETV6(exon2/3/4)). Neither the reciprocal ABL1(exon1a/1b)/ETV6(exon6) nor evidence for an ABL1(exon1a/1b)/ETV6(exon2/3/4) fusion, as would be expected from an insertion, were found.
There is increasing evidence that ETV6 plays the role of a tumor suppressor gene because the non-rearranged ETV6 allele is frequently deleted in childhood ALL cases with an ETV6/RUNX1 fusion [6,10,11] and ETV6 point mutations have also been described in hematological malignancies . In addition, ETV6 mutations have been identified in familial leukemia cases . Therefore, we sequenced the complete coding region of ETV6 in our patient. This analysis did not reveal a mutation (data not shown).
Based on the detection of the ETV6-ABL1 fusion transcript, off-label, evidence-based Imatinib therapy (400mg/d) was commenced [3,5,14–19]. She showed a complete normalization of her blood counts after 4 weeks. We developed a quantitative real time PCR assay (RQ-PCR) for the ETV6-ABL1 fusion transcript. Our minimal residual disease (MRD) monitoring revealed a 4 Log10 fold reduction in the levels of the ETV6-ABL1 fusion transcript over a period of 11 months. There was a moderate increase of MRD levels of about 1.5 Log10 fold 6 months later (Fig. 1D).
Discussion The ETV6 gene on the short arm of chromosome 12 is transcribed from the telomere to the centromere, whereas ABL1 is transcribed from the centromere to the telomere on the long arm of chromosome 9. The different transcriptional orientations of ETV6 and ABL1 are most likely the reason for the rarity and complexity of ETV6-ABL1 rearrangements. In most of the reported ETV6-ABL1 cases, these so-called cryptic rearrangements were not detected by conventional cytogenetics. FISH analyses showed that the ETV6-ABL1 fusion resulted either from an insertion of the 3′ region of the ABL1 gene into the ETV6 locus or an insertion of the 5′ region of the ETV6 gene within the ABL1 gene locus. FISH screening for the BCR-ABL1 rearrangement is part of the routine diagnostic work-ups for MPD (CML and MPNs) and ALL. Depending on the type of FISH probe used, this assay also allows the detection of unusual rearrangements at the BCR and ABL1 loci. Therefore, cryptic ETV6-ABL1 rearrangements, resulting from an insertion of the 3’ ABL1 region within the ETV6 locus, can be detected during the routine diagnostic work-up as an additional or split ABL1 signal. However, the ETV6-ABL1 fusions resulting from an insertion of the 5’ ETV6 region within the ABL1 locus will be overlooked, unless an ETV6 rearrangement specific FISH assay is performed. In cases with a t(9;12;Var), the involvement of 9q34 or 12p13 is an indication that an ETV6-ABL1 rearrangement might be present, which can then be confirmed by specific FISH assays and ETV6-ABL1 fusion specific RT-PCR. However, cases like the one present here, pose a problem since our initial BCR-ABL1 FISH assay was completely normal and the results of our initial cytogenetics analysis did not indicate an involvement of the ABL1 locus either. The only hint came from the chromosomal rearrangement involving band p13 of chromosome 12 on chromosome 9. With ETV6-ABL1 being a rather rare entity, FISH probes specific for the detection of the ETV6-ABL1 fusion are not commercially available. However, a FISH assay using a mixture of the standard FISH probes for the BCR-ABL1 and the ETV6-RUNX1 fusions on the metaphase chromosomes can detect and characterize even cryptic and rare ETV6-ABL1 rearrangements. Recently, multiplex RT-PCR assays have been shown to be efficient tools for the detection of a wide range of leukemia specific fusion transcripts. Several ETV6-ABL1 cases were detected by such multiplex RT-PCR approach [2,4]. Table 1 gives an overview of the ETV6-ABL1 positive cases described in the literature so far. This table is an up-date and extension of the table of Zuna et al. .