Chromosomal translocation engineering to recapitulate primary events of human cancer.

Mouse models of human cancers are important for understanding determinants of overt disease and for "preclinical" development of rational therapeutic strategies; for instance, based on macrodrugs. Chromosomal translocations underlie many human leukemias, sarcomas, and epithelial tumors. We have developed three technologies based on homologous recombination in mouse ES cells to mimic human chromosome translocations. The first, called the knockin method, allows creation of fusion genes like those typical of translocations of human leukemias and sarcomas. Two new conditional chromosomal translocation mimics have been developed. The first is a method for generating reciprocal chromosomal translocations de novo using Cre-loxP recombination (translocator mice). In some cases, there is incompatible gene orientation and the translocator model cannot be applied. We have developed a different model (invertor mice) for these situations. This method consists of introducing an inverted cDNA cassette into the intron of a target gene and bringing the cassette into the correct transcriptional orientation by Cre-loxP recombination. We describe experiments using the translocator model to generate MLL-mediated neoplasias and the invertor method to generate EWS-ERG-mediated cancer. These methods mimic the situation found in human chromosome translocations and provide the framework for design and study of human chromosomal translocations in mice.

Analysis of ETV6/AML1 abnormalities in acute lymphoblastic leukaemia: incidence, alternative spliced forms and minimal residual disease value.

The t(12;21)(p13;q22) translocation, resulting in the fusion of the ETV6 and AML1 genes, occurs in 20-25% of paediatric B-lineage acute lymphoblastic leukaemias (ALL). The identification of the fusion product has important prognostic and therapeutic implications as the translocation has been associated with a favourable clinical outcome. The aim of this study was threefold: (i) to assess the frequency and clinical association of the fusion gene in patients with and without a cytogenetically detectable chromosome 12 and/or 21 abnormality or failed cytogenetic results, (ii) to characterize alternative forms of ETV6/AML1 transcripts, and (iii) to use ETV6/AML1 as a molecular marker for the investigation of minimal residual disease (MRD). ETV6/AML1 fusion was detected in 22 (39%) of 56 cases studied by reverse transcriptase polymerase chain reaction (RT-PCR). ETV6/AML1 fusion was found in nine out of 16 (56%) cases with a cytogenetically visible chromosome 12 abnormality, but also in nine out of 29 patients (31%) without a chromosome 12 abnormality or patients with failed cytogenetics (four out of 11 patients, 36%), making this the most common cytogenetic abnormality in childhood ALL. Alternatively spliced ETV6/AML1 forms were investigated in 14 of the positive patients. Exon 5 of ETV6 was fused in frame to all AML1 exons, except exon 4. Fusion to exon 6 of AML1 resulted in one amino acid change. The presence of ETV6/AML1 was associated with a lower white blood cell count (Student's t-test; P = 0.009) and common (c)ALL phenotype (chi(2) test; P > 0.001), but no better disease-free survival. Our study shows that (i) RT-PCR is the most effective approach for the detection of t(12;21) in childhood ALL, (ii) the association of ETV6/AML1 and chromosome 12 and/or 21, seen in 56% of our cases, further confirms existing data, (iii) overall survival of patients with t(12;21) was not better than other cytogenetics groups, and (d) MRD analysis using ETV6/AML1 fusion is specific, but not sensitive enough to avoid false negative results.