Cloning and characterization of the Ewing's sarcoma and peripheral neuroepithelioma t(11;22) translocation breakpoints

Abstract

Ewing's sarcoma (ES) and peripheral neuroepithelioma (PN) are related tumors, possibly of neural crest origin, which are cytogenetically characterized by the specific translocation t(11;22)(q24;q12). The cos5 locus, previously identified in the vicinity of the chromosome 22 breakpoint of this translocation, was shown by in situ hybridization on interphase nuclei to lie between VIIIF2 and LIF, two loci located on either side of the breakpoint and at a distance of less than 2,000 kb. The progressive expansion of this locus by chromosome walking led to the construction of a 300 kb contig, which finally crossed the breakpoint. The subsequent cloning of the two translocation junction fragments of a PN, followed by the molecular characterization of the translocation breakpoints of 20 ES and PN, showed that most chromosome 22 breakpoints are clustered within a small, 2 kb region. In contrast, the chromosome 11 breakpoints are scattered over a region of at least 40 kb. The translocation leads to the synthesis of chimeric transcript that links sequences from chromosomes 22 and 11. Finally, no evidence was found of any specific difference in the position of ES and PN translocation breakpoints.

RET activation in adult and childhood papillary thyroid carcinoma using a reverse transcriptase-n-polymerase chain reaction approach on archival-nested material

“…Large retrospective studies can also be performed. The method is particularly applicable to determining the biological role of the recently discovered chromosomal rearrangements/translocations giving rise to chimeric fusion transcripts in a variety of solid tumours (Galili et al, 1993;Crozat et al, 1993;Zucman et al, 1993;Schoenmakers et al, 1995). Studies of these translocations have usually been performed on only small numbers of tumours because of the requirement for fresh tissue for standard molecular biological studies.…”

“…Whereas oncogene point mutations commonly occur in a range of tumour types, translocations are often restricted to specific tumour types, for example in the lymphomas and leukaemias (Dalla-Favera et al, 1982;Bartram et al, 1983). More recently translocations have been characterised in solid tumours including sarcomas and here too they can be specific for one morphological type, for example myxoid liposarcoma, alveolar rhabdomyosarcoma and Ewings sarcoma, each with their own distinct fusion gene found in all or nearly all tumours (Crozat et al, 1993;Galili et al, 1993;Zucman et al, 1993;Schoenmakers et al, 1995). Other chromosomal rearrangements such as the RET rearrangement although restricted to thyroid papillary carcinoma have been found in only a proportion of cases.…”

Primary synovial sarcoma of the heart: a cytogenetic and molecular genetic analysis combining RT-PCR and COBRA-FISH of a case with a complex karyotype

“…6 Owing to the involvement of chromosome 22q12, known to be a breakpoint in the t(11;22)(q24;q12) in Ewing's sarcoma, a cosmid probe set covering the EWS region on chromosome 22 was used. Two cosmid probes covering the 5 0 (G9) and 3 0 (F7) parts of the breakpoint regions 7 were labeled with fluorescein isothiocyanate (FITC) or Cy3 d-UTPs using random prime labeling kit (Invitrogen) according to the manufacturer's instructions. On normal chromosome 22, the simultaneous hybridization of the two differentially labeled cosmids results in a colocalization of the green and red signals.…”

Esthesioneuroblastoma is not a member of the primitive peripheral neuroectodermal tumour-Ewing’s group

“…Probes F7 cosmid probes, located distal to the 22 chromosome breakpoint, and 1p3, which proximally flanks and overlaps half the breakpoint on chromosome 11 Zucman et al, 1992), were labelled by nick translation. Biotin-11-dUTP (Boehringer Mannheim, Germany) was incorporated in F7, and digoxigenin-11-dUTP (Boehringer Mannheim, Germany) in 1p3; 50 ng of both probes were then annealed to 5 µg of unlabelled human competitor DNA to suppress the repetitive sequences, and to 5 µg of carrier salmon sperm DNA.…”

“…RT-PCR was performed to verify the occurrence of the EWS/FLI-1 fusion transcript, of the EWS/ERG by RNA originated from the less frequent t(21;22)(q22;q12) translocation reported in pPNETs-ETs (Sorensen et al, 1994), and of EWS/FEV transcript related to the very rare variant t(2;22)(q33;q12) (Peter et al, 1997). FISH on interphase nuclei was carried out in three cases using cosmid probes flanking the fusion gene (EWS/FLI-1) Zucman et al, 1992) and with a centromeric probe for chromosome 8 (D8Z2) in order to determine the presence of the t(11;22)(q24;12) translocation (Desmaze et al, 1994) and trisomy 8 respectively. Moreover, Southern blot analysis, with a cDNA probe spanning the breakpoint cluster region of EWS, Summary Esthesioneuroblastoma (ENB) is a rare, site-specific, locally aggressive neuronal malignancy so far thought to belong to primitive peripheral neuroectodermal tumour-Ewing's tumour (pPNETs-ETs).…”