Molecular genetic characterization of the EWS/ATF1 fusion gene in clear cell sarcoma of tendons and aponeuroses

Clear cell sarcoma (CCS) is a rare malignant soft tissue tumor particularly associated with tendons and aponeuroses. The cytogenetic hallmark is the translocation t(12;22)(q13; q 12) resulting in a chimeric EWS/ATFI gene in which the Y-terminal part of EWS at 22q is replaced by the Y-terminal part of ATFI at 12q. To date, only 13 cases of CCS have been analyzed for fusion genes at the transcription level, and there is no information about the breakpoints at the genomic level. In the present study, we describe the molecular genetic characteristics of CCS from 10 patients. Karyotypes were obtained from 10 cases, 7 of which showed the characteristic t(12;22). As an initial step in the characterization of the EWS/ATFI and ATFI/EWS chimeras, we constructed an exon/intron map of the ATFI gene. The entire ATFI gene spanned >40 kb and was composed of 7 exons. Intron 3, in which most of the genomic breakpoints occurred, was to a large extent (83%) composed of repetitive elements. RT-PCR amplified EWS/ATFI cDNA fragments in all patients and ATFI/EWS cDNA fragments in 6 of 10 patients. Four types of EWS/ATFI chimeric transcript, designated types 1-4, were identified. The most frequent chimeric transcript (type 1) was an in-frame fusion of exon 8 of EWS with exon 4 of ATFI. This was the only chimeric transcript in 5 patients but found together with other variants in 3 tumors. The type 2 transcript of EWS/ATFI, an in-frame fusion of exon 7 of EWS with exon 5 of ATFI, was detected in 4 patients, as the only transcript in I case and together with other variants in 3 cases. An in-frame fusion of exon 10 of EWS with exon 5 of ATFI (type 3) was found in I patient as the only transcript, and an out-of-frame fusion of EWS exon 7 with ATFI exon 7 (type 4) was detected in I patient together with type I and type 2 transcripts. Sequencing of the amplified ATFI/EWS cDNA fragments showed in 5 patients that ATFI exon 3 was fused with EWS exon 10, resulting in an out-of-frame chimeric transcript. In I case, nt 428 of ATFI (exon 4) was fused with EWS exon 8; at the junction, there was an insertion of 4 nucleotides, also resulting in an out-of-frame transcript. Genomic extra long PCR and sequence analysis mapped the genomic breakpoints to introns 7, 8 and 9 of EWS and intron 3 and exon 4 of ATFI. While a simple end-to-end fusion was observed in 2 cases, additional nucleotides were found at the junctions in 2 other cases. In addition, topoisomerase I consensus sequences were found close to the junctions, suggesting that this enzyme may participate in the genesis of the EWS/ATFI fusion. (C) 2002 Wiley-Liss, Inc.