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

Abstract

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; q12) resulting in a chimeric EWS/ATF1 gene in which the 3 -terminal part of EWS at 22q is replaced by the 3 -terminal part of ATF1 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/ATF1 and ATF1/EWS chimeras, we constructed an exon/ intron map of the ATF1 gene. The entire ATF1 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/ATF1 cDNA fragments in all patients and ATF1/EWS cDNA fragments in 6 of 10 patients. Four types of EWS/ATF1 chimeric transcript, designated types 1-4, were identified. The most frequent chimeric transcript (type 1) was an inframe fusion of exon 8 of EWS with exon 4 of ATF1. This was the only chimeric transcript in 5 patients but found together with other variants in 3 tumors. The type 2 transcript of EWS/ATF1, an in-frame fusion of exon 7 of EWS with exon 5 of ATF1, was detected in 4 patients, as the only transcript in 1 case and together with other variants in 3 cases. An in-frame fusion of exon 10 of EWS with exon 5 of ATF1 (type 3) was found in 1 patient as the only transcript, and an out-of-frame fusion of EWS exon 7 with ATF1 exon 7 (type 4) was detected in 1 patient together with type 1 and type 2 transcripts. Sequencing of the amplified ATF1/EWS cDNA fragments showed in 5 patients that ATF1 exon 3 was fused with EWS exon 10, resulting in an out-of-frame chimeric transcript. In 1 case, nt 428 of ATF1 (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 ATF1. 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/ATF1 fusion.

Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma

“…32,33 To date, four separate fusion transcripts have been described in the literature. 17,18 The most common fusion transcript, found in approximately 87% of trans location-positive cases with fusion structure data, 17 consists of an EWS exon 8 fusion to ATF1 codon 65, as described by Zucman et al 34 The other three EWS-ATF1 variant transcripts are rare, accounting for approximately 10% of cases. 35 The EWS-ATF-1 36 whose mechanism of action appears to be targeted repression of the CBP/p300 transcriptional coactivator, resulting in loss of function of p53.…”

“…26 However, in contradistinction to malignant melanoma, clear cell sarcoma harbors EWS gene rearrangements in up to 90% of cases. [11][12][13][14][15][16][17][18][19]27 Thus, translocations involving EWS are diagnostically useful for distinguishing clear cell sarcoma from its most important mimic malignant melanoma. 19 Although not a hypothesis tested in this study, clear cell sarcoma could conceivably be distinguished from benign melanocytic proliferations that mimic malignant melanoma, such as cellular blue nevi, deep penetrating nevi, and proliferative nodules arising within congenital nevi, as these lesions have not been reported to contain EWS gene rearrangements.…”

“…2,[6][7][8][9][10] In contrast to malignant melanoma, a unique and recurrent t(12;22)(q13;q12) involving the EWS gene has been identified in 70% to over 90% of clear cell sarcomas by cytogenetics, reverse-transcriptase polymerase chain reaction (RT-PCR) looking for the resultant EWS-ATF1 fusion protein, and by fluorescence in situ hybridization (FISH). [11][12][13][14][15][16][17][18][19] Clear cell sarcoma is rare and the true incidence of this neoplasm is not well established; however, the increased utilization of molecular studies for the …”

Absence of mutations of the BRAF gene in malignant melanoma of soft parts (clear cell sarcoma of tendons and aponeuroses)

“…The clinical data and the cytogenetic and molecular identification of the fusion gene have been reported [13].…”

Angiomatoid Fibrous Histiocytoma: The Current Status of Pathology and Genetics

“…64,65 Primary pulmonary myxoid sarcomas bear a striking histologic resemblance to extraskeletal myxoid chondrosarcoma but lack the characteristic translocations of extraskeletal myxoid chondrosarcoma that fuse NR4A3 on chromosome 9q22 to a variety of partner genes. [66][67][68][69][70][71][72][73] Primary pulmonary myxoid sarcomas generally express only vimentin but are occasionally weakly and focally immunoreactive for epithelial membrane antigen. The reticular pattern characteristic of PPMS is sometimes seen in AFH, which may cause diagnostic confusion in AFHs arising in the pulmonary region.…”