title: Multiplex RT-PCR assay for the differential diagnosis of alveolar rhabdomyosarcoma and Ewing's sarcoma.
abstract: Cytogenetic analysis has defined specific translocations associated with two of the most common small round cell tumors of childhood, t(11;22) in Ewing's sarcoma and t(2;13) in alveolar rhabdomyosarcoma. We and others have previously demonstrated the diagnostic utility of a reverse transcriptase polymerase chain reaction (RT-PCR) assay for the detection of the t(11;22) encoded EWS/FLI-1 chimeric message in Ewing's sarcoma. More recently, we have cloned the t(2;13)(q35;q14) translocation and have shown that it results in the fusion of the PAX3 gene on chromosome 2 to FKHR, a novel member of the fork-head family of transcription factors on chromosome 13. To define the morphological spectrum of childhood sarcomas that express the t(2;13) encoded PAX3/FKHR chimeric message, we have performed RT-PCR analysis on samples from 44 primary pediatric sarcomas and 8 sarcoma cell lines. PAX3/FKHR chimeric messages were detected in 24 of 27 alveolar, 2 of 12 embryonal, and 0 of 1 pleomorphic rhabdomyosarcoma and in 1 of 2 ectomesenchymomas. In contrast, none of 8 Ewing's sarcomas or 2 undifferentiated sarcomas expressed this message. Chimeric transcripts were detected in all cases with cytogenetic evidence of the (2;13) translocation, and in each case the chimeric PAX3/FKHR message had the identical junction sequence, suggesting that genomic chromosome breaks were clustered in a single intron in both genes. By combining the PAX3/FKHR RT-PCR assay with primers for detection of the Ewing's sarcoma t(11;22) encoded EWS/FLI-1 chimeric transcript, we have developed a multiplex RT-PCR reaction that allows the rapid and accurate identification of either translocation in a biopsy sample.
authors.affiliation: Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105.
authors.forename: J R
authors.initials: JR
authors.lastname: Downing
authors.forename: A
authors.initials: A
authors.lastname: Khandekar
authors.forename: S A
authors.initials: SA
authors.lastname: Shurtleff
authors.forename: D R
authors.initials: DR
authors.lastname: Head
authors.forename: D M
authors.initials: DM
authors.lastname: Parham
authors.forename: B L
authors.initials: BL
authors.lastname: Webber
authors.forename: A S
authors.initials: AS
authors.lastname: Pappo
authors.forename: M G
authors.initials: MG
authors.lastname: Hulshof
authors.forename: W P
authors.initials: WP
authors.lastname: Conn
authors.forename: D N
authors.initials: DN
authors.lastname: Shapiro
mesh_terms: D001483:Base Sequence; D002675:Child, Preschool; D002678:Chimera / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D003937:Diagnosis, Differential; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D018232:Rhabdomyosarcoma, Alveolar / Q000175:diagnosis* / Q000235:genetics; D012512:Sarcoma, Ewing / Q000175:diagnosis* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014178:Translocation, Genetic
chemical_list: D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015345:Oligonucleotide Probes; D014157:Transcription Factors