Novel fluorescent ligase detection reaction and flow cytometric analysis of SYT-SSX fusions in synovial sarcoma.
Synovial sarcomas (SS) are characterized by the t(X;18)(p11;q11) translocation and its resultant fusion gene, SYT-SSX. Two homologues of the SSX gene (ie, SSX1 and SSX2) are involved in the vast majority of SS and the SYT-SSX1 type of fusion has been associated with inferior clinical outcome. Thus, detection of the presence and type of SYT-SSX fusion is critical for diagnosis and prognosis in SS. Identification of SYT-SSX fusion type is typically accomplished by reverse-transcription polymerase chain reaction (RT-PCR) followed by a post-PCR analytic method. As mRNA nucleotide sequences of the SSX1 and SSX2 segments involved in the SYT-SSX fusion are nearly identical, post-PCR methods must be highly discriminatory. We describe a novel method to identify and differentiate these two chimeric transcripts using RT-PCR followed by fluorescent thermostable ligase detection reaction (f-LDR), microparticle bead capture and flow cytometric detection. Evaluation of this unique approach in 11 cases of SS without prior knowledge of SYT-SSX status, six cases of control sarcomas (CS) and three hematopoietic cell lines, revealed that the f-LDR technique was rapid, unambiguous, and highly specific. The f-LDR results were compared to XmnI enzyme digestion patterns and sequencing of PCR products, revealing a 100% concordance for all cases of SS with regards to SYT-SSX transcript type. In addition, there was a strong association of transcript type detected by f-LDR and morphological subclassification of SS, as previously reported. We conclude that this f-LDR method with flow-based detection is a robust approach to post-PCR detection of specific nucleotide sequences in SS and may be more broadly applicable in molecular oncology.
Gaffney
Robyn
R
University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Chakerian
Artemis
A
O'Connell
John X
JX
Mathers
Joan
J
Garner
Kelly
K
Joste
Nancy
N
Viswanatha
David S
DS
D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D008025:Ligases / Q000737:chemistry*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013584:Sarcoma, Synovial / Q000175:diagnosis* / Q000235:genetics*; D012983:Soft Tissue Neoplasms / Q000175:diagnosis* / Q000235:genetics*
D005456:Fluorescent Dyes; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; C116512:SYT-SSX fusion protein; D012313:RNA; C028649:endodeoxyribonuclease XmnI; D015252:Deoxyribonucleases, Type II Site-Specific; D008025:Ligases