Detection of SYT-SSX Fusion Transcripts in Synovial Sarcoma by Reverse Transcription-Polymerase Chain Reaction Using Archival Paraffin-Embedded Tissues

Tsuji, Seiji; Hisaoka, Masanori; Morimitsu, Yosuke; Hashimoto, Hiroshi; Shimajiri, Shohei; Komiya, Setsuro; Ushijima, Masahiro; Nakamura, Toshio

doi:10.1016/s0002-9440(10)65695-7

Cited by 135 publications

(99 citation statements)

References 18 publications

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“…The RNA yield was more than adequate from all FNA samples. All RT-PCRs were carried out on prospective fresh samples in our study although the SYT-SSX fusion transcript may be demonstrated from archival paraffin-embedded tissues 2,19 and archival cytology material in up to 90% cases. 19 Moreover additional prognostic information may be obtained if one further categorizes the transcript as SYT-SSX1 or of SYT-SSX2 types.…”

Section: Discussionmentioning

confidence: 99%

Synovial sarcoma: Diagnosis on fine‐needle aspiration by morphology and molecular analysis

Srinivasan

Gautam

Gupta

et al. 2009

Cancer Cytopathology

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Section: Discussionmentioning

confidence: 99%

Synovial sarcoma: Diagnosis on fine‐needle aspiration by morphology and molecular analysis

Srinivasan

Gautam

Gupta

et al. 2009

Cancer Cytopathology

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“…At present, 3 different types of translocations have been identified in SS, involving SSX1, SSX2 (Crew et al, 1995;de Leeuw et al, 1995) and SSX4 . A strong association between SYT-SSX fusion type and morphology has been documented (Kawai et al, 1998;Nilsson et al, 1999;Antonescu et al, 2000), even if exceptions occur (Crew et al, 1995;Kashima et al, 1997;Tsuji et al, 1998;Mancuso et al, 2000).…”

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confidence: 96%

Identification of a novel spliced variant of the SYT gene expressed in normal tissues and in synovial sarcoma

et al. 2001

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Summary Synovial sarcoma (SS) is cytogenetically characterized by the translocation t(X;18)(p11.2-q11.2) generating a fusion between the SYT gene on chromosome 18 and one member of the SSX family gene (SSX1; SSX2; SSX4) on chromosome X. Here, we report for the first time that 2 forms of SYT mRNA are present in both normal tissues and SSs. By amplifying the full-length SYT cDNA of two SSs, we detected 2 bands, here designated N-SYT and I-SYT. The latter, previously undescribed, contains an in-frame insertion of 93 bp. Its sequencing revealed a 100% homology with the mouse SYT gene. These two SYT forms were present, although in different amounts, in all human normal tissues examined, including kidney, stomach, lung, colon, liver and synovia. Coexistence of N-SYT and I-SYT (both fused with SSX) was detected in a series of 59 SSs (35 monophasic and 24 biphasic) and in a SS cell line. A preliminary analysis of the differential expression levels of N-SYT and I-SYT in SSs revealed that the latter was consistently overexpressed, suggesting a role in SS pathogenesis. 1087-1094 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.1710, available online at http://www.idealibrary.com on http://www.bjcancer.com MATERIALS AND METHODS Tumours and patients59 SSs were analysed by RT-PCR on frozen material. 31 showed the non-random translocation SYT-SSX1, 16 the SYT-SSX2 and one the SYT-SSX4; in the remaining 11 tumours no specific fusion transcript has been yet detected and these are the subject of further studies. Among the SYT-SSX1 tumours, 16/31 (51.6%) were monophasic and 15/31 (48.4%) biphasic; in the SYT-SSX2 group 12/16 (75%) were monophasic and 4/16 (25%) biphasic. The single SYT-SSX4 tumour was monophasic. There were 21 primary tumours, 15 local relapses and 23 metastases; in 2 cases (patients n. 27 and 42) we analysed both the primary tumour and the metastasis, and in 4 cases (patients n. 25, 28, 41 and 55) we analysed relapses and/or metastases ( Table 2).The human synovial sarcoma CME cell line was kindly provided by Dr B Kazmierczak (Renwick et al, 1995); these cells had the typical (X;18) translocation and showed the SYT-SSX2 fusion transcript.6 normal tissue samples (kidney, stomach, liver, lung, colon and synovia) derived from different patients surgically treated in our Institute were also analysed. RNA extraction and reverse-transcription reaction (RT-PCR)Total RNA was extracted using the RNAzol method (GIBCO BRL, Life Technology) from snap-frozen tissue samples stored at -80˚C. One µg of RNA was reverse-transcribed to cDNA with oligo(dT) and random examers primers, using Superscript II reverse transcriptase (GIBCO BRL, Paisley, UK), according to the manufacturer's conditions. One µl of cDNA was used as template for each PCR reaction, which was performed using AmpliTaq (Perkin Elmer), according to the manufacturer's conditions. All the amplification products were stained with ethidium bromide and analysed on 1.8% agarose gel. SYT-SSX PCRThe detection of the putative SYT-SSX1 or SYT-SSX2 fusion transcript ...

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“…5,10,11 However, exact determination of the SYT-SSX type in SS is more aptly determined by molecular approaches. As the SYT-SSX fusion gene is actively transcribed, standard reverse-transcriptase polymerase chain reaction (RT-PCR) methods have been developed to specifically detect the chimeric mRNA in tissue biopsies of SS.…”

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confidence: 99%

“…As the SYT-SSX fusion gene is actively transcribed, standard reverse-transcriptase polymerase chain reaction (RT-PCR) methods have been developed to specifically detect the chimeric mRNA in tissue biopsies of SS. [7][8][9][10][11][12][13][14] To delineate the SSX1 and SSX2 fusion products, several approaches have been described. Direct sequencing of the PCR products has been used to distinguish the two types of fusion products (4,(7)(8)(9)12).…”

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confidence: 99%

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Novel Fluorescent Ligase Detection Reaction and Flow Cytometric Analysis of SYT-SSX Fusions in Synovial Sarcoma

Gaffney

Chakerian

O’Connell

et al. 2003

The Journal of Molecular Diagnostics

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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 reversetranscription 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. Synovial sarcoma (SS) is the fourth most common soft tissue sarcoma following malignant fibrous histiocytoma, liposarcoma and rhabdomyosarcoma. 1 These tumors predominantly affect adolescents and young adults with a predilection for periarticular regions of the extremities. Morphologically, two patterns of SS are recognized: a monophasic variant, composed entirely of a spindle cell proliferation and a biphasic variant, consisting of glands and spindle cells. Regardless of morphological type, SS is associated with the t(X;18)(p11;q11) translocation in greater than 90% of cases. 2 The presence of the t(X;18) thus constitutes a tumor specific marker for SS and can be helpful when excluding the diagnosis of other spindle cell proliferations. Specifically, the t(X;18) results in fusion of the upstream segment of the SYT gene at 18q11 to sequences of one of two related highly homologous genes at Xp11 (SSX1 or SSX2), forming a chimeric SYT-SSX gene. [3][4][5] Evaluation of the gene product of SSX suggests that it may function as a DNA binding factor, which becomes aberrantly functional with fusion to the SYT gene. 5 Although several additional SSX gene family members have been described, 6 the vast majority of SS harbor either the SYT-SSX1 or SYT-SSX2 fusions. A study by Inagaki et al 7 demonstrated a relationship w...

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Detection of SYT-SSX Fusion Transcripts in Synovial Sarcoma by Reverse Transcription-Polymerase Chain Reaction Using Archival Paraffin-Embedded Tissues

Cited by 135 publications

References 18 publications

Synovial sarcoma: Diagnosis on fine‐needle aspiration by morphology and molecular analysis

Synovial sarcoma: Diagnosis on fine‐needle aspiration by morphology and molecular analysis

Identification of a novel spliced variant of the SYT gene expressed in normal tissues and in synovial sarcoma

Novel Fluorescent Ligase Detection Reaction and Flow Cytometric Analysis of SYT-SSX Fusions in Synovial Sarcoma

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