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00019606-200203000-00004ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2002 Lippincott Williams & Wilkins, Inc.11March 2002 p 16-21Molecular Detection of the Synovial Sarcoma Translocation t(X;18) by Real-Time Polymerase Chain Reaction in Paraffin-Embedded MaterialOriginal ArticlesHostein, Isabelle Ph.D.; Menard, Armelle Ph.D.; Bui, Bin Nguyen M.D.; Lussan, Cathy Med. Tech.; Wafflart, Jean M.D.; Delattre, Olivier M.D.; Peter, Martine Ph.D.; Benhattar, Jean Ph.D.; Guillou, Louis M.D.; Coindre, Jean-Michel M.D.From the Departments of Molecular Oncology (I.H., A.M.), Medicine (B.N.B.), Radioanalysis (C.L., J.W.), and Somatic Genetics (O.D., M.P.), Institut Curie, Paris, France; University Institute of Pathology, CHUV, Lausanne, Switzerland (J.B., L.G.); and Department of Pathology, Institut Bergonié, Bordeaux, France (J.-M.C.).Address correspondence and reprint requests to Isabelle Hostein, Ph.D., Molecular Oncology Laboratory, Institut Bergonié 229, cours de l'Argonne, 33076 Bordeaux Cedex, France (e-mail: [email protected]).This work was supported by the Ligue Nationale Contre le Cancer, Committee of Gironde and Landes.AbstractThe t(X;18) translocation is known to be a useful marker for the diagnosis of synovial sarcoma. In this study, the authors describe a new real-time reverse transcriptase–polymerase chain reaction (RT-PCR) method to detect SYT/SSX fusion transcripts using paraffin-embedded and frozen tumor specimens. A series of 38 soft tissue sarcomas were analyzed. Diagnosis was based on clinical, histologic, and immunohistochemical examination. The fusion transcripts were detected in 16 of 17 synovial sarcoma samples (the 17th sample was not suitable for molecular analysis). No t(X;18)-fusion transcript was PCR-amplified in the 21 nonsynovial sarcoma mesenchymal tumors. Therefore, real-time PCR amplification appears to be a powerful, rapid, specific, and sensitive technique that can be used routinely to diagnose the synovial sarcoma t(X;18) translocation. In addition, the t(X;18) can be detected not only on frozen but also on paraffin-embedded tumor samples.Synovial sarcomas represent only 10% of adult soft tissue sarcomas (1,2), and the diagnosis is often difficult, especially for the monophasic spindle cell and for the poorly differentiated subtypes. Most synovial sarcomas occur in the extremities of young adults, but they can be encountered in older patients or in unexpected locations such as the head, neck, mediastinum, pleura, retroperitoneum, and even in parenchyma such as lung, heart, prostate, and kidney (3). Many soft tissue sarcomas bear specific reciprocal translocations, and their detection is more and more often used for diagnostic purposes (3). The translocation t(X;18) (p11.2; q11.2) is typically associated with synovial sarcoma (4) and is now considered the main criterion to define synovial sarcoma. This translocation results in the fusion of the SYT gene located on chromosome 18 with one of the three SSX-related genes (SSX1, SSX2, or SSX4), which are located on chromosome X (5,6). Recently, a correlation was reported between SYT/SSX1 or SYT/SSX2 and morphologic type (7) and/or patient outcome in synovial sarcoma (8,9), but this should be confirmed in a larger series.Molecular biology and especially reverse transcriptase–polymerase chain reaction (RT-PCR) are useful tools for pathologists to confirm the histopathologic diagnosis of sarcomas, especially in situations with unusual aspects, or to confirm a doubtful diagnosis. They have been widely used not only for different translocations in the field of hematologic diseases (10–13) but also in soft tissue tumors (14–16). Among the molecular techniques, RT-PCR is the most specific and sensitive method to detect the t(X;18) translocation. Several studies have shown the efficiency of PCR amplification for t(X;18) detection in synovial sarcoma tumors using frozen samples (17,18).However, it is well known that in most laboratories, tumor samples are fixed in formol or Holland Bouin fluid. In daily routine diagnosis and when frozen tissue is not always available, it is also important to be able to exploit such material. Nucleic acid (DNA or RNA) extraction from paraffin-embedded fixed tissues has been widely used for PCR analysis, although the sensitivity of the method is lower in fixed than in frozen tissues (10,19,20). Regarding synovial sarcomas, good results have been obtained with this technique using paraffin-embedded tissue (19).Conventional PCR amplification requires post-PCR amplicon detection steps based on gel electrophoresis and PCR product accurate sizing or Southern blot analysis by using a P32-labeled probe hybridization to confirm amplification specificity. These steps are time-consuming and usually require several days. To date, the detection of the t(X;18) translocation has been performed by conventional RT-PCR. This technique will progressively be used as a routine diagnostic tool, so every step will have to be standardized to avoid false-negative or false-positive results resulting from the sensitivity of the PCR technique. Recently, the Taqman real-time PCR assay, a new method avoiding post-PCR analysis, was developed. This technique is rapid, specific, and sensitive and can be used routinely as an automated technique to detect a PCR amplification (21). In fact, it combines PCR amplification and probe hybridization (22,23). The probe is 5´ end-labeled with a reporter dye and 3´ end-labeled with a quencher dye. During extension, the hybridized probe is degraded by the Taq polymerase 5´ nuclease activity. This results in a fluorescent dye emission that can be monitored in real time with the ABI PRISM 5700 Sequence Detector (Applied Biosystems, Foster City, CA). Amplification curves plotting the dye fluorescent emission versus the PCR cycle number allow determination of a threshold value. The threshold represents the baseline signal at which an increase in reporter fluorescence can first be detected. For each sample, a Ct (cycle number at the threshold) value can be measured from the amplification curve, related to the threshold value.In this article, we report our experience with a new real-time PCR assay applicable to paraffin-embedded tissues for detecting the t(X;18)-fusion.MATERIALS AND METHODMaterialThirty-eight tumors were examined. For 17 of them (12 were fixed with Holland Bouin fluid and 5 with 4% buffered formalin), a histopathologic diagnosis of synovial sarcoma was ascertained by conventional microscopy and immunohistochemical analysis, and for some tumors by cytogenetic and molecular analysis (Table 1): 7 tumors were biphasic (specimens 22, 24, 25, 31–33, 36) and 10 were monophasic synovial sarcomas. Immunohistochemistry was used for every specimen of the monophasic type. The following antibodies were used: cytokeratin (monoclonal KL1, diluted 1:50, Immunotech), epithelial membrane antigen (monoclonal clone E29, diluted 1:50, Dako), S100 protein (polyclonal rabbit, diluted 1:500, Dako), desmin (monoclonal, clone D33, diluted 1:40, Dako), smooth muscle actin (monoclonal, clone 1A4, diluted 1:4,000, Sigma), CD34 (monoclonal, clone Q Bend 10, diluted 1:200, Immunotech, Glostrup, Denmark). Immunostaining was carried out with the streptavidin-biotin-peroxidase technique. Tissue sections underwent microwave oven heating before staining. Cytokeratin was positive on a subset of tumor cells in five tumors and epithelial membrane antigen in nine tumors. One tumor (specimen 34) was negative for both cytokeratin and epithelial membrane antigen, but cytogenetic analysis showed typical t(X;18). CD34, desmin, and smooth muscle actin were negative in all samples. S100 protein was positive in one sample. Twenty-one specimens corresponded to other types (19 were fixed with Holland Bouin fluid and 2 with formol). In 22 samples, a molecular analysis had previously been done by conventional RT-PCR (19). Paraffin-embedded and frozen material was available in 11 of 17 synovial sarcoma tumors. For six tumors, only paraffin-embedded material was available. For 6 of the 21 nonsynovial sarcoma tumors, paraffin-embedded and frozen tumor material was available.JOURNAL/dimp/04.03/00019606-200203000-00004/table1-4/v/2021-02-17T195830Z/r/image-tiffHistologic, cytogenetic, and molecular features in 38 soft tissue tumorsRNA extraction from paraffin-embedded and frozen tissuesTissues were deparaffinized twice in toluene, then washed twice with absolute ethanol. For Holland Bouin-fixed tissues, sections were washed with lithium carbonate to remove picric acid. Then tissues were washed with TNE 1× (Tris, pH 8, 10 mmol/L; EDTA, pH 8, 1 mmol/L; NaCl, pH 8, 100 mmol/L). Finally, sections were resuspended with 500 μL ATL buffer (Qiagen, Hilden, Germany) added with proteinase K (Qiagen) (final concentration 0.8 mg/mL) and incubated for 4 hours at 60°C.RNA from fixed or frozen tissues were extracted according to the method of Chomczynzki and Sacchi (24) using 1.5 mL Trizol-LS reagent (Gibco BRL, Carlsbad, CA) for 500 μL cellular lysate. The solution was placed under moderate shaking for 30 minutes to 1 hour and RNA was extracted according to the manufacturer's instructions (Qiagen). The RNA pellet was resuspended in 50 μL RNase-free water and stored at −80°C.RT-PCR methodReverse transcription of 5 μg RNA was performed in a total volume of 20 μL with 50 mmol/L Tris-HCl (pH 8.3), 40 mmol/L KCl, 5 mmol/L MgCl2, 0.5% Tween, 0.5 mmol/L dNTP Mix, 10 mmol/L dithiothreitol, specific reverse primer (65 ng SSX-B or 50 ng GAPDH-reverse primer;Table 2), 12 U RNAse inhibitor (Promega, Madison, WI), and 10 U Expand reverse transcription (Roche Diagnostics, Manathien, Germany). Samples were incubated at 42°C for 1 hour, then at 95°C for 5 minutes.JOURNAL/dimp/04.03/00019606-200203000-00004/table2-4/v/2021-02-17T195830Z/r/image-tiffOligonucleotide primer and probe sequencesThe PCR reaction was performed in triplicate in a total volume of 50 μL with the PCR Core Reagent kit (Applied Biosystems) consisting of 4 mmol/L MgCl2, 0.4 mmol/L of dATP, dCTP, and dGTP, and 0.8 mmol/L of dUTP, 1.5 U Taq Gold polymerase, 0.5 U uracil-N-glycosylase, 4 μL of the cDNA reaction, 65 ng sense primer, 65 ng reverse primer, and 5 pmole probe. Probe and primer sequences are detailed in Table 2. Thermal cycling conditions were 2 minutes at 50°C for amperase activation, 10 minutes at 95°C for Taq polymerase activation, then 50 cycles of three PCR steps consisting of 30 seconds at 95°C, 45 seconds at 63°C, and 75 seconds at 72°C. GAPDH transcript expression has been studied as a housekeeping gene to control the RNA ability to be reverse-transcribed and PCR-amplified for all the tumors (25).RESULTSThirty-eight tumors were tested for t(X;18)-fusion transcript expression by real-time PCR after specific RT. GAPDH transcript amplification was detected in 36 of 38 tumor samples. Ct value ranged from 17 to 36 (mean 28). For the two tumors (specimens 21 and 22) in which GAPDH PCR amplification was not detected, a second RNA extraction attempt was performed but was unsuccessful.The t(X;18) gene transcripts were detected in 16 of 17 synovial sarcomas (94%). Results could not be assessed for specimen 22 because no GAPDH transcript amplification was observed, although this tumor proved to be positive by conventional RT-PCR (see Table 1). Figure 1 represents a typical real-time PCR amplification graph plotting the cycle number versus the fluorescence intensity obtained from paraffin-embedded tissue. The threshold was fixed at 0.05. The Ct value was approximately 32 for t(X;18) fusion transcript and 24 for GAPDH amplification. Ct values for SYT/SSX amplification ranged from 19 to 33 (mean 22) for frozen tumors and from 25 to 39 (mean 31) for fixed tissues. The Ct value for SYT/SSX amplification ranged from 29 to 39 (mean 32.7; n = 8) for tissues fixed with Holland Bouin fluid and from 25 to 30 (mean 28.7; n = 4) for tissues fixed with formol. All specimens that proved to be positive for the SYT-1/SSX-B PCR amplification using frozen material were also positive in corresponding paraffin-embedded tissue. Figure 2 compares plot amplification performed for the same paraffin-embedded and frozen tumor.JOURNAL/dimp/04.03/00019606-200203000-00004/figure1-4/v/2021-02-17T195830Z/r/image-tiff Amplification plots of real-time reverse transcriptase–polymerase chain reaction (RT-PCR) assay for t(X;18)-fusion transcript and GAPDH detection (patient 26) by the PRISM 5700 Sequence detector. The graph shows fluorescent emission data in linear scale (Rn) collected during the extension phase of each PCR cycle. Each PCR amplification was done in triplicate. The graph was redrawn based on the device values and is not the actual raw output of the device.JOURNAL/dimp/04.03/00019606-200203000-00004/figure2-4/v/2021-02-17T195830Z/r/image-tiff Amplification plots of real-time reverse transcriptase–polymerase chain reaction (RT-PCR) assay for t(X;18)-fusion transcript (patient 38) obtained from RNA isolated from the same fixed and frozen synovial sarcoma tumor. The graph was redrawn based on the device values and is not the actual raw output of the device.Of the 21 nonsynovial sarcoma tumors (desmoid tumor, leiomyosarcoma, liposarcoma, gastrointestinal stromal tumor, and malignant fibrous histiocytoma; see Table 1), 20 were negative for t(X;18) transcript gene amplification and 1 (from patient 21, leiomyosarcoma) could not be interpreted because of lack of GAPDH amplification. Neither conventional RT-PCR nor karyotyping was performed for this sample.Overall, of the 36 tumors suitable for molecular analysis, molecular diagnosis by real-time PCR was in agreement with the histologic diagnosis in 100% of specimens.DISCUSSIONOn both fixed and frozen tissues, RT-PCR has been used to detect the translocation t(X;18) in synovial sarcomas (14,19). To facilitate routine application of this technique, we developed a new Taqman real-time RT-PCR method to detect the t(X;18)-fusion transcript that can be applied to both paraffin-embedded and frozen tumors. Thirty-eight tumors were analyzed for t(X;18)-fusion transcript amplification and for GAPDH amplification. Of the 38 tumors, 2 were not suitable for real-time PCR analysis because of GAPDH amplification failure, the latter being used as a positive internal control (patients 21 and 22).t(X;18)-fusion transcripts were detected in all 16 analyzable and histologically diagnosed synovial sarcomas. Amplification curves were quite similar, thus reflecting a good reproducibility. With 100% sensitivity in our hands, the t(X;18)-fusion transcript real-time RT-PCR detection method appears to be a powerful technique.RNA extracted from both frozen and paraffin-embedded material was suitable for t(X;18)-fusion transcript real-time RT-PCR amplification. Interestingly, the mean Ct values were higher in fixed than in frozen specimens. This difference in Ct value is due to the difference in RNA quality between fixed and frozen samples. RNA extracted from frozen tissues is of higher quality than that from fixed tissues, and RNA quality is related to tissue preservation and fixation (19). Nevertheless, with 50 rounds of PCR performed, the t(X;18) translocation in paraffin-embedded synovial sarcomas was always detected: Ct values ranged from 25 to 39. Moreover, t(X;18) was detected for each tumor, both in frozen and in the corresponding paraffin-embedded tissue. Classically, only tissues fixed with buffered formalin are suitable for molecular diagnosis. However, our findings show that tissues fixed with Holland Bouin fluid are also suitable for SYT/SSX RT-PCR analysis, in keeping with the results of Guillou et al. (19), although Ct values from formalin-fixed tissues are lower than those from tissues fixed with Holland Bouin fluid.The main limitation of our method is RNA quality. This is exemplified in specimen 22, in which the RNA was probably completely degraded, preventing amplification of GAPDH and SYT/SSX transcripts.The main problem with PCR is the risk of PCR amplicon contaminations from carryover. A real-time PCR technique lowers the risk because no post-PCR handling manipulations are necessary, and the use of uracil-N-glycosylase prevents contamination. The enzyme hydrolyzes uracil–glycosidic bonds at dU-containing DNA sites, releasing uracil and creating an alkali-sensitive apyrimidic site in the DNA. 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Hypertension 2000; 35:1002–8.[Context Link][Full Text][CrossRef][Medline Link]Paraffin-embedded tissue; Real-time reverse transcriptase-polymerase chain reaction; Soft tissue sarcoma; Synovial sarcoma; t(X;8) translocation00019606-200203000-0000400019606_1998_7_184_hostein_polymerase_|00019606-200203000-00004#xpointer(id(citation_FROM_JRF_ID_d1096e844_citationRF_FLOATING))|11065404||ovftdb|SL000196061998718411065404citation_FROM_JRF_ID_d1096e844_citationRF_FLOATING[Full 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of real-time reverse transcriptase–polymerase chain reaction (RT-PCR) assay for t(X;18)-fusion transcript and GAPDH detection (patient 26) by the PRISM 5700 Sequence detector. The graph shows fluorescent emission data in linear scale (Rn) collected during the extension phase of each PCR cycle. Each PCR amplification was done in triplicate. The graph was redrawn based on the device values and is not the actual raw output of the device. Amplification plots of real-time reverse transcriptase–polymerase chain reaction (RT-PCR) assay for t(X;18)-fusion transcript (patient 38) obtained from RNA isolated from the same fixed and frozen synovial sarcoma tumor. The graph was redrawn based on the device values and is not the actual raw output of the device.Molecular Detection of the Synovial Sarcoma Translocation t(X;18) by Real-Time Polymerase Chain Reaction in Paraffin-Embedded MaterialHostein Isabelle Ph.D.; Menard, Armelle Ph.D.; Bui, Bin Nguyen M.D.; Lussan, Cathy Med. Tech.; Wafflart, Jean M.D.; Delattre, Olivier M.D.; Peter, Martine Ph.D.; Benhattar, Jean Ph.D.; Guillou, Louis M.D.; Coindre, Jean-Michel M.D.Original ArticlesOriginal Articles111p 16-21