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Annals of Clinical & Laboratory Science 32:219-224 (2002)
© 2002 Association of Clinical Scientists

TaqMan Junction Probes and the Reverse Transcriptase Polymerase Chain Reaction: Detection of Alveolar Rhabdomyosarcoma, Synovial Sarcoma, and Desmoplastic Small Round Cell Tumor

Thomas J. Cummings, Nicholas M. Brown and Timothy T. Stenzel
Department of Pathology, Duke University Medical Center, Durham, North Carolina

Address correspondence to: Thomas J. Cummings, M.D., Duke University Medical Center, Department of Pathology, Box 3712, Durham, NC, USA 27710; tel 919 684 6592; fax 919 681 7634; e-mail: cummi008{at}mc.duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The reverse transcriptase polymerase chain reaction (RT-PCR) provides a technique to diagnose a group of sarcomas and small round cell tumors that contain specific chromosomal translocations and chimeric gene fusion products. We adapted real-time qualitative RT-PCR to utilize dual-labeled, fluorogenic, TaqMan probes, which hybridize to targets that overlap the junction of the chimeric gene fusions in alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT). Assays were confirmed on cell lines and tissue samples; appropriate negative amplification assays were obtained when each tumor-specific probe and primer set was used on different neoplasms and cell lines that were not expected to harbor the specific translocations and chimeric gene fusions. Although our cases are few, we speculate that as more molecular variants of ARMS, SS, and DSRCT are discovered, clinical correlations based on precise molecular features will be required and fusion site specificity will be assured by the use of junction-based TaqMan probes.

(received 19 February 2002; accepted 17 March 2002)

Keywords: RT-PCR, TaqMan junction probes, chromosomal translocations, chimeric gene fusion products


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT) are neoplasms with specific reciprocal translocations and chimeric gene fusions. The ability to diagnose these neoplasms has been enhanced by the ability to amplify and detect their gene fusion products by the reverse transcriptase polymerase chain reaction (RT-PCR).

ARMS is a "small round cell tumor" that primarily involves the soft tissues of young people. The majority of cases of ARMS involve two specific translocations. The translocation t(2;13)(q35q14) involves the PAX3 gene on chromosome 2q35 and the FKHR gene on 13q14, resulting in a novel chimeric gene fusion [1–3]. This translocation accounts for approximately 75% of cases. Half of the remaining cases usually result from translocation t(1:13)(p36;q14), fusing the PAX7 gene on chromosome 1p36 with the FKHR gene [4].

SS favors the lower limbs of young adult males, but lesions involving both sexes and wide ranges of ages and sites have been reported. Two main groups have been described histologically: the biphasic and monophasic spindle cell types. Both types share a t(X;18) translocation, resulting in the production of two specific chimeric gene fusions: SYT-SSX1 t(X;18)(p11.23;q11), and SYT-SSX2 t(X;18) (p11.21;q11). Although biphasic tumors typically are associated with the SYT-SSX1 gene fusion, and monophasic tumors with SYT-SSX1 and SYT-SSX2 gene fusions, definitive clinical and pathological correlations are uncertain [5–11].

DSRCT is a malignant small cell neoplasm that favors adolescents and young adult males and also has a diverse clinicopathologic spectrum. It is characterized by a t(11;22)(p13;q12) translocation, resulting in the EWS-WT1 chimeric gene fusion in most all cases [12–16].

Successful application of the TaqMan technique requires specific hybridization between the TaqMan probe and the target to generate a fluorescent signal, thereby negating non-specific amplification owing to mispriming events. If the target sequence is present, the probe anneals between the forward and reverse primers. Two fluorescent dyes, a reporter and a quencher dye, are attached to the 5' and 3' ends of the TaqMan probe. When both dyes are attached to the probe, the fluorescent emission of the reporter dye is absorbed by that of the quencher dye.

During the extension phase of the PCR cycle, the Taq DNA polymerase cleaves the reporter dye from the probe and the reporter dye emits its characteristic fluorescence [17,18]. The reaction-specific TaqMan probes obviate further post-PCR testing, reducing costs and contamination. In patient care, agarose gel electrophoresis is a rapid and simple adjunct that supplements the RT-PCR amplification results; we advocate its use without reservation.

In contrast, SYBR Green is a double-stranded DNA (dsDNA) binding dye that detects all of the dsDNA products that are generated during PCR. However, amplification of specific and non-specific products generates signals and thus reduces specificity [19]. These mispriming events can lead to spurious bands on electrophoretic gels [18]; the false-positive signals may require post-PCR studies to confirm amplicon product specificity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
The ARMS (PAX3-FKHR) cell line SJRH30 [RMS 13] was obtained from American Type Culture Collection (ATCC, Manassas, VA); the cell line A2243 (SYT-SSX2) was kindly donated by Dr. S. Aaronson. A total of 5 clinical cases was studied, including 3 cases of SS (SYT-SSX1), 1 case of ARMS (PAX3-FKHR), and 1 case of DSRCT (EWS-WT1); these cases were obtained from the tumor bank of the Molecular Diagnostics Laboratory at Duke Hospital.

Total RNA was extracted using the QUIamp® RNA mini-kit (Quiagen, Valencia, CA). RNA (1 mg) was reverse transcribed to complementary DNA (cDNA) using the SuperscriptTM First-Strand Synthesis System for RT-PCR (Gibco-BRL Life Technologies). All cases were run with a "housekeeping" gene (ß2-microglobulin) to assure nucleic acid preservation.

Forward and reverse oligonucleotide primers for ARMS (PAX3-FKHR) [20] and for SYT-SSX1 and SYT-SSX2 [5] were previously reported. The forward primer for DSRCT was designed using Perkin Elmer/Applied Biosystems (PE/ABI) Primer ExpressTM software (PE/ABI, Foster City, CA), and the reverse primer was previously reported [20].

TaqMan probes were designed using PE/ABI software. All probes were designed to overlap the junction of the novel chimeric gene fusions, and were labeled by a 5'-FAM reporter fluorescent dye and a 3' TAMRA quencher fluorescent dye (Table 1Go). All primers and probes complied with PE/ABI guidelines for primer and probe design. The forward and reverse primer concentrations were optimized over the concentration range from 50 to 900 nM; in all cases a 900 nM primer concentration was utilized. Similarly, a TaqMan probe optimization experiment was performed in which the probe concentration was varied from 50 to 300 nM; the optimal probe concentration was 200 nM.


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  		Table 1. Sequences of the forward and reverse oligonucleotide primers and of the dual-labeled junctional fluorogenic TaqMan probes used in RT-PCR assays.
 
For all reactions, a final volume of 25 µl containing 12.5 µl of TaqMan® Universal PCR Master Mix (PE/ABI, Roche, Branchburg, NJ), 2.5 µl of forward primer, 2.5 µl of reverse primer, 2.5 µl TaqMan probe, 3 µl cDNA template, and 2 µl distilled H2O was used. All oligonucleotide primers and TaqMan probes were obtained from PE/ABI (Foster City, CA). Reactions were placed in MicroAmp 96-well optical plates and covered with MicroAMP 8 well optical caps (PE/ABI, Foster City, CA). Reactions were run on the ABI Prism 7700 Sequence Detection System (PE/ABI, Foster City, CA) using the following thermal cycling conditions: 2 min at 50°C, 10 min at 95°C, and 50 cycles of amplification at 95°C for 15 sec and 65°C for 60 sec. Reaction conditions were programmed and data was analyzed on a Power Macintosh G3 (Apple Computer, Cupertino, CA) directly linked to the ABI Prism 7700 Sequence Detection System. Amplification plots were obtained and the products of the RT-PCR reaction were separated by electrophoresis on 3% agarose gels stained with ethidium bromide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Fig. 1Go shows a qualitative amplification plot obtained with a patient sample, using real-time RT-PCR with TaqMan junction probe of the PAX3-FKHR gene fusion. Similar reproducible plots were obtained for all cases of EWS-WT1, SYT-SSX1, and SYT-SSX2 gene fusions. Gel electrophoresis bands were obtained in ARMS, DSRCT, and SS at the following expected base pair (bp) markers: 408 bp PAX3-FKHR (ARMS, patient sample), 354 bp EWS-WT1 (DSRCT, patient sample), 331 bp SYT-SSX1 (SS), and 331 bp SYT-SSX2 (SS, cell line) (Fig. 2Go).



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  		Fig. 1. Representative RT-PCR amplification plot.

 


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  		Fig. 2. Gel electrophoresis analysis of RT-PCR with junction TaqMan probe, showing 354-bp product of DSRCT (lane 1), 331-bp product of SYT-SSX2 (lane 2) (SYT-SSX1 has an identical 331-bp product and is not shown), and 408-bp product of PAX3-FKHR (lane 3). The size marker (lane M) utilized a 100-bp DNA ladder marker.

 
RT-PCR was successfully performed on snap-frozen tumor samples stored in the tumor bank of the Molecular Diagnostics Laboratory at Duke Hospital in 1 case of ARMS, 1 case of DSRCT, and 3 cases of SS. These assays demonstrated the PAX3-FKHR, EWS-WT1, and SYT-SSX1 gene fusion products, respectively. Primer and probe sets were tested in each of these neoplasms and cell lines, in addition to other neoplasms, including malignant fibrous histiocytomas, peripheral nerve sheath tumors, and hematopoietic neoplasms. Appropriately negative amplification assays were obtained when each tumor-specific probe and primer set was used on the different neoplasms and cell lines that were not expected to harbor the specific translocations and chimeric gene fusions. In each case, no amplification was seen.

Results were compared with RT-PCR assays without the TaqMan junction probes that substituted SYBR® Green PCR Master Mix (PE/ABI, Foster City, CA) for TaqMan® Universal PCR Master Mix (PE/ABI, Roche, Branchburg, NJ). The difference between assays with the TaqMan probes and SYBR® Green was the absence of spurious or non-specific bands from mispriming events when the highly specific TaqMan probes were added to the reaction.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
A multiplex RT-PCR method using TaqMan probes downstream or 3' to the site of the gene fusion has recently been developed for solid tumors with specific translocations and chimeric gene products [21]. TaqMan junction probes have been used in RT-PCR reactions to detect gene fusions in the hematopoietic neoplasms chronic myeloid leukemia (CML) and acute myeloid leukemia [22,23]. CML is characterized cytogenetically by t(9;22)(q34;q11) resulting in the novel chimeric gene fusion BCR-ABL. The BCR-ABL fusion RNA transcripts usually have a b3a2 or b2a2 junction, depending on whether the BCR gene break occurs between exons 2–3 or 3–4. In 5–10% of cases, both b3a2 and b2a2 transcripts are formed as a result of alternative splicing [24]. Rare cases exhibit atypical junctions involving b3 or b2 with ABL exon a3 resulting in smaller PCR products that can fail to be detected if the primer design is inappropriate [24].

Junction TaqMan probes have been used to detect specifically the b2a2 and the b3a2 variants of BCR-ABL, in order to monitor their expression in patients with CML treated by bone marrow transplantation or peripheral blood stem cell transplantation [22]. This method allows independent detection of either isoform in a highly specific manner. The EWS-FLI1 transcript in Ewing’s sarcoma shows marked variability in intronic breakpoints resulting in a heterogeneous combination of alternative forms of fusion products [25,26]. Approximately 90% of EWS-FLI1 fusion types involve EWS exon 7 fused to FLI1 exon 4–8, and the remaining 10% involve EWS exon 9 or 10 [27,28]. There are at least 18 different structural possibilities of these gene fusions, and it is becoming apparent that functional differences among the fusion genes and heterogeneity in chimeric transcript structure may define distinct clinical subsets of patients based on precise molecular classification [27–30].

Rare recurrent molecular variant gene fusions have been described in the EWS-WT1 fusion of DSRCT [16] and in the SYT-SSX1 and SYT-SSX2 fusions of SS [31]. Sarcoma translocation fusion protein peptides that span the breakpoint region have been shown to serve as tumor antigens and therefore as candidates for cancer immunotherapy [32]. Theoretically, non-junction probes designed to recognize a sequence from only one of the two fused genes may detect product despite variations in intronic breakpoints and exonic compositions, and thus sacrifice specific molecular profiling. A junction probe, however, precisely recognizes genetic material from each of the two involved genes.

Use of reaction-specific TaqMan probes obviates the need for further post-PCR testing with Southern blotting, sequencing, or hybridization, and thus negates potential post-PCR product contamination. Agarose gel electrophoresis is a rapid and simple adjunct to supplement the RT-PCR amplification results, and we advocate its use without reservation. Compared to other molecular cytogenetic methods, such as fluorescence in situ hybridization (FISH), the TaqMan technique is rapid and permits precise detection of the exon-to-exon gene fusion site. Although FISH is an important diagnostic tool and has been successfully utilized to detect the chromosomal abnormalities of these neoplasms, the required probes generally are neither validated, standardized, nor commercially available [33].

Our design was successfully performed on human tissue and cell lines in cases of ARMS, SS, and DSRCT, and utilized identical reaction conditions for each assay, permitting multiplex analysis. Although we have too few cases to make clinical correlations, this is a future objective. As more molecular variants are discovered in ARMS, SS, and DSRCT, clinical correlations based on precise molecular features will be required and fusion site specificity can be assured by use of junction-based TaqMan probes.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Dr. Timothy Fields, Dr. Michael Datto, and Mr. Bartley Adams for technical advice and assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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