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Detection of Fusion Gene Transcripts in Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Tissue Sections of Soft-Tissue Sarcomas After Laser Capture Microdissection and RT-PCR

Jin, L.; Majerus, J.; Oliveira, A.; Inwards, C. Y.; Nascimento, A. G.; Burgart, L. J.; Lloyd, R. V.

Diagnostic Molecular Pathology: December 2003 - Volume 12 - Issue 4 - pp 224-230
Original Article

The diagnosis of small round cell sarcomas is often very difficult, especially when only small biopsy specimens are available for examination. Recent studies have shown that some sarcomas have specific recurrent chromosomal translocations producing chimeric gene fusions, which can be detected by reverse transcription-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH), or cytogenetic analysis. In this study, 12 cases of well-defined sarcomas including Ewings sarcoma/primitive neuroectodermal tumors (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumors (DSRCT) were used to collect specific numbers of cells by laser capture microdissection (LCM), subsequently used for RT-PCR to detect specific chimeric gene transcripts. Tumor cells from fresh-frozen (FS) tissue sections and paraffin-embedded (PS) tissue sections from the same cases were compared directly to evaluate the sensitivity of FS and PS sections as the starting material for analysis. Samples were used for RNA extraction, RT-PCR analysis, and Southern hybridization with fluorescein-labeled internal probes followed by enhance chemiluminescence (ECL) detection. The fusion gene transcripts could be detected using 50 cells from FS materials in all cases and from 1 cell in 9 of 12 cases. For PS, a positive signal could be detected using 200 to 1000 cells in all cases, while weaker signals were detected using 50 cells in most cases. These results indicate that the fusion gene products from small round cell sarcomas can be detected by RT-PCR with 10 to 200 cells from FS and PS tissues. The sensitivity of RT-PCR with FS was 10- to 50-fold greater than with PS. These results also suggest that RT-PCR analysis for sarcoma fusion gene products can be successfully performed when only a few cells are available for analysis, although this is not recommended for routine clinical use.

The diagnosis of small round cell sarcomas can be very difficult by morphology alone, especially when only small biopsy specimens are available for examination. Specific recurrent chromosomal translocations have been found in many small round cell sarcomas. These translocations produce specific chimeric gene fusion transcripts that have helped to define and characterize sarcomas and can be used as diagnostic markers. 1–11 Some common chimeric gene fusion products that have been reported include EWS/FLI-1 and EWS/ERG in ES/PNET, 1–3 SYT/SSX1 and SYT/SSX2 in synovial sarcomas, 4–6 PAX3/FKHR and PAX7/FKHR in ARMS, 7,8 and EWS/WT1 in DSRCT. 9,10

Fusion proteins resulting from these translocations may be detected by immunohistochemistry such as with ES/PNET or DSRCT, 11,12 but specific antibodies are still very limited. Other antibodies such as CD99, keratin, epithelial membrane antigen, and desmin can assist in establishing the diagnosis; nonetheless, they lack specificity. Southern blot methods can be used to detect the gene rearrangements, 13 but this approach requires a substantial amount of tumor tissue for DNA extraction and does not work well with paraffin-embedded tissues. Gene translocation in sarcomas can be detected by FISH with the site-specific probes labeled with fluorescent dyes, 14 but the specific subtypes of translocations that may indicate differences in prognosis 6 are not distinguished by FISH. RT-PCR analyses have been shown to be sensitive, specific, and reproducible in the differential diagnosis of soft tissue sarcomas using FS and PS tissues. 15–18

Although the quality of mRNA obtained from FS is generally considered to be superior to that obtained from PS, a quantifiable difference between these 2 approaches for detection of specific transcripts has not been systematically analyzed. LCM is a recently developed method used for isolation of homogeneous cell populations from tissue sections or cell preparations. 19–22 LCM methods had been used to study synovial sarcoma recently. 23 However, a direct comparison of the sensitivity of RT-PCR between fresh and paraffin tissues after LCM collection has not been reported.

In this study, we analyzed 12 cases of well-defined sarcomas, including ES/PNET, synovial sarcoma, ARMS, and DSRCT from both FS and PS tissue sections using LCM and RT-PCR analyses. The results indicate that the sensitivity of RT-PCR using FS was 10- to 50-fold higher compared with PS for the detection of specific chimeric gene fusion transcripts.

From the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

Reprints: Dr. RV Lloyd, Mayo Clinic Department of Laboratory Medicine and Pathology, 200 First Street, SW Rochester, MN.

© 2003 Lippincott Williams & Wilkins, Inc.