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.
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.
Jin
L
L
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
Majerus
J
J
Oliveira
A
A
Inwards
C Y
CY
Nascimento
A G
AG
Burgart
L J
LJ
Lloyd
R V
RV
D015139:Blotting, Southern; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D006801:Humans; D007834:Lasers; D042282:Microdissection; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012509:Sarcoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012983:Soft Tissue Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016707:Tissue Fixation; D014178:Translocation, Genetic
D017931:DNA Primers; D015342:DNA Probes; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D012334:RNA, Neoplasm