Desmoplastic Small Round-Cell Tumor (DSRCT) by Reverse Transcriptase PCR (RT-PCR), Paraffin
Supporting the diagnosis of desmoplastic small round-cell tumor
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Desmoplastic small round-cell tumor (DSRCT) is a member of the small round-cell tumor group that also includes rhabdomyosarcoma, synovial sarcoma, lymphoma, Wilms tumor, and Ewing sarcoma. DSRCT is a type of sarcoma that affects mainly children and adolescent males, usually in the form of widespread intra-abdominal growth not related to any specific organ system. The tumor is composed of angulated nests of small round cells with an abundant desmoplastic stroma. The tumor cells show multiphenotypic differentiation and are usually positive for cytokeratin and desmin.(1-4) These tumors can express renal, epithelial, muscle, and endocrine markers.
While treatment and prognosis depend on establishing the correct diagnosis, the diagnosis of sarcomas that form the small round-cell tumor group can be very difficult by light microscopic examination alone, especially true when only small-needle biopsy specimens are available for examination. The use of histochemical and immunohistochemical stains (eg, desmin, cytokeratin, and WT1) can assist in establishing the correct diagnosis, they cannot distinguish between DSRCT and other small round-cell tumors. Expertise in soft tissue and bone pathology are often needed.
Studies have shown that some sarcomas have specific recurrent chromosomal translocations. These translocations produce highly specific gene fusions that help define and characterize subtypes of sarcomas and are useful in the diagnosis of these lesions.(1-4)
DSRCT is associated with a unique chromosomal translocation t(11:22)(p13;q12) that involves the EWSR1 and the WT1 genes. EWSR1 is the breakpoint site of translocations associated with Ewing sarcoma and WT1 is a gene altered in some Wilms tumors. The translocation results in a fusion of the 2 genes with expression of a chimeric EWSR1-WT1 product. The most common breakpoints involve the intron between EWSR1 exon 7 and 8 and the intron between WT1 exons 7 and 8. Analyses of these transcripts have shown an in-frame fusion of RNA encoding the amino-terminal domain of EWSR1 to the zinc finger of the DNA-binding domain of WT1.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
A positive EWSR1-WT1 result is consistent with a diagnosis of desmoplastic small-round-cell tumor (DSRCT).
Sarcomas other than DSRCT, and carcinomas, melanomas, and lymphomas are negative for the fusion products.
A negative result does not rule out a diagnosis of DSRCT.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Reliable results are dependent on adequate specimen collection and processing. This test has been validated on formalin-fixed, paraffin-embedded tissues; other types of fixatives are discouraged. Improper treatment of tissues, such as decalcification, may cause PCR failure. False-negative results may occur in tumor specimens when tumor cells comprise <5% of the cell population. Tumor cells may be enriched by macrodissection to avoid false-negative results
Clinical diagnosis or therapy should not be based solely on this assay. The results should be considered in conjunction with clinical information, histologic evaluation, and additional diagnostic tests.
A total of 36 cases that were diagnosed as desmoplastic small round-cell tumor by soft tissue pathology experts and by immunohistochemical staining were analyzed. These consisted of 4 frozen tissue specimens and 32 paraffin sections. Three of the cases from frozen and paraffin tissue sections were the same cases for direct comparison. All specimens were also analyzed by Southern hybridization using a sequence internal to the primers as a probe. A few specimens were also analyzed by sequencing the PCR products.
The EWSR1-WT1 product was detected in all 4 (100%) fresh tissues and in 30/32 (94.7%) of the paraffin sections. Southern hybridization detected 1 additional case in the paraffin section that was not detected on the ethidium bromide gel. Sequencing of the fresh tissues and paraffin sections confirmed the sequence of the breakpoint of the fusion transcript of EWSR1 exon 7 to WT1 exon 8. Comparable RT-PCR results were obtained for the 3 cases that were analyzed by frozen and paraffin tissues. Analysis of other small round-cell tumors (alveolar rhabdomyosarcoma, synovial sarcoma, and Ewing sarcoma) (n=12) were all negative for the EWSR1-WT1 fusion transcript.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Barr FG, Chatten J, D'Cruz CM, et al: Molecular assays for chromosomal translocations in the diagnosis of pediatric soft tissue sarcomas. JAMA 1995;273:553-557
2. Gerald WL, Miller HK, Battifora H, et al: Intra-abdominal desmoplastic small round-cell tumor: Report of 19 cases of a distinctive type of high-grade polyphenotypic malignancy affecting young individuals. Am J Surg Pathol 1991;15:499-513
3. Gerald WL, Rosai J, Ladanyi M: Characterization of the genomic breakpoint and chimeric transcripts in the EWS-WT1 gene fusion of demoplastic, small round-cell tumor. Proc Natl Acad Sci USA 1995;92:1028-1032
4. Jin L, Majerus J, Oliveira A, et al: 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. Diagn Mol Pathol 2003;12:224-230