Ewing Sarcoma/Primitive Neuroectodermal Tumors (ES/PNET) by Reverse Transcriptase PCR (RT-PCR), Paraffin
Supporting a diagnosis of Ewing sarcoma and primitive neuroectodermal tumors
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Ewing sarcoma (ES) and primitive neuroectodermal tumor (PNET), a closely related tumor, are members of the small round-cell tumor group that also includes rhabdomyosarcoma, synovial sarcoma, lymphoma, Wilms tumor, and desmoplastic small round-cell tumor. ES is the second most common malignant tumor of bone in children and young adults. It is an aggressive osteolytic tumor with a high risk of metastasizing. ES can also present as a soft tissue tumor mass. These tumors are usually bland and undifferentiated with relatively low mitotic indexes, which is misleading in light of the rapid growth commonly observed clinically.
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, MIC2 [CD99], desmin, myogenin, myoD1, WT1) can assist in establishing the correct diagnosis, but these markers are not entirely specific for ES/PNET. 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 that are useful in the diagnosis of these lesions.(1-4)
The balanced t(11;22)(q24;q12) chromosomal translocation produces the EWSR1-FLI1 fusion transcript and is present in 95% of ES and PNET. Because the EWSR1-FLI1 fusion transcript is a common finding in ES/PNET, in soft tissues these 2 lesions are essentially identical. Less common are the t(21;22)(p22;q12) or EWSR1-ERG transcript, present in <5% of ES/PNET tumors, and the t(7:22)(p22;q12) or EWSR1-FEV transcript, present in <1% of these tumors. These fusion transcripts can be detected by reverse-transcriptase PCR (RT-PCR), by FISH, chromogenic in situ hybridization, or by classical cytogenetic analyses. The RT-PCR and FISH procedures are the most sensitive methods to detect these fusion transcripts.(3)
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-FLI1 or EWSR1-ERG result is consistent with a diagnosis of Ewing sarcoma and primitive neuroectodermal tumor (ES/PNET).
Sarcomas other than ES/PNET, and carcinomas, melanomas, and lymphomas are negative for the fusion products.
A negative result does not rule out a diagnosis of ES/PNET.
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 and 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 43 specimens that were diagnosed as ES/PNET by soft tissue pathology experts and by immunohistochemical staining were analyzed. These consisted of 16 frozen tissue specimens and 27 paraffin-embedded tissue specimens. Three of the cases from frozen and paraffin sections were the same for direct comparison. The EWSR1-FLI1 fusion was detected in 14 of 16 cases (87.5%) and EWSR1-ERG fusion in 2 of 16 (12.5%) frozen tissues. Of the 27 cases of paraffin-embedded tissues analyzed, 23 of 27 cases (85.2%) had an identified fusion product. EWSR1-FLI1 fusion was detected in 18 of 23 cases (78.3%) and EWSR1-ERG fusion in 5 of 23 cases (21.7%).
Southern hybridization with internal probes followed by chemiluminescent detection confirmed the findings in all cases. Sequencing the PCR product also identified the breakpoint of the fusion product in all 8 cases in which it was performed. Similar RT-PCR results were obtained in the 3 cases analyzed from frozen tissues and paraffin-embedded tissues. Analysis of other small round-cell tumors (alveolar rhabdomyosarcoma, desmoplastic small round-cell tumor, synovial sarcoma) (n=12) were negative for the ES/PNET transcripts.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Delattree O, Zucman J, Melot T, et al: The Ewing family of tumors-A subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med 1994;331:218-222
2. 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
3. Ladanyi M, Bridge JA: Contribution of molecular genetic data to the classification of sarcomas. Hum Pathol 2000;31:532-538
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
5. Zucman J, Melot T, Desmaze C, et al: Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO J 1993;12:4481-4487