Ewing Sarcoma/Primitive Neuroectodermal Tumors (ES/PNET) by Reverse Transcriptase PCR (RT-PCR), Paraffin
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Supporting a diagnosis of Ewing sarcoma and primitive neuroectodermal tumors
Additional Tests Lists test(s) that are always performed, at an additional charge, with the initial test(s)
|Test ID||Reporting Name||Available Separately||Always Performed|
|60254||AP Special Studies Review||No||Yes|
Testing Algorithm Delineates situation(s) when tests are added to the initial order. This includes reflex and additional tests.
This test is performed in conjunction with 60254 AP Special Studies Review. Additional testing may be performed after review by pathologist. Upon approval from the requesting clinician, 60254 AP Special Studies Review could be changed to 5439 Surgical Pathology Consultation, if determined to be more appropriate.
Special Instructions and Forms Describes specimen collection and preparation information, test algorithms, and other information pertinent to test. Also includes pertinent information and consent forms to be used when requesting a particular test
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Gel Electrophoresis
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Ewings Sarcoma, RT-PCR, Paraffin
EWS (Ewing Sarcoma)
EWS (Ewing Sarcoma)
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
A pathology/diagnostic report and a brief history are required.
Preferred: Formalin-fixed, paraffin-embedded (FFPE) tissue
Acceptable: Unstained slides; slides may be stained and/or scraped
1. Process all specimens into FFPE blocks prior to submission.
2. If submitting slides, a minimum of ten, 4- to 5-micron thick, unstained slides are required.
1. A quality specimen is essential for evaluation. Submit only tissue containing tumor cells; minimal tissue is required for evaluation.
2. Special stains performed outside Mayo Medical Laboratories and included with the case may be repeated and charged at the reviewing pathologist's discretion. Testing requested by the referring physician may not be performed if deemed unnecessary by Mayo Clinic pathologist.
Forms: If not ordering electronically, submit a Pathology/Cytology Request Form (Supply T246) with the specimen.
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
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 EWS-FLI-1 fusion transcript and is present in 95% of ES and PNET. Because the EWS-FLI-1 fusion transcript is a common finding in Ewing sarcoma and PNET, in soft tissues these 2 lesions are essentially identical. Less common are the t(21;22)(p22;q12) or EWS-ERG transcript, present in <5% of ES/PNET tumors, and the t(7:22)(p22;q12) or EWS-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 result is consistent with a diagnosis of Ewing sarcoma (ES) and primitive neuroectodermal tumor (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.
Clinical diagnosis and/or therapy should not be based solely on this assay. The results should be considered in conjunction with clinical information, histologic evaluation, and/or additional diagnostic tests.
A total of 43 specimens that were diagnosed as Ewing sarcoma (ES) and primitive neuroectodermal tumor (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 EWS-FLI1 fusion was detected in 14 of 16 cases (87.5%) and EWS-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. EWS-FLI1 fusion was detected in 18 of 23 cases (78.3%) and EWS-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. Analyses of other small-round-cell tumors (alveolar rhabdomyosarcomas, desmoplastic small-round-cell tumor, synovial sarcomas) (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, by 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
Method Description Describes how the test is performed and provides a method-specific reference
The paraffin-embedded tissue is deparaffinized, lysed, and digested. RNA is extracted using either the TRIzol kit (Invitrogen) or High Pure FFPE RNA Micro kit (Roche). DNase digestion is performed. RNA is converted to cDNA via reverse transcription (RT); the cDNA is amplified via PCR. Primers specific for the Ewing sarcoma EWSR1-FLI1 and EWSR1-ERG transcripts are used. Controls are run with each specimen to assess possible contamination issues and overall test performance. The PCR products are separated by gel electrophoresis and stained with ethidium bromide. The agarose gel is viewed under ultraviolet light and photographed to document the results. In some cases, the specimen is further analyzed by FISH and/or sequencing.(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)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday; 8 a.m.-5 a.m.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
1 week/7 days
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
81401-EWSR1/FLI1 (t(11;22)) (eg, Ewing sarcoma/peripheral neuroectodermal tumor), translocation analysis, qualitative, and quantitative, if performed
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
|Result ID||Reporting Name||LOINC Code|
|83363||Ewings Sarcoma, RT-PCR, Paraffin||In Process|