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Sarcoma Gene Fusion Panel (NGS)

Test Codes

EPIC: LAB1230869

Department

Molecular Pathology

Instructions

Sarcoma fusion panel by Next Generation Sequencing (NGS) using anchored multiplex PCR-NGS and Illumina sequencing platform. This method permits the identification of a rearrangement even if the fusion partner is not known and is not included in the panel.

This test is designed for sequencing RNA for the detection of gene fusions/rearrangements in the following genes (specific targeted regions of each gene can be found here):

ALK, BCOR, BRAF, CAMTA1, CCNB3, CIC, CSF1, CTNNB1, EGFR, EPC1, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1, FGFR2, FGFR3, FOS, FOSB, FOXO1, FUS, GLI1, HMGA2, JAZF1, MBTD1, MDM2, MEAF6, MET, MGEA5, MKL2, MYOD1, NCOA1, NCOA2, NCOA3, NR4A3, NTRK1, NTRK2, NTRK3, NUTM1, PAX3, PDGFB, PDGFRA, PHF1, PLAG1, PRKCA, PRKCB, PRKCD, RAF1, RET, ROS1, SS18, STAT6, TAF15, TCF12, TFE3, TFG, USP6, VGLL2, YAP1, YWHAE

Specimen Collection Criteria

• Paraffin-embedded tissue.
  A paraffin block must be submitted. Submit 10% formalin-fixed, paraffin-embedded block with corresponding H&E slide.
• Unstained sections of 5-µm thickness mounted on glass slides can also be used (minimum 5 sections for large tissue and 10 sections for small tissue such as core biopsy). Tissue should be well fixed and well processed. Recommended tissue size 5.0 mm2. Minimum tumor cellularity 30%.
• Once extracted, RNA will be assessed for quantity and quality. If either is deemed insufficient for analysis, testing will be cancelled with client notification.
• All specimens must be accompanied by a completed requisition and must contain the patient’s name, date of birth, collection date, ordering physician, and source of specimen.

Physician Office/Draw Specimen Preparation

Do not freeze specimens. Maintain tissue blocks and unstained slides at room temperature (20-26°C or 68-78.8°F).

Preparation for Courier Transport

Transport: Tissue blocks and unstained slides at room temperature (20-26°C or 68-78.8°F).

Rejection Criteria

• Tissue decalcified with agents other than Mol Decal (EDTA).
• Fixatives other than 10% neutral buffered formalin.
• Improper labeling or inadequate information.
• Less than 30% tumor cellularity.
• Poor quality and/or quantity of extracted RNA.
• Frozen specimens.
• Unlabeled tubes or samples.

Testing will be cancelled on specimens meeting the above criteria with client notification. Under certain circumstances (i.e., lack of alternative specimens), testing may proceed with approval from the medical director or designee.

Inpatient Specimen Preparation

Specimens at Royal Oak may be sent to the Surgical Pathology tube station #201. In-house specimens are also picked up by a Surgical Pathology assistant every hour on the hour.

In-Lab Processing

Unstained sections of 5-µm thickness are cut from selected tissue blocks. The number of sections cut and the need for macro-dissection are determined by the pathologist, based upon the amount of available tissue and the tumor cellularity.

Storage

Solid Tissue Specimen Stability for Testing:

Room Temperature (20-26°C or 68-78.8°F): Indefinitely
Refrigerated (2-8°C or 36-46°F): Unacceptable
Frozen (-20°C/-4°F or below): Unacceptable

Specimen Storage in Department Prior to Disposal:

Solid Tissue: Room Temperature (20-26°C or 68-78.8°F): Returned to originating lab upon completion of testing.

Specimen Storage (DNA libraries post testing):

Frozen (-25 to -15°C): 2 months

Laboratory

Royal Oak Molecular Pathology Laboratory

Performed

Once or twice per week, dependent upon test volume. Results available in 10-15 days.

Reference Range

No variants detected or only likely benign variants detected.

Test Methodology

Tissue section slides are reviewed by a pathologist and relevant tumor is selected for analysis. RNA is isolated from the sample and quantified. Recovered RNA extracts are prepared for sequencing with ArcherDx Invitae FusionPlex^® Sarcoma v2 library preparation kit and sequenced on the Illumina^® MiSeq^™ instrument using paired-end sequencing. Analysis is performed using Archer Analysis Unlimited (AAU)^® software. A personalized interpretive report is generated that lists the fusions detected and are classified based on a standardized classification scheme for somatic fusions and provides detailed interpretative comments.

Test Limitations:

• Test performance is optimal with specimens containing at least 30% tumor cells and nucleic acid concentrations of at least 10 ng/ul. Values below these thresholds will result in suboptimal test performance.
  
  
• This test is designed to detect somatic fusion only. The status of potential germline fusions cannot be verified because parallel testing is not performed on paired normal tissues. Additional testing is necessary for any potential hereditary risk.
  
  
• Variants occurring in the listed genes but outside of the targeted regions will not be detected by this assay. See the links below for the list of genomic regions covered by each panel.
  
  
• Rare false positives and negatives may occur due to errors in sequencing chemistry. Quality assurance criteria are established to minimize such occurrences.
  
  
• Fusions which are present in the data but with supporting reads below the established analytical sensitivity will not be reported unless confirmed by orthogonal testing, due to the increased risk of false positive results with such findings.
  
  
• Variant interpretations are based upon data from public databases available at the time of case sign out and do not reflect new information that becomes available after that date.
  
  
• Decisions regarding patient care must be based upon independent judgement of the treating physician, taking into consideration all applicable information about the patient’s condition, including but not limited to patient and family history, physical examination, information from other diagnostic tests, and patient preferences, in accordance with the applicable standard of care.
  
  
• Drug associations provided in this report do not guarantee that any particular agent will be effective in the treatment of a specific patient.

Test results should be interpreted in the context of clinical findings, tumor sampling, and other laboratory data. If results obtained do not match other clinical or laboratory findings, please contact the laboratory director.

Clinical Utility

Bone and soft tissue tumors are rare entities and comprise more than 100 different tumor types, which can show morphological and immunohistochemical overlap that poses challenges in diagnostics. Integration of morphological, immunohistochemical, and molecular methods is necessary for precise diagnosis and subsequent clinical management of sarcomas. Identification of chromosomal translocations and fusion genes has substantially contributed to diagnosis, disease subclassification, and determining therapy.

CPT Codes

81456, G0452

Contacts

Last Updated

8/19/2024