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Myeloid Neoplasms NGS Panel

The myeloid neoplasm NGS Panel is a next generation sequencing test that detects variants in both DNA and RNA samples including single-nucleotide variants (SNV) and fusions. It is optimized to provide high sensitivity and specificity for low-frequency somatic variants. See “Clinical Utility” below for specific biomarkers.

Test Codes

EPIC: LAB1230965

Department

Molecular Pathology

Specimen Collection Criteria

Bone Marrow (Preferred for myeloid neoplasms panel):

• 1.0 mL bone marrow aspirate in a Lavender-top EDTA tube. (Minimum: 0.5 mL)      

Peripheral Blood (Secondary option for myeloid neoplasms panel):

• 1.0 mL peripheral blood sample in a Lavender-top EDTA tube (Minimum 1.0 mL)

Surgical Specimens and Cytology Cell Blocks (Formalin Fixed, 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. Average tissue size 5.0 mm².
• 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

Bone Marrow and Peripheral Blood:  

Do not freeze specimens. Maintain specimens refrigerated (preferred) (2-8°C or 36-46°F) or at room temperature (20-26°C or 68-78.8°F) and transport fresh samples to the Laboratory within 4-6 hours of collection.

Paraffin blocks and unstained slides should be kept at room temperature (20-26°C or 68-78.8°F).

Preparation for Courier Transport

Transport: Bone marrow or peripheral blood refrigerated (preferred) (2-8°C or 36-46°F) or at room temperature (20-26°C or 68-78.8°F). Paraffin blocks and unstained slides should be kept at room temperature (20-26°C or 68-78.8°F).

Rejection Criteria

• Specimens collected in heparin (Green-top), clot tubes (Red-top) ACD anti-coagulant tubes or SST tubes.
• Centrifuged specimens.
• Bone marrows greater than 7 days.
• Tissue decalcified with agents other than Mol Decal (EDTA)  
• Fixatives other than 10% neutral buffered formalin.  
• Improper labeling or inadequate information.   
• Poor quality and/or quantity of extracted DNA/RNA. 
• Frozen or unfixed 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. 

In-Lab Processing

Maintain fresh specimens (i.e., bone marrow and peripheral blood) refrigerated (2-8°C or 36-46°F) prior to testing. 

Paraffin blocks and unstained slides should be kept at room temperature (20-26°C or 68-78.8°F). 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 medical director or designee, based upon the amount of available tissue and the tumor cellularity.

Storage

Bone Marrow Specimen Stability for Testing:

Room Temperature (20-26°C or 68-78.8°F): 6 hours
Refrigerated (2-8°C or 36-46°F): 7 days
Frozen (-20°C/-4°F or below): Unacceptable.

FFPE 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:

Bone Marrow:
Refrigerated (2-8°C or 36-46°F): 7 days

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

Specimen Storage (Extracted nucleic acid):

DNA: Frozen (-25 to -15°C)
RNA: Frozen (-63 to -70°C)

Laboratory

Royal Oak Molecular Pathology Laboratory

Performed

Once or twice per week, dependent upon test volume.
Results available within 21 days.

Reference Range

No variants detected or only likely benign variants detected.

Test Methodology

DNA and RNA are isolated from the sample and quantified. Recovered DNA/RNA extracts are prepared for sequencing with the Illumina TruSight^® Oncology 500 DNA Kit and Illumina TruSight^® Tumor 170 RNA Kit and sequenced using paired-end sequencing on the Illumina NextSeq 500/550 instrument with the Illumina NextSeq™ 500/550 High Output Reagent V2 300 Cycle sequencing kit. Alignment and variant calling of both RNA and DNA libraries occurs using the Illumina TruSight Oncology 500 v2.2 pipeline. The variant results are annotated and compiled by Velsera Clinical Genomics Workspace (CGW) to generate a clinical report. A personalized interpretive report is generated that lists the variants detected in each gene, classified based on a standardized classification scheme for somatic variants, and provides detailed interpretative comments.

Test Limitations:

• Test performance is optimal at DNA concentrations of at least 3.3 ng/ul and RNA concentration of at least 10ng/ul. Values below these thresholds will result in suboptimal test performance.
• This test is designed to detect somatic variants only. The status of potential germline variants 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 link below for the list of genomic regions covered by the panel.
• Rare false positives and negatives may occur due to errors in sequencing chemistry. Quality assurance criteria are established to minimize such occurrences.  
• Variants which are present in the data but with allele frequency/copy number/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.
• SNVs and indels with allele fraction below the established analytical sensitivity are not reproducibly detected by this assay. Large indels (insertions greater than 18 bp or deletions greater than 28 bp) will not be detected by this assay.
• Due to the nature of this assay, the majority of variants reported will be of uncertain clinical significance and novel (not previously identified by the TSO 500 assay in this lab). Orthogonal confirmatory testing is reserved for novel variants with well-defined clinical significance (i.e. tier I variants). Confirmatory testing of other variants (tier II/III) that may affect clinical decision making is left to the discretion of the treating physician and subject to the availability of suitable confirmatory methods.
• Variants with supporting evidence consisting only of small case series, case reports, or preclinical data will be reported as variants of uncertain significance with no further interpretative comments.
• Germline variants that are frequent in the general population (>1% frequency in population databases) and considered benign or likely benign (i.e. unrelated to cancer) may be identified but will not be reported. 

• The exons in the following list have consistently shown poor performance in validation studies and may not reliably detect variants: KMT2A NM_001197104.1 (exon 1); STAT5B NM_012448.3 (exon 7); BCR NM_004327.3 (exon 1,18); CEBPA NM_004364.3 (exon 1), FLT3 NM_004119.2 (exon 1); WT1 NM_024426.4 (exon 1); JAK2 NM_004972.3 (exon 15); IDH2 NM_002168.2 (exon 1); PTEN NM_000314.4 (exon 3); RUNX1 NM_001754.4 (exon 3); SETBP1 NM_015559.2 (exon 6); STAG2 NM_001042750.1 (exon 3, 12, 22, 27)
• This test may not detect insertions greater than 18 base-pairs; however, validation studies have confirmed high sensitivity for detecting FLT3 duplications up to 24 bp. It is recommended to perform FLT3-ITD by a more sensitive assay like PCR with electrophoresis-based product sizing.
• 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 judgment 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.
• Single nucleotide variants (SNVs) with allele fraction of at least 4% and depth of coverage at least 100X are detected with 96.67% sensitivity, 100% specificity, 100% PPV, and 98.28% NPV.
• Insertion-deletion variants (indels) with an allele fraction of at least 4% and depth of coverage at least 100X are detected with 93.18% sensitivity, 99.31% specificity, 98.40% PPV, and 96.95% NPV.
• RNA fusions are detected with 94.74% sensitivity, 100% specificity, 100% PPV, and 99.41% NPV with a high confidence filter and additional minimum requirements of either 20 unique split reads or at least 3 unique split reads with at least 28 unique paired reads supporting the fusion event.
• Special care should be taken when reviewing indels in a homopolymer region.
• Clinical utility of the TSO500 assay for fusion calling is limited for clinically relevant fusions in hematological disorders, the following fusions are consistently not detected due to limitations of the variant calling analysis. Furthermore, the minor BCR-ABL1(e1a2) transcript cannot be detected in the presence of another major BCR-ABL1 fusion transcript due to the overlapping nature of the fusion reads.

1. FIP1L1-PDGFRA.
2. MYST3-CREBBP
3. RUNX1-RUNX1T1
4. PML-RARA
5. BCR-ABL1 (e1a2) 

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

This test is designed to detect multiple classes of DNA and RNA mutations including single-nucleotide variants (SNV), multi-nucleotide variants ≤ 3 (MNV), small insertions (1-18 bp)/deletions (1-24 bp) (indels).

Myeloid Neoplasms NGS Panel (49 genes):
Regions Sequenced (Myeloid Neoplasms Table)

ABL1, ABL2, ASXL1, BCOR, BCORL1, BCR, BRAF, CALR, CBL, CEBPA, CSF3R, DDX41, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MLL (KMT2A), MPL, MYD88, NF1, NPM1, NRAS, PHF6, PPM1D, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2 (MFSD11), STAG2, STAT3, STAT5B, TET2, TP53, U2AF1, WT1 and ZRSR2.

Genes Analyzed for Fusions

ABL1, BRAF, FLT3, JAK2, MLL (KMT2A), KIT and TP53.

CPT Codes

81450, G0452

Contacts

Last Updated

10/8/2025