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Tumor Type-Specific and Large Pan-Cancer Panels by Next Generation Sequencing

Comprehensive Cancer, Lung, Myeloma, Myeloid Neoplasms, Gastrointestinal Stromal Tumor (GIST), Sarcoma, Melanoma, Colorectal and Neuro Panels Tumor Mutation Panels by Next Generation Sequencing (NGS) using the Illumina TruSight Oncology 500 assay (TSO500). Sub-panels available: lung, myeloma, myeloid neoplasms, GIST, sarcoma, melanoma, colorectal, and neuro-oncologic

Test Codes

Panel Name	Soft code-5 character
Lung Cancer NGS Panel	MLUNG
Melanoma NGS Panel	MMEL
Colorectal Cancer NGS Panel	MCRC
Sarcoma NGS Panel	MSARC
Gastrointestinal Stromal Tumor NGS Panel	MGIST
Neuro NGS Panel	MNEUR
Myeloma NGS Panel	MMYLM
Myeloid neoplasms NGS Panel	MMYLD
Comprehensive Cancer NGS Panel	M500G

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 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.
• Tissue decalcified with agents other than Mol Decal (EDTA)  
• Fixatives other than 10% neutral buffered formalin.  
• Improper labeling or inadequate information.   
• Less than 12.5% tumor cellularity
• Poor quality and/or quantity of extracted DNA/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

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): 48 hours
Frozen (-20°C/-4°F or below): Unacceptable

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 (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, DNA or both 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 for all biomarkers with specimens containing at least 25% tumor cells (SNVs and indels at 12.5% tumor cells. Fusions/Splice Variants at 7.5% tumor cells. CNV, MSI and TMB at 25% tumor content) and DNA concentrations of at least 3.3 ng/ul and RNA concentration of at least 4.7ng/ul. Values below these thresholds will result in suboptimal test performance.
• The content of this test is not optimized for tumor types other than those listed in the gene subpanels.  
• 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 links below for the list of genomic regions covered by each panel.

For Solid Tumors:

• Single nucleotide variants (SNVs) with allele fraction of at least 3% and depth of coverage at least 100X are detected with 96.08%          sensitivity, 99.65% specificity, 95.69% PPV, and 99.68% NPV. Only variants detected at allelic frequency greater than or equal to 5% will be reported due to the difficulty of confirming variants at allelic frequency lower than 5%, unless confirmed by another method or stated in the report.
• Insertion-deletion variants (indels) with allele fraction of at least 3% and depth of coverage at least 100X are detected with 89.81% sensitivity, 96.58% specificity, 95.10% PPV, and 92.76% NPV. Only indels detected at allelic frequency greater than or equal to 5% will be reported due to the difficulty of confirming variants at allelic frequency lower than 5%, unless confirmed by another method.
• CNVs with at least 5 copies are detected with 72.58% sensitivity, 99.44% specificity, 95.74% PPV, and 95.42% NPV. Only CNVs greater than 5 copies will be reported, due to high rate of false positives at lower amplification.
• MSI is detected with 100% clinical sensitivity, 100% specificity, 100% PPV and 100 NPV.
• TMB is detected with95.06% clinical sensitivity, 95.06% specificity, 95.06% PPV and 95.06% NPV.
• RNA fusions are detected with 100% sensitivity, 99.41% specificity, 91.67% PPV, and 100% 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.
• Splice variants are detected with 100% sensitivity, 100% specificity, 100%  PPV, and 100% NPV with a minimum requirement of 25 unique split reads.

For Hematologic Malignancies (blood and bone marrow):

• Single nucleotide variants (SNVs) with allele fraction of at least 3% and depth of coverage at least 100X are detected with 91.30% sensitivity, 100% specificity, 100% PPV, and 99.23% NPV. Only variants detected at allelic frequency greater than or equal to 5% will be reported due to the difficulty of confirming variants at allelic frequency lower than 5%, unless confirmed by another method or stated in the report.
• Insertion-deletion variants (indels) with allele fraction of at least 3% and depth of coverage at least 100X are detected with 88.89% sensitivity, 100% specificity, 100% PPV, and 81.82% NPV. Only indels detected at allelic frequency greater than or equal to 5% will be reported due to the difficulty of confirming variants at allelic frequency lower than 5%, unless confirmed by another method or stated in the report.
• RNA fusions are detected with 95.83% 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:
  
  
  1. FIP1L1-PDGFRA.
  2. MYST3-CREBBP
  3. RUNX1-RUNX1T1
  4. PML-RARA
  5. BCR-ABL (e1a2)

• 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 following genes and genomic regions consistently show poor performance in this assay and will not reliably detect variants: List of Genes (Poor Performers Table).
• Variant calls in the HLA-A, KMT2B, KMT2C, and KMT2D genes are filtered out due to potential mis-mapping, using Illumina’s analysis pipeline, as a result of sequence homology with other genomic regions.
• 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. 
• High tumor mutational burden (TMB-high) indicates the presence of an elevated number of somatic, non-synonymous mutations in the coding regions of a tumor cell genome, expressed as mutations per megabase (mut/MB) of sequenced tumor DNA (PMID: 28481359, PMID: 30505710, PMID: 30505709).
• While TMB has historically been assessed by whole-exome sequencing (WES), recent studies have demonstrated that TMB can be effectively estimated using targeted sequencing panels covering 1.1 Mb or more of genomic content (PMID: 28420421, PMID: 30238975). The validity of targeted panels for determination of TMB status allows for the assessment of this pan-cancer biomarker by a wider range of methods and panels in community-based laboratories, facilitating greater patient access to this testing. Currently, pembrolizumab is FDA-approved for pediatric and adult patients with unresectable and metastatic solid tumors with ≥ 10 mut/MB as determined by the FoundationOneCDx assay (fda.gov). However, optimal cutoff values for classification of TMB values into high and low categories are not yet standardized across different methods and panels (PMID: 30664300) and the definition of high TMB varies across studies from ≥7.4 to 20 mut/Mb when measured with a large NGS panel (PMID: 29658845, PMID: 29337640, PMID: 31258479, PMID: 30505709, PMID: 32864131). However, TMB results obtained with the TSO500 assay have been evaluated and compared with WGS and FoundationOneCDx (PMID: 32306292, PMID: 35218944). In these studies, TSO500 with a TMB-high cutoff of ≥10 mut/MB demonstrates a high degree of concordance and clinical utility when compared with WES and FoundationOneCDx.  While the clinical utility of TMB is still being defined, continuing efforts to standardize TMB calling between laboratories and manufacturers are ongoing.
• Microsatellite instability (MSI) refers to the hypermutability of tumor DNA that is caused by germline or somatic defects in the DNA mismatch repair pathway. MSI status is divided into MSI-High (MSI-unstable) tumors, which have changes in microsatellite repeat length due to defective DNA mismatch repair activity, and microsatellite stable (MSS) tumors which lack these defects of DNA repair. In the clinical setting MSI is traditionally analyzed directly with PCR (MSI-PCR), and indirectly by immunohistochemistry for the mismatch repair proteins (MMR). However, NGS allows for the analysis of a greater number of microsatellite loci than MSI-PCR, presenting opportunities to identify new MSI profiles in previously uncharacterized cancer types. Although NGS can interrogate thousands of microsatellite loci, compared to the 5–7 loci that are tested by PCR, the optimal cut-off value for MSI-High (MSI-H/MSI-unstable) in diverse tumor types is controversial. To date, optimal thresholds for the classification of NGS-derived MSI values into stable and high categories are not yet standardized across different methods and panels (PMID: 36465367). Several studies have reported concordance of MSI status between PCR and NGS based testing in solid tumors (PMID: 34625576, PMID: 35172512, PMID: 31530574, PMID: 27863258). A recent large study comparing TSO500 to MSI-PCR and MMR determined that an MSI-H cutoff of ≥ 20% unstable sites and an equivocal zone of 7% ≤ X ˂ 20% for TSO500 demonstrates a high degree of concordance with MSI-PCR and MMR (PMID: 35405622). In keeping with these results, any patient values within this equivocal zone will be tested further by orthogonal MSI-PCR and/or MMR for final classification of MSI status.
• 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.

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), splice variants (in MET and EGFR), fusions and copy number variants (CNV). The test is also capable of quantitative assessment of both microsatellite instability (MSI) and tumor mutational burden (TMB). The pan-cancer biomarkers NTRK1/NTRK2/NTRK3 gene fusions are also included with all solid tumor panels

The test can be ordered as either the comprehensive 517 gene analysis, or one of eight tumor type-specific gene panels that analyze only the mostly clinically relevant genes for that tumor type, as shown below.

* Genes analyzed for fusions.
§ Genes analyzed for copy number variants.

1. Lung Cancer NGS Panel (27 genes):

   AKT1, ALK*^§, BRAF*^§, DDR2, EGFR*^§, ERBB2*^§, FGFR1*^§,  FGFR2*^§, FGFR3*^§, HRAS, KRAS^§, MAP2K1, MET*^§, MLH1, MSH2*, MSH6, NRAS^§, NTRK1*, NTRK2*, NTRK3*, PIK3CA*^§, PMS2, PTEN, RET*^§, ROS*, SMAD4, TP53.
   Regions Sequenced in Lung Cancer NGS Panel (Lung Table)

2. Myeloma NGS Panel (27 genes):
   AKT2, AKT3, ATM, BIRC3, BRAF, CCND1, CDKN1B, CDKN2A, CXCR4, EGFR, FGFR3, IDH1, IDH2, IRF4, IKZF1, JAK2, KDM6A, KRAS, MYC, MYD88, NRAS, PIK3CA, PIM1, RB1, STAT3, TP53.
   Regions Sequenced in Myeloma NGS Panel (Myeloma Table)

3. Myeloid Neoplasms NGS Panel (46 genes):

   AKT1, ALK*^§, BRAF*^§, DDR2, EGFR*^§, ERBB2*^§, FGFR1*^§,  FGFR2*^§, FGFR3*^§, HRAS, KRAS^§, MAP2K1, MET*^§, MLH1, MSH2*, MSH6, NRAS^§, NTRK1*, NTRK2*, NTRK3*, PIK3CA*^§, PMS2, PTEN, RET*^§, ROS*, SMAD4, TP53.
   Regions Sequenced in Myeloid Neoplasms NGS Panel (Myeloid Neoplasms Table)

4. Gastrointestinal Stromal Tumor NGS Panel (10 genes):

   BRAF*^§, KIT*^§, PDGFRA*^§, NTRK1*, NTRK2*, NTRK3*, RET*, SDHB, SDHC, SDHD.
   Regions Sequenced in Gastrointestinal Stromal Tumor NGS Panel (GIST Table)

5. Sarcoma NGS Panel (29 genes):

   ALK*^§, APC, BRAF*^§, CDK4*^§, CTNNB1, ETV6, EWSR1*, FOXO1, GLI1, IDH1, IDH2, KIT*^§, MDM2^§, MYOD1, NAB2, NF1, NTRK1*, NTRK2*, NTRK3*, PAX3*, PAX7*, PDGFRA*^§, PDGFRB*^§, RET*^§, SDHB, SDHC, SMARCB1, TFE3, WT1.
   Regions Sequenced in Sarcoma NGS Panel (Sarcoma Table)

6. Melanoma NGS Panel (16 genes):

   BRAF*^§, CTNNB1, GNA11, GNAQ, KIT*^§, MAP2K1, NF1, NRAS^§, NTRK1*, NTRK2*, NTRK3*, PDGFRA*^§, PIK3CA*^§, PTEN, RET*^§, TP53.
   Regions Sequencing in Melanoma NGS Panel (Melanoma Table)

7. Colorectal Cancer NGS Panel (17 genes):
   AKT1, BRAF*^§, HRAS, KRAS, MET*^§, MLH1, MSH2*, MSH6, NRAS^§, NTRK1*, NTRK2*, NTRK3*, PIK3CA*^§,  PMS2, PTEN, RET*^§, SMAD4, TP53.

   Regions Sequenced in Colorectal Cancer NGS Panel (Colorectal Table)

8. Neuro-oncologic NGS Panel (50 genes):

   AKT1, BRAF*^§, HRAS, KRAS, MET*^§, MLH1, MSH2*, MSH6, NRAS^§, NTRK1*, NTRK2*, NTRK3*, PIK3CA*^§,  PMS2, PTEN, RET*^§, SMAD4, TP53.
   Regions Sequenced in Neuro-oncologic NGS Panel (Neuro-oncologic Table)

9. Comprehensive Cancer NGS Panel (517 genes):
   Please review last page for comprehensive list.
   Regions Sequenced in Comprehensive Cancer NGS Panel (Comprehensive Table)

CPT Codes

Panel Name	CPT Codes
Lung Cancer NGS Panel	81445
Melanoma NGS Panel	81445
Colorectal Cancer NGS Panel	81445
Sarcoma NGS Panel	81445
Gastrointestinal Cancer NGS Panel	81445
Neuro NGS Panel	81445
Myeloma NGS Panel	81450
Myeloid neoplasms NGS Panel	81450
500 CANCER GENE PANEL NGS	81455

Contacts

Last Updated

10/30/2023