Predictive

NGS comprehensive tumor profiling for melanoma performed by Diagnostic Laboratory Services is a next-generation sequencing assay able to identify known single nucleotide polymorphisms, copy number alterations, transcript fusion mutations, and microsatellite instability to determine matched FDA approved; NCCN guided therapy. Over the past 10 to 15 years, targeted agents have … NGS comprehensive tumor profiling for melanoma performed by Diagnostic Laboratory Services is a next-generation sequencing assay able to identify known single nucleotide polymorphisms, copy number alterations, transcript fusion mutations, and microsatellite instability to determine matched FDA approved; NCCN guided therapy. Over the past 10 to 15 years, targeted agents have been a fundamental part of therapeutic decision-making. There has been a remarkable improvement in the overall survival of patients where there is a molecular target identified. In 2019, the impact trial was published in the Journal of hematology and oncology. Their look at 3487 patients who underwent tumor profiling showed statistically significant differences in response rate, progression-free survival, and overall survival in months at 3- years and 10- years. Approximately 300,000 patients underwent tumor profiling. At least one treatable molecular variant was found in 1307 patients, who eventually received an associated matched therapy. Objective response rates when there was a match were 16.4% versus 5.4% (p-value <0.0001). Median progression-free survival for matched and unmatched patients was 4.0 and 2.8 months (p-value <0.0001). Overall survival showed differences in matched and unmatched patients, 9.3 versus 7.3 months, with a 3- year survival rate of 15% in matched patients and 7% in unmatched patients. At 10 years, this survival rate was 6% versus 1% ( p-value <0.0001). In multivariate analysis, there was a shorter overall survival in patients with non-matched therapies (p-value <0.001). View more

Guidance for selecting a drug therapy and/or dose

Advanced Cancer Patients

Sample Negative Report Help
Revised report requested by NCBI

Sample Positive Report Help
Revised report requested by NCBI

Sample VUS Report Help
Sample VUS Report

Next-Generation Sequencing

TruSight Oncology 500

Tests performed
Entire test performed in-house

Clinical samples were correlated with 3 external sources: Reports from outside commercial laboratories running similar tumor CGP assays on an Illumina based sequencer, a partnered laboratory running the same TSO500 assay on the NextSeq550 and single orthogonal methods targeting key genomic hotspots (e.g. RT-PCR, FISH, IHC). A variety of sample … Clinical samples were correlated with 3 external sources: Reports from outside commercial laboratories running similar tumor CGP assays on an Illumina based sequencer, a partnered laboratory running the same TSO500 assay on the NextSeq550 and single orthogonal methods targeting key genomic hotspots (e.g. RT-PCR, FISH, IHC). A variety of sample types were sourced including commercial controls and a wide range of clinical samples. Technicians were rotated on sample extraction and sequencing. Inter-run and Intra-run reproducibility was measured. For these above modifications, the correlation method bias and its estimation is expected to be low. One hundred-seventeen (117) samples were run. Eighty-nine (89) clinical samples were acquired from different tumor types (Figure 1 [table abbrev]: SYN-synthetic controls of actionable target types; NSCLC-Non-small cell lung cancer; HNN-Head&Neck; CRC-Colorectal; UGI-Upper gastrointestinal; GU-Genitourinary; GYN-Gynecologic, Non-ovarian; GIST-Gastrointestinal stromal tumor; CNS-Central nervous system). All were formalin-fixed paraffin-embedded tissue in a mixture of surgical resection, biopsy and cytology cases. Archival dates ranged from 1 to 9 years. This assay is able to report DNA variants meeting or exceeding QC thresholds with a MEDIAN TARGET COVERAGE of at least 250 reads, a MEAN TARGET COVERAGE of 250 reads; at least 50% of ALIGNED READS; at least 50% percent of ON-TARGET READS AT 0.4X OF THE MEAN COVERAGE; at least 50% of ON-TARGET READS AT 100X; at least 50% of ON-TARGET READS AT 250X. RNA variants meeting or exceeding QC thresholds with a MEDIAN COEFFICIENT OF VARIATION IN GENE COVERAGE of 45%, at least 14,000,000 TOTAL ON-TARGET READS, at least 70% ON-TARGET READS and a SCALED (based on gene length) MEDIAN GENE COVERAGE of 2000 reads. This sequencing chemistry shows consistent sensitivity. Our lower threshold of detection is set at 2%. Published sensitivities on this platform are 1.3% with FFPE cell lines diluted down to variant allele frequencies of 5% 3.8% 2.5% and 1.30%. Our validation has demonstrated positive percent agreements of 93.5% for SNVs, 97.3% for INDELS, 99.5% for amplifications, 80.0% for splice site variants and 97.0% for fusions. The platform reports consistent detection with variable input amounts ranging from 40 ng down to 10ng. On-board sequencing metric files are used to monitor enrichment. The MetricsOutput.tsv file reports the PCT_READ_ENRICHMENT (%) parameter that measures enrichment of on-target reads for each sample. This is sample- and input-dependent. For samples passing DNA and RNA QC thresholds, this value was repeatedly in the 80-90% range. The TSO500 sequencing chemistry demonstrates increased specificity. The onboard variant calling software uses unique molecular identifier tags to remove sequencing error artifacts and calls in low confidence regions resulting in a 500 to 1000 times reduction in false-positive detection. Our validation has demonstrated false detection rates of 0.06% for SNVs, 3.1% for INDELS, 0.5% for amplifications, 8.3% for splice site variants and 5.9% for fusions. The TSO500 microsatellite instability (MSI) component utilizes a tumor-only strategy interrogating 130 homopolymer intronic regions. MSI assessment in 54 colorectal cancers compared with the gold-standard, paired tumor-normal PCR-fragment analysis showed 100% concordance. When MSI was analyzed across multiple different tumor types the assay demonstrated a 98.98% accuracy compared to tumor-normal assessment by standard PCR. Tumor mutation burden is also assessed on the TSO500 assay. Reported TMB analysis of 108 formalin-fixed paraffin-embedded tissue samples demonstrated significant agreement compared to whole-exome sequencing (R2 = 0.931) and in our validation compared to orthogonal samples (Pearson r=1.00; p=1.3e-44). View more

Formalin-fixed paraffin-embedded tissue. The different procedures of tissue procurement were documented (e.g. Surgical resection versus cytology). Surgical and Cytology reports were cataloged for review. See Internal Test Validation Method below. RNA component of DLS_PS_52 failed with notable necrosis and hemorrhage. All other cases passed RNA QC thresholds. DLS_PS_41 and DLS_PS_63 … Formalin-fixed paraffin-embedded tissue. The different procedures of tissue procurement were documented (e.g. Surgical resection versus cytology). Surgical and Cytology reports were cataloged for review. See Internal Test Validation Method below. RNA component of DLS_PS_52 failed with notable necrosis and hemorrhage. All other cases passed RNA QC thresholds. DLS_PS_41 and DLS_PS_63 failed due to notable comedo necrosis. Acid-based decalcification is a significant processing interference. DLS_PS_20 and DLS_PS_6.2 failed due to exposure to decalcification solution. All other cases passed DNA QC thresholds. In the tested range of failed samples, the limit of detection was 12.20 ng +/- 11.61 (CI=9.70) for DNA and 18.33 ng +/- 18.93 (CI=47.02) for RNA. The lower limit of sample able to generate acceptable results was 36.34 ng +/- 9.68 (CI=8.95) for DNA and 35.52 ng +/- 10.02 (CI=12.44) for RNA. A minimum input of 40 ng of DNA and 40 ng of RNA is recommended by the manufacturer. Subsequent work published by Illumina, Inc in an industry white paper (TA p.95) shows that small variant analysis, MSI and TMB results can reliably be extended to 20 ng of DNA and 20 ng of RNA. This was confirmed in our LOD study. Successful sequencing results were produced at an input of 14.4 ng of DNA and 17.6 ng of RNA. Our minimum input will be in accordance with the manufacturer's recommendations. View more

Is proficiency testing performed for this test? Help
Yes

Method used for proficiency testing: Help
Inter-Laboratory

PT Provider: Help
College of American Pathologists, CAP

Description of PT method: Help
CAP sourced interlaboratory comparisons and surveys, for general SNV and small INDELs. We additionally challenge the CAP PT program in copy number variants, tumor mutation burden and microsatellite instability.

Description of internal test validation method: Help
Clinical samples were correlated with 3 external sources: Reports from outside commercial laboratories running similar tumor CGP assays on an Illumina based sequencer, a partnered laboratory running the same TSO500 assay on the NextSeq550 and single orthogonal methods targeting key genomic hotspots (e.g. RT-PCR, FISH, IHC). A variety of sample … Clinical samples were correlated with 3 external sources: Reports from outside commercial laboratories running similar tumor CGP assays on an Illumina based sequencer, a partnered laboratory running the same TSO500 assay on the NextSeq550 and single orthogonal methods targeting key genomic hotspots (e.g. RT-PCR, FISH, IHC). A variety of sample types were sourced including commercial controls and a wide range of clinical samples. Technicians were rotated on sample extraction and sequencing. Inter-run and Intra-run reproducibility was measured. For these above modifications, the correlation method bias and its estimation is expected to be low. One hundred-seventeen (117) samples were run. Eighty-nine (89) clinical samples were acquired from different tumor types (Figure 1 [table abbrev]: SYN-synthetic controls of actionable target types; NSCLC-Non-small cell lung cancer; HNN-Head&Neck; CRC-Colorectal; UGI-Upper gastrointestinal; GU-Genitourinary; GYN-Gynecologic, Non-ovarian; GIST-Gastrointestinal stromal tumor; CNS-Central nervous system). All were formalin-fixed paraffin-embedded tissue in a mixture of surgical resection, biopsy and cytology cases. Archival dates ranged from 1 to 9 years. View more

Software used to interpret novel variations Help
Genome Oncology (Cleveland, Ohio). Links to Functional Prediction software via dbNSFP: SIFT, SIFT 4G, Polyphen-2 HumDiv, Polyphen-2 HumVar, Mutation Taster. Clinical Knowledge bases: ClinVar, JAX-CKB, COSMIC, cBioPortal, OncoKB, My_Cancer_Genome. Variant Databases: dbSNP, 1000 Genomes, gnomAD, Varsome. Literature searches through Genomenon's Mastermind.

Laboratory's policy on reporting novel variations Help
Variants of known or potential pathogenicity and variants of unknown significance are listed in the report. The type of alteration, clinical significance, therapeutic implication, VAF, gnomAD score, Fold-change are provided. The report pushes to the Hospital EMR or is faxed to the ordering physician's Fax number on file. Exceptional results … Variants of known or potential pathogenicity and variants of unknown significance are listed in the report. The type of alteration, clinical significance, therapeutic implication, VAF, gnomAD score, Fold-change are provided. The report pushes to the Hospital EMR or is faxed to the ordering physician's Fax number on file. Exceptional results are texted to the physician via a HIPAA-compliant messaging application. View more