Test Information: Tumor-normal exome sequencing is used to identify clinically significant genetic variants in a patient’s tumor that may have therapeutic, prognostic, and/or diagnostic implications. This test targets the exons, which are the protein coding regions of the genome. Variants included on the report are limited to those classified as either Tier 1, Tier 2 or Tier 3 with evidence towards somatic origin or with potential clinical significance.
Secondary findings are likely pathogenic and pathogenic variants identified in one or more of the genes included in the American College of Medical Genetics (ACMG) list of genes that are suggested as returnable results for individuals undergoing whole exome/genome sequencing (PMID: 27854360). Secondary findings are only reported if the patient opts in to receive them.
Methodology: This test has been designed to assess variants occurring within protein coding regions and splice junctions of RefSeq genes. Nucleic acid from the submitted tumor and “normal” specimens is enriched for coding and adjacent noncoding (splice) regions of the genes using the IDT xGen Exome (Integrated DNA Technologies, Coralville, IA). The library products are sequenced with 2 by 150 bp reads on either the Illumina NextSeq or NovaSeq sequencing instruments (Illumina, San Diego, CA). After alignment to the reference genome (GRCh37/hg19), off target, low quality, and duplicate reads are removed from the analysis. The targeted regions are assessed for average depth of coverage and other data quality thresholds. Variants are called using several different variant calling algorithms, including ones developed by Genosity.
This test detects single nucleotide substitutions (SNVs) and small insertions, deletions, and indels located in the coding sequences and known splice regions in the genes targeted by the test. Tumor mutation burden is presented as the number of mutations per mega base of the analyzed region.
This is an oncology-driven analysis and variants included on the report are limited to those identified in genes reported in the patient’s tumor type or any tumor type if patient’s tumor type is unknown or unclear or knowledge of the associations of genes with the patient’s tumor type is limited (list available upon request). Data generated from the “normal” sample is used to create a filter to identify variants that are only present in the tumor sample. Sequence alterations are described according to the Human Genome Variation Society (HGVS) nomenclature guidelines. Variants are classified according to the joint consensus recommendations for the interpretation and reporting of sequence variants in cancer by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists Variants (PMID: 27993330). Variants with strong or potential clinical significance (Tier 1 and Tier 2 variants) are reported. Variants of unknown clinical significance (Tier 3 variants) with evidence towards somatic origin are reported, Tier 3 variants without evidence towards somatic origin are not reported. Benign or likely benign variants (Tier 4 variants) are not reported.
Secondary findings are only reported if elected by the patient. Under certain circumstances, such as a single pathogenic variant identified in a gene with autosomal recessive inheritance, a likely pathogenic or pathogenic variant in one of these genes may not be reported. The secondary findings gene list is subject to change over time as it is updated by the ACMG. The current gene list for secondary findings consists of 59 genes (PMID: 27854360): ACTA2, ACTC1, APC, APOB, ATP7B, BMPR1A, BRCA1, BRCA2, CACNA1S, COL3A1, DSC2, DSG2, DSP, FBN1, GLA, KCNH2, KCNQ1, LDLR, LMNA, MEN1, MLH1, MSH2, MSH6, MUTYH, MYBPC3, MYH11, MYH7, MYL2, MYL3, NF2, OTC, PCSK9, PKP2, PMS2, PRKAG2, PTEN, RB1, RET, RYR1, RYR2, SCN5A, SDHAF2, SDHB, SDHC, SDHD, SMAD3, SMAD4, STK11, TGFBR1, TGFBR2, TMEM43, TNNI3, TNNT2, TP53, . TPM1, TSC1, TSC2, VHL, WT1.
Incidental findings are genetic changes that are of medical importance but that are not directly relevant to the indication for testing. Potential germline pathogenic variant (PGPV) is considered as one type of incidental findings. They are reported at the discretion of the laboratory and are only reported if they have significant healthcare implications for the patient.
The test was developed, and its performance characteristics were determined by Genosity. The report and methods used to generate the report, have not been cleared or approved by the U.S. Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary. This test is used for both clinical and clinical research purposes. Pursuant to the requirements of federal regulations listed in the Clinical Laboratory Improvement Amendments of 1988 (CLIA), this laboratory has established and verified this test's accuracy and precision. However, the possibility of a false positive or false negative result incurred during any phase of the testing cannot be completely excluded.
Limitations: A normal or inconclusive result does not eliminate the possibility of a genetic basis for disease in this individual and does not guarantee present or future health. Some types of genomic variants are not detectable by the technologies used to perform this test. This test is not intended to detect the following types of genetic variants: deletions/insertions of >15 bp, structural variation, variants in promotor regions and other non-coding regions, copy number variations, variations occurring within repetitive sequences and repeat expansion mutations, variants in regions with high homology or in genes with pseudogenes, mitochondrial genome mutations, epigenetic effects, mosaic events, and other complex aberrations. In addition, not all regions of the exome are fully covered by this analysis and variants in regions of low coverage may not be detected.
The interpretation of this test is based on the assumption that the clinical information provided to the laboratory is accurate. Not all variants identified have been analyzed. In addition, the interpretation of these results is based on currently available scientific knowledge. Not all disease-associated genes have been identified and the clinical significance of variation in many genes is not well understood. Variant interpretation may change over time if more information becomes available. It is recommended that genetic test results be periodically reinterpreted. Healthcare providers can contact the laboratory to determine if there have been any changes to the interpretation of a variant or test result.
Although rare, the accuracy of results may also be impacted due to sample mix-up, DNA contamination, poor DNA quality, or the presence of pre-malignant or malignant cells in the “normal” sample, as well as in the setting of bone marrow transplantation or a recent blood transfusion.
Laboratory Information: Genosity is a College of American Pathologists (CAP)-accredited and Clinical Laboratory Improvement Amendments (CLIA)-certified clinical diagnostic laboratory (CAP#: 8289311; CLIA #31D2142534). This test was performed at Genosity located at 485F US Route 1 South, Suite 110, Iselin, NJ 08830.