Cautions

This test cannot differentiate between somatic and germline alterations. Additional testing may be necessary to clarify the significance of results if there is a potential hereditary risk.

RNA is particularly labile and degrades quickly. Rapid preservation of the tumor sample after collection reduces the likelihood of degradation, but sometimes, there are biological factors, such as tumor necrosis, that interfere with obtaining a high-quality RNA specimen despite rapid preservation.

Variants and fusions of uncertain significance may be identified.

A negative result does not rule out the presence of a variant that may be present below the limits of detection of this assay. The analytical sensitivity of this assay for sequence reportable alterations is 5% mutant allele frequency with a minimum coverage of 500X in a sample with 20% or more tumor content.

The sensitivity of this assay for gene fusions depends on several variables including decreased sensitivity with decreased tumor percentage, and decreased sensitivity with decreased level of expression of a variant. A negative result does not rule out the presence of a gene fusion, splice variant, or BCOR exon 15 internal tandem duplication that may be present but below the limits of detection of this assay. The analytical sensitivity of this assay for rearrangements is a minimum coverage of 5 unique variant molecules in a sample with at least 10% tumor content.

Point mutations and small deletion-insertion mutations (delins) will be detected in the ALK, APC, BAP1, BCOR, BRAF, CDKN2A, CTNNB1, DICER1, EED, EGFR, FGFR4, GNA11, GNA14, GNAQ, GNAS, H3-3A, H3-3B, KIT, MDM2, MED12, MYOD1, NF1, PDGFRA, PDGFRB, PTPRD, ROS1, SMARCB1, SUZ12, TERT-promoter, TP53, and TSC2 genes. This test may detect single exon deletions but does not detect multi-exon deletions, duplications, larger-scale genomic copy number variants, copy neutral loss of heterozygosity, or epigenetic modifications such as promoter methylation. Delins of 1000 bp or less are detectable with at least 50 or more supporting reads.

This assay can detect in-frame and out-of-frame fusions involving 1445 genes. Sensitivity for detecting out-of-frame fusions such as exon-intron, intron-intron or big insertions, may be lower due to bioinformatics detection limitations. This assay will only detect fusions involving at least 1 gene in the defined gene fusion target list of interest. This assay may not detect fusions involving deep intron or intergenic regions and will not detect chromosomal rearrangements that do not create a fusion transcript (i.e. enhancer repositioning).  Variants not expressed, or expressed at very low level, are not detected by this assay.

Variant allele frequency (VAF) is the percentage of sequencing reads supporting a specific variant divided by the total sequencing reads at that position. In somatic testing, VAF should be interpreted in the context of several factors including, but not limited to: tumor purity/heterogeneity/copy number status (ploidy, gains/losses, loss of heterozygosity) and sequencing artifact/misalignment.(1,2)

Rare variants, or alterations derived from the production of a gene fusion, may be present that could lead to false-negative or false-positive results.

The presence or absence of a variant or fusion may not be predictive of response to therapy in all patients.

Test results should be interpreted in the context of clinical, tumor sampling, histopathological, and other laboratory data. If results obtained do not match other clinical or laboratory findings, contact the laboratory for discussion. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

Reliable results are dependent on adequate specimen collection and processing. This test has been validated on cytology slides and formalin-fixed, paraffin-embedded tissues; other types of fixatives are discouraged. Improper treatment of tissues, such as decalcification, may cause polymerase chain reaction failure.