This test may be ordered for patients with the following tumour types:
- Sarcomas
- Cholangiocarcinomas
- Brain tumours
- Rare solid tumours
- Certain hematological malignancies
This test is a capture-based targeted resequencing assay designed to detect RNA alterations, including fusions, and other aberrant splicing events of interest, single nucleotide variants (SNVs), small insertions/deletions (indels), and internal tandem duplications in a select number of genes clinically relevant to solid tumours.
This test is performed on RNA extracted from fresh or frozen tissue, formalin-fixed paraffin-embedded (FFPE), peripheral blood, or bone marrow specimens. Analysis is carried out using an RNA prep enrichment kit from Illumina (Illumina catalog#20040537), and regions are captured using the exome panel enrichment oligos from Illumina (Illumina catalog#20020183). Paired-end massively parallel sequencing of 75-bp fragments is performed with an Illumina NextSeq550 instrument. cDNA sequences are aligned and compared to reference genome GRCh38 to identify fusions and other aberrant splicing events of interest, SNVs, indels, and internal tandem duplications in a select list of genes (see appendix).
The following run-level and specimen-level quality control metrics are targeted for fresh or frozen tissue, peripheral blood, or bone marrow specimens: Q30 performance achieved during validation, generation of equal or higher than 8.0 million FASTQ reads, and equal or higher than 75% of reads aligned to targeted regions. The following run-level and specimen-level quality control metrics are targeted for formalin-fixed paraffin-embedded (FFPE) specimens: Q30 performance achieved during validation, generation equal or higher than 4.0 million FASTQ reads, and equal or higher than 75% of reads aligned to targeted regions. Additionally, a set of 15 proxy quality control (pQC) genes (ABI1, CD74, CNBP, CTNNB1, FLNA, GTF2I, HSP90AA1, NONO, PRKAR1A, SEC31A, SEPTIN2, STAT6, TAF15, TCF12, ZNF207) are used to evaluate the quality of the RNA samples (PMID: 36958423). The level of expression of the pQC genes was used to establish a threshold for quality metrics. Only samples with at least 11 of the 15 pQC genes showing acceptable expression levels are accepted for analysis.
These laboratory results were generated using a capture-based test; results should be viewed in conjunction with other clinical information and diagnostic findings. The NGS assay is limited to testing for the presence of exon-exon rearrangements and other aberrant splicing events of interest, SNVs, indels, and internal tandem duplications. This panel does not comprehensively test for structural variants within all genes present in the genome and does not include the entire coding regions of genes specifically targeted for the detection of SNVs, indels, and internal tandem duplications. The list of specific exons for each gene targeted by this assay is available upon request.
The analytical sensitivity of this test is estimated to be 100% for fresh or frozen tissue, peripheral blood, and bone marrow with 20% or greater disease involvement. The analytical sensitivity of this test is estimated to be 98.17% for FFPE, with 20% or greater disease involvement. However, this methodology may not detect structural variants, SNVs, indels, or internal tandem duplications that have low abundance due to low-level sub-clonal populations or low expression levels. In addition, poor-quality RNA can decrease the number and quality of reads, which subsequently can reduce assay sensitivity and can potentially lead to false-negative results.
Variants are interpreted and categorized based on their clinical impact, as per standards and guidelines in the field (PMID: 27993330): Tier I, variants with strong clinical significance (level A and B evidence); Tier II, variants with potential clinical significance (level C and D evidence); Tier III, variants with unknown clinical significance; and Tier IV, benign or likely benign variants. Assessments are limited by the information available at the time of report issued for any given phenotype, gene, locus, or variant.
Structural variants classified as benign or likely benign observed at a significant frequency among all tested samples or in population databases with no existing published evidence of cancer association are not included.
Turnaround time: 15 working days
Specimens accepted:
- 10 x 10 μm scrolls in 1.5 mL tube
- 10 x 10 μm unstained slides
- A tumour cell content (TCC) of 20% or more is required. This information is mandatory to assess the validity of the test.
Please see our Preparation and Shipping Guidelines for additional details.
Submit your test request using our solid tumour requisition.