This test is an amplicon-based targeted resequencing assay designed to detect recurrent somatic DNA alterations in 14 genes that are clinically relevant to brain tumours. These include single-nucleotide DNA variants (SNVs) and small insertions/deletions (indels).
Panel content
| Gene | Transcript |
|---|---|
| ACVR1 | NM_001111067.4 (Arg206, Gln207, Arg258, Gly328 and Gly 356) |
| BRAF | NM_004333.6 (all residues in exons 11, 12, 13 and 15) |
| CTNNB1 | NM_001904.4 (all residues in exon 3) |
| EGFR | NM_005228.5 (Arg108, Ala289 and Gly598) |
| FGFR1 | NM_023110.3 (Asn546 and Lys656) |
| H3-3A | NM_002107.7 (Lys28 (also known as Lys27) and Gly35 (also known as Gly34)) |
| H3C2 | NM_003537.4 (Lys28 (also known as Lys27)) |
| H3C3 | NM_003531.3 (Lys28 (also known as Lys27)) |
| IDH1 | NM_005896.4 (Arg132) |
| IDH2 | NM_002168.4 (Arg140 and Arg172) |
| PDGFRA | NM_006206.6 (Lys385) |
| PIK3CA | NM_006218.4 (Glu542, Glu545 and His1047) |
| TERT | NM_198253.3 (promotor positions C228 (chr5:1,295,228) and C250 (chr5:1,295,250)) |
| TP53 | NM_000546.6 (all coding exons and flanking intronic regions (+/- 10 bp)) |
This test is performed on DNA obtained from tumour specimens using the AmpliSeq for Illumina technology. Targeted DNA regions are amplified by multiplex PCR. Paired-end massively parallel sequencing of 150-bp fragments is performed with an Illumina MiSeq instrument. DNA sequences are aligned and compared to reference genome GRCh37 to identify SNVs and indels.
This test does not detect all possible variants in the genes tested. Only regions with recurrent (i.e. hotspot) somatic DNA alterations are covered by this assay. Furthermore, a negative result does not rule out the presence of an alteration that may be present but below the limits of detection of this assay. The minimum percentage of tumour nuclei required in the submitted sample is 10%; lower percentages may result in false negatives. All samples with less than 10% tumour nuclei will be reported as inconclusive. This test does not allow definitive differentiation between germline and somatic variants.
Test sensitivity can vary depending on DNA quality, tumour cell percentage, and tumour heterogeneity. Only clinically relevant variants (i.e. Tier I and II variants) are reported. For the SNVs and indels, the threshold for variant calling is set at five or more reads, at a minimal sequencing depth of 250 reads, which are the recommended criteria to confidently call variants at an allele frequency of 5% (PMID: 28341590). At least 95% of targeted bases must be covered by 250 reads or more to qualify a sample as valid for analysis. This test is an amplicon-based assay. Therefore, variants located in primer binding sites can result in inaccurate diagnosis due to allelic dropout.
The presence or absence of an alteration may not be predictive of response to therapy in all patients.
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.
Turnaround time: 10 working days
Specimens accepted:
- 10 x 5 μm scrolls in 1.5 mL tube (minimum of 5 scrolls)
- 6 x 5 μm unstained slides
- A tumour cell content (TCC) of 10% or more is required. This information is mandatory to assess the validity of the test.
Submit your test request using our solid tumour requisition.