The Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) has been specifically designed to work with DNA extracted from formalin fixed paraffin embedded (FFPE) tissue, which is often fragmented and of low yield. The panel amplifies short target regions of DNA to reduce the impact of fragmentation on variant detection. The panel is also optimised for small amounts of DNA (minimum 10ng) and is therefore suitable for most small biopsy samples.
Sample Requirements
As with all Molecular Pathology requests, when testing is required, an appropriate FFPE tissue block (containing target tumour regions) should be sent directly to the laboratory. The corresponding H&E stained slide and pathology report is also required to enable estimation of tumour percentage and targeted DNA extraction to increase the overall tumour percentage, if required.
Assay Limitations
The Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) is designed to target clinically relevant “hotspot” regions of the target genes; entire genes are not analysed. Therefore, the panel may not detect, or detect with reduced sensitivity, some variants located out with the target regions. A full list of reference gene transcripts and specific target regions are available here.
The limit of detection (LoD) of the Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) has been calculated to be 5% for small nucleotide variants and approximately 10% for insertions and deletions. Variants present at an allele frequency below the LoD will not expected be detected. The likelihood of missing a low level variant increases in samples with a low tumour burden. An estimated tumour percentage is included on all reports; samples with <10% tumour burden are considered suboptimal and a false negative result cannot be excluded.
Due to the wide range of regions/amplicons analysed using this NGS panel, it would not be feasible to calculate a limit of detection for every possible variant. Differences in the LoD may occur in some genomic regions due to factors intrinsic to DNA structure. Furthermore, the Cancer Hotspot NGS kit may fail to accurately characterise variants located in long homopolymer regions due to limitations in the sequencing technology. Sequences that are GC rich and areas that form secondary structures may also prove challenging to sequence. Failure to detect variants located at the end of amplicons may cannot be excluded.
Samples are reported as having “optimal coverage” where there are a minimum of 250 reads for all reportable amplicons, except for “EGFR amplicon 6“, which requires a minimum of 200 reads. The Association of Molecular Pathology and the College of American Pathologists recommend a minimum of 250 reads per tested amplicon, corresponding to a false positive or false negative rate of <0.5% when there is a 5% limit of detection. A lower read coverage of EGFR amplicon 6 is accepted because although this region often has a lower read depth than others, the assay has been shown to reproducibly detect variants located in this region at 5% allele frequency when the read depth is >200. Please note, for most samples the read depth is significantly higher than 250 across all target regions.
Reporting of Results
Results are reported for clinically relevant genes based on the patient’s tumour type and clinical history. Results for additional genes included in the panel (Figure 1) can be interpreted on request. Please contact a member of our team to discuss.
Variant interpretation is performed according to Li, M. et al. The Journal of Molecular Diagnostics, 2017 Volume 19 , Issue 1 , 4 – 23. Tier I (variants with strong clinical significance) and Tier II (variants with potential clinical significance) variants will be reported, while Tier III (variants of unknown clinical significance) and Tier IV (benign or likely benign) variants will not be reported. Variants are reported according using HGVS nomenclature with respect to an appropriate reference sequence. Please note, result interpretation will be correct at the time of reporting, however the significance of variants may change over time.
The NGS panel is designed for the detection of somatic variants only. While germline variants may be present, it is not possible to differentiate germline variants using this protocol technology. If the presence of a germline variant is suspected, please contact South East Scotland Regional Genetics Service to discuss further.
Turnaround Time
NGS results are expected to be available within 14 calendar days of sample receipt. If testing is urgent please contact the laboratory team to discuss.
Accreditation Status
NGS testing using the Cancer Hotspot Panel v2 (CHPv2) has been validated in-house and is accredited to ISO 15189:2012.