For each patient whose WGS report discussed at the GTAB, there should be a structured step-by-step review of results as follows.
The first step is to discuss the clinical context of the patient. This includes information regarding whether the sample submitted for WGS was from a resection or biopsy sample. Was the pathological diagnosis the same as that clinically suspected when the tissue was sent for WGS? Does the patient have localised disease and if so, have they undergone surgical resection? Does the patient have locally advanced or metastatic disease, and if so, what line of treatment are they receiving? Has the clinical context changed from when the biopsy was taken? Are they clinically fit enough for further treatment?
When reviewing the WGS report, firstly, all demographic information should be confirmed, followed by a review of the quality metrics of the sample and the sequencing undertaken, including cellularity, tumour content and sequencing coverage.
Somatic domain 1 and 2 variants should be reviewed as these may have a clinical impact with regard to prognosis or therapeutics. The report contains relevant information to appraise the variant, including the gene it is identified in, the cDNA and protein change, and the predicted consequence (i.e. frameshift variant or missense variant). Certain metrics are critical for variant interpretation, such as supporting reads (depth of coverage) and variant allele frequency (VAF), and should be reviewed when evaluating the called variant, along with the COSMIC ID (if present for this variant) and links to clinical trials at the gene, and for some tumour types, at the variant level. If a variant is not well characterised, further investigation or evidence may be required by a member of the GTAB, before or after the meeting to aid interpretation and advise on whether validation is appropriate. Structural variants, including translocations and CNVs, should also be reviewed, particularly if there is a known standard-of-care testing for a particular tumour type, such as EML4-ALK in non-small cell lung cancer or ERBB2 amplification in breast cancer.
As discussed above, only known pathogenic mutations in cancer genes with a 3-star ClinVar rating are flagged to the clinical team. A senior clinical scientist should review and interpret any report tier 3 variants. This ensures that only highly penetrant mutations as reviewed by an expert panel are reported, alleviating concerns about identifying variants of unknown significance (VUS). Any findings are highlighted to the clinical genetics team to contact the patient and arrange an appointment. Importantly, a discrepancy in suspected and eventual tumour type may mean some germline variants are not reported because of lack in evidence in one tumour type compared with another. This would necessitate a repeat analysis. Secondly, if a GTAB is reviewing a cancer panel test rather than WGS, the germline is unlikely to be sequenced. In this case, it is important that the clinical scientists and clinical geneticists identify common germline variants if found in the panel and organise appropriate germline testing. In the case of deceased patients, the guidance recommends that only pertinent germline variants need to be reviewed due to the potential impact for relatives.
Tumour mutational burden
The whole genome report provides a wealth of information, including pan-genomic markers detailed in the supplementary information of the genome report . The first of these is tumour mutational burden (TMB) which is quantified in coding SNVs per megabase. Recent work has shown that TMB may serve as a predictive or prognostic biomarker of response to immunotherapy, due to its correlation with neoantigens, which are expressed on the tumour cell surface and recognised by the immune system. If TMB is greater than 10 coding SNVs per megabase, this may prompt consideration of immunotherapy, within a trial setting. It may also support the finding of a mismatch repair deficiency/microsatellite instability on standard-of-care testing. The 100,000 Genomes WGS report includes a rainfall plot which visually displays mutational burden across the genome and highlights areas of hypermutation or ‘kataegis’ , but the clinical relevance of these areas of hypermutation is not yet fully understood.
A second pan-genomic marker includes mutational signatures. These are patterns of mutational combinations associated with particular mutational processes that may be intrinsic or extrinsic . There are over 30 mutational signatures with the vast majority of unknown aetiology. However, some are clinically relevant, including those associated with tobacco exposure, UV light and failures of DNA repair mechanisms including mismatch repair and double-strand break repair. They therefore may simply act to support the known pathological diagnosis, e.g. tobacco exposure in a small cell lung cancer, or may provide insights into the possible aetiology of a cancer such as failure of DNA repair mechanisms . In the 100,000 WGS report, this is displayed by both a bar graph showing the percentage of each mutational signature within the tumour (Fig. 2a) and a histogram of the percentage of each type of base substitution (in a trinucleotide context) (Fig. 2b).
In the case of the 100,000 Genomes WGS report, somatic information, including sequencing coverage, small variants and structural variants, is visually represented via a circos plot (Fig. 2c).
Following the review of a 100,000 Genomes WGS report, the GTAB may issue one of five outcomes in their clinical report (Table 2) . They will also provide additional information to the treating clinician including advice on validation method (see above) and relevant clinical trials. The 100,000 Genomes report provides links to clinical trials with information about inclusion criteria and recruiting status, but this should always be verified by the GTAB and treating clinician.