Germline-focussed tumour analysis should be carried out in all laboratories as part of routine analysis of a large tumour panel.
Germline-focussed tumour analysis can be delivered via an automated pipeline so as not to add substantial additional manual work, cost or delay to tumour analysis.
Variants in should be flagged which are (i) predicted to result in protein truncation in genes acting through loss-of-function and/or (ii) classified as Pathogenic/Likely Pathogenic via a well-maintained, comprehensive and curated clinical resource (ClinVar is recommended).
Germline-focussed tumour analysis can be restricted to variants of VAF >30% (SNVs) or >20% (small insertions/deletions). Local validation will be required to confirm the accuracy of tumour VAF estimates, especially for PCR-based NGS methodologies.
Samples known or suspected to be hypermutated should be included for germline-focussed tumour analysis.
Germline-focussed tumour analysis in the off-tumour context should be restricted to ‘High Actionability-CSGs’ (Box 1).
Recessively acting ‘High Actionability-CSGs’ (currently MUTYH alone) should be included for germline-focussed tumour analysis but reporting and germline follow-up testing should be undertaken only on detection of two pathogenic variants.
Germline-focussed tumour analysis of ‘standard actionability’-CSGs should be restricted to the on-tumour setting.
‘Standard actionability’-CSGs included for germline-focussed tumour analysis can be restricted to genes of high penetrance.
Germline-focussed tumour analysis can be restricted to gene-scenarios for which the germline conversion rate is >10%. For selected genes, it may therefore be appropriate to restrict germline-focussed tumour analysis to just those tumours arising age <30 years.
Formal variant review and classification should be undertaken by an experienced clinical scientist before initiation of patient re-contact and/or germline testing.
Before analysis of their germline sample for the pathogenic variant, adequate information should be provided to the patient regarding the implications of germline testing, along with documentation of their consent.
The tumour-observed pathogenic variant should be analysed in an appropriate germline sample (lymphocytes, saliva/buccal swab, normal tissue) in a laboratory accredited for germline analysis.
A patient in whom a germline pathogenic variant is detected should be referred to a specialist genetics service for long term follow-up and management of the family.
A normal/negative tumour sequencing result should not be taken as equivalent to a normal/negative germline result unless robust analysis of dosage has been carried out. This distinction is particularly important for genes such as BRCA1 and MSH2, for which whole exon deletion/duplications constitute a substantial proportion of pathogenic variants.
Re-evaluation of this workflow, revised analyses and update of these recommendations should be undertaken at least 2-yearly. Reanalysis should include updated data regarding pathogenicity of variants and penetrance of CSGs, along with review of thresholds for ‘germline conversion rates’ and VAF cut-offs.
