Standardisation of Somatic Variant Analysis using an AMP based Framework Kirsty Russell Trainee Clinical Scientist (Bioinformatics – Genomics) Bristol Genetics Laboratory kirsty.russell@nbt.nhs.uk http://www.nbt.nhs.uk/genetics

Overview Somatic Variant Classification AMP guidelines Development of AMP Based framework Somatic Variant Analysis at BGL Case Study Future Developments http://www.nbt.nhs.uk/genetics

Somatic NGS & Variant Analysis Increase in diagnostic somatic NGS Test directory requires Cancer NGS Panels Analysis of 100k genome results Discrepancies between labs in how variants are classified and reported Majority use 5 Class system (ACMG) Not suitable for somatic variants Li et al (2017) http://www.nbt.nhs.uk/genetics

AMP guidelines http://www.nbt.nhs.uk/genetics

AMP Tiered Variants Variants are assessed based on the clinical significance / actionability A variant is actionable if it has a known therapy or has prognostic or diagnostic relevance. Variants can be therapeutic or prognostic/diagnostic Assessment based on specific tumour type http://www.nbt.nhs.uk/genetics

Framework Development http://www.nbt.nhs.uk/genetics

Framework Development http://www.nbt.nhs.uk/genetics

Rule Set Development Defines the combinations which lead to different tier classifications. Example: 1xSPS + 1xSPM + 0xSBS/SBM = Tier IID Developed and optimised using 10 solid tumour variants from several cancer types http://www.nbt.nhs.uk/genetics

Duplication/ insertion Framework Validation Final framework validated through analysis of 62 unique variants identified using Solid Tumour NGS panel - Further refinement of rules Sent to several labs for feedback Gene No of Variants BRAF 5 EGFR 18 KRAS 10 NRAS 4 KIT 14 PDGFRA 6 PIK3CA Variant Type No of Variants SNVs 67 Deletion 30 Duplication/ insertion 12 Delins 10 http://www.nbt.nhs.uk/genetics

Somatic Variant Analysis at BGL Using this AMP framework for all somatic NGS analysis Solid Tumour panel 100k genome return of results More in future In House Somatic Pipeline Database Evidence Somatic UV Form Completion Evidence Gathering Functional analysis Literature Search http://www.nbt.nhs.uk/genetics

Evidence Therapeutic Databases Somatic Variant Databases Cancer Genome Interpreter (Somatic Variant Report), My Cancer Genome, ClinicalTrials.gov, UK Clinical Trials Gateway Somatic Variant Databases COSMIC, CIViC, DOCM, IARC TP53 Population Databases gnoMAD Germline Databases HGMD, ClinVar, InSiGHT, CaVaDa Functional Analysis In silico & literature Literature Searching http://www.nbt.nhs.uk/genetics

KRAS:c.35G>T p.(Gly12Val) Lung Case Study KRAS:c.35G>T p.(Gly12Val) Lung http://www.nbt.nhs.uk/genetics

Case Study: Database Evidence http://www.nbt.nhs.uk/genetics

Case Study: Therapeutic Evidence Some evidence shows lack of response to first line EGFR tyrosine kinase inhibitors Some evidence shows association with a poor prognosis Serves as inclusion in phase 2/3 trials Known to confer decreased sensitivity to anti EGFR antibodies in Colorectal http://www.nbt.nhs.uk/genetics

Case Study: Functional Evidence Highly Conserved Nucleotide & Amino Acid Align GVGD, SIFT and Polyphen – Deleterious Mutation type activating in an oncogene Variant in a cancer pathway associated with tumour type Predicted driver (OncoDriveMut) http://www.nbt.nhs.uk/genetics

Case Study: Result KRAS:c.35G>T p.(Gly12Val) AMP Tier: IIC Variant of potential clinical significance for which there is some evidence towards therapeutic/prognostic status and serves as inclusion in Phase 2/3 clinical trials. 3 x SPS + 3 x SPM + 2 x SPP = Tier IIC http://www.nbt.nhs.uk/genetics

However… Case Study: Result KRAS:c.35G>T p.(Gly12Val) AMP Tier: IIC Variant of potential clinical significance for which there is some evidence towards therapeutic/prognostic status and serves as inclusion in Phase 2/3 clinical trials. However… 3 x SPS + 3 x SPM + 2 x SPP = Tier IIC http://www.nbt.nhs.uk/genetics

http://www.nbt.nhs.uk/genetics

Case Study: Result KRAS:c.35G>T p.(Gly12Val) AMP Tier: IB Variant of clinical significance listed in professional guidelines regarding its therapeutic and prognostic significance and serves as inclusion in Phase 2/3 clinical trials. 1x SPSV + 3 x SPS + 3 x SPM + 2 x SPP = Tier IB http://www.nbt.nhs.uk/genetics

Future Development Working group started to work on standardisation across laboratories First teleconference was 22nd June 2018 Capture what different laboratories are doing Questionnaire to be sent around labs Sharing of SOPs Set of variants to be sent round labs for classification Refinement of framework for haematological malignancy First working group meeting Autumn ‘18 http://www.nbt.nhs.uk/genetics

Everyone who has joined the Somatic Variant Analysis Working Group! Acknowledgements Bristol Genetics Laboratory Oncology Team Chris Wragg Claire Faulkner Paula Waits Helen Williamson Kenneth Smith Manchester Centre for Genomic Medicine George Burghel Everyone who has joined the Somatic Variant Analysis Working Group! West Midlands Regional Genetics Laboratory Paula Page Joanne Mason David Hill Kim Reay http://www.nbt.nhs.uk/genetics