Standardisation of variant interpretation across scorers. Matthew Smith Molecular Pathology Diagnostic Service University Hospital Birmingham

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1 Standardisation of variant interpretation across scorers Matthew Smith Molecular Pathology Diagnostic Service University Hospital Birmingham

2 During development of Somatic Tumour Panel The classification system employed by the laboratory How to determine which classification a variant should be given The types of evidence used to classify a variant How this evidence is collated and recorded. MPDS adopted the 4 Tier classification (AMP) Resulted in a policy to help classify and standardise variant interpretation along side a framework of documenting evidence UKAS How do you documented differences in interpretation?

3 Tier 1: Variants of Strong Clinical Significance Tier 2: Variants of Potential Clinical Significance Tier 3 Variants of Unknown Clinical Significance Tier 4 Benign or Likely Benign Evidence to support interpretation NICE approved therapy Professional Regulatory Guidelines Established variant used in clinical management NICE approved therapy for different types + Limited evidence from multiple smaller scale studies with some consensus No supporting evidence regarding variant in cancer site/histology Observed at significant allele frequency in the general population Published evidence of supporting no cancer association Randomized control trials Well powered studies with consensus from experts in the field. Pre-clinical trials or a few case reports without consensus Multiple studies with strong consensus Degree of Professional Judgement What will the differences between tiers 2 and 3 mean to an oncologist

4 Evidence Variant database search MANDATORY, internal variant database, dbsnp, COSMIC, MY CANCER GENOME, OPTIONAL - LOCUS SPECIFIC DATABASES, CLINCIAL TRIALS Literature Search Regulatory guidelines, Licencing guidelines, Clinical Trial data, prospective/retrospective biomarker analysis. Multi-centre or single centre studies Optional Insilico Prediction Prediction of affect on protein function (min 3 different programs) Prediction of affect on splicing min (min 3 different programs)

Database searches Literature searches In silico analysis Process in place in case

5 Documenting the Process Each patient has a Analysis report. Within this is a section for documenting evidence to support variant interpretation Database searches Literature searches In silico analysis Process in place in case checker 1/checker 2 disagree Variant calling, panel performance, QC data, variant interpretation all located in same report.

6 PUBLIC DATABASE SEARCHES dbsnp Date database accessed Further dbsnp details if required Variant found on dbsnp dbsnp build rs number Frequency Number of genomes sampled Accuracy of data confirmed by 2nd checker (UHB initials in box provided) Date Literature search Suggested search terms to use in combination GENE NAME, VARIANT NAME (both HGVS and non-standard nomenclature), TISSUE, HISTOLOGY, CANCER TYPE, RESPONSE, PROGNOSIS Search terms used and search engines used, recommended google, google scholar and pubmed Date searches performed Summary Reference papers and provide a summary of evidence supporting interpretation

7 In summary 4 Tier classification The collation of evidence is performed by an initial investigator (checker 1). The investigator performs a database search. The investigator performs a literature search Based on the evidence found the initial investigator makes an interpretation of the variant and assigns it to one of the four Tiers. The evidence is reviewed and collaborated by a second investigator (checker 2). The second investigator can add further evidence if required The second investigator makes an interpretation of the variant and assigns it to one of the four Tiers In the event the investigators disagree then the following actions are taken Both investigators review the evidence together to determine if the initial call can be amended. The reason for any change in interpretation can be documented in provided box in the patient analysis file All evidence is passed to a third investigator to review the evidence. The third investigator provides a classification of the variant. The final decision is documented in the interpretation tab in the patient analysis file Need to adopt variant interpretation software universal approach

Somatic Variant Analysis at BGL + ACGS Somatic Bioinformatics BPG Update ACGS Somatic Variant Interpretation Workshop 21 st September 2018 Kirsty Russell

8 Somatic Variant Analysis at BGL + ACGS Somatic Bioinformatics BPG Update ACGS Somatic Variant Interpretation Workshop 21 st September 2018 Kirsty Russell Healthcare Scientist / Bioinformatician Bristol Genetics Laboratory kirsty.russell@nbt.nhs.uk Exceptional healthcare, personally delivered

9 Standardisation of Somatic Variant Analysis using an AMP based Framework Exceptional healthcare, personally delivered

10 Overview AMP guidelines Development of AMP Based framework Somatic Variant Analysis at BGL Exceptional healthcare, personally delivered

11 AMP guidelines Exceptional healthcare, personally delivered

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

12 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 Exceptional healthcare, personally delivered

13 Framework Development Exceptional healthcare, personally delivered

14 Framework Development Exceptional healthcare, personally delivered

15 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 Exceptional healthcare, personally delivered

Framework Validation Final framework validated through analysis of 62 unique variants identified using Solid Tumour NGS panel Sent to several labs for feedback Gene No of Variants BRAF 5 EGFR 18

16 Framework Validation Final framework validated through analysis of 62 unique variants identified using Solid Tumour NGS panel Sent to several labs for feedback Gene No of Variants BRAF 5 EGFR 18 KRAS 10 NRAS 4 KIT 14 PDGFRA 6 PIK3CA 4 Variant Type No of Variants SNVs 67 Deletion 30 Duplication/ insertion 12 Delins 10 Exceptional healthcare, personally delivered

17 Somatic Variant Analysis at BGL Using this AMP framework for all somatic NGS analysis Solid Tumour panel Myeloid panel (in validation) 100k genome return of results More in future In House Somatic Pipeline Database Evidence Somatic UV Form Completion Evidence Gathering Functional analysis Literature Search Exceptional healthcare, personally delivered

18 Evidence Therapeutic 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 Exceptional healthcare, personally delivered

19 Somatic Bioinformatics BPG Update Exceptional healthcare, personally delivered

20 Overview Questionnaire Results Scope of guidelines VAF LOD Types of Analysis Confirmations Neoplastic Cell Content Next Steps Exceptional healthcare, personally delivered

21 What would you like to be included as part of the guidelines? Reporting Putative Germline Variants Validation Requirements Quality Control Analysis Methods Variant Prioritisation Confirmations Exceptional healthcare, personally delivered

22 What Variant Allele Frequency (VAF) is your pipeline validated to call down to and for what type of cancer? Haematological Solid Tumour ctdna < 5% 5-10% >=10% Exceptional healthcare, personally delivered

23 Exceptional healthcare, personally delivered

24 Exceptional healthcare, personally delivered

25 Exceptional healthcare, personally delivered

26 Next Steps Full Questionnaire Results presented at ACGS National Bioinformatics Meeting Oct 18 Establish scope of the guidelines Stick to specifically bioinformatics or widen scope? Initial working group meeting Exceptional healthcare, personally delivered

Acknowledgements Bristol Genetics Laboratory Oncology Team Chris Wragg Claire Faulkner Paula Waits Helen Williamson Kenneth Smith ACGS Somatic Bioinformatics BPG Group Joo Wook Ahn Jan Taylor Pavlos

27 Acknowledgements Bristol Genetics Laboratory Oncology Team Chris Wragg Claire Faulkner Paula Waits Helen Williamson Kenneth Smith ACGS Somatic Bioinformatics BPG Group Joo Wook Ahn Jan Taylor Pavlos Antoniou Patrick Lombard Susie Cook Sanjeev Bhaskar David Brawand Everyone who has joined the Somatic Variant Analysis Working Group and fed back on framework Exceptional healthcare, personally delivered

28 Amplicon based NGS panel for Myeloid Malignancies HMDS Jan Taylor/Paul Evans

29 Gene Panel

30 Test selection based on initial sample screening i. Samples selected based on immunophenotyping and morphology. ii. Cases with a diagnosis of suspected AML, MDS, CMML or MDS/MPN overlap. iii. Cases screened as cytopenia with at least one lineage showing evidence of dysplasia iv. Referred cases requesting myeloid HTS only

31 Filtering Rules Technical Filter step: <100 read depth, <0.05 VAF Known exclusions Retain known hotspots Annotation filter: Synonymous Intronic/splice Known polymorphisms QC filter picard metrics

32 Variant identification Compare in-house to MiSeq Reporter consensus VAF >20%, RD >500 call VAF >10% missense repeat VAF >5% frameshift, nonsense repeat SRSF2/MPL/ASXL1 hotspots confirmed by Sanger

33 Reporting and Annotation Use of Alamut Batch, Alamut Visual and COSMIC Variants are reported either as hotspot (via COSMIC ID), likely pathogenic (frameshift, nonsense) Variant of unknown significance

37 Clinical Interpretation HTS report is a supplementary data file attached to the HMDS integrated report Clinical interpretation is carried out by a Consultant Haematopathologist in the context of other test results Trial options/eligibility is discussed at myeloid MDT

Many similarities between guidelines Use of HGVS nomenclature and reference sequences on all reports Not using any piece of evidence in isolation In silico programmes (missense and splicing) Should

databases and cancer-specific databases Need to know where data has come from, e.g.

38 Many similarities between guidelines Use of HGVS nomenclature and reference sequences on all reports Not using any piece of evidence in isolation In silico programmes (missense and splicing) Should not be based on 1 programme and only be used if results are generally consistent Only a prediction, likely to overestimate and only 1 piece of supporting evidence Warnings regarding population databases and cancer-specific databases Need to know where data has come from, e.g. ClinVar Needs to be a concise report summarising evidence and have a very clear conclusion (standardised wording) Shouldn t just be a list of variants Lessons learned from germline analysis Knowledge of genes / disorder can help with classification Large NGS panels don t always generate more answers, can lead to more questions Need to educate users regarding terminology used

39 Types of somatic reports Treatment e.g. BRCA, CLL, lung cancer Diagnostic e.g. AML, CML Prognostic e.g. AML, ALL, MDS Questions for somatic guidelines How much evidence required to meet: Levels A-D Whether activating mutation, LOF When to request a sample for germline testing? Confirmation of variants, especially low level variants?

40 TP53 case presentation ACGS Somatic Variant Interpretation Workshop Paula Page 21/9/18

41 Case details 57yr old male Marrow (July 2018):?MDS??aplastic anaemia, easy fatigability and macrocytic anaemia Flow: Consistent with MDS-EB1 (9.4% blasts) SNP array showed abnormal clone with del(20q) - Good cytogenetic risk group (IPSS-R) Myeloid NGS panel requested

42 NGS results Use Nextera version of Illumina TruSight Myeloid Sequencing panel Analyse in silico panel of 24 genes: Identified 2 clinically significant (Tier I) variants in TP53: c.542g>a p.(arg181his), 53% VAF - In DNA binding domain - Absent from population databases (or at very low level) - 38 entries in COSMIC (2H&L) - Present in IARC - Annotated in ClinVar as Pathogenic/Likely pathogenic. Reported in Li-Fraumeni syndrome (not classic) - Functional work available: Defective/reduced promoter binding activity c.298c>t p.(gln100*) in TP53, 6% VAF - Nonsense in DNA-binding domain - Absent from population databases - Recurrent in COSMIC (43 entries, 2H&L)

43 NGS report Deletions of 20q and TP53 variants both recurrent in MDS. However, del(20q) classified in the good cytogenetic risk group (IPSS-R), whereas TP53 variants usually associated with -5/5q-, 7q-, complex karyotypes and adverse prognosis. Presence of del(20q) together with a TP53 variant represents an unexpected finding and prognosis is therefore uncertain. The TP53 c.542g>a p.(arg181his) variant with a VAF of 53% may be somatic or germline in origin. This particular variant has been reported in both somatic cancer and hereditary Li-Fraumeni syndrome. Recommend patient referred to Clinical Genetics for genetic counselling and to discuss the potential for investigation of TP53 germline mutation status.

44 Additional info Unexpected finding: result rang out - Germline testing ASAP, may affect treatment - Long waiting time for patients to obtain clinical genetics appointment Arranged meeting with Consultant Clinical Geneticist to discuss this patient and our approach to investigating potential germline TP53 variants - In these cases it s appropriate for Haematology consultants to decide if germline testing is performed before a clinical genetics referral if it would alter clinical management. Received PB in August 2018: Extracted DNA from CD3+ T-cells

45 Germline report Result supports a diagnosis of Li-Fraumeni syndrome dependent on clinical criteria and family history. Recommend patient and their family referred to Clinical Genetics. Following genetic counselling, presymptomatic testing available to at risk relatives. Offspring have up to a 50% risk of inheriting this mutation. - Any further testing (presymptomatic) will only be performed if referred from a clinical geneticist. Highlights need for considered/multi-disciplinary approach to dealing with potential pertinent germline findings Need to be able obtain suitable material to analyse germline Need for clear clinical guidelines for management for both Haematologists and Clinical Genetics

46 Congential Rhabdomyosarcoma Associated with Phakomatosis Pigmentokeratotica and Mosaic RASopathy Dr Mamoona Munir Molecular Pathology, West Midlands Regional Genetics Laboratory

Patient Sebaceous naevi of the face and scalp Congenital

further development of pigmented moles consistent with

testing was normal for Beckwith-Weidemann syndrome and

47 Patient Sebaceous naevi of the face and scalp Congenital rhabdomyosarcoma of the right thigh Café au lait macules, further development of pigmented moles consistent with phakomatosis pigmentokeratotica (PPK) Previous genetic testing was normal for Beckwith-Weidemann syndrome and Neurofibromatosis type 1, also no detection of FOX01 gene rearrangement. Clinician referred this patient for HRAS gene testing because of the rhabdomyosarcoma and the PPK Representative images

Somatic Activating Mosaic Mutation Diagnostic Results Syndrome Pathway Affected Tissue (DNA or fresh/frozen) ± Blood

background wild-type DNA Pathogenic variant detected in affected tissue in 67% of patients tested This change has not

Frequently reported in ectomesenchymoma tumours HRAS mutations at the same codon have been reported in Costello

48 Somatic Activating Mosaic Mutation Diagnostic Results Syndrome Pathway Affected Tissue (DNA or fresh/frozen) ± Blood DNA NGS; Nextera Rapid Capture AKT1, HRAS, KRAS, NRAS, PIK3CA, PTEN Sensitivity of assay is ~5% sequence variation in background wild-type DNA Pathogenic variant detected in affected tissue in 67% of patients tested This change has not been reported in rhabdomyosarcomas before. Frequently reported in ectomesenchymoma tumours HRAS mutations at the same codon have been reported in Costello syndrome Gly13Arg is the most common pathogenic mutation described in sebaceous naevi >90% of cases Presence of the variant in sebaceous naevi further supports a clinical diagnosis of mosaic RASopathy

We are now receiving many referrals of patients with both common and unique clinical symptoms, making classification challening.

49 Summary Mosaic RASopathies are disorders caused by somatic mutations in genes involved in the RAS/MAPK signalling pathway. Our patient does not fit in to a previously defined mosaic RASopathy, but overlaps between previously identified RASopathies. We are now receiving many referrals of patients with both common and unique clinical symptoms, making classification challening. Currently no guidelines suitable for classifying somatic mosaic disorders. This case highlights the importance of sharing clinical and genetic data to facilitate diagnosis and expanding options for patient management.

50 Acknowledgements Dr Pauline Rehal Dr Derek Lim Professor Celia Moss Dr Malobi Ogboli Dr Natasha Harper Dr Alessandro Rettino Graham Halford

51 Interpreting Somatic Sequence Variants in Solid Tumours ACGS Somatic Variant Interpretation Workshop 21 st September 2018 Nicola Roberts Manchester Genomic Diagnostic Laboratory

52 Our Services Single gene tests NSCLC: EGFR Melanoma: BRAF, KIT, NRAS GIST: KIT, PDGFRA NGS BRCA1/2: Ovarian NGS Somatic Panel/Glioma Panel Colorectal subpanel (KRAS, NRAS, BRAF, PIK3CA) Phase 1/TARGET Plasma: resistance mutations Glioma Haematological malignancies- MDL

53 Variants passing standard quality checks Assess run frequency and VAF against other samples (TCC also considered) - Discount artefacts Tumour Suppressor or Oncogene (consider functional consequence) gnomad, HGMD, COSMIC, IARC, MyCancerGenome Internal database, Literature, Jackson Lab, GENIE etc Classification by AMP and ACMG Assess clinical significance (tumour type) Passenger Record Class 3 Report? Referral category? Pathogenic (class 4/5) Report

54 Reporting Somatic Variants Colorectal Panel/EGFR/Melanoma/GIST treatment decision/prognosis report potential actionable variants with clinical interpretation Somatic Panel/Glioma Panel treatment decision/prognostic/diagnostic report all variants related to cancer type TARGET treatment decision/clinical trial selection report all variants related to cancer type and reports discussed at MDT 100K GEL interpret and report all domain 1 and some domain 2 variants classified by ACMG and AMP reported after discussion in MDT

55 Interesting cases KRAS c.180_183delinsp.(gin61delinsvalglncysasnglu) Colorectal adenocarcinoma Classified likely pathogenic (other mutations at same codon activating) In conclusion, the neoplastic cells in this pathology sample have an unreported KRAS mutation this result may reduce the likelihood of response to EGFR based therapy, but should be treated with caution KRAS c. 193_198delins p.(ser65_ala66delinsleuasplystyr) Low grade glioma Classified likely pathogenic (not previously observed, functional studies show insertions in this region are oncogenic) These results should be integrated with clinical and pathological findings before use as a predictor of response to treatment and/or prognosis.

56 Interesting cases PIK3CA c.3193delinsat p.(his106511efsextter3) Colorectal adenocarcinoma Variant not previously observed Similar protein extending mutations described as somatic in CRC Classified uncertain significance Conclude- Therefore, the presence of this mutation could be considered for use for clinical trial selection or to guide clinical management

57 Summary and Questions Important to consider pathogenicity and clinical actionability Mutation vs Variant: what nomenclature should we use? How to report results in dysplastic samples? assuming the dysplastic cells are representative of the neoplastic cells in this patient s tumour, this pathology sample has detectable mutant KRAS which is predicted to be unresponsive to EGFR-based therapy. How to report low frequency variants? mutation was present at a low level (approximately 4% mutant admixture). This may indicate that the mutation is present in a minority tumour clone.

58 The Royal Marsden Clinical Genomics Dörte Wren Senior Clinical Scientist

MiSeq Reporter analysis pipeline De-multiplexing In-house analysis pipeline Fastq generation Barcode de-multiplexing for amplicon panel BWA mem alignment

8) Picard sort and de-duplication Samtools sort and deduplication GATK v3 indel reaglinment per sample pair Remove un-aligned reads MuTect2 somatic

59 MiSeq Reporter analysis pipeline De-multiplexing In-house analysis pipeline Fastq generation Barcode de-multiplexing for amplicon panel BWA mem alignment (v0.7.5) BWA aln/sampe alignment (v0.6.8) Picard sort and de-duplication Samtools sort and deduplication GATK v3 indel reaglinment per sample pair Remove un-aligned reads MuTect2 somatic variant caller GATK v2 indel reaglinment per sample per chr ASCAT CNV estimation Samtools merge per chr bam files and indexing Manta SV detection Illumina somatic variant caller Picard QC metrics and reporting Picard QC metrics and reporting Lina Yuan, Lead Bioinformatician

60 In-house analysis pipeline Sample overview & QC

61 In-house analysis pipeline : Variant calls & Analysis Information from COSMIC, GnomAD, dbsnp, Drugbank Two Checkers: Discrepancies between calls highlighted & comments logged

62 Variant review- Manual VarSome/Alamut Cosmic IARC BIC/BRCAshare GnomAD, Exac MyCancerGenome OncoKB, CIViC, cbioportal PubMed/Lit searches In-house results

63 Qiagen Clinical Insight Interpret QCI-I Based on the Ingenuity Knowledge Base (acquired by Qiagen in 2013, QKB) Complies with ACMG and NCCN guidelines Clinical Trials: ClinicalTrials.gov but highlights inclusion/exclusion criteria focussing on genetic markers In addition to what is provided by HGMD and ClinVar for example QCI-I includes curation of primary literature Review included whole genetic profile cross-assessing and linking variants not interpreting them in isolation Annotation in earlier samples will inform future results Laboratories can modify rules for variant classification to include new findings Hybrid of AI and dedicated team of experts curating the evidence

64 Metadata for filtering and interpretation Diagnosis/primary tumour site Generic : Thrombocytosis/Anaemia WES- patient phenotype QIAGEN's proprietary phenotype-driven ranking (PDR) algorithm Cytogenetic alterations/snv/cnv/fusions Can have virtual panels for ROI filtering

68 Variant overview

69 Variant Assessment- Pathogenicity/Actionability

70 Variant Assessment- Frequencies Specific variant and gene Data from COSMIC Population frequencies based on: 1. GnomAD 2. ESP genomes Allele Frequency Community

71 Variant Assessment- Prognosis Evidence

72 Variant Assessment- Clinical Trials Annotation of exclusion/inclusion criteria

73 Reporting Everything that is mark as reportable during assessment Report comments provided can be modified Can remove as needed variants, treatments and clinical trials Comments, assessments etc are logged by Username & date Upload into other systems feasible (?how flexible)

Variant interpretation exercise. ACGS Somatic Variant Interpretation Workshop Joanne Mason 21/09/18

Variant interpretation exercise ACGS Somatic Variant Interpretation Workshop Joanne Mason 21/09/18 Format of exercise Compile a list of tricky variants across solid cancer and haematological malignancy.