NGS genetic analysis for National Lung Matrix Trial Stratified Medicine technology hubs and Illumina 22 June 2015

Contents Overview of sample workflow in technology hubs Timings QC steps Overview of the SMP2 NGS panel and analysis Explanation of Nextera protocol Overview of the validation at Illumina Analysis / How aberrations are detected How the aberrations will be reported Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

TH Operations Clinical Hub Technology Hub Matrix Trial / CRUK XML XML Sample prep and assessment Clinical Hub XML receipt and logging in Allocation of test Macro-dissection and DNA extraction Testing Technology Hub Receipt of results Monthly KPIs to CR-UK Archive results to FTP Patient enters appropriate trial arm Matrix Trial / CRUK XML You will hear quite a lot about this entire process today. This is an OVERVIEW. All three THs have established very similar operations, but not identical. In each case utilising their existing diagnostic workflows and expertise – would have been very difficult to establish from “scratch” Sample journey: XML & request receipt – blood and tumour samples (not always at the same time) – booking-in using usual sample reception, checks of sample identifiers and assignment of tests. Sorting out of discrepancies…… Macrodissection and DNA extraction (Cardiff: 2 FTE+) – huge time resource, but essential for good quality analyses. No easy automation!! Molecular analysis – organised by gene, NOT tumour. In Cardiff, team of 5 or 6 GTs. All analyses checked by two. Integrated into main lab work (e.g. KRAS, EGFR…) NGS developed throughout and then launched in June 2013 for final ~700-800 samples. (NGS development expected from TSB). Results recombined by tumour. Checking, checking, checking. Transcription errors avoided. Can not be reported until all results ready. Result returned to CH by XML KPI data direct to CR-UK: No. of samples, failures, RTs, clinical trials – major resource required (not funded) XML XML Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Sample processing timings at the TH Pre-testing Post-testing Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Pre-testing Day 1 Day 2 Day 3 Day 4 Day 5-10 Paired sample receipt Booked in Triaged Assign extraction / send for quantification Day 2 DNA extraction Day 3 DNA quantification >50ng – proceed to testing <50ng – send Pre-Test QC fail report for tumour and bank blood DNA Day 4 Select next 5 pairs ready for testing Prepare worksheets, etc Day 5-10 Nextera enrichment MiSeq run Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

QC step Work done by Illumina to identify a QC step Three samples run at different concentrations. Lower than 30ng input DNA, no sequencing data is generated. 50ng input DNA to the NGS analysis is optimal. 50ng 30ng 20ng 10ng Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Factors affecting transitioning from Day 3 to Day 4 Batching 5 pairs processed per NGS run 10 pairs processed max per week (2 MiSeq runs) (40 samples per month) Sample numbers Too few – waiting for samples to activate a batch Too many – samples in a queue waiting to start testing Timings Protocol for enrichment is complex – 5 day protocol Safe stopping points that are built into a working week Sample pair that is QC ready on a Thursday will wait until following Wednesday to start testing Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Post-testing Day 11 Day 12 Day 13 Day 14 Day 15 Raw data retrieved from MiSeq Scientist 1 processes data and performs 1st analysis Day 12 Scientist 2 performs 2nd check Scientist 3 validates test result entry onto laboratory database Day 13 Reports written Day 14 Reports checked and authorised by senior scientist XML generated Day 15 XML reports available for retrieval by CH from sFTP site From start of test it takes ~10 days to turnaround a sample. The whole process from sample receipt should take ~15 days but because of the issues highlighted in the previous slide this is variable Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Nextera hybridisation CR-UK NGS Panel 2 Nextera hybridisation 28 gene Must have matched blood sample and tumour % information for analysis stage Detects SNVs, insertions/deletions, CNV, translocations Developed in partnership with Illumina Panel linked to Matrix Trial Increased gene spectrum Increased mutation spectrum AKT1 ALK BRAF CCND1 CCND2 CCND3 CCNE1 CDK2 CDK4 CDKN2A EGFR FGFR2 FGFR3 Her2* HRAS KRAS MET NF1 NRAS NTRK1 PIK3CA PTEN RB1 RET ROS1 STK11 TSC1 TSC2 Single test that requires less DNA, analyses more genes, all types of mutational events, better quality result Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Nextera library prep Patient DNA is processed to generate a sequencing library collection of DNA fragments derived from the patient DNA fragments are flanked by adaptor sequences Allow fragments to ‘stick’ to the flow cell surface Transposons fragment and tag the DNA with primers Indexes are added to each sample so they can be separated Uses an enrichment or capture approach positively select fragments of interest from your sequencing library

SMP2 Nextera NGS panel validation overview Validation was performed at Illumina Assessment of how panel works All types of variant were validated, but not CNV as no clinical samples were available Used sample previously tested by THs in their routine clinical pathways Cell line DNA provided by Horizon Discovery translocations known variant frequency Technology transfer to TH TH ran 3 Nextera panels in house Panel 1, cell lines Panel 2, previously analysed blood and tumour Panel 3, ‘real’ SMP2 samples No issues with the transfer of the wet protocol. Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Validation conclusions Combining the results of the validation for SNV and indels; we know with 95% certainty that the TH can detect SNV and indels at >10% overall allele frequency in at least 95% of cases. CNV are detectable in samples with high tumour percentage and if the CNV is large. Confident in calling >5 copy number increases TH will request a retrospective FISH slide to confirm low level or suspected copy number variants. Homozygous deletions are not detectable in samples with <60% of neoplastic nuclei, because of contamination from normal tissue. The TH will request a retrospective FISH slide to confirm the deletion is homozygous. Translocations detected but caution required with poorer quality samples where less read depth achieved Prospective validation by FISH analysis, through analysis of a number of translocation negative samples. Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

How aberrations are detected Development of analysis tool by the THREE TH 4 stages to analysis 1 Raw Data output from MiSeq run 2 Analysis of raw data using commercially available (Illumina Variant Studio), Open source tools (Manta), or manually 3 Collation of detected variants Excel spreadsheet containing algorithms developed by TH based bioinformaticians Information on variants detected and coverage achieved across the panel test 4 Assessment of pathogenicity / eligibility for Matrix Trial Scientific assessment Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Detecting SNV and indels is automated. Patient raw data from MiSeq (blood and FFPE) MiSeq reporter Windows BWA alignment to human genome (generate BAM files) 2 1 Flag and remove duplicate reads that span chromosomes Coverage calculator (FFPE) Somatic variant caller SNV and indels in FFPE and blood (generates gVCF files) Min 3 events Manta variant calling Structural changes (FFPE) Min 3 events Using coverage at each base per gene calculates; Average depth per gene Min and max depth per gene Mean depth per gene Variant studio Germline variants removed from FFPE 2 Germline variants removed from FFPE Virtual machine Unix Pass filter 2 2 3 Excel SNV calling; Filter known SNP out Filter 10% allele freq and min 10 reads Manual raw data check if needed CNV calling; Plot graph of mean coverage per gene/mean coverage per sample Filter gains >5 fold Filter loss <0.5 fold Structural variant calling; See if Manta has pulled out a variant. WT calling; Add tumour % Look at % of bases in each gene coverage to required depth Consider hotspot genes Pass or fail gene Bioinformatics pipeline has required significant development by THs with Illumina Detecting SNV and indels is automated. CNV and translocation detection is not automated as there is no commercially available software. Detecting CNV will always be complex with NGS due to contamination of normal tissue. 4 Compare to tier variant list from Pharma Report in XML

How aberrations will be reported General rules Variants are being reported in an XML format. Split into 3 tiers by the pharmaceutical partners. Tier 1= trial eligible Tier 2= trial eligible Tier 3= not trial eligible These lists will be maintained throughout the programme through quarterly meetings to look at evidence for moving variants between tiers Confidence scoring system developed to answer the question of how confident can you be that in a given tumour sample no variants have been missed that are above 10% frequency. This considers; Minimum sequencing coverage across the region of interest Tumour % of sample Can only be applied to SNVs and in/dels Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Tumour %, depth of coverage, WT confidence, variant frequency are all linked The number of times a DNA base or gene is sequenced is called the depth/coverage or number of reads/read depth This is what makes NGS a quantitative assay 100% tumour material has a KRAS c.35G>A p.(Gly12Asp) present at 20% In 100 reads at base c.35, 75 reads =G and 25 reads = A NGS allows us to look for low level variants ie ‘needle in a haystack’ Based on validation, test sensitivity set at 10% Confidently call variants where we detect 10 reads in 100 100% tumour material where we have 100 uniform reads at any given site we can be confident that we have not missed any variant that was present above 10% Most material sent for testing will be less than 100% tumour even if macrodissected so this will either Decrease sensitivity of detection if keep a given read depth eg 100 reads Maintain sensitivity by increasing read depth Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Tumour % information critical for analysis Variant present at 10% frequency – what % of reads would be mutant Mutant reads 10 mutant reads minimum so minimum read depth required is 5% 0.5% 1/200 2000 10% 1% 1/100 1000 20% 2% 1/50 500 40% 4% 1/25 250 50% 1/20 200 100% 1/10 100 Tumour % information critical for analysis Really important to define this to the nearest 10%. Coverage calculator works out % of bases within gene covered to the depth required Gene is passed or failed based on criteria below

How aberrations will be reported Wild type samples or failed samples Wild type samples will say how confident the TH are this is a true WT for SNV and indels. Samples that fail either NGS or QC step are eligible for a Matrix Trial re- biopsy. TH will never say no translocation or no copy number variation as still developing confidence value for this. Test Result Test Report Wild type No variant detected High/medium confidence Gene test failed No result Fail. Repeat sample requested if available. QC step failed Not tested Failed QC step- insufficient sample. Repeat sample requested if available. Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

How aberrations will be reported Variants detected The general format for variants that will be in the ‘Test Result’ field are; Type of variant Test Result Single nucleotide variants and small indels (Mutation/Sequence change/In dels) c.codon number and base change p.(amino acid number and change) Translocation Gene_Gene_exons Copy number variant- deletions Whole gene deletion homozygous Whole gene deletion heterozygous Exons deletion homozygous Exons deletion heterozygous Copy number variant - amplification Whole gene amplification ± confirmed by FISH Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

How aberrations will be reported Variants detected The ‘Test Report’ field will contain text around the variant tier, therefore whether the variant makes the patient potentially trial eligible. There will also be text to say if a FISH confirmation confirmed the variant. If FISH slides were requested by the TH but could not be obtained, this will be in the comments field of the XML. Test Report Meaning- trial eligibility Tier 1 Potentially trial eligible Tier 2 Tier 3 Confirmed by FISH Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

How aberrations will be reported Examples of reporting – WHOLe panel Gene Test Status Test Result Test Report AKT1 Success c.49G>A p.(Glu17Lys) Tier 1 AKT1 variant ALK EML4-ALK Tier 3 ALK translocation BRAF c.1799T>A p.(Val600Glu) Tier 3 BRAF variant CCND1 No variant detected Medium confidence Wild Type CCND2 Complete Fail No result Fail. Repeat sample requested if available. CCND3 CCNE1 CDK2 CDK4 Amplification CDKN2A Whole gene deletion homozygous Tier 1 CDKN2A homozygous deletion confirmed by FISH EGFR c.2235_2249del15 p.(Glu746_Ala750del)  Tier 3 EGFR 14bp duplication FGFR2 FGFR2-TACC2 Tier 3 FGFR2 translocation FGFR3 Her2 Exon 5 and 6 deletion heterozygous Tier 3 Her2 exon 5 and 6 heterozygous deletion HRAS High confidence Wild Type KRAS MET Exon 5 deletion heterozygous Tier 3 MET exon 5 heterozygous deletion NF1 Partial fail c.135T>A p.(Asn45Lys) Tier 2 NF1 variant NRAS c.183A>C p.(Gln61His) Tier 1 NRAS variant NTRK1 CD74-NTRK1.C3N13 Tier 3 NTRK1 translocation PIK3CA c.1624G>A p.(Glu542K) and c.1616C>G p.(Pro539Arg) Tier 1 and tier 2 PIK3CA variant PTEN c.106G>C p.(Gly36Arg) Tier 2 PTEN variant RB1 RET Partial Fail Low confidence Wild Type ROS1 CD74-ROS1_C6:R34 Tier 1 ROS1 translocation STK11 c.523_528del6 (delAAGGAC) p.(Lys175_Asp176del) Tier 2 STK11 6bp deletion TSC1 c.2647G>A p.(Ala883Thr) Tier 2 TSC1 variant TSC2 Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

How aberrations will be reported Monday 22 June 2015 Lung Cancer Research Stratified Medicine Educational Event- Birmingham

Stats so far … 147 samples received 48 (33%) failed before testing - not enough DNA 78 reported full 28 gene panel 9/78 (12%) all genes failed post testing 21 in progress Detected sequence variants 49/78 patients (63%) 78 sequence variants detected in 49 patients 39/49 patients with Tier 1 mutations Some with multiple Tier 1 mutations

78 Variants Variants detected in 17/28 genes on panel AKT1 ALK BRAF 2 CCND1 2 CCND2 CCND3 CCNE1 1 CDK2 CDK4 2 CDKN2A 6 EGFR 8 FGFR2 2 FGFR3 Her2* HRAS KRAS 15 MET 2 NF1 10 NRAS NTRK1 PIK3CA 10 PTEN 1 RB1 7 RET ROS1 2 STK11 6 TSC1 1 TSC2 1 Variants detected in 17/28 genes on panel

Thank you cruk.org CRUK SMP2 Technology Hubs Illumina Birmingham- Mike Griffiths, Jennie Bell, Fiona MacDonald, Pauline Rehal, Alessandro Rettino, Sam Clokie Cardiff- Rachel Butler, Ian Williams, Michelle Wood, Helen Roberts RMH- David Gonzalez de Castro, Lisa Thompson, Keeda Dover, Brian Walker, Lisa Grady Illumina David McBride Mark Ross