1. Mutation Screening of KRAS in FFPE samples using Pyrosequencing Phil Chambers CR-UK Genome Variation Laboratory Service St. James’s University Hospital Leeds

2. Number of samples with mutations KRAS codon number Data from the Cancer Genome Project COSMIC database KRAS  KRAS has 6 exons  Exon 1 is non-coding. Exons 2, 3, and 4 are invariant coding exons  Exon 5 undergoes alternative splicing  Mutation hotspots at codons 12 and 13 (exon 2) and 61 (exon 3) Codon 12 Codon 13 Codon 61

3. KRAS  GTPase which plays a vital role in cell signalling  KRAS mutations play a role in many human cancers:  15-30% lung adenocarcinomas  20-50% colorectal carcinomas  Activating mutations cause KRAS to accumulate in the active, GTP-bound state

4. KRAS and monoclonal antibody therapy for colorectal cancer  More than 300,000 new patients are diagnosed with colorectal cancer (CRC) in the USA and European Union each year  Response rates, progression-free survival rates and overall survival have improved significantly in the last decade  Mainly as a result of:  New combinations of standard chemotherapy  New agents targeted at molecular events-small molecule inhibitors and monoclonal antibodies  Therapies directed towards epidermal growth factor receptor (EGFR) are of particular interest

5. KRAS and monoclonal antibody therapy for colorectal cancer  Chimeric immunoglobulin cetuximab:  Binds to EGFR and blocks ligand-induced phosphorylation  Is active in metastatic CRC expressing EGFR detected by IHC  Only 8-23% of patients achieved an objective response  Cancer Research 2006: v66, p3992-3995:  Presence of a KRAS mutation was significantly associated with the absence of response to cetuximab (0% of responders vs. 68.4% of non-responders; P = 0.0003)  Overall survival of patients without a KRAS mutation was significantly higher (median16.3 vs. 6.9 months; P = 0.016)  KRAS mutations are a predictor of resistance to cetuximab therapy and are associated with a worse prognosis

6. Arteaga, C. L. Oncologist 2002;7(Suppl 4):31-39 The EGFR signalling network

7. What is Pyrosequencing?  Sequencing-by-synthesis technology suitable for analysing short-to-medium stretches of DNA  Assays give real-time quantitative results  Flexible assay design  Assays are simple and robust with inbuilt controls  Does not use fluorescent labels or gels/polymers

8. Pyrosequencing assays PCR primer  Three primers required: Regular PCR primer PCR primer with a 5’ biotin label Sequencing primer  Two types of assay: SNP genotyping and sequence analysis (SQA)  Assay design favours short amplicons Pyrosequencing primer Region of interest PCR primer

9. Pyrosequencing workflow PCR Immobilisation – 5 minutes Isolation of ssDNA – 1 minute Annealing of sequencing primer - 2 minutes Pyrosequencing analysis – 10-60 mins/96 samples

10. Pyrosequencing technology PPi ATP Time Light

11. Quantitative SNP analysis  Very short amplicon, therefore excellent for FFPE samples  Following ssDNA preparation, assay completed in 10 minutes  Straightforward data analysis using proprietary software  Table of peak heights can be exported for manual analysis wild-type heterozygote Quantitative determination of mutant allele Negative controls Reference peaks

12. Quantitative sequence analysis  KRAS codons 12 and 13  Analysis of short - medium stretches of DNA  Assay design more challenging  Very short amplicon, therefore excellent for FFPE samples  Following ssDNA preparation, assay completed in 20 minutes  Table of peak heights exported for analysis in Excel c.35 G>A WT Controls Reference peaks Quantitative determination of mutant allele

13. Spreadsheet-assisted analysis of sequence analysis data  Interpretation of sequence analysis data:  Done poorly by proprietary software, especially for diploid organisms  Inefficient and inaccurate when done by visual inspection  Low level variants are especially difficult to analyse  Assisted by calculation of peak height ratios and standard deviations if a variant is detected in this assay this peak height ratio will be 1.1 peak heights >mean +1 standard deviation are also flagged spreadsheet-assisted analysis combined with visual inspection

14. Pyrosequencing summary  Flexible, simple assay design  Short amplicons  Straightforward data analysis  Quantitative  Rapid  Good quality control features  Self and mis-priming can be a problem  Accuracy of quantification calculations in homopolymer regions  Short read sequencing  Data interpretation in diploid organisms

15. Why is Pyrosequencing suitable for analysing KRAS in FFPE samples?  90-95% of mutations occur in 2 hotspots  All mutations in each hotspot can be detected in one amplicon  Pyrosequencing favours short PCR amplicons  Problems caused by chemical modification of cytosine residues are not observed  Our data indicates the success of the technique

16. Mutation screening of KRAS in FFPE samples  KRAS mutation hotspots amplified in two amplicons:  codons 12 and 13: 80bp  codon 61: 86bp  Analysed using the Pyrosequencing SQA mode  711 FFPE samples  DNA extracted using Proteinase K and phenol:chloroform  43% (308/711) patients had a KRAS mutation  0.7% (5/711) of samples failed analysis  50 samples re-extracted with Qiagen DNA FFPE kit:  1 failed analysis  no change in sensitivity and specificity of mutation detection  Similar data for other sample batches

17. Gene Collector Protocol overview (Fredriksson et al. NAR 2007, v35 p47) Multiplex PCR (Pfu polymerase) Blunt-ended products suitable for ligation by circularization Collector probes guide circularization, closed circles formed by thermostable ligase Enrichment of circular DNA by exonuclease treatment and rolling circle amplification

18. Acknowledgements  Cancer Research UK Genome Variation Laboratory Service Chris Booth Jo Lowery Helen Snowden Jo Morgan Graham Taylor  Leeds Institute of Molecular Medicine Susan Richman Sophie Grant Phil Quirke