Mutation Screening of KRAS in FFPE samples using
Pyrosequencing
Phil Chambers
CR-UK Genome Variation Laboratory Service
St. James’s University Hospital
Leeds
Num
ber
of
sam
ples
w
ith m
utat
ions
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
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
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
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
Arteaga, C. L. Oncologist 2002;7(Suppl 4):31-39
The EGFR signalling network
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
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 interestPCR primer
Pyrosequencing workflow
PCR
Immobilisation – 5 minutes
Isolation of ssDNA – 1 minute
Annealing of sequencing primer - 2 minutes
Pyrosequencing analysis – 10-60 mins/96 samples
Pyrosequencing technology
PPi
ATP Time
Light
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
heterozygote
Quantitative determination of mutant allele
Negative controls Reference peaks
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
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 <0.9, >1.1
peak heights >mean +1 standard deviation are also flagged
spreadsheet-assisted analysis combined with visual inspection
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
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
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
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
Acknowledgements
Cancer Research UK
Genome Variation Laboratory ServiceChris Booth
Jo Lowery
Helen Snowden
Jo Morgan
Graham Taylor
Leeds Institute of Molecular Medicine
Susan Richman
Sophie Grant
Phil Quirke
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