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