Implementing a K-RAS
mutation testing service for
colorectal cancer
Cate Faulkes
Molecular Biology Department
Royal Surrey County Hospital
April 2010
Royal Surrey County Hospital
NHS Foundation Trust
Colorectal cancer
3rd most common cancer worldwide.
 Approximately 25% of patients present
with metastatic colorectal cancer (mCRC).
 Metastatic disease develops in 40-50% of
newly diagnosed patients.
 Treatment is typically by surgery and
chemotherapy.
 Now includes EGFR targeted monoclonal
antibodies e.g. cetuximab & panitumumab.

Van Cutsem et. al. 2009, Karapetis 2008
EGFR signalling pathway disrupted by
EGFR targeted monoclonal antibodies
EGFR targeted mAbs
competitively inhibit ligand
binding thus inhibiting the
phosphorylation cascade
leading to activation of
transcription factors.
 This therefore inhibits cellular
processes such as cell
proliferation and migration.
 Also has cytotoxic effect via
antibody dependent cellular
cytotoxicity.

Vincenzi B et. al. 2010, Bardelli & Siena
2010, commons.wikimedia.org
Not everyone with mCRC responds to
EGFR targeted therapies


Only 10-20% of tumours
respond to EGFR targeted
therapy.
35-45% of colorectal
tumours have at least one
activating mutation in the
K-RAS gene, thus
constitutively activating
cell signalling downstream
of EGFR, so resistant to
EGFR targeted therapies.
Not everyone with mCRC responds to
EGFR targeted therapies
Without
Cetuximab
Overall
survival
rate
With
Cetuximab
Median
4.8
months
Median
9.5
months
Progression Median
free
1.9
survival
months
rate
Median
3.7
months




In patients with wild type K-RAS
tumours, such drugs double
overall and progression free
survival rates.
Mutations occur predominantly
in codons 12 and 13 and less
commonly in codons 61 and
146.
Current NICE guidelines
recommend testing for K-RAS
mutations in all mCRC cases.
K-RAS mutations don’t explain
all non-responders: 5-10%
thought to be due to B-RAF
mutations, others unexplained.
Kareptis et. al. 2008, Chang et. al. 2009,
Bardelli, Siena 2009 & Siena 2010
Introduction of a K-RAS testing service
at RSCH
Prior to September 2009 all K-RAS testing
for patients at RSCH was carried out by
commercial lab or part of drug trial.
 Introduced in-house testing following
requests from local oncologists, to
improve service to patients, meeting NICE
guidelines for treatment.
 Rapid turnaround times crucial when
making decisions regarding targeted
therapies. Samples do not need to be sent
away and the results can be made
available by LIMS (WinPath).

Methods considered
Approx
sensitivity
Dideoxy
Pyro
High
sequencing sequencing Resolution
Melt Analysis
(HRM)
DxS
TheraScreen
® K-RAS
mutation kit
20%
5%
5%
1%
CE marked No
Yes
No
Yes
Detects all Yes
mutations
Yes
Yes
No: 7 most
common
Cost
Mid range
Cheap
Cheap
Expensive
Capital
purchase
Yes
Yes
No but need
2nd method
to verify seq
changes
No
Why choose DxS TheraScreen® K-RAS
mutation kit: mutation frequency

Cosmic
(www.sanger.ac.uk)
Detects the 7 most common
mutations in codons 12 and 13 of
K-RAS gene by real time PCR
(approx 97%).
Why choose DxS TheraScreen® K-RAS
mutation kit: Sensitivity
Germline
wild type
K-RAS
Tumour (H&E)
Somatic activating K-RAS mutation in
tumour:
 Not germline so not wt, het or hom.
 Normal bowel tissue present in
sample.
 Percentage of tumour within the
sample is variable.
 K-RAS mutation not necessarily
present in 100% of tumour.
 Therefore mutated DNA is
percentage of wild type so sensitive
assay required.
 DxS TheraScreen® K-RAS mutation
kit has 1% sensitivity.
www.shands.org
How DxS TheraScreen® K-RAS
mutation kit works




1 control assay and 7 mutation assays each with ARMS
primers, a FAM labelled Scorpion probe and HEX labelled
exogenous control reaction to ensure no inhibition
present. Mixed standard is positive control for all assays.
Control assay used to test sample suitability.
7 mutation assays
and control assay
run simultaneously.
Difference between
mutation Ct and
control Ct (ΔCt)
determines whether
sample has K-RAS
mutation or is wild
type.
Sample 12 VAL
Mixed standards
Sample
control assay
ΔCt=1.48
Comparison study
Tested 13 samples, 11 of which agreed with results from
other laboratories.
 Both discrepancies were samples originally tested as wild
type but our assay detected a mutation.
 1 non-concordant sample was re-cut and re-extracted then
retested at RSCH and the lab in question. Both results
agreed with our original results, showing a mutation.
 The other remains unresolved, however may be due to a
sensitivity issue as it was originally tested by
pyrosequencing and we found a low level mutation.
Conclusion:
CE marked kit run on Roche Lightcycler 480 with Adapt
software containing algorithms for determining mutation
status. Assay was considered fit for purpose.

Workflow
Pathologist reviews H&Es, selects
appropriate block & ascertains tumour
load.
DNA extracted from FFPE tissue using
Qiagen DNA mini kit.
Sample assessment protocol (DxS kit)
to ascertain DNA suitability.
Mutation detection assay (DxS kit).
Results reported.
Expected
turnaround
time within 7
working days.
Percentage turnaround times for K-RAS
testing at RSCH (37 cases)

87% cases reported
within 7 days receipt.
Arrived 23.12.09
Low DNA concre-extracted
QC problem
with kit
K-RAS mutation frequency of 50 patients
tested at RSCH (includes comparison study)

38% patients
tested carry KRAS
mutation which
falls within 3545% K-RAS
mutations cited in
literature.
Summary




DxS kit is a rapid, robust and sensitive method.
Our testing service enables treatment choices to
be made as early as possible for the patient.
Oncologists are pleased with the shorter turn
around times.
If anyone is interested in using our service please
contact Dr Louise Lavender (01483 571122
ext6839, louiselavender@nhs.net).
The Future
Develop an HRM method for codons 12
and 13 and test alongside DxS
TheraScreen® K-RAS mutation kit.
 Any sample wild type by DxS
TheraScreen® K-RAS mutation kit, but
mutant by HRM confirm by sequencing or
pyrosequencing.
 Devolop HRM method for codon 61 of KRAS gene.

Acknowledgements
Dr Gary Middleton (Oncologist): patient
samples.
 Dr Stefano De Sanctis (Histopathologist):
Tumour assessment.
 Dr Louise Lavender (Head of Molecular
Biology) : Support.
 Dave Brettle and Bruno Ping: DNA
extraction.

References






Van Cutsem E, et al. Cetuximab and chemotherapy as initial
treatment for metastatic colorectal cancer. N Engl J Med. 2009;
360:1408-1417.
Bardelli A & Siena S. Molecular mechanisms of resistance to
Cetuximab and Panitumumab in colorectal cancer, J Clin Oncol.
2010;Jan:e published.
Vincenzi B, et al. Cetuximab: From bench to bedside, Curr
Cancer Drug Targets. 2010;Jan:e published.
Karapetis CS et al. K-RAS mutations and benefit from
cetuximab in advanced colorectal cancer, N Engl J Med.
2008;359:1757-1765.
Chang YS et al. Fast simultaneous detection of K-RAS mutations
in colorectal cancer. BMC Cancer. 2009;9:175-185.
Siena S, et al. Biomarkers predicting clinical outcome of
epidermal growth factor receptor-targeted therapy in metastatic
colorectal cancer. J Natl Cancer Inst. 2009;101:1-17.