Download What is K-RAS?

The Role of KRAS Mutation
Testing in the Management of
Colorectal Cancer
Mark D. Pool, M.D.
Copyright © 2009 Mark D. Pool
All Rights Reserved
Epidermal Growth Factor
Receptor (EGFR)

Transmembrane growth factor receptor with
tyrosine kinase activity
HER1 (ERBB1), belongs to HER/ErbB family
 Selectively binds 10 different ligands
 After binding, forms dimers that causes
autotransphosphorylation through intrinsic tyrosine
kinase on cytoplasmic domain

Activation of EGFR
• Triggers signaling cascade via
RAS/RAF/MEK/MAPK and PI3K/AKT pathways
• Via these pathways, critical cell functions affected
–
–
–
–
–
–
Survival
Proliferation
Angiogenesis
Cytoskeleton organization and motility
Vesicle trafficking
Calcium signaling
EGFR

Two classes of EGFR inhibitors
Anti-EGFR mAbs: cetuximab, panitumumab
 Small molecule inhibitors of tyrosine kinase activity
(EGFR-TKIs): erlotinib, gefitinib

EGFR



EGFR is overexpressed in more than 85% of
tumors from patients with metastatic CRC.
Only a subset of patients with mCRC achieve a
clinical benefit from treatment with EGFR
inhibitors.
Why?
KRAS Mutation and CRC


Poor correlation between EGFR expression by
IHC and treatment response
Downstream “effectors” may be as important as
receptor expression
RAS
• RAS genes are the most common targets for
somatic gain-of-function mutations in human
cancers
• Activating RAS mutations occur in 30% of
human cancers
– Specific RAS genes are mutated in different cancers
• KRAS prevalent in pancreatic, colorectal,
endometrial, lung, and cervical cancers
RAS Proteins


Small GTPases that act as molecular switches by
coupling cell membrane growth factor receptors
to intracellular signaling pathways to
transcription factors that control various cellular
processes
3 genes, 4 proteins


HRAS, NRAS, KRAS (4A and 4B)
Localized to the inner surface of cell membrane
RAS


Interacts with more than 20 effector proteins
Specific mutations within KRAS can result in
the KRAS protein being inherently activated
independent of upstream growth factor receptor activation
KRAS Mutations and Pathogenesis
• KRAS mutations occur early in colorectal
carcinogenesis
• Occur in 35% to 40% of CRC
• 95% concordance between paired primary
cancers and metastases
KRAS Mutation and CRC

Only certain mutations lead to constitutive,
growth-factor-receptor-independent activation
of KRAS


Mostly occur at codons 12 and 13
Impair intrinsic GTPase activity of KRAS and
prevent GTPase activating proteins from
promoting conversion of GTP (active) to GDP
(inactive)
Frequency of Significant KRAS
Mutations
Codon
Amino Acid
Substitution
Amino Acid
Change
Incidence, %
12
12
12
12
12
12
13
Others
Gly12Asp
Gly12Val
Gly12Cys
Gly12Ser
Gly12Ala
Gly12Arg
Gly13Asp
Aspartate
Valine
Cysteine
Serine
Alanine
Arginine
Aspartate
32.5
22.5
8.8
7.6
6.4
0.9
19.5
1.8
KRAS Mutations as Prognostic
Marker
• RASCAL study (1998) multivariate analysis
presence of KRAS mutation associated with
increased risk of recurrence and death
– Only Gly12Val
– RASCAL II (2001) confirmed but only in Dukes C
(Stage 3) tumors
• Others studies have not confirmed
KRAS Mutations as Prognostic
Marker
• Role of KRAS must be interpreted in context
of other molecular and signaling abnormalities
– Any mutation in KRAS, BRAF, or PI3KCA
associated with shorter 3-year survival
– BRAF mutation and MSS-tumors: negative
prognostic effect?
– KRAS mutation status of lymph nodes in stage 2
patients may identify increased risk of recurrence
KRAS Mutation as Predictive Marker


Recent studies have examined the correlation
between KRAS mutations and response to antiEGFR mAbs inhibitors in patients with mCRC.
Efficacy limited to patients whose tumors with
wild-type KRAS gene.
Results of the NCIC CTG CO.17
Trial
Outcome
Mutant KRAS
ORR (CR+PR)
Median PFS
Median OS
Wild-type KRAS
ORR (CR+PR)
Median PFS
Median OS
Therapy
Best
Cetuximab
Supportive
Hazard Ratio
P value
0
1.8 months
4.6 months
1.2%
1.8 months
4.5 months
NA
NA
0.96
0.89
0
1.9 months
4.8 months
13%
3.7 months
9.5 months
NA
0.99 (0.73-1.35)
0.98 (0.70-1.37)
0.40 (0.34-0.59)
0.55 (0.41-0.74)
Karapetis, CS et al. KRAS mutations and benefit from cetuximab in
advanced colorectal cancer. New Engl J Med 2008;359:1757-1765.
NA
<0.001
<0.001
Results of the OPUS Trial
Outcome
Unselected Patients
ORR
mPFS
Mutant KRAS
ORR
mPFS
Wild-type KRAS
ORR
mPFS
Therapy
FOLFOX alone
FOLFOX +
cetuximab
P value
36%
7.2 months
46%
7.2 months
0.064
0.617
49%
8.6 months
33%
5.5 months
0.1
0.019
37%
7.2 months
61%
7.7 months
0.011
0.016
Bokemeier C, et al. Fluorouracil, leucovorin, and oxaliplatin with and without
cetuximab in the first-line treatment of metastatic colorectal cancer.
J Clin Oncol 2008;27:663-671.
Results of the CRYSTAL Trial
Outcome
FOLFIRI
alone
Unselected
patients
ORR
mPFS
mOS
Mutant
KRAS
ORR
mPFS
mOS
Wild-type
KRAS
ORR
mPFS
mOS
Therapy
Hazard ratio
FOLFIRI+cetuximab
P value
39%
8.0 months
18.6 months
47%
8.9 months
19.9 months
1.40 (1.12-1.77)
0.85 (0.72-0.99)
0.93 (0.81-1.07)
0.004
0.048
0.31
36%
8.1 months
17.7 months
40%
7.6 months
17.5 months
0.80 (0.44-1.45)
1.07 (0.75-1.40)
1.03 (0.74-1.44)
0.90
0.75
0.85
43%
8.7 months
21.0 months
59%
9.9 months
24.9 months
1.91 (1.24-2.93)
0.68 (0.50-0.93)
0.84 (0.64-1.11)
0.003
0.02
0.22
Van Cutsem E, et al. Cetuximab and chemotherapy as initial treatment for
metastatic colorectal cancer. New Engl J Med 2009;360:1408-1417.
KRAS Mutation and CRC

Improved progression-free and overall survival



~12-20 weeks PFS, 8-12 weeks OS
Both drugs are ineffective when the patient's
tumor has a KRAS mutation.
NCCN and ASCO recommend that KRAS
mutation testing be part of the evaluation of
patients with metastatic CRC.
KRAS Mutation Testing

Direct sequencing by PCR (Sanger sequencing)

“Gold standard” but requires large amount of tumor
DNA to detect mutation
Direct Sequencing of KRAS exon 2
KRAS Mutation Testing
• Allele-specific methods: Thera Screen (DxS)
– Specific primers are used to detect each of the
common KRAS mutations
– More sensitive than direct sequencing detecting as
little as 1% to 10% of mutant DNA from total
DNA in sample
Frequency of Significant KRAS
Mutations
Codon
Amino Acid
Substitution
Amino Acid
Change
Incidence, %
12
12
12
12
12
12
13
Others
Gly12Asp
Gly12Val
Gly12Cys
Gly12Ser
Gly12Ala
Gly12Arg
Gly13Asp
Aspartate
Valine
Cysteine
Serine
Alanine
Arginine
Aspartate
32.5
22.5
8.8
7.6
6.4
0.9
19.5
1.8
KRAS Testing Using the DxS Kit
KRAS Mutation Testing
Role of Pathologists



Specimen collection and handling
Selection of appropriate tumor tissue for
testing
Methodology and quality control

“In-house” versus reference lab