k-ras and beyond josep tabernero, md vall d’hebron university hospital barcelona, spain
TRANSCRIPT
K-Ras and Beyond
Josep Tabernero, MDVall d’Hebron University
HospitalBarcelona, Spain
Disclosures
• Participated in Advisory Boards of Merck, Amgen, Imclone, Sanofi-Aventis, Onyx, and Roche
K-Ras and B-Raf in CRC• Constitutive mutations of K-Ras
predict resistance to anti-EGFR MoAbs in CRC:
– refractory1 setting– first-line2-3 setting– basis for regulatory approval
(EMEA) & national guidelines (NCCN)
• Role of mutations of other signal transducer proteins is being evaluated:
– i.e. B-Raf: refractory setting4
1Lièvre, A. Cancer Res; 66:3992-3995, 20062Van Cutsem, E. et al. N Engl J Med; 360:1408-1417, 2009
3Bokemeyer, C. et al. J Clin Oncol; 27:663-671, 20094Di Nicolantonio, F. et al. J Clin Oncol; 26:5702-5712, 2008
EGFR
RAS
RAF
MEK
MAPKAkt
PI3K
Cell Proliferation
Cell Survival
K-Ras, B-Raf, N-Ras and PIK3CA mutations and cetuximab efficacy
A4020 – Poster Board #: 11; Diether Lambrechts et al.
The role of KRAS, BRAF, NRAS, and PIK3CA mutations as markers of resistance to cetuximab in chemorefractory metastatic colorectal cancer.
Lambrechts: Patients and Methods
Endpoint Performance of 4 tumor based tests: K-Ras, B-Raf, N-Ras and PIK3CA mutation status
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded)
PatientsSample size
Refractory mCRC treated with Irinotecan + Cetuximab276 tumors 580 tumors (European consortium)
Assay Sequenom MALDI TOF MassArray system
Lambrechts: Results (1)Mutations included % coverage of
potential mutations (Cosmic)
Mutation rate detected
KRAS G12S, G12R , G12C, G12D , G12A , G12V , G13D, A146T, G13A, G13V, A59T, Q61K , Q61E, Q61P, Q61R, Q61L, Q61H
99.2% 36.5% ( 622 samples)
BRAF V600E ,K601E, D594G ,V600M 97% 5%(589 samples)
NRAS Q61P,Q61L,Q61H,Q61H,Q61Q,Q61E,G13S,G13C,G13R,Q61K,Q61R, G12D,G12S ,G12C
97% 6% (261 samples worked)
PI3K H1047R, H1047L , K179T, P539R,Q546K,Q546E, E81K, R88Q,G106V,N345K, R93W, S158L, H160N,R38H,E542K, E542Q,E545K,E545Q, G118D, G12D,K567R,H1047Y, P134S, R108H, C420R,H701P,K184E, C901F,M1004I, G1049R, G1007R, G1049S
86% 13%(578 samples)
• K-Ras, B-Raf and N-Ras mutually exclusive• 17.7% K-Ras mt and 10.4% K-Ras wt had a
PIK3CA mutation (p= 0.009 Pearson Chi square)
• 6% B-Raf mutants and 13% B-Raf wt had a PIK3CA mutation
(p= 0.412 Fisher’s Exact test) • Representative series: outcomes in
accordance with the literature – mPFS 18 wks, mOS 38 wks (≈BOND)
Lambrechts: Results (1)
Lambrechts: Results (2) - RRKRAS CR + PR SD + PD total p
WT 130 (36%) 226 (64%) 356 p<.001
Mut 11 (5%) 192 (95%) 203
BRAF CR + PR SD + PD total p
WT 141 (26%) 399 (74%) 540 p=.035
Mut 2 (8%) 24 (92%) 26
NRAS CR + PR SD + PD total p
WT 50 (21%) 179 (79%) 239 p=.317
Mut 1 ( 6%) 14 (94%) 15
PI3K CR + PR SD + PD total p
WT 128 (27%) 357 (73%) 485 p=.028
Mut 10 (14%) 60 (86%) 70
PI3KIn KRAS wt
CR + PR SD + PD total p
WT 117 (38%) 195 (62%) 312 p=0.107
Mut 8 (24%) 26 (76%) 34
Lambrechts: Results (3) – PFS & OS
KRAS WT MUT
N=369 N=212
Median PFS 24 weeks 12 weeks
HR (95% CI) 0.542 (0.452-0.650)
P value (log rank)
<0.001
Lambrechts: Results (3) – PFS & OS
KRAS WT MUT
N=369 N=212
Median PFS 24 weeks 12 weeks
HR (95% CI) 0.542 (0.452-0.650)
P value (log rank)
<0.001
BRAF WT MUT
N= 561 N= 28
Median PFS 19 weeks 7.8 weeks
HR (95% CI) 0.410 (0.275-0.610)
P value (log rank) <0.001
Lambrechts: Results (3) – PFS & OS
KRAS WT MUT
N=369 N=212
Median PFS 24 weeks 12 weeks
HR (95% CI) 0.542 (0.452-0.650)
P value (log rank)
<0.001
BRAF WT MUT
N= 561 N= 28
Median PFS 19 weeks 7.8 weeks
HR (95% CI) 0.410 (0.275-0.610)
P value (log rank) <0.001
PI3K WT MUT
505 73
Median PFS 19 weeks
12.5
HR (95% CI) 0.772 (0.601-0.991)
P value (log rank) 0.036
Lambrechts: Results (3) – PFS & OS
KRAS WT MUT
N=369 N=212
Median PFS 24 weeks 12 weeks
HR (95% CI) 0.542 (0.452-0.650)
P value (log rank)
<0.001
BRAF WT MUT
N= 561 N= 28
Median PFS 19 weeks 7.8 weeks
HR (95% CI) 0.410 (0.275-0.610)
P value (log rank) <0.001
PI3K WT MUT
505 73
Median PFS 19 weeks 12.5
HR (95% CI) 0.772 (0.601-0.991)
P value (log rank) 0.036
All KRAS wt
PI3K WT MUT
323 36
Median PFS 24 weeks 23 weeks
HR (95% CI) 0.848(0.599-1.201)
P value (log rank) 0.338
Lambrechts: Results (4) – Multivariate
PFS Cox regresion HR 95%CI P value
KRAS 0.523 0.434 – 0.631 p<.001
BRAF 0.328 0.217 – 0.497 p<.001
Pi3K 0.798 0.620 – 1.027 p=.079
OS Cox regresion HR 95%CI P value
KRAS 0.549 0.452 – 0.667 p<.001
BRAF 0.378 0.250 – 0.572 p<.001
Pi3K Not retained
p=.187
OR Logistic regresion
OR 95%CI P value
KRAS 0.093 0.048 – 0.177 p<.001
BRAF 0.140 0.032 – 0.604 p=.008
Pi3K Not retained
p=.136
• K-Ras impact ≈ literature1
• N-Ras impact: not mature, full series to be analyzed, currently mt incidence 6%
• B-Raf ≈ literature2. Most powerful negative predictor
• PIK3CA: little effect, no effect if restricted to K-Ras wt, not retained in multivariate analysis Discrepancy with the literature3,4 (although limited number of patients)
Lambrechts: Conclusions
1Lièvre, A. Cancer Res; 66:3992-3995, 20062Di Nicolantonio, F. et al. J Clin Oncol; 26:5702-5712, 2008
3 Sartore-Bianchi, A et al Cancer Res; 69:1851-7, 20094Ann Oncol ;20:84-90, 2008
Lambrechts: Implications• Strengths:
– Unique and consistent population– Large database– Not influenced by other treatments
Lambrechts: Implications• Weakness:
– Not all the mutations have the same addictive role
– Other possible deregulations not considered so far: PTEN mutations, PTEN loss of function, Src mutations, p53 mutations, …
– Other potential predictors: • Role of the ligands• Polymorphisms1-3:
– EGFR, EGF, … – Fc receptors (ADCC): FcgammaRIIa-H131R and
FcgammaRIIIa-V158F 1Lurge, J et al. Clin Cancer Res 1;14:7884-95,20082Zhang, W wt al. J Clin Oncol 20;25:3712-8,2007
3Bibeau, F et al. J Clin Oncol; 27:1122-9,2009
Amphiregulin/Epiregulin
A4016 – Poster Board #: 7; Derek J Jonker et al.
High epiregulin (EREG) gene expression plus K-ras wild-type (WT) status as predictors of cetuximab benefit in the treatment of advanced colorectal cancer (ACRC): Results from NCIC CTG CO.17—A phase III trial of cetuximab versus best supportive care (BSC).
A4019 – Poster Board #: 10; Hans Prenen et al.Use of amphiregulin and epiregulin mRNA expression in primary tumors to predict outcome in metastatic colorectal cancer treated with cetuximab.
A4021 – Poster Board #: 12; Fotios Loupakis et al.Amphiregulin (AR) expression in the prediction of benefit from cetuximab plus irinotecan in KRAS wild-type metastatic colorectal cancer (mCRC) patients.
1Singh, AB et al. Cell Signal; 17:1183-1193,20052Shelly, M et al. J Biol Chem; 273:10496-10505,1998
3Khambata-Ford, S. et al. J Clin Oncol; 25:3230-3237, 2007
Amphiregulin/Epiregulin• EGFR ligands:
– 1 in C. Elegans– 4 in Drosophila– 7 in mammals: EGF, TGF-α,
HB-EGF, amphiregulin (AREG), betacellulin, epiregulin (EREG) and epigen1
– EREG and AREG bind more weakly to EGFR than EGF but much more potently and prolonged
– EREG preferentially activates heterodimers2
• High gene expression levels of EREG and AREG predict response to cetuximab3
Jonker: Patients and MethodsEndpoint Three tumor based tests: K-Ras mutation
status and EREG & AREG (not shown) expression
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded material) – Study NCIC CTG CO.17
PatientsSample size
Refractory mCRC treated with Cetuximab or BSCK-Ras 394/572 (69%); EREG 385/572 (67%)
Assay EREG gene expression by quantitative RT-PCR
Jonker: Background
NCIC CTG CO.17: mCRC Cetuximab vs BSC
HR OS: ITT 0.7 K-Ras wt 0.55
1Jonker, DJ et al. NEJM; 357:2040-8,2007 2Karapetis, CS. et al. NEJM;359:1757-65,2008
Jonker: Results (1)• EREG in K-Ras wt as a continuous
variable: prognostic and predictive
Study armAdjusted HR (95% CI) for 1 unit
increase in EREG normCT (toward normal)
P value
Test for treatment / biomarker interaction (adjusted p value)
CET/BSC 1.17 (1.04-1.32) 0.01HR 1.03 (0.88-1.20) p=0.75
BSC 1.13(1.01-1.27) 0.04
EREG and OS in patients with K-Ras wild-type
EREG and PFS in patients with K-Ras wild-type
Study armAdjusted HR (95% CI) for 1 unit
increase in EREG normCT (toward normal)
P value
Test for treatment / biomarker interaction (adjusted p value)
CET/BSC 1.13 (1.01-1.26) 0.03HR 1.14 (0.98-1.33) p=0.08
BSC 0.96 (0.87-1.07) 0.48
Jonker: Results (2)• EREG in K-Ras wt as a categorical
variable (high vs low): predictive but not prognostic– In K-Ras wt patients on BSC, high EREG
expression did not correlate with OS using:• pre-specified threshold: adjusted HR 0.82
[0.58-1.15], p=0.24• minimum p threshold: adjusted HR 0.85
[0.59-1.22], p=0.38
Jonker: Results (3)• Combimarker: K-Ras wt and high EREG
– Pre-especified threshold1
– Minimum threshold: 169/384 (44%)• response rate was 15.5 vs 0% for cetuximab
vs BSC• median PFS was 5.1 vs 1.9 months for
cetuximab vs BSC (HR, 0.33; p<0.0001)• median OS was 9.9 vs 5.0 months for
cetuximab vs BSC (HR, 0.46; p<0.001)• Implications in patients to be treated:
– All comers 394 (100%) HR: 0.7– K-Ras wt 230 (58%) HR: 0.55– Combimarker 169 (44%) HR: 0.46
1Khambata-Ford, S. et al. J Clin Oncol; 25:3230-3237, 2007
Jonker: Results (4)• Combimarker: K-Ras wt and high EREG
– Minimum threshold: 169/384 (44%)
Low EREG by minimum-p threshold
Cetuximab + BSC
BSC alone
HR 0.93 [0.51-1.71], p=0.81P
rop
ortio
n al
ive
0
20
40
60
80
100
Time from randomization (months)
03026
22518
41615
61310
885
1053
High EREG by minimum-p threshold
Pro
por
tion
aliv
e
0
20
40
60
80
100
Time from randomization (months)
08485
28073
47654
66626
84319
102814
121810
1485
Cetuximab + BSC
HR 0.46 [0.32-0.65], p<0.0001
BSC alone
Prenen: Patients and MethodsEndpoint Three tumor based tests: K-Ras mutation
status and EREG and AREG expression
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded)
PatientsSample size
Irinotecan refractory mCRC treated with Irinotecan + Cetuximab220 tumors + 67 tumors (external validation)
Assay EREG and AREG gene expression by quantitative RT-PCR
Prenen: Results (1)• EREG expression is higher in K-Ras wt
than in K-Ras mut tumors (p=0.0002)
Prenen: Results (2)• EREG and AREG expression as a continuous
variable is predictive of response in K-Ras wt but not in mut tumors
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Epi versus OR without splines (wt)
Epi
Pro
babi
lity
of R
espo
nse
pointCI
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Amphi versus OR without splines (wt)
Amphi
Pro
babi
lity
of R
espo
nse
pointCI
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Epi versus OR with splines (wt)
Epi
Pro
babi
lity
of R
espo
nse
pointCI
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Amphi versus OR with splines (wt)
Amphi
Pro
babi
lity
of R
espo
nse
pointCI
EREG AREG
p=.0005 p=.0017
Prenen: Results (3)• EREG and AREG expression as a categorical
variable is predictive of RR, DCR, PFS, OS in K-Ras wt tumors
• However, the cut-offs points are different by ROC-analysis for each end-point
Odd ratio
EREG RR 5.04
DCR 20.7
AREG OR 5.46
DCR 6.86
Prenen: Results (3)• Combination of K-Ras wt and EREG or AREG and
OS
EREG HR OS: 0.42 (95% CI 0.28 – 0.63)
p<.001
• Strengths:– Large series
• One randomized study: 394 pts.• One multicentric cohort series: 287 pts.
– Not influenced by other treatments– Proof of concept of AREG & EREG well
established, beyond K-Ras
Jonker & Prenen: Implications
• Weakness:– Do not discriminate between AREG &
EREG– Underestimate other relevant mutations– Reproducibility: magnitude and cut-off– Variability in the categorization and loss
of power
Jonker & Prenen: Implications
Loupakis: Patients and MethodsEndpoint Three tumor based tests: K-Ras and B-
Raf mutation status and AREG expression
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded)
PatientsSample size
Refractory mCRC treated with Irinotecan + Cetuximab87 tumors (4 centers in Italy)
Assay AREG expression by IHC (Mo Ab cl 31221, RD) H-Score (0-300)Mutations K-Ras & B-Raf (not described)
• RR: - ITT: 16%- K-Ras wt: 25%; K-Ras wt + B-Raf wt:
30%• AREG: High expression associated with B-Raf wt
(p=.0005) but not with K-Ras wt • AREG in K-Ras wt and B-Raf wt: no relation with RR,
PFS and OS• In the multivariate analysis only B-Raf status keep
the prognostic value Difficult to conciliate with the literature due the low frequency of B-Raf mut (5-10%)
• AREG by IHC not standardized
Loupakis: Results - Implications
Polymorphisms
A4022 – Poster Board #: 13; Dongyun Yang et al.Pharmacogenetic analysis in metastatic colorectal cancer (mCRC) patients (pts) treated with second-line irinotecan (IR)+/- cetuximab (CB): The EPIC experience.
Yang: Methods and Results
Endpoint Two tumor based tests: K-Ras mutation status and EGFR-CA repeats in Intron 1
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded)
PatientsSample size
Oxaliplatin-refractory mCRC treated with Irinotecan + Cetuximab84 pts treated in the US (Ir/Cmab) - EPIC study
Assay EGFR-CA repeats in Intron 1 (PCR)Mutations (method not defined)
Yang: Results - Methods
• K-Ras mutation status was not significantly associated with PFS or response• EGFR-CA- repeat in intron 1 in arm be associated with PFS (p=0.031)• Results difficult to interpret: few patients in variant 20/20
Yang: ImplicationsUS patients Ir/Cmab
(n=84)Ir (n=102) p
RR 13.1 5.9 -
mTTP (m) 3.0 2.7 -
Total Ir/Cmab (n=84)
Ir (n=102) p
RR 16.4 4.2 <.05
mTTP (m) 4.0 2.6 <.05
• Behavior of homozygous variants (20/20 & <20/<20) is different to the heterozygous (20/<20)• Biology?• Sample size
Methodology in K-Ras mutations determination
A4018 – Poster Board #: 9; Andreas Jung et al.The German quality assurance system for the molecular-pathological detection of KRAS-mutations in colorectal cancer.
Jung: Patients and MethodsEndpoint Quality audit of K-Ras mutation status test
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded)
PatientsSample size
mCRC patients from German-speaking countries (Austria, Germany and Switzerland)10 patients; 50 institutions
Assay K-Ras mutation analysis by DDS (disesoxy sequencing – Sanger), ARMS (amplification refractory mutation sequencing) and MPA (melting point analysis, pyrosequencing)
• 10 patients: 74 different K-Ras determinations• Limited number of patients and analysis• The authors raise concerns on the difficulties
to establish quality assurance systems • The authors state there is no
technique/method superior to another?• Delay in the result higher than expected (>14
days) • 15% conflicting results: not disclosed• SOP: critical step
Jung: Results - Implications
IGF-1/IGF1R axis in the treatment with anti-EGFR MoAbsA4017 – Poster Board #: 8; Mario Scartozzi et al.
Correlation of insulin-like growth factor 1 (IGF-1) expression and clinical outcome in K-RAS wild-type colorectal cancer patients treated with cetuximab-irinotecan.
Scartozzi: Methods and Results
Endpoint Two tumor based tests: K-Ras mutation status and Insulin-like growth factor (IGF-1) expression
Utility Predictive biomarker
Specimen Tumor specimens (paraffin-embedded)
PatientsSample size
Refractory mCRC treated with Irinotecan + Cetuximab62 tumors (4 centers in Italy)
Assay IGF-1 expression by IHC (Cell Signaling)Mutations (method not defined)
Total 62 pts. IGF-1 - IGF-1 + p
RR PR 7 (50%) 1 (5%) .004
mTTP (m) 11 3.2 .03
• Combined IGF-1 IHC expression and K-Ras mutation analysis may represent an effective strategy for a better selection of responding colorectal tumors for cetuximab treatment
• Caveats:
• IHC considered positive if 2
• These results should be externally validated
• Reproducibility of IHC for IGF-1, IGFBPs and IGF-1R is cumbersome
• Potential role for anti-EGFR and anti-IGF1R combinations:
• Activation of IGF-1/IGF1R reduces sensitivity to EGFR TKI in cancer cells. IGF-1R inhibition restores sensitivity to EGFR TKIs1,2
Scartozzi: Results - Implications
1Jones, HE et al. Br J Cancer 95;172-180, 20062Guix, M et al. J Clin Invest. 118:2609–2619, 2008
Conclusions• Each of these studies constitute and
Academic effort to personalize the treatment in patients with mCRC by tuning the target population beyond the standard of care (K-Ras status)
• In order to completely define the ultimate role of the different predictive factors an international collaboration is needed
Conclusions
• Predictive factors accepted:– K-Ras status
• Far advanced:– B-Raf status
• To be defined:– N-Ras, PIK3CA status – Loss of PTEN – Ligands: AREG, EREG– Polymorphisms: EGFR, EGF, Fc receptors (ADCC):
FcgammaRIIa-H131R and FcgammaRIIIa-V158F – Others
Acknowledgements
• ASCO Program Committee• Poster presenters for providing their
presentations in a timely fashion• Eduardo Vilar, MD and Javier
Hernández, PhD for their thoughtful comments
• Audience