k-ras and beyond josep tabernero, md vall d’hebron university hospital barcelona, spain

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