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K-Ras and Beyond Josep Tabernero, MD Vall d’Hebron University Hospital Barcelona, 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 EGFR RAS RAF PI3K Akt MEK MAPK Cell Cell Survival Proliferation 1Lièvre, A. Cancer Res; 66:3992-3995, 2006 Cutsem, E. et al. N Engl J Med; 360:1408-1417, 2009 3Bokemeyer, C. et al. J Clin Oncol; 27:663-671, 2009 4Di Nicolantonio, F. et al. J Clin Oncol; 26:5702-5712, 2008 2Van 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 Utility Performance of 4 tumor based tests: K-Ras, BRaf, N-Ras and PIK3CA mutation status Predictive biomarker Specimen Tumor specimens (paraffin-embedded) Patients Sample size Refractory mCRC treated with Irinotecan + Cetuximab 276 tumors 580 tumors (European consortium) Sequenom MALDI TOF MassArray system Assay Lambrechts: Results (1) KRAS BRAF Mutations included % coverage of potential mutations (Cosmic) Mutation rate detected G12S, G12R , G12C, G12D , G12A , G12V , G13D, A146T, G13A, G13V, A59T, Q61K , Q61E, Q61P, Q61R, Q61L, Q61H 99.2% 36.5% V600E ,K601E, D594G ,V600M 97% ( 622 samples) 5% (589 samples) NRAS PI3K Q61P,Q61L,Q61H,Q61H,Q61Q,Q61E,G13S,G13C ,G13R,Q61K,Q61R, G12D,G12S ,G12C 97% 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% 6% (261 samples worked) 13% (578 samples) Lambrechts: Results (1) • 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 (2) - RR KRAS 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 PI3K 128 (27%) CR357 (73%) SD + PD WT 485 + PR In KRAS wt Mut WT 10 (14%) Mut 60 (86%) 117 (38%) 8 (24%) p p=.028 total 70 195 (62%) 312 26 (76%) 34 p p=0.107 Lambrechts: Results (3) – PFS & OS KRAS Median PFS HR (95% CI) P value (log rank) WT MUT N=369 N=212 24 weeks 12 weeks 0.542 (0.452-0.650) <0.001 Lambrechts: Results (3) – PFS & OS KRAS BRAF Median PFS Median PFS HR (95% CI) HR (95% CI) P value (log rank) P value (log rank) WT MUT WT MUT N=369 N=212 N= 561 N= 28 24 weeks 12 weeks 19 weeks 7.8 weeks 0.542 (0.452-0.650) 0.410 (0.275-0.610) <0.001 <0.001 Lambrechts: Results (3) – PFS & OS KRAS WT MUT N=369 N=212 WT MUT WT MUT 24 weeks 12 N= 561 N= 28weeks 505 73 HR (95% 0.542 (0.452-0.650) Median PFS CI) 19 weeks 7.8 weeks Median PFS 19 weeks 12.5 valueCI) (log rank) <0.001 HR P(95% 0.410 (0.275-0.610) HR (95% CI) 0.772 (0.601-0.991) P value (log rank) <0.001 P value (log rank) 0.036 BRAF PI3K Median PFS Lambrechts: Results (3) – PFS & OS KRAS WT MUT N=369 N=212 MUT All KRAS wt WT Median PFS 24 weeks weeks MUT PI3K PI3K N= 561 WT WTN=12 28 MUT HR (95% 0.542323 (0.452-0.650) Median PFS CI) 19 weeks 505 7.8 weeks 73 36 BRAF valueCI) (log HR P(95% Median PFSrank) Median PFS P value HR (log (95% CI) CI) HRrank) (95% P value (log rank) P value (log rank) <0.00112.5 19 0.410 weeks (0.275-0.610) 24 weeks 23 weeks 0.772 <0.001 (0.601-0.991) 0.848(0.599-1.201) 0.036 0.338 Lambrechts: Results (4) – Multivariate 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 PFS Cox regresion HR KRAS p=.136 95%CI P value 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 Lambrechts: Conclusions • 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) 1Lièvre, 2Di A. Cancer Res; 66:3992-3995, 2006 Nicolantonio, F. et al. J Clin Oncol; 26:5702-5712, 2008 3 Sartore-Bianchi, A et al Cancer Res; 69:1851-7, 2009 4Ann 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;14:7884-95,2008 W wt al. J Clin Oncol 20;25:3712-8,2007 3Bibeau, F et al. J Clin Oncol; 27:1122-9,2009 2Zhang, 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. 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 1Singh, AB et al. Cell Signal; 17:1183-1193,2005 M et al. J Biol Chem; 273:10496-10505,1998 3Khambata-Ford, S. et al. J Clin Oncol; 25:3230-3237, 2007 2Shelly, Jonker: Patients and Methods Endpoint Utility Three tumor based tests: K-Ras mutation status and EREG & AREG (not shown) expression Predictive biomarker Tumor specimens (paraffin-embedded material) – Study NCIC CTG CO.17 Patients Refractory mCRC treated with Cetuximab or BSC Sample size K-Ras 394/572 (69%); EREG 385/572 (67%) Assay EREG gene expression by quantitative RT-PCR Specimen Jonker: Background NCIC CTG CO.17: mCRC Cetuximab vs BSC HR OS: ITT 0.7 K-Ras wt 0.55 1Jonker, 2Karapetis, DJ et al. NEJM; 357:2040-8,2007 CS. et al. NEJM;359:1757-65,2008 Jonker: Results (1) • EREG in K-Ras wt as a continuous variable: prognostic and predictive EREG and OS in patients with K-Ras wild-type Adjusted HR (95% CI) for 1 unit increase in EREG normCT (toward normal) P value CET/BSC 1.17 (1.04-1.32) 0.01 BSC 1.13(1.01-1.27) 0.04 Study arm Test for treatment / biomarker interaction (adjusted p value) HR 1.03 (0.88-1.20) p=0.75 EREG and PFS in patients with K-Ras wild-type Adjusted HR (95% CI) for 1 unit increase in EREG normCT (toward normal) P value CET/BSC 1.13 (1.01-1.26) 0.03 BSC 0.96 (0.87-1.07) 0.48 Study arm Test for treatment / biomarker interaction (adjusted p value) HR 1.14 (0.98-1.33) p=0.08 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 [0.591.22], 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%) High EREG by minimum-p threshold 100 Cetuximab + BSC 80 Proportion alive Proportion alive 100 Low EREG by minimum-p threshold 60 40 BSC alone 20 Cetuximab + BSC 80 60 40 BSC alone 20 HR 0.46 [0.32-0.65], p<0.0001 HR 0.93 [0.51-1.71], p=0.81 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 84 85 80 73 76 54 66 26 43 19 28 14 18 10 8 5 30 26 25 18 16 15 13 10 8 5 5 3 Time from randomization (months) Time from randomization (months) Prenen: Patients and Methods Endpoint Utility Three tumor based tests: K-Ras mutation status and EREG and AREG expression Predictive biomarker Specimen Tumor specimens (paraffin-embedded) Patients Sample size Irinotecan refractory mCRC treated with Irinotecan + Cetuximab 220 tumors + 67 tumors (external validation) EREG and AREG gene expression by quantitative RT-PCR Assay 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 EREG p=.0005 AREG 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 Odd ratio EREG AREG RR 5.04 DCR 20.7 OR 5.46 DCR 6.86 • However, the cut-offs points are different by ROCanalysis for each end-point 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 Jonker & Prenen: Implications • 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 Loupakis: Patients and Methods Endpoint Utility Three tumor based tests: K-Ras and B-Raf mutation status and AREG expression Predictive biomarker Specimen Tumor specimens (paraffin-embedded) Patients Sample size Refractory mCRC treated with Irinotecan + Cetuximab 87 tumors (4 centers in Italy) AREG expression by IHC (Mo Ab cl 31221, RD) H-Score (0-300) Mutations K-Ras & B-Raf (not described) Assay Loupakis: Results - Implications • 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 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) Patients Sample size Oxaliplatin-refractory mCRC treated with Irinotecan + Cetuximab 84 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: Implications US patients Ir/Cmab (n=84) Ir (n=102) p RR 13.1 5.9 - mTTP (m) 3.0 2.7 - Ir/Cmab (n=84) Ir (n=102) p RR 16.4 4.2 <.05 mTTP (m) 4.0 2.6 <.05 Total • 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 Methods Endpoint Quality audit of K-Ras mutation status test Utility Predictive biomarker Specimen Tumor specimens (paraffin-embedded) Patients mCRC patients from German-speaking countries Sample size (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) Jung: Results - Implications • 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 IGF-1/IGF1R axis in the treatment with anti-EGFR MoAbs A4017 – 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) Patients Sample size Refractory mCRC treated with Irinotecan + Cetuximab 62 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 11 3.2 .03 mTTP (m) Scartozzi: Results - Implications • 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 1Jones, 2Guix, HE et al. Br J Cancer 95;172-180, 2006 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 (KRas 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