Molecular Predictors in Clinical Decision Making for Colon Cancer

Molecular Predictors in Clinical Decision Making for Colon Cancer
Axel Grothey Professor of Oncology Mayo Clinic Rochester

Disclosures Consulting activities (honoraria went to the Mayo Foundation) Amgen Bayer Pfizer Roche/Genentech Sanofi-Aventis BMS Eli Lilly/Imclone I WILL include discussion of investigational or off-label use of a product in my presentation.

CRC: worldwide incidence
Third leading cancer death in the world Second in the US and Western Europe Landis SH, et al. CA Cancer J Clin 1999;49:8–31 CRC = colorectal cancer 3

(Familial CRC of syndrome “X”)
Hereditary AC-1 without MMR (Familial CRC of syndrome “X”) Lynch Syndrome Sporadic FAP; AFAP Mixed Polyposis Syndrome Ashkenazi I1307K CHEK2 (HBCC) MYH TGFBR1 PJS FJP CD BRRS = as yet undiscovered hereditary cancer variants Hamartomatous Polyposis Syndromes

Genetic Syndromes: Magnitude of the Problem
Annual worldwide incidence of CRC is 1,023,152*: • Lynch syndrome (LS) accounts for  2-5% (20,460-51,160 cases). • < 1% (10,230 cases) constitute FAP. •  20% (204,630 cases) are familial (2 or more first- degree relatives with CRC. *International Agency for Research on Cancer. Globocan Available at: H. Lynch, ASCO 2009

Genetic Heterogeneity in HNPCC
MSH6 MLH1 MSH2 PMS2 PMS1 Chr 7 Chr 3 Chr 2 HNPCC is associated with germline mutations in any one of at least five genes

SPORADIC CRC 15% microsatellite instable tumors (MSI)
P53-BAX- WNT- TGFb- Hyper- methylation pathway Late adenoma Carcinoma MLH1 MSH 2/3/6 TGFIIb BAX TCF4 WISP3 Hypo- methylation Normal epithelium Early adenoma Intermed. adenoma APC b-catenin KRAS 18q loss SMAD2/4 WNT- pathway MAPK- pathway TP53 Late adenoma Carcinoma TGFb- pathway P53-BAX- pathway 85% chromosome instable tumors (CIN)

CRC: Demographics and Presentation
Estimated 2009 U.S. incidence (new cases): 147,000 Estimated 2008 U.S. mortality: 49,900 12% stage I* 18.6% stage IV* Many patients have already progressed to stage III or IV disease before receiving treatment. These later stages of disease require aggressive treatment, involving surgery, chemotherapy, and sometimes radiation. Commonly used screening techniques for the diagnosis of CRC include1 Fecal occult blood test Flexible sigmoidoscopy Colonoscopy Double-contrast barium enema Digital rectal exam (for rectal cancer only) Using a combination of these procedures, patients with CRC receive an initial workup to determine disease stage (I-IV), which guides subsequent treatment decisions. While staging techniques vary worldwide, clinicians in the United States rely on Dukes’ and TNM (tumor/node/metastasis) staging techniques for disease staging.2 Stage II disease demonstrates invasion through the muscularis propria without nodal involvement or distant metastases.2 The role of adjuvant therapy for stage II colon cancer remains controversial. Patients with stage III disease have nodal involvement without distant metastases and are treated using adjuvant chemotherapy.2 Stage IV disease has metastatic involvement of distant lymph nodes and/or distant organs.2 Most patients present with stage II (24.5%) or III (32.6%) disease.3 Unresectable and metastatic stage IV disease is generally fatal. 24.5% stage II* 32.6% stage III* 12.3% of patients presented with recurrent CRC. *2002 data. American Cancer Society, 2005; Datamonitor, 2003. 1. Treatment Algorithms: Colorectal Cancer. 5th ed. Datamonitor. January 2003:59. 2. Treatment Algorithms: Colorectal Cancer. 5th ed. Datamonitor. January 2003:73. 3. Treatment Algorithms: Colorectal Cancer. 5th ed. Datamonitor. January 2003:50. 8

Palliative Therapy of Colorectal Cancer

Advances in the Treatment of Stage IV CRC
1980 1985 1990 1995 2000 2005 Best supportive care (BSC) 5-FU Irinotecan Capecitabine median overall survival Oxaliplatin Cetuximab Bevacizumab Panitumumab

Treatment paradigms for mCRC
Some patients with stage IV disease can be cured by an interdisciplinary approach In the palliative setting: FOLFOX = XELOX = FOLFIRI (XELIRI has problems with toxicity) Most patients tolerate a chemotherapy doublet, but not all need it The addition of biologics to chemotherapy has improved outcomes, but not as much as we hoped We are on the verge of individualized therapy based on molecular predictive factors

Personalized Cancer Therapy: Key Challenges
Tumor heterogeneity Primary vs metastasis Established tumor vs micrometastasis Whack-a-mole pathway modulation Combination therapy complicates choice of appropriate biomarkers Identification of biomarkers lags behind standard of care and agents used in clinical trials Most biomarkers identified in retrospective analysis without prospective validation Complex, step-wise trial designs to validate usefulness of biomarkers

Retrospective analysis
Biomarker Validation Prognostic marker Retrospective analysis Predictive marker Randomized Clinical Trials The validation of prognostic markers does not necessitate prospective randomized clinical trials and is valuably performed in assessing prospective series of patients treated with standard treatment The validation of predictive markers is on the contrary best performed in the frame of randomized clinical trials. But what Trial designs are should be employed for this purpose?

5-FU: Predictive Markers
RNA DPD FU FUH2 FUrd FUMP FUDP FUTP TP FUdR FUPA FdUMP FdUDP FdUTP LV FBAL TS dUMP dTMP DNA 5,10-CH3THF DHF

Danenberg Tumor Profile Scale
DPD, TS and TP Gene Expression vs Response to 5-FU/LV in Colorectal Cancer 1.2 Response Non response 1 DPD 0.8 TS Danenberg Tumor Profile Scale 0.6 TP 0.4 0.2 7 135 137 150 154 165 204 289 361 374 574 438 91 121 152 164 189 196 217 220 270 278 288 359 396 401 458 526 559 582 583 585 105m Patient ID Number Salonga et al. Clin Cancer Res 2000

Single marker analysis - Chemotherapy
Agent Marker Prognostic Predictive Efficacy Toxicity 5-FU TS + DPD (+) TP Irinocetan UGT1A1 Oxaliplatin GSTP1 ERCC1 XPD (ERCC2)

Biologic Agents in Colorectal Cancer = Monoclonal Antibodies
Fab Fc Murine Ab “momab” Chimeric Mouse-Human Ab “ximab” Humanized Ab “zumab” Human Ab “mumab” EGFR (17-1A) Cetuximab Bevacizumab Panitumumab VEGF

Nomenclature of Monoclonal Antibodies
-mab monoclonal antibody -mo-mab mouse mab -xi-mab chimeric mab -zu-mab humanized mab -mu-mab human mab -tu-xx-mab tumor-directed xx mab -li-xx-mab immune-directed xx mab -ci-xx-mab cardiovascular-directed xx mab -vi-xx-mab virus-directed xx mab Inf-li-xi-mab Beva-ci-zu-mab Ri-tu-xi-mab Pani-tu-mu-mab

mAbs Target Tumor Cell-Bound EGFR
Ligand Extracellular EGF-R Ras PI3K PTEN Raf Intracellular Akt MEK MAPK Cell survival DNA Cell Motility Proliferation Angiogenesis Metastasis

RAS (RAt Sarcoma virus)
• Three genes encode highly homologous proteins: H-RAS, N-RAS, and K-RAS • Point mutations in RAS genes occur in 30% of all cancers • K-RAS mutations present in 40% of CRC • Codons 12, 13, and 61 are most commonly involved • Mutations result in constitutive activation of RAS-RAF-MAPK signaling pathway leading to cell proliferation and enhanced cell survival Based on pfs data that makes it look like folfir/c favorable vs. folfox/cetux Early, prelim data TTF vs. PFS not yet available Immature survival data OS with cetux not yet reached, chemo alone similar to expectPFS is not final --need a few months

KRAS as Biomarker for Panitumumab Response in Metastatic CRC
PFS log HR significantly different depending on K-ras status (P < .0001) Percentage decrease in target lesion greater in patients with wild-type KRAS receiving panitumumab Patients With Wild-Type KRAS Patients With Mutant KRAS 1.0 Pmab + BSC Median in Wks Mean in Wks 1.0 BSC alone Events/N (%) Pmab + BSC 0.9 0.9 Median in Wks Mean in Wks BSC alone 0.8 Events/N (%) 115/124 (93) 12.3 19.0 0.8 0.7 114/119 (96) 7.3 9.3 0.7 76/84 (90) 7.4 9.9 0.6 0.6 95/100 (95) 7.3 10.2 Proportion With PFS HR: 0.45 (95% CI: ) 0.5 CRC; colorectal cancer; HR, hazard ratio; PFS, progression-free survival Stratified log rank test: P < .0001 Proportion With PFS 0.5 0.4 HR: 0.99 (95% CI: ) 0.4 0.3 0.3 0.2 0.2 0.1 0.1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Weeks Weeks Amado et al. JCO 2008

NCIC CTG CO.17: Randomized Phase III Trial in mCRC Cetuximab vs BSC (no cross-over)
KRAS mut KRAS wild-type All patients BSC n=83 Cetux n=81 n=113 n=117 n=285 n=287 RR 0% 1.2% 12.8% 6.6% PFS (mos) 1.8 1.9 3.8 OS (mos) 4.6 4.5 4.8 9.5 6.1 <0.0001 <0.0001 <0.0001 0.0046 Karapetis et al. NEJM 2008

NCIC CTG C0.17: Overall Survival by KRAS Status in BSC Arm
MS (months) 95% CI Mutated 4.6 3.6 – 5.5 Wild-Type 4.8 4.2 – 5.5 HR % CI (0.74,1.37) Log rank p-value: 0.97 Karapetis et al. NEJM 2008

CRYSTAL Study (1st Line)
FOLFIRI + Cetuximab N = 599 EGFR-expressing metastatic CRC R PFS Stratified by: Regions ECOG PS FOLFIRI N = 599 Primary Endpoint: PFS (independent review) Secondary Endpoints: RR, DCR, OS, Safety, QoL Sample Size: patients randomized, ITT: 1198 pts Van Cutsem et al. NEJM 2009

CRYSTAL trial: Primary endpoint PFS ITT population independent review
1.0 RR 0.8 0.9 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 FOLFIRI + Cetuximab 46.9% FOLFIRI 38.7% P = PFS estimate 8.9 mo 1-year PFS rate 23% vs 34% 8.0 mo HR = 0.851 P = 2 4 6 8 10 12 14 16 18 20 Subjects at risk FOLFIRI alone 599 492 402 293 178 83 35 16 7 4 1 Cetuximab + FOLFIRI 499 392 298 196 103 58 29 12 5 Progression-free survival time (months) Van Cutsem et al. NEJM 2009

Progression-free survival estimate
Relating KRAS status to efficacy Primary endpoint: PFS – KRAS wild-type 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 2 4 6 8 10 12 14 16 18 Months Progression-free survival estimate Cetuximab + FOLFIRI FOLFIRI KRAS wild-type (n=348) HR=0.68; p= mPFS Cetuximab + FOLFIRI: 9.9 months mPFS FOLFIRI: 8.7 months 1-yr PFS rate 25% vs 43% Van Cutsem et al. NEJM 2009

CRYSTAL: recent efficacy update after additional KRAS testing
KRAS wild-type FOLFIRI FOLFIRI + cetuximab P value n 350 316 RR (%) 39.7 57.3 < mPFS (mos) 8.4 9.9 0.0012 mOS (mos) 20 23.5 0.0094 Δ 1.5 Δ 3.5 KRAS mutated FOLFIRI FOLFIRI + cetuximab P value n 183 214 RR (%) 36.1 31.3 0.34 mPFS (mos) 7.7 7.4 0.26 mOS (mos) 16.7 16.2 0.75 Van Cutsem et al. ESMO-ECCO 2009, ASCO GI 2010 Courtesy: C. Punt

OPUS: Study design Cetuximab + FOLFOX4 N=168 EGFR-detectable mCRC R
Primary endpoint Overall confirmed response rate (as assessed by independent review) Secondary endpoints PFS time OS time Rate of curative surgery for metastases Safety Stratification by: ECOG PS 0/1, 2 Bokemeyer et al. JCO 2008

OPUS - Relating KRAS status to efficacy Secondary endpoint: PFS – KRAS wild-type 1.0 mPFS Cetuximab + FOLFOX: 7.7 mos FOLFOX: 7.2 mos HR=0.57; p=0.016 0.9 0.8 0.7 0.6 Progression-free survival estimate 0.5 0.4 Difference between arms in KRAS wt only Belt at point 0.5, thus small difference in medians but compelling Hazard Ratio 0.3 0.2 FOLFOX Cetuximab + FOLFOX 0.1 0.0 2 4 6 8 10 12 Months Bokemeyer et al. JCO 2008

OPUS - Relating KRAS status to efficacy Secondary endpoint: PFS – KRAS mutant 1.0 mPFS Cetuximab + FOLFOX: 5.5 mos FOLFOX: 8.6 mos HR=1.83; p=0.019 0.9 0.8 0.7 0.6 Progression-free survival estimate 0.5 0.4 Difference between arms in KRAS mt only Cetuximab addition to FOLFOX in KRASmt more or less implausible, suggesting better PFS for treatment without cetuximab 0.3 0.2 FOLFOX Cetuximab + FOLFOX 0.1 0.0 2 4 6 8 10 12 Months Bokemeyer et al. JCO 2008

PRIME: FOLFOX +/- P-mab PFS by KRAS Mutation Status
WT KRAS MT KRAS Events n (%) Median (95% CI) months Panitumumab + FOLFOX 199 (61) 9.6 (9.2–11.1) FOLFOX 215 (65) 8.0 (7.5–9.3) Events n (%) Median (95% CI) months Panitumumab + FOLFOX 167 (76) 7.3 (6.3 – 8.0) FOLFOX 157 (72) 8.8 (7.7 – 9.4) HR = 0.80 (95% CI: 0.66–0.97) P-value = 0.02 HR = 1.29 (95% CI: 1.04 – 1.62) P-value = 0.02 Douillard, et al. ECCO-ESMO 2009

COIN (cetuximab): First-line Study
Continuous* XELOX or FOLFOX MRC-sponsored study supported by Merck (109 UK/Irish Hospitals) Arm A First-line mCRC (n= 2445) Continuous XELOX or FOLFOX + cetuximab R Arm B Intermittent** XELOX or FOLFOX Arm C Primary endpoints: OS in patients with K-ras wild-type tumours Secondary endpoints include: OS in K-ras mutant; “all” wild-type (K-ras, N-ras, B-raf); “any” mutant, ITT PFS Response rate Quality of life Health economic evaluation 65% XELOX; 35% FOLFOX (patient/physician choice) *Treatment until disease progression or unacceptable toxicity **Stop and Go treatment (12 wks then restart at progression) Maughan, et al. ECCO-ESMO 2009 33

HR point estimate = 0.959 95% CI 0.84–1.09 p=0.60
COIN study: KRAS WT PFS Survival probability Arm A (XELOX/FOLFOX) Arm B (XELOX/FOLFOX + cetuximab) 1.00 0.75 0.50 0.25 Arm A Arm B Diff. Median PFS, months 8.6 +0.07 HR point estimate = % CI 0.84–1.09 p=0.60 6 12 18 24 30 36 42 Time (months) No. at risk Arm A Arm B 367 361 245 249 92 103 41 42 18 22 11 9 6 1 Maughan, et al. ECCO-ESMO 2009

Rationale for Combining EGFR- and Angiogenesis- Inhibitors
EGFR Inhibitors Angiogenesis Inhibitors Tumor cell growth Synthesis of angiogenic proteins Response of endothelial cells to angiogenic proteins Targets - - - Angiogenic proteins bFGF VEGF TGF- Endothelial cells Tumor Herbst et al. J Clin Oncol. 2005;23:2544.

CapOx + BEV + Cetuximab (COB-C, n=368)
CAIRO2: Study design CapOx + BEV (COB, n=368) EGFR-detectable mCRC R CapOx + BEV + Cetuximab (COB-C, n=368) Primary endpoint Progression-free survival Secondary endpoints RR OS time Toxicity Translational research Oxaliplatin d/c’d after 6 cycles i.e. after 18 weeks = 4.5 mos Tol et al. NEJM 2009

CAIRO2 – Summary Efficacy results
COB n = 368 COB-C n = 368 P-value Median PFS (mos) (HR; 95% CI) 10.7 ( ) 9.4 ( ) 0.01 Median OS (mos) 20.3 ( ) 19.4 ( ) 0.16 Response rate (CR + PR) 50% 52.7% 0.49 Disease control rate (CR + PR + SD) 94% 94.6% 0.72 Tol et al. NEJM 2009

CAIRO2 - KRAS genotyping (n=501)
KRAS wild-type n = 314 (61%) KRAS mutated n = 196 (39%) p value Median PFS (months) COB 10.6 12.5 0.80 COB-C 10.5 8.1 0.04 0.30 0.003 Median OS (months) 22.4 24.9 0.82 21.8 17.2 0.06 0.64 0.03 Tol et al. NEJM 2009

mAbs Target Tumor Cell-Bound EGFR
Ligand Extracellular EGF-R Ras PI3K PTEN Raf Intracellular Akt MEK MAPK Cell survival DNA Cell Motility Proliferation Angiogenesis Metastasis

Distribution of Mutations in COIN
Maughan, et al. ECCO-ESMO 2009

Advances Towards Personalized Therapy: What about other Molecular Biomarkers?
EGFR gene copy number (FISH) EGFR ligand overexpression – epiregulin and amphiregulin BRAF mutations PI3K/Akt mutations PTEN expression Expression of alternative, vertical signaling pathways - e.g. IGF-1R

Fluorescence in-situ hybridization (FISH) for detecting EGFR expression
• FISH analysis can show increases in EGFR copy number • Significantly increased copy number may be associated with clinical response • One study demonstrated – 8/9 responders had increased EGFR copy number vs 1/21 non-responders • Costly and technically challenging testing method • Further studies required Moroni et al, Lancet Oncol 2005

Epiregulin and Amphiregulin Levels Correlate With Disease Control
1000 2000 3000 4000 5000 6000 7000 Affymetrix mRNA level 500 1500 2500 3500 CR/PR SD Non-Responders Epiregulin p = 1.5e-05 Amphiregulin p = 2.5e-05 Subjects Khambata-Ford S et al, JCO 2007

Epiregulin/Amphiregulin Levels Correlate With PFS in mCRC
100 EREG p = 100 AREG p = EREG expression cutoff 500 High Low AREG expression cutoff 100 High Low 80 80 60 60 Patients Free of Tumor Progression (%) Patients Free of Tumor Progression (%) 40 40 20 20 100 200 300 400 100 200 300 400 Time (days) Time (days) Hazard ratio = 95% CI = to Hazard ratio = 95% CI = to Khambata-Ford S et al, JCO 2007

Mutations of PI3K pathway genes in CRC
Role of PI3K Pathway 40% of CRC tumors have mutations in PI3K pathway1 PI3K pathway dysregulation predicts Cetuximab resistance in CRC cell lines2 Of 36 tumors with PI3KCA mutations, 27 also had alteration in KRAS1 Patients treated with Cetuximab3 4/31 PI3KCA mutations (4/16 non-responders) 4/31  PTEN gene copy number 3/30 PTEN mutations (3/15 non-responders) PI3KCA mut: early or late event? RTKs PIP2 PIP3 PIK3CA P P P P P P PIP3 P P P P IRS2 P p85 PDK1 AKT2 PTEN P ? Mutations of PI3K pathway genes in CRC PAK4 P Tumours PKK1 AKT2 PAK4 AKT2/ PAK4 amp IRS2 amp INSRR ERBB4 PTEN PIK3CA CSX3 T354M wt CX10 MX20 D527E CX7 S302G HX66 R371H CO86 A279T 800del/ 968del HX63 E329K CO78 15 fold CO82 8 fold CO84 12 fold CO69 7 fold HX160 6 fold MX5 T1014M CO87 I1030M MX9 del CX28 Y88C HX170 L325H/LOH HX199 R741/F341V R88Q HX219 A86P/LOH HX242 R47S 36 cases MUT 90 cases 1. Parsons, et al. Nature Jhawer, et al. Cancer Res 2008; 3. Perrone, et al. Ann Oncol 2009

PIK3CA Point Mutations Exon 9 Exon 20 CRC Hotspots
Bader et al., Nat Rev Cancer 2005

> 85% received at least 1 prior Rx
PFS and PIK3CA Mutational Status in mCRC Patients Treated With Panitumumab/Cetuximab 110 pts > 85% received at least 1 prior Rx Cetuximab 13% Panitumumab 20% Cetuximab/Irinotecan 67% Sartore-Bianchi A et al, Cancer Res 2009

Cetuximab/Irinotecan 184 Total Patients 200
Fig. 1 Cetuximab 16 Cetuximab/Irinotecan 184 Total Patients 200 Prenen H et al, Clin Cancer Res 2009

Cape +/- Perifosine Rand Phase II
Perifosine 50 mg PO QD Capecitabine 825 mg/m2 BID d Placebo PO QD Patients with 2nd or 3rd line mCRC No prior Rx with CAP in metastatic setting Prior Rx with 5-FU or 5-FU based regimen Cycle = 21 Days Primary Objective: To compare time to progression (TTP) of P-CAP vs. CAP as 2nd or 3rd line Rx Secondary Objective: To compare overall response rate (CR + PR) and overall survival To evaluate the safety of P-CAP vs. CAP Richards et al., ASCO 2010

Median Time to Progression (TTP)
ALL EVALUABLE PATIENTS 5-FU REFRACTORY PATIENTS Median TTP: P-CAP: 28 weeks [95% CI (12, 48)] Median TTP: CAP: 11 weeks [95% CI (9, 15.9)] p-value = Median TTP: P-CAP: 18 weeks [95% CI (12, 36)] Median TTP: CAP: 10 weeks [95% CI (6.6, 11)] p-value = Hazard ratio: 0.284 (0.127, 0.636) Hazard ratio: 0.186 (0.066, 0.521) Richards et al., ASCO 2010

Median Overall Survival (OS)
ALL EVALUABLE PATIENTS 5-FU REFRACTORY PATIENTS Median OS: P-CAP: 17.7 mos [95% CI (8.5, 24.6)] Median OS: CAP: 10.9 mos [95% CI (5, 16.9)] p-value = Median OS: P-CAP: 15.1 mos [95% CI (7.3, 22.3)] Median OS: CAP: 6.6 mos [95% CI (4.7, 11.7)] p-value = Hazard ratio: 0.410 (0.193, 0.868) Hazard ratio: 0.313 (0.122, 0.802) Phase III trial (1:1 Cape/Peri vs Cape) started Richards et al., ASCO 2010

PTEN Expression and Cetuximab Efficacy
PTEN + median PFS = 4.7 months PTEN – median PFS = 3.3 months 1.0 0.8 0.6 0.4 0.2 0.0 PTEN + (n=33) PTEN – (n=22) Responders (CR+PR+SD) 12 (36%) 1 (5%) Non-responders 21 (64%) 21 (95%) Log-rank test: p=0.005 HR=0.49; 95% CI: 0.20–0.75 PFS estimate PTEN+ n=55 PTEN- Months Fisher’s Exact Test p=0.008 Concordance primary tumor sample/metastasis: 27/45 (60%) CR = complete response; PR= partial response SD = stable disease Loupakis et al, ASCO 2008 Loupakis et al, J Clin Oncol 2009

PTEN and KRAS Status: Effect on Efficacy
PTEN + KRAS wild-type median = 5.5 months All other median PFS = 3.8 months PTEN + KRAS wild-type (n=17) All other (n=28) Responders (CR+PR+SD) 8 (47%) 1 (4%) Non responders 9 (53%) 27 (96%) 1.0 0.8 0.6 0.4 0.2 0.0 Log-rank test: p= HR=0.42; 95% CI: 0.17–0.65 PFS estimate PTEN+ n=45 PTEN- Fisher’s Exact Test p=0.008 Months Loupakis et al, ASCO 2008

Sartore-Bianchi et al, Cancer Res 2009
Challenges with PTEN Expression in primary tumors does not reflect expression in metastases PTEN is not mutated in mCRC, its expression can be regulated by methylation, miRNAs, and/or other regulatory mechanisms Difficulty in standardizing IHC in different labs Supplemental Figure 1: Representative examples of PTEN positive (A, B) and negative (C, D) cases. The cases reported in A and C panels were evaluated at Ospedale Niguarda Ca’ Granda (Milan, Italy) whereas those in B and D at the Institute of Pathology in Locarno (Switzerland). Sartore-Bianchi et al, Cancer Res 2009

Non-responder: BRAF mutation 10%
Potential relationship between KRAS status and response to EGFR monoclonal antibodies, alone or in combination with irinotecan, in chemorefractory patients Non-responder: BRAF mutation 10% Responds to standard dose 22% Non-responder: Loss of PTEN or PI3K mutation % unknown KRAS wild-type Responds to increased dose ~5% Non-responder: Reason unknown % unknown KRAS mutant Non-responder: KRAS mutant 40% Wong and Cunningham, J Clin Oncol 2008

Tumor cell proliferation
BRAF Mutations in CRC BRAF is primary effector of KRAS signaling BRAF mutations: Occur most frequently in exon 15 (V600E) Found in 4%-14% of patients with CRC Mutually exclusive with KRAS mutations EGF Tumor Cell P P Ras P P Raf MEK Tumor cell proliferation and survival Erk Yarden. Nat Rev Mol Cell Biol. 2001;2:127; Di Nicolantonio. J Clin Oncol. 2008;26:5705; Artale. J Clin Oncol. 2008;26:4217.

Wild-type BRAF is required for response to EGFR inhibitors in mCRC
Patients with KRAS wild-type status 100 80 60 40 20 BRAF wild-type BRAF mutant p=0.0010 100 80 60 40 20 BRAF wild-type BRAF mutant p<0.0001 PFS (%) OS (%) ,000 1,200 1,400 Time since start of treatment (days) Time since start of treatment (days) Di Nicolantonio et al., J Clin Oncol 2008

CRYSTAL trial update: outcome in KRAS wild-type/ BRAF mutated mCRC
KRAS wt/BRAF wt (n=566) KRAS wt/BRAF mt (n=59) FOLFIRI (n= 289) FOLFIRI + Cetuximab (n= 277) (n=33) (n=26) mOS (mo) 21.6 25.1 10.3 14.1 HR [95% CI] p-valuea 0.83 [0.687–1.004] 0.0549 0.91 [0.507–1.624] 0.7440 mPFS (mo) 8.8 10.9 5.6 8.0 0.68 [0.533–0.864] 0.0016 0.93 [0.425–2.056] 0.8656 RR (%) [95% CI] 42.6 [36.8–48.5] 61.0 [55.0–66.8] 15.2 [5.1–31.9] 19.2 [6.6–39.4] p-valueb <0.0001 0.9136 aStratified log-rank test; bCochran-Mantel-Haenszel test Van Cutsem et al, ASCO GI 2010

Bevacizumab: OS independent of biomarker status in first-line mCRC
Placebo + IFL Bev + IFL Biomarker N n Median (months) n Median (months) HR (95% CI) HR All subjects 267 120 17.45 147 26.35 0.57 (0.39–0.85) KRAS mutation status Mutant Wild-type 78 152 (0.37–1.31) (0.34–0.99) BRAF mutation status Mutant Wild-type 10 217 3 97 7 120 (0.01–1.06) (0.34–0.82) KRAS and BRAF mutation status Mutant Wild-type 88 125 (0.37–1.20) (0.34–0.82) p53 mutation status Mutant Wild-type 27.70 NR (0.30–0.95) (0.32–1.42) p53 overexpression Positive Negative (0.45–1.10) (0.15–0.70) Overall survival also independent of p53, VEGF (plasma and tissue) and TSP-2 Bev = bevacizumab TSP-2 = thrombospondin-2 Jubb, et al. JCO 2006 Ince, et al. JNCI 2005 59 59

VEGF Polymorphisms and Predictive Value in E2100 (Pac +/- Bev Metastatic Breast Cancer)
Kaplan-Meier curve for overall survival (OS) in experimental arm by genotype; (A) vascular endothelial growth factor (VEGF)-2578 C/A; (B) VEGF-1154 G/A Caveats: Predictive for OS, but not PFS Did not include Pac Rx alone group Schneider, et al. J Clin Oncol; 2008

Phase III Trial of IFL +/-Bevacizumab in mCRC: PFS
1.0 HR=0.54, P< mPFS: 6.2 vs 10.6 mo 0.8 0.6 Proportion progression-free 0.4 0.2 IFL + placebo IFL + bevacizumab 10 20 30 Months Hurwitz et al. N Engl J Med 2004 Hurwitz et al. ASCO, Late breaking abstract 3646 and oral presentation.

CRYSTAL trial: FOLFIRI +/- Cetuximab: PFS ITT population, independent review
1.0 RR 0.8 0.9 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 FOLFIRI + Cetuximab 46.9% FOLFIRI 38.7% P = PFS estimate 8.9 mo 8.0 mo HR = 0.851 P = 2 4 6 8 10 12 14 16 18 20 Subjects at risk FOLFIRI alone 599 492 402 293 178 83 35 16 7 4 1 Cetuximab + FOLFIRI 499 392 298 196 103 58 29 12 5 Months Van Cutsem et al. NEJM 2009

Anti-VEGF Therapy: Tool to assess predictive marker
George Sledge

Grade 3/4 Hypertension Is Associated With Improved Median OS in E2100
25.3 mo vs mo p=0.002 Schneider et al; J Clin Oncol, 2008

Hypertension is a Biomarker of Efficacy in Patients with Metastatic Renal Cell Carcinoma Treated with Sunitinib Brian Rini et al (2010)

Analysis of early hypertension and clinical outcome with bevacizumab
“…The primary HTN endpoint was an SBP increase 20 mmHg or DBP increase 10 mmHg within the first 60 days of Tx. ….Conclusions: HTN during Tx does not predict clinical benefit from BV based on PFS or OS.….” Hurwitz et al. ASCO 2010

Cytokine increase on BEV therapy
Kopetz et al., JCO 2010

Multiple Potentially Active Biologics
Anti-VEGF bevacizumab VEGF-Trap vatalanib sorafenib Sunitinib cediranib axitinib IMC1121b Anti-EGF-R cetuximab Panitumumab erlotinib gefitinib lapatinib Further targets (selection) IGF-R mTOR PKC PI3K/AKT Dll4/Notch Hedgehog JAK/Stat C-met MEK TRAIL MMPs CDKs

Non-Specificity of VEGFR Kinase Inhibitors - The Human Kinome Tree -
The larger the red circle, the more effective the drug is for the target Markers that may be predictive for one TKI, may not be predictive for another Karaman et al., Nature Biotechnol. 2008

Bevacizumab vs EGFR Antibodies in Advanced CRC - Simplified
Agent Strength Weakness Bevacizumab Delay in tumor progression Gain in time Toxicity profile No single agent activity Weak effect on RR EGFR antibodies Single agent activity Consistent increase in RR Activity independent of line of therapy Predictive marker Gain in time to progression moderate

Conclusions CRC has morphed into 2 distinct entities:
KRAS wt and KRAS mut CRC No EGFR therapy in KRAS mut CRC! If response is primary goal (symptoms, conversion therapy), cetuximab is reasonable first-line option in KRAS wt CRC In palliative situation, BEV-containing regimen remains preferable, even in KRAS wt CRC More impressive gain in time, less toxicity Treat to progression (and perhaps beyond?) Dual antibodies not outside clinical trials in first-line therapy Might be different in salvage scenario We need new drugs in CRC – and KRAS mut CRC can be the target for new drug development

Adjuvant Therapy of Colon Cancer

AJCCv6 TNM Staging Definitions
TNM / AJCC v7 Effective Jan 2010 AJCCv6 TNM Staging Definitions Primary tumor (T) Tis Carcinoma in situ T1 Tumor invades submucosa T2 Tumor invades muscularis propria T3 Tumor invades through muscularis propria or subserosa T4 Tumor directly invades other organs or structures Regional lymph nodes (N) N0 No regional lymph node metastases N1 Metastases in 1–3 regional lymph nodes N2 Metastases in 4 or more regional lymph nodes Distant metastases (M) M0 No distant metastases M1 Distant metastases T4a: perf. visceral peritoneum T4b: invasion of organs N1a: 1 N+ N1b: 2-3 N+ N2a: 4-6 N+ N2b: >7 N+ AJCC = American Joint Committee on Cancer. National Comprehensive Cancer Network (NCCN), 2008; Greene et al., 2002.

AJCC v7 Stage II Stage III Gunderson et al, JCO 2009

Beyond 5-FU in the adjuvant setting
Completed studies: Capecitabine (X-ACT) – non-inferior to 5-FU Oxaliplatin (MOSAIC, NSABP C-07, XELOXA) Irinotecan (CALGB 89803, ACCORD-2, PETACC-3) Bevacizumab (NSABP C-08, AVANT) Cetuximab in KRAS wt CC (N1047) Ongoing studies: Bevacizumab (QUASAR-2, E5202) Cetuximab in KRAS wt CC (PETACC-8)

Adjuvant Trials in Colon Cancer with Bevacizumab
FOLFOX4 6m AVANT Stage II/III colon cancer (N=3450) XELOX 6m + Bevacizumab 12m FOLFOX4 6m + Bevacizumab 12m NSABP C-08 mFOLFOX6 6m Stage II/III colon cancer (N=2710) 25% Stage II Reported at ASCO2009 mFOLFOX6 6m + Bevacizumab 12m Drevs et al. Proc Am Soc Clin Oncol. 2003;22:284. Abstract 1142. Steward et al. Proc Am Soc Clin Oncol. 2003;22:274. Abstract 1098. Trarbach et al. Proc Am Soc Clin Oncol. 2003;22:285. Abstract 1144.

R NSABP C-08 N=2710 pts 25% stage II mFOLFOX6 q2wk X 6 mo
BEV* q2wk X 1 yr N=2710 pts 25% stage II *5mg/kg Wolmark et al ASCO 2009

NSABP C-08 – DFS Ev 3yDFS mFF6+B mFF HR P

NSABP C-08 HR over Time 0.08 0.05 0.02 0.004 0.0004

AVANT Adjuvant Colon Cancer Study
24 Weeks 48 Weeks FOLFOX4 q2wk n=3451 Stage III or high-risk stage II colon cancer 1:1:1 FOLFOX4 q2wk Bev 5 mg/kg, q2wk Bev 7.5 mg/kg q3wk Stratified by stage and region XELOX q3wk Bevacizumab 7.5 mg/kg q3wk 3451 patients were enrolled between November 2004 and June 2007 Primary analysis: compare DFS between control and each treatment arm in stage III patients Safety data presented at ESMO September 2009 by P. Hoff 82

Revised, Biomarker-driven Design of N0147
Z E Arm A mFOLFOX6 P R E G I S T KRAS WT Arm D mFOLFOX6 + Cetuximab Stage 3 Colon Cancer (N = 3768) Centralized K-ras analysis R E G I S T Arm G Adjuvant therapy per primary oncologist Report therapy given Annual status through year 8 KRAS Mut Alberts/Goldberg et al., ASCO 2010

Alberts/Goldberg et al., ASCO 2010
Phase III Trial N FOLFOX +/- Cetuximab by KRAS status in Stage III Colon Cancer: DFS KRAS WT KRAS Mut Arm 3 Year Rates (95% CI) HR P-value FOLFOX N=902 75.8% ( %) 1.2 ( ) 0.22 FOLFOX + Cmab N=945 72.3% ( %) Arm 3 Year Rates (95% CI) HR P-value FOLFOX N=374 67.2% ( %) 1.2 ( ) 0.13 FOLFOX + Cmab N=343 64.2% ( %) Alberts/Goldberg et al., ASCO 2010

Reason For Failure of Biomarker-driven Adjuvant Trial in Colon Cancer: EMT?
Epithelial phenotype Mesenchymal phenotype E-cadherin Cytokeratin Laminin-1 MUC-1 EGFR N-cadherin Vimentin Fibronectin ETS a-SMA Loss of epithelial and gain of mesenchymal markers Carcinoma in situ EMT Hematogenous dissemination Metastasis Kalluri & Weinberg, J Clin Invest 2009

Should patients with stage II colon cancer receive adjuvant therapy?

QUASAR: OS in patients with “no clear indication for chemo” (mostly stage II) 5-FU/LV vs surgery alone 100 Observation (n=1622) Chemotherapy (n=1617) 80 60 5-yr OS difference: 2.9% % of Patients 40 Patients with stage II or III colorectal cancer who had undergone complete resection of disease and received adjuvant chemotherapy (predominantly 5-FU/low-dose LV) had improved 5-year OS vs patients who underwent observation alone (80.3% vs 77.4%, P=.02). This improved OS resulted in a reduction in the risk of death of 17% (relative risk = 0.83 [95% CI, 0.71 to 0.97]). P = .02 5-year OS, Observation = 77.4% vs Chemotherapy = 80.3% Relative risk = 0.83 (95% CI, ) 20 1 2 3 4 5 6 7 8 9 10 Years QUASAR group, Lancet 2007 Gray et al. ASCO Abstract At: Accessed November 2004.

“High-risk” Stage II Colon Cancer
Clinical-pathological parameters T4 tumors Less than 10 (12) LNs examined Obstruction/perforation Lymphatic or vascular invasion Undifferentiated histology Molecular parameters Single marker vs signature - TBD

Defective MMR - Colon cancer
Characterized by presence of MSI & loss of MLH1, MSH2, MSH6 or PMS2 expression ~15% of Sporadic CC, >90% loss of MLH1 Clinical Correlations: Right sided, Female, Early stage, Better prognosis Tumors: Poorly differentiated, Signet-ring-cell, Lymphocytic infiltration, near diploid MMR-D cells resistant to 5-FU1,2 Colon cancer is now recognized as arising from at least 2 distinct pathways. In this study we focus attention on the defective mismatch repair pathway. These tumors are characterized by the presence of MSI, where tumors have multiple errors in repetitive DNA sequences, know as microsatellites, throughout their genome. These tumors also demonstrate loss of protein expression for one of the DNA mismatch repair genes MLH1, MSH2, MSH6, or PMS2. Defective mismatch repair tumors comprise approximately 15% of sporadic colon cancers, of which greater than 90% are due to loss of MLH1 by promoter hypermethylation. Clinically, dMMR tumors tend to occur in the right colon, have a tendency to occur in females, are diagnosed at an earlier stage, and have a more favorable prognosis. dMMR tumors tend to be poorly differentiated, with a signet-ring-cell histology, demonstrate lymphocytic infiltration, and tend to be diploid. Multiple pre-clinical studies have demonstrated that cells with defective mismatch repair are resistant to 5-FU. 1Carethers, 1999; 2Arnold 2003

Mismatch Repair Deficiency (MMR-D): Unique Biological Subgroup of Colon Cancer
IHC for MMR protein status MLH1+ MSH2- MLH1- MSH2+ A defective DNA mismatch repair (MMR) mechanism is a key biologic characteristic of ~15% of stage II colon cancer patients. This biological characteristic, also called MMR deficiency, represents one of two distinct mechanisms for producing colon tumors, with the other mechanism being chromosomal instability – both mechanisms lead to accumulation of genetic changes in tumors which ultimately drive tumor formation. In normal cells, the presence of a multi-protein “machine” (panel at left) allows for repair of routinely encountered errors in DNA replication. This machine ceases to function if any of its components (e.g. MLH1, MSH2 or the other proteins depicted) is missing. Loss of expression of MMR proteins in tumor, especially MLH1 and MSH2 (accounting for >95% of patients with this characteristic), results in an inability to faithfully replicate DNA, which in turn leads to accumulation of mutations which drive tumorigenesis. One readily detectable manifestation of tumor MMR deficiency is an inability to faithfully replicate microsatellite DNA sequences (short stretches of repetitive sequence DNA found throughout the genome), such that the length of these microsatellites in tumor DNA is not faithfully preserved. Differences in the lengths of microsatellite DNA sequences observed in MMR deficient tumors relative to normal tissue has thus been termed microsatellite instability (MSI). Colon tumors with MMR deficiency can be identified either by (1) IHC for the MMR proteins (top right panel) – MMR proficient = both MLH1 and MSH2 staining positive and MMR deficient = either MLH1 or MSH2 staining negative, or (2) PCR assessment of DNA markers for MSI, where high-grade MSI (MSI-H) demonstrates lack of faithful replication of DNA in tumor relative to normal tissue. In tumors exhibiting the MSI-H phenotype (bottom right panel), tumor DNA is of different lengths compared to normal tissue. MMR deficiency (MMR-D) directly results in the MSI-H phenotype. Thus, MMR-D is considered synonymous with MSI-H. It is worth noting that concordance studies of MMR status assessment by IHC for MMR proteins vs PCR for MSI have shown that the two methods have >90% concordance overall. A very recent study by CALGB, involving ~700 colon cancer patients from CALGB 89803, rigorously demonstrated that IHC for MLH1 and MSH2 had >97% concordance with PCR for MSI (using a 10 marker panel) (Bertagnolli et al JCO 2009). References: Bertagnolli MM, Niedzwiecki D, Compton CC, et al: Microsatellite Instability Predicts Improved Response to Adjuvant Therapy with Irinotecan, Fluorouracil, and Leucovorin in Stage III Colon Cancer: Cancer and Leukemia Group B Protocol J Clin Oncol ; 27: Imai and Yamamoto. Carcinogenesis 2008 Umetani, Annals of Surgical Oncology 2000 Rosen et al. Modern Pathology (2006) 19, Thus, IHC for MMR proteins and PCR for MSI detect two manifestations of the same tumor biology: MMR-D is synonymous with MSI-H MMR-P is synonymous with MSI-L/MSS PCR on tumor DNA for MSI (microsatellite instability) Imai and Yamamoto. Carcinogenesis 2008 Umetani, Annals of Surgical Oncology 2000 Rosen et al. Modern Pathology (2006) 19,

DFS/OS in Stage II MMR-D Patients (N=102)
5-yr DFS N = 47 5-yr OS N = 55 On this slide we present the results of disease free survival in patients with dMMR tumors, by stage, from the pooled dataset. In patients with stage II disease, there was a strong trend toward worsened disease free survival with treatment compared to control, with a p-value of 0.05 and a hazard ratio of No benefit from treatment was observed in these patients with stage III disease, with the survival curves essentially overlapping and a p-value of 0.86. Untreated 87% Treated % HR: 2.80 ( ) p=0.05 Untreated % Treated % HR: 3.15 ( ) p=0.03 Sargent JCO 2010

PETACC 3: Multivariate Analysis Prognostic Factors Stage II
Markers HR [95% CI] P value T4 v. T3 2.58 [ ] MSI-H v. MSS 0.28 [ ] 0.0089 18qLOH 1.37 [ ] 0.38 BRAF, v-raf murine sarcoma viral oncogene homolog B1; CRC, colorectal cancer; 5-FU/LV, 5-fluorouracil, leucovorin; K-ras, Kirsten rat sarcoma viral oncogene homologue; LoH, loss of heterozygosity; PETACC, Pan-European Trials in Alimentary Tract Cancer; SMAD4, SMA- and MAD-related protein 4 Roth AD, et al. ASCO 2009, JCO 2010.

Genomic Health: Development and Validation of an 18-Gene RT-PCR Colon Cancer Assay
Colon Cancer Technical Feasibility 761 genes Development Studies Surgery Alone NSABP C-01/C-02 (n=270) CCF (n = 765) Development Studies Surgery + 5FU/LV NSABP C-04 (n=308) NSABP C-06 (n=508) 375 genes 18 genes Selection of Final Gene List & Algorithm Validation of Analytical Methods ASCO 2009 Clinical Validation Study – Stage II Colon Cancer QUASAR (n>1200) Test prognostic, but not predictive! Kerr et al., ASCO 2009, abstr. 4000

Multivariate Analysis
QUASAR RESULTS: Recurrence Score, T Stage, and MMR Deficiency are Independent Predictors of Recurrence in Stage II Colon Cancer Multivariate Analysis Kerr et al., ASCO 2009, abstr. 4000

T3 and MMR proficient (76%) Risk of recurrence at 3 years
QUASAR Results: Recurrence Score, T Stage, and MMR Deficiency are Key Independent Predictors of Recurrence in Stage II Colon Cancer 45% 40% T4 stage (13%) 35% 30% 25% T3 and MMR proficient (76%) Risk of recurrence at 3 years 20% 15% 10% T3 and MMR deficient (11%) 5% 0% 10 20 30 40 50 60 70 Recurrence Score Kerr et al., ASCO 2009, abstr. 4000

Decision Algorithm in Adjuvant Therapy
Resected Colon Ca Stage II Stage III T4 and/or <12 LNs yes High-Risk * no yes Low-Risk dMMR FOLFOX XELOX Marker signature? no Intermed. Risk * 5-FU/LV or Capecitabine No therapy! ? *pts not considered candidates for oxaliplatin Grothey, Oncology 2010

How A Cell Works

Molecular Predictors in Clinical Decision Making for Colon Cancer

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