Promising Future Biomarkers For Colorectal Cancer: Focus on Targeted Therapies Lee M. Ellis, MD Depts of Surgical Oncology and Cancer Biology UT MD Anderson Cancer Center Houston, Texas USA

Prognostic marker –associated with clinical outcome, independent of specific treatment Predictive marker –identifies groups receiving different degrees of benefit from a therapy –specific for a given treatment (i.e., Ras for EGFR MoABs in CRC, HER2 expression for trastuzumab) Surrogate / activity marker –modulated by treatment –may be on target tissue (tumor) or surrogate tissue (i.e., lymphocytes, skin) –might or might not correlate with clinical activity A Few Definitions A “marker” is not a “marker” is not a “marker”

CRYSAL Trial We Need to Do Better!! Van Cutsem et al. NEJM 2009

EGFR –Downstream signaling pathways RAS (Lenz) Raf PTEN/PI3K –AACR Presentations (including EGFR amplifications) VEGF –? Biomarkers for Targeted Therapies in CRC I apologize in advance for some mixing and matching as some studies report more than one marker.

Survival (anti-apoptosis) Gene transcription Cell-cycle progression Angiogenesis Invasion and metastasis Chemotherapy / radiotherapy resistance Proliferation pY Ligand Antibodies to EGFR cetuximab, panitumumab EGFR-TK pY EGF Receptor: Its Role in CRC Therapy Meyerhardt & Mayer, N Engl J Med 2005 Venook, Oncologist 2005 RASRAF MEK MAPK PI3K AKT STAT PTEN pY

“Bypass” Pathways and EGFR Resistance Clin Ca Res, Jan 2005

Now that Mutated K-Ras is an Established Marker of Resistance, the Next Advance will be in the identification of resistance markers in K-Ras Wild-type tumors CRC K-Ras WT K-Ras MT B-Raf PI3K PTEN EGFR copy # EGFR MoAB ?

Bypass Pathways and EGFR Resistance Camp et al Clin Ca Res 2005

B-Raf and Resistance to EGFR MoABs Mutated in 3-15% CRC Ras and Raf mutations exclusive of each other Gain-of-function mutations –Inability to convert the active form to inactive confirmation Schubbert et al. Nat Rev Ca, 2007

“We hypothesized that in K-ras wild-type patients, B-raf mutations could have prognostic/predictive value.” All pts had progressed on at least one line of therapy –~50% received monotherapy with EGFR MoAB –~50 MoAB with chemotherapy In patients with tumors with Mut B-raf, there were NO objective responses Nicolantonio et al. JCO 2008 Wild-Type BRAF is Required for Response to..(EGFR MoABS)

Wild-type K-ras All Patients B-Raf Predicts for Benefit of Anti-EGFR Therapy in Patients with WT Ras and the Entire Cohort PFS OS Ras and PFS

Bypass Pathways and EGFR Resistance Camp et al Clin Ca Res 2005

Planchon et. al., J Cell Sci, 2008 Classic PTEN Pathway: Inhibition of the Activation of AKT PTEN is a phosphatase that blocks activation of Akt / survival pathway –Loss of function of PTEN associated with an increase in cell survival signaling

Planchon et. al., J Cell Sci, 2008 PTEN Pathway – Functions in the Cytoplasm and Nucleus Nuclear PTEN plays a role in chromosome stability, DNA repair, cell cycle arrest and cellular stability.

44th ASCO Annual Meeting, 2008 “PTEN …. Intensity was scored according to a four-tier system: 0, no staining; 1, weak; 2, moderate; and 3, strong. We attributed one, two, or three additional points if the percentage of positive cells was less than 25%, 25% to 50%, or greater than 50%, respectively. Specimens were defined as positive if the score was 4 or greater.” Loupakis et al. JCO 2009

Logrank Test: p=0.005 HR = 0,49 95% CI: PTEN+ PTEN- PTEN Expression and PFS Only PTEN in the metastasis was predictive of efficacy. –PTEN in the primary tumor was NOT predictive of efficacy. PTEN in the primary tumor and liver metastasis was concordant in only 60% of cases pAKT was NOT predictive of efficacy. Loupakis et al. JCO 2009

Challenges with PTEN Expression in primary tumors does not reflect expression in metastases –Although it is not lost or mutated in CRC, its expression can be regulated by methylation or miRNA It will be hard to standardize 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.

Bypass Pathways and EGFR Resistance Camp et al Clin Ca Res 2005

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

Prenen, H. et al. Clin Cancer Res 2009 Fig. 1 Cetuximab 16 Cetuximab/Irinotecan184

Sartore-Bianchi, A. et al. Cancer Res 2009 PFS and PIK3CA Mutational Status in mCRC Patients Treated With Panitumumab and Cetuximab Cetuximab13% Panitumumab20% Cetuximab/Irinotecan67% 110 pts > 85% received at least 1 prior Rx

EGFR –Downstream signaling pathways RAS (Lenz) Raf PTEN/PI3K –AACR Presentations (including EGFR amplifications) VEGF –? Biomarkers for Targeted Therapies in CRC

CAIRO -2 STUDY CAPOX + Bevacizumab +/- Cetuximab 755 Pts Tissue in 545  EGFR copy number 7% no difference in PFS among arms Loss PTEN 42% no difference in PFS Tol et al. Proc AACR, 2009 Abstract 691 EGFR amplification and PTEN did NOT predict for response to chemo + cetuximab therapy

BRAF & EGFR Amplification in Metastatic CRC with Wild-type K-Ras 173 Pts FactorRRPFSOS Raf+/-8 vs 31 wks7 vs 15 mos EGFR Amp + PTEN loss--12 vs 16 mos Ras Wt (116) Ras Mut (57) PTEN NegativePositive EGFR Amplification Raf Mt WT Kras/ WT BRAF/ EGFR Amp = 80% RR Laurent-Puig et al, Proc AACR 2009, A1897 Cetuximab based therapy

Do Mutations / Aberrations in the Primary Tumor Reflect the Metastasis?

PathwayPrimary TumorMetastasis K-Ras Mut16/3715/37 B-Raf Mut2/36 PTEN Loss8/3812/38 EGFR Amplification 25/3629/36 Overall, mutations do not change between the primary and the metastasis. But….expression levels or gene amplification may change.

The Role of PREDICTIVE Markers for Efficacy of EGFR MoABs The sure thingProbably Yes Maybe, jury is still outNo K-RasB-RafPI3K mutationsEGFR by IHC EGFR amplification PTEN Gene expression arrays Excluding patients from EGFR MoAB Rx by use of multiple predictive factors will greatly increase the efficacy of EGFR MoABS It is imperative to PROSPECTIVELY include biomarkers and tissue procurement in clinical trials − When possible, biomarkers in primary tumors and liver metastasis should be compared Mutational status gives you a “black and white” answer and is more likely to be reproducible relative to other biomarkers (IHC, etc)

EGFR –Downstream signaling pathways RAS (Lenz) Raf PTEN/PI3K –AACR Presentations (including EGFR amplifications) VEGF –? Biomarkers for Targeted Therapies in CRC

What Holds Promise in Anti-VEGF Therapy? None currently identified in CRC!

Hahn, O. M. et al. J Clin Oncol; 2008 Kaplan-Meier estimates for progression-free survival (PFS) of patients with low and high baseline volume transfer constant of contrast agent (Ktrans) and blood plasma volume fraction (Vp)

Schneider, et al. J Clin Oncol; 2008 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 VEGF Polymorphisms and Predictive Value in ECOG-2100 (Pac +/- Bev Metastatic Breast Cancer) Caveats: Predictive for OS, but not PFS Small numbers Did not include Pac Rx alone group

Prognostic marker –associated with clinical outcome, independent of specific treatment Predictive marker –identifies groups receiving different degrees of benefit from a therapy –specific for a given treatment (i.e., Ras for EGFR MoABs in CRC, HER2 expression for trastuzumab) Surrogate / activity marker –modulated by treatment –may be on target tissue (tumor) or surrogate tissue (i.e., lymphocytes, skin) –might or might not correlate with clinical activity A Few Definitions A “marker” is not a “marker” is not a “marker”

The Harvest of a Decade of Biomarker Studies for Anti-angiogenic Therapy George Sledge

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

Hypertension and Overall Survival: Axitinib Rini, BI et al. Proc. ASCO 2008, Abstract 3543

ReferenceNMalignancyTreatmentMain Findings Scartozzi et al. 39;25;1 1 Colorectal cancer 5-FU, irinotecan + bevacizumab 20% grade 2-3 HT; response in patients with grade 2-3 HT: 75% (n = 8) versus 32% (n = 31), P = 0.04 Rixie et al.40; 32mRCCSunitinib22.5% grade 3 HT; response in patients with grade 3 HT: OR 5.69 (95% CI ), P = 0.03 Rini et al.52Cytokine- resistant mRCC AxitinibOverall survival in patients with DBP ≥90 mmHg: not reached (n = 32) versus 12.9 months (n = 20) Rini et al.62Sorafenib- resistant mRCC AxitinibOverall survival in patients with DBP ≥90 mmHg: not reached (n = 39) versus 8.4 months (n = 23) Spano et al.69Pancreatic carcinoma Gemcitabine + Axitinib 6% grade 3 HT, 22% all-grade HT; overall survival in patients with DBP ≥90 mmHG: 13.0 months (95% CI ) versus 5.6 months (95% CI ) Friberg et al.52Pancreatic carcinoma Gemcitabine + bevacizumab 19% grade 3 HT; overall survival in patients with early HT (<56 days of treatment): 13.7 months versus 8.7 months (P = 0.007) Rini et al.32MelanomaAxitinibOverall survival in patients with DBP ≥90 mmHg: 13 months (n = 19) versus 6.4 months (n = 13) Rini et al.32NSCLCAxitinibOverall survival in patients with DBP ≥90 mmHg: 15 months (n = 19) versus 11.6 months (n = 13) Rini et al.60Thyroid cancer AxitinibOverall survival in patients with DBP ≥90 mmHg: not reached (n = 39) versus 21.6 months (n = 21) Schneider et al. 345Breast cancerPaclitaxel + bevacizumab Overall survival in patients with grade 3-4 HT (n = 52): 38.7 months versus 25.3 months (n = 293), P = 0.02 Mir et al. Ann Onc 2009 Relationship Between HTN and the Efficacy of Anti-VEGF Rx

Numerous Studies Testing Dose Escalation of VEGF Inhibitors A Dose Escalation Study of Sorafenib (BAY , NSC ) in Normotensive Patients with Advanced Malignancies- Michael Maitland, Univ of Chicago

Angiogenic Cytokines Are Increased Prior to Disease Progression In Metastatic Colorectal Cancer Patients Treated With Bevacizumab Scott Kopetz, Paulo M. Hoff, Cathy Eng, Michael Overman, Katrina Y. Glover, David Z. Chang, Robert A. Wolff, James L. Abbruzzese, Lee M. Ellis, John V. Heymach The University of Texas, M.D. Anderson Cancer Center, Houston, Texas Centro de Oncologia, Hospital Sírio Libanês, Sao Paulo, Brazil

Sample Collection Study Aim 1: Subsequent samples were obtained every two weeks n=40 with evaluable samples at the selected time points Study Methods Bevacizumab FOLFIRI Bevacizumab Cycle 1 Day 1 Cycle 2 Day 15 Cycle 3 Day 29 After BevacizumabAfter FOLFIRI + BevBaseline Cycle 4+ Day 43 Bevacizumab FOLFIRI Every 2 weeks

Cytokines Increased Prior to Progression

Conclusions: Future PREDICTIVE Biomarkers for Targeted Therapies in mCRC EGFR MoABs –K-Ras ---- current and validated! –B-Raf –Not ready for prime time PI3K mutations? PTEN? EGFR amplifications? Gene arrays (under study)? VEGF MoAB –Wide open –Can we learn from other disease sites? VEGF polymorphisms? –I predict that imaging will be the answer

ASCO 2009…Tons of Colorectal Cancer Predictive Biomarker Abstracts Type: General Poster Session Time: Sunday May 31, 8:00 AM to 12:00 PM Location: Level 2, West Hall C Type: Poster Discussion Time: Monday June 1, 8:00 AM to 12:00 PM Location: Level 2, W240A Discussion: Monday June 1, 11:00 AM to 12:00 PM Location: Level 2, West Hall E1