The HER Superfamily of Receptors and Related Pathways in Gastroesophageal Cancers Jordan Berlin, M.D. Ingram Associate Professor of Medicine Vanderbilt-Ingram Cancer Center AKA: ToGA: The starting point

Disclosures For this talk –Advisory board and research support: Roche/Genentech –Advisory Board and research support: Amgen –Research Support: Lilly/Imclone I have a lot of relationships, but no others relevant

ToGA trial design HER2-positive advanced GC (n=584) 5-FU or capecitabine a + cisplatin (n=290) R a Chosen at investigator’s discretion GEJ, gastroesophageal junction 5-FU or capecitabine a + cisplatin + trastuzumab (n=294) Stratification factors −advanced vs metastatic −GC vs GEJ −measurable vs non-measurable −ECOG PS 0-1 vs 2 −capecitabine vs 5-FU Phase III, randomized, open-label, international, multicenter study 1 Bang et al; Abstract 4556, ASCO patients screened HER2-positive (22.1%)

Rationale for trastuzumab in HER2-positive GC There is no universal standard treatment, but –fluoropyrimidine (capecitabine / 5-FU) / platinum (cisplatin / oxaliplatin)-based chemotherapy considered as reference regimen epirubicin or docetaxel sometimes added biologicals are under investigation Unmet need for new treatment options in advanced gastric cancer Some gastric adenocarcinomas are HER2 positive Trastuzumab is effective against HER2-overexpressing GC cell lines in vitro and in vivo Fujimoto-Ouchi et al 2007; Gravalos & Jimeno 2008

Primary end point: OS Time (months) No. at risk Event FC + T FC Events HR % CI 0.60, 0.91 p value Median OS T, trastuzumab

Secondary end point: PFS Event No. at risk Time (months) FC + T FC Events HR % CI 0.59, 0.85 p value Median PFS

11 3 OS in IHC2+/FISH+ or IHC3+ (exploratory analysis) Time (months) FC + T FC Events HR % CI 0.51, 0.83 Median OS Event No. at risk

CALGB / ECOG E1206: Schema Stratification: ECOG 0-1 vs 2 ADC vs. SCC ARM A: (ECF + cetuximab); 1 cycle = 21 days Cetuximab 400  250mg/m2 IV, weekly Epirubicin 50 mg/m2 IV, day 1 Cisplatin 60mg/m2 IV, day 1 Fluorouracil 200mg/m2/day, days 1-21 ARM B: (IC + cetuximab); 1 cycle = 21 days Cetuximab 400  250mg/m2 IV, weekly Cisplatin 30 mg/m2 IV, days 1 and 8 Irinotecan 65 mg/m2 IV, days 1 and 8 ARM C: (FOLFOX + cetuximab); 1 cycle = 14 days Cetuximab 400  250mg/m2 IV, weekly Oxaliplatin 85 mg/m2 IV, day 1 Leucovorin 400 mg/m2, day 1 Fluorouracil 400 mg/m2 IV bolus, day 1 Fluorouracil 2400 mg/m2 IV over 46hrs (days 1-2) Enzinger PC and Burtness B, et al. ASCO 2010

Cetuximab: chimerized monoclonal antibody - EGFR (oropharyngeal cancer, NSCLC, and colorectal cancer) EGFR expression – 3/4 of ADC and SCC 1-5 EGFR expression correlates with prognosis in esophagogastric ADC and SCC 1-5 KRAS mutations occur in approx. 2% of esophageal cancers 6 CALGB / ECOG E1206: Background 1-5 Mukaida. Cancer 1991; Itakura. Cancer 1994; Yacoub. Mod Pathol 1997; Torzewski. Anticancer Res 1997; Koyama. J Cancer Res Clin Oncol 1999; 6 Lea. Carcinogenesis 2007

CALGB 80403/ECOG 1206: Survival

So what happened? ToGA worked and there is no real signal from ECOG/CALGB trial –Is this a failure of the “science?” –Does HER 2 overexpression mean something different than HER 1 (EGFR)?  Data from breast, colon and lung cancer don’t suggest they are the same. Her 2 is a driver, but HER 1 is not in most cases –Am I reading too much into CALGB/ECOG trial?  Authors of REAL3 and EXPAND hope so

Next Steps TOGA identified that for a small subset of GE patients, trastuzamab helps improve survival but not miraculously We need to identify further targets, optimization of identified targets and how to combine targeted therapies How do we select?

HER family HER 1, 2, 4 transmembrane protein with extracellular ligand binding domains and intracellular tyrosine kinase domain HER 3 membrane bound with ligand binding domain (neuregulin, for example) but no kinase domain –HER 3 heterodimerization may be key effector of HER2 activation Activation by ligand results in homo- or heter-dimerizaiton and activation of PI3k and/or MAP K pathways

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Underlying etiology Find a driver of cell growth such as Her 2 One place to start is with the start of the cancer Helicobacter Pylori –Increasing evidence that CagA + H. Pylori stimulates malignant transformation –Some data exists on methods by which H. Pylori stimulates this process

H Pylori CAG A + H. Pylori binds to CD 74 receptor –This results in IL-8 production (other cytokines) through NF-kappa B activation –Induces CD 74 expression –MEK inhibition may decrease the IL-8 production Beswick EJ, et al J Immunol 2005;175; Sekiguchi H, et al Biosci Biotechnol Biochem. 2010;74:

What can CAG-A induce? It has been associated with activation of –EGFR (red because it appears to come up with all the below)  MAP kinase  AKT  ERK  PI3 kinase Nagy TA, et al J Infect Dis 2009; 199:641–5 Tabassam FH, et al Cellular Micro 2009:11, 70–82 Chen Y-C, et al W J Gastro 2006; 12:

Underlying etiology as a target Based on this evidence, EGFR should be a key target –Also, Ras mutations are rare (<2%) H. Pylori is not the only etiology of gastric cancer and not the cause of esophageal cancer –Likely more important in distal gastric cancer –ECOG/CALGB studied esophagus and GE junction Is it possible EGFR inhibition is more relevant in H. Pylori associated disease? Is it possible that if EGFR is an inciting event, by the time we find the cancer, it is too late to block EGFR and make a difference?

Target differences by location: Not a new concept HER 2 overexpression by location –GE junction: 24-32% –Corpus/antrum: 10-18% HER 2 overexpression by histology –Diffuse: 2-7% –Intestinal: 16-34%

Is it possible that activation of a kinase may have different effects in different cells? Colon cancer and EGFR as an example: –EGFR inhibitors don’t work (and in some settings may harm) if Ras is mutated  Is PI3 kinase irrelevant in this setting?  Or is it that unbalanced stimulation of PI3K by EGFR results in harm in some cases when Ras is also activated? To butcher Orwell, –“All pathways are equally relevant but some are more equally relevant than others”, but which pathway is more equally relevant varies

MET activation results in activation of beta catenin pathwasy, PI3kinase pathway, STAT pathway and NOTCH pathway MET appears to prolong Ras induction and Ras mediates MET activation Every protein and its brother stimulates PI3K and MAPkinase pathways

C-MET I get to talk about it because they said I should talk about all the proteins that interact with the HER family and C-MET does Overexpressed in both gastric and esophageal cancers –In esophageal cancer, expression increases in the pathway from metaplasia to adenocarcinoma Can confer poor prognosis Preclinical studies show some cell lines susceptible to MET inhibition C-MET can activate HER signaling through cross- talk with HER family receptors Anderson MR, et al Clin Cancer Res 2006;12: Arkenau H-T J Cancer Res Clin Oncol 2009;135:855–866

C-MET inhibition C-Met inhibition has worked in both gastric and esophageal cell lines –C-Met inhibition in esophageal cancer  In at least one cell line (Flo-1), MET increased PI3K/AKT and PI3K inhibition mimicked the effects of MET inhibition  That means that in Flo-1 cells, PI 3 kinase mediates MET activity  Annotating the cell lines may help us better understand what works and when Watson GA, et al Neoplasia. 2006, 8:949 – 955

C-MET Inhibition Clinically, minimal data, but not impressive –Can the lab tell us why? One example:  Took C-MET addicted cell lines and made them resistant to inhibition –Displayed C-MET gene amplification –Progressively developed Wt Kras amplification –Cells became resistant to C-Met inhibition but dependent on K-Ras –Too bad we can’t inhibit Ras Cepero V, et al Cancer Res 2010; 70; 7580–90.

Further complexity Src: a non-receptor tyrosine kinase –Commonly activated in gastric cancer –Induces activation of ERK and AKT –In cell lines sensitive to Src inhibition, this results in decreased phosphorylation of ERK and AKT and increased apoptosis In a study of 16 gastric cancer cell lines, 14 with activated Src –Dasatinib inhibited Src activated cell lines as long as MET was not activated –MET inhibition inhibited Src resistant cell lines with Src activation –Neither method inhibited cell lines without Src activation Okamoto W, et al. Mol Cancer Ther 2010;9:

HER and MET Studies have now shown that activation of HER family receptors confers resistance to MET inhibition –Activation of EGFR by providing ligand or by adding mutated, constitutively activated EGFR led to AKT and MAPK activation and resistance to MET inhibition –HER 2/HER3 upregulation conferred secondary resistance to CMET inhibition –MET independent activation of HER family receptors reactivates MAPK and AKT phosphorylation Bachleitner-Hoffman, et al Mol Cancer Ther 2008;7:3499–508 Corso et al. Molecular Cancer 2010, 9:121

What can we learn from the limited C-MET story I’ve shown? C-Met, like HER family has complex interactions with other signaling pathways Upregulation of C-MET doesn’t always mean C- MET sensitivity There are many mechanisms of resistance to C- MET –Mutations in another pathway, –Upregulation of pathway proteins –Gene overexpression –Stimulation by ligand of another, complimentary (HER) pathway In turn, C-MET activation can confer resistance to inhibition of another pathway

Bringing it Back to HER Why is trastuzamab good, but no miracle? –Cross-talk between pathways is clearly important –Genetic variations in the interacting pathways occur (eg AKT, PTEN, PI3kinase) –Mutations in interacting pathways occur (Ras 2%, AKT 2%, PI3K 16%) –All of the above can confer either primary or secondary resistance Hildebrandt MAT, et al J Clin Oncol 2009;27: Barbi et al. Journal of Experimental & Clinical Cancer Research 2010, 29:32 Soung YH, et al Oncology 2006;70:285–289

HER is at the top of the cascade, why not go to the bottom?

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The bottom (or close to it) Yoon, et al showed that different co-mutations led to differential responsiveness of lung cancer lines to MEK inhibition In HER activated, MET resistant cell lines, MAPK and AKT inhibition were both required to inhibit cell growth PI3K mutations confer resistance to MEK inhibitors, especially in presence of Ras mutation And the list goes on Yoon Y-K, et al MOLECULAR CARCINOGENESIS 49:353–362 (2010) Bachleitner-Hoffman, et al Mol Cancer Ther 2008;7:3499–508 Wee S, et al Cancer Res 2009;69:

Conclusions I Cancer is “driven” to grow The HER superfamily and its downstream effectors are drivers of many gastroesophageal cancers (and other cancers too) Targeting this superfamily will likely yield results But to truly win, we have to get smarter

Gettin’ Smarter Characterize our cell lines Determine which variables predict for sensitivity to which inhibitors Test our patients for these variables and treat based on these variables Determine mechanisms of resistance so we are ready to adjust It’s time to split, not lump –Challenge will be in trial design