Accepted Manuscript Title: Biomarkers for personalized medicine in GI cancers Author: Shuang Yin Zhang, Shuang Qin Zhang, Ganji Purnachandra Nagaraju, Bassel F. El-Rayes PII: DOI: Reference:

S0098-2997(15)00037-0 http://dx.doi.org/doi:10.1016/j.mam.2015.06.002 JMAM 602

To appear in:

Molecular Aspects of Medicine

Received date: Accepted date:

27-3-2015 2-6-2015

Please cite this article as: Shuang Yin Zhang, Shuang Qin Zhang, Ganji Purnachandra Nagaraju, Bassel F. El-Rayes, Biomarkers for personalized medicine in GI cancers, Molecular Aspects of Medicine (2015), http://dx.doi.org/doi:10.1016/j.mam.2015.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

BIOMARKERS FOR PERSONALIZED MEDICINE IN GI CANCERS Shuang Yin Zhang1, Shuang Qin Zhang2, Ganji Purnachandra Nagaraju1, and Bassel F. El-Rayes1 1

Department of Hematology Oncology, Winship Cancer Institute, Emory University, Atlanta, GA; 2Department of Medicine, Section of Hematology and Oncology, University of Chicago, Chicago, IL Word Count:

Abstract Text Table

87 7,678 1

Key words:

Biomarkers, personalized medicine, gastrointestinal malignancy, gastric cancer, pancreatic cancer, colorectal cancer

Correspondence: Bassel F. El-Rayes 1365 Clifton Rd NE Atlanta, GA 30322, USA Phone: 1-404-778-1900, Fax: 1-404-686-4411 E-mail: [email protected]

Disclosure of Potential Conflict of Interest: BFE receives research support from Roche/Genentech, Lily/ Imclone, BMS, Synta Pharmaceuticals, Pfizer, Novartis, Cleave Biotech, Kirin Pharmaceuticals and Boston Biomedical Incorp (BBI). BFE also participates as a consultant to Roche and to Genentech. Other Authors declare no competing financial interests.

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Abstract: Gastrointestinal malignancies are a major health care challenge due to the high incidence and overall poor outcome. A biomarker is a molecular characteristic of a tumor that may be utilized in the initial risk assessment and the subsequent management of the patient. This review focuses on the most pertinent prognostic and predictive biomarkers used in the clinical management of gastric, pancreas, and colon cancer. The available assays, limitations and clinical use for each biomarker are reviewed. The clinical trials evaluating novel biomarkers in GI cancers are discussed.

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1. Introduction A biomarker is a characteristic or a factor that “could be the indicators of normal biologic processes, pathologic processes, or pharmacologic responses to therapeutic interventions” (Biomarkers Definitions Working, 2001). A cancer biomarker has been defined as “a molecular, cellular, tissue, or process-based alteration that provides indication of current, or more importantly, future behavior of the cancer”(Hayes et al., 1996). The utility of cancer biomarkers spans the entire healthcare spectrum from basic science laboratories to bedside applications. In the clinical setting, cancer biomarkers are currently used for screening, risk stratification, prevention, diagnosis, treatment, and surveillance(Vanessa Deschoolmeester, 2012). A prognostic biomarker serves as an indicator of how the patient is projected to react to their disease burden, regardless of the therapy they may receive. Prognostic biomarkers do not provide information regarding how the patient will respond after administration of the therapy. In personalized medicine, prognostic biomarkers may allow selection of patients based on risk of disease recurrence for neoadjuvant or adjuvant therapy or may identify patients at higher risk of rapid disease progression for more intensive treatment regimen. A predictive biomarker serves as an indicator of how the patient is projected to respond to therapy. In personalized medicine, predictive biomarkers can be used to exclude therapies that the tumor will not respond to or select therapies that have a high chance for tumor response. Predictive markers may be utilized as a target for therapy as well as a tool to predict the effectiveness of treatment(Oldenhuis et al., 2008). The development of cancer biomarkers must surmount two primary challenges: the discovery of candidate markers and the validation of those candidates for specific uses. The discovery process entails possessing the necessary technology to identify, explore, reliably measure and explain the complex biochemistry of a potential biomarker and its consistent role in a disease process. The validation process refers to the costly and arduous journey of proving that a surrogate marker accurately reflects a clinical endpoint with sufficient sensitivity, specificity, and positive predictive value in a disease(Moses, 2007). Personalized cancer therapy is tailoring treatment recommendations for each individual patient based on their genetic characteristics and their tumor related biomarkers. The aim of personalized therapy is to either increase the efficacy of treatment or lower the potential side effects by selecting the appropriate treatment based on the unique features of the patient and the tumor. The aim of this paper is to review the use of prognostic or predictive tumor biomarkers for personalized therapy that are currently in use or in development in the area of GI malignancies.

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2. Gastric Cancer Gastric malignancy is the second leading cause of cancer death worldwide(Gravalos and Jimeno, 2008; Wagner and Moehler, 2009). Based on clinicopathologic features, gastric cancer can be classified into two major subtypes: the intestinal and the diffuse form. The intestinal form of gastric cancer is typically located in the corpus and antrum of the stomach. The intestinal type develops in the background of chronic atrophic gastritis usually related to h. pylori infections. In contrast, the diffuse form of gastric cancer is found in the proximal stomach and gastroesophageal junction (GEJ) with a high incidence in the obese, young male population suffering from chronic reflux disease. This type typically confers a comparatively worse prognosis(Wagner and Moehler, 2009). Treatment options for gastric cancer remain limited. In the localized, non-metastatic setting, multi-modality therapy with surgical resection and chemotherapy continues to serve as the mainstay of treatment. In the metastatic setting, the combination of fluoropyrimidine and a platinum analogue either alone or in combination with a third drug such as an anthracycline or taxane are the most commonly used regimens. The median survival for patients with advanced stage gastric cancer remains at 8-10 months(Lorenzen and Lordick, 2011). HER2-neu HER2 is a proto-oncogene encoded by ERBB2 on chromosome 17q21. It is a 185-kDa tyrosine kinase receptor belonging to the epidermal growth factor receptor (EGFR) family, which comprises of HER1 (or EGFR), HER2, HER3, and HER4. HER2 has an extracellular ligandbinding domain, a transmembrane domain, and an intracellular domain(Akiyama et al., 1986). Though there is no identified ligand for HER2, heterodimerization of HER2-HER3 activates downstream signaling pathways including Ras/Raf/mitogen-activated protein kinase (Ras/Raf/MAPK), phosphatidylinositol-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR), leading to activation of gene transcriptional programs(Okines et al., 2011). Aberrant amplification or overexpression of HER2 plays a critical role in the initiation and progression of HER2-driven tumors(Hsieh and Moasser, 2007). Clinically, HER2 status is assessed by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH). Reliable HER2 testing is critical for appropriate use of anti-HER2 therapy. Despite controversy regarding which method yields a higher predictive value for anti-HER2 response, the general consensus is that complementary use of IHC and FISH is the ideal option(Gomez-Martin et al., 2013; Stenzinger et al., 2012; Yan et al., 2011). Initial assessment of HER2 status by IHC seems to be the preferred methodology(Ruschoff et al., 2010) with FISH being used for the intermediate cases by IHC. Increased HER2 expression has been reported in a wide range (13-23%) of gastric carcinomas with variability attributed to different methods used, subjectivity in interpretation of results with FISH as compared to IHC, tumor location, histological subtype and inherent intra-tumoral heterogeneity(De Vita et al., 2010; Lorenzen and Lordick, 2011; Yoon et al., 2012). Trastuzumab is a recombinant humanized IgG1 monoclonal antibody that targets the extracellular domain of HER2 and inhibits HER2-mediated signaling. In the landmark Trastuzumab for Gastric Cancer (ToGA) study, patients with gastric or GEJ tumors with HER2 overexpressionwere randomized to chemotherapy alone (fluoropyrimidine plus cisplatin) [n=296] ortrastuzumaband chemotherapy [n=298]. Patients were deemed HER2 positive if tumor samples were scored 3+ by IHC or if they were FISH positive; IHC 2+ were retested by 4

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FISH to confirm positivity. Notably, there was a high degree of concordance between IHC and FISH(Bang et al., 2010). Three thousand six hundred and sixty five (3665) patients were screened to identify 810 patients with HER2 overexpression with a total of 594 patients satisfying all study criterions and ultimately participating in the trial. The addition of trastuzumab yielded significantly improved overall survival (13.8 vs 11.1 months), progression free survival (6.7 vs 5.5 months), time to progression (7.1 vs 5.6 months), and durable response (6.9 vs 4.8 months). There was no difference in incidence of grade 3 or 4 adverse events between the two cohorts(Bang et al., 2010). Based on prior preclinical findings(Fujimoto-Ouchi et al., 2007; Matsui et al., 2005; Tanner et al., 2005) and results from the ToGA study, Trastuzumab in combination with chemotherapy was approved for first line treatment of metastatic HER2positive gastric cancer patients in the United States in October 2010(Pazdur, 2013). Lapatinib is a dual reversible tyrosine kinase inhibitor of HER2 and EGFR and has shown activity against Her2 positive breast cancer. Lapatinib has been evaluated in two randomized trials in HER2 positive gastric cancer. In the phase III TyTAN trial, patients received lapatinib plus paclitaxel or single agent paclitaxel in the second line setting for treatment of HER2 positive gastric cancer. The trial failed to meet its primary endpointimprovement in OS(Satoh et al., 2014). The TRIO-013/LOGiC phase III trial evaluated the addition of lapatinibto capecitabine and oxaliplatin in gastric adenocarcinoma. The addition of lapatinib did not have a significant impact on OS(J. Randolph Hecht, 2013). In addition to trastuzumab and lapatinib, other strategies targeting HER2 are under investigation. Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that combines trastuzumab with targeted delivery of DM1. Based on preclinical evidence of T-DM1 xenograft tumor models(Barok et al., 2011), a multicenter phase II/III study of T-DM1 versus Taxane in patients with previously treated advanced HER2 positive gastric cancer has been launched (GATSBY trial, NCT01641939). Pertuzumab is a monoclonal antibody that targets the extracellular heterodimerization domain of HER2 with subsequent inhibition of HER2/HER3 heterodimerization. In the preclinical setting, pertuzumab displayed efficacy in combination with trastuzumab and T-DM1(Yamashita-Kashima et al., 2011). A multicenter phase III trial of pertuzumab in combination with trastuzumab and chemotherapy in patients with HER2 positive metastatic gastric cancer is ongoing (JACOB trial, NCT01774786). Taken together, HER2 is a predictive biomarker and has a central role in the management of advanced stage gastric/GEJ cancer. C-Met The c-Met proto-oncogene encodes the c-Met receptor, a 190-kD heterodimericglycoprotein composed of an extracellular alpha-chain and a transmembrane beta-chain. When the c-Met receptor binds to its ligand, the hepatocyte growth factor (HGF), a signal is transmitted through the cytoplasmic tyrosine kinase domain of the transmembrane beta-chain.Activation of c-Met leads to downstream signaling through the RAS/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, signal transducer and activator of transcription (STAT), Ras-related C3 botulinum toxin substrate 1 (RAC1)-cell division cycle 42 (CDC42) and p21 activated protein kinase (PAK) pathways(Giordano et al., 1989; Wu et al., 1998). Under normal physiological conditions, the cMET-signaling pathway is responsible for developmental morphogenesis(Trusolino et al., 2010). Dysregulation of this pathway plays a critical role in tumor survival, growth, and metastasis(Webb et al., 1998). In gastric cancer, c-MET dysregulation can occur through constitutive activation or overexpression in the presence or absence of gene amplification(Jeffers et al., 1998; Webb et al., 1998).Clinically, there is currently no standard approach for evaluation of c-Met activation. 5

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Commonly used approaches include protein overexpression by immunohistochemistry (IHC) and gene amplification by RT-PCR/fluorescence in situ hybridization (FISH). By these techniques, cMet has been reported to be overexpressed in 18-82% of gastric cancer tissues while gene amplification was detected in 4-20% of patients(Hara et al., 1998; Janjigian et al., 2011; Kuniyasu et al., 1992). Findings of prevalence have clearly varied among studies, and perhaps may be due to differences in methods and techniques. Based on extensive investigation, there appears to be consensus that high MET gene amplification and expression in gastric cancer serve as an independent negative prognostic factor for this patient population(Nakajima et al., 1999; Tang et al., 2004). Yu et al performed a systemic review and meta-analysis of 15 studies that assessed survival in gastric cancer by cMet status (n=2210 patients). High c-Met expression was significantly associated with poorer overall survival in patients with gastric cancer, independent of disease stage(Yu et al., 2013). Peng et al evaluated 14 studies (n= 2,258 patients) by meta-analysis and similarly concluded that MET overexpression directly correlated with poor overall survival (HR 2.57)(Peng et al., 2014). These findings are consistent with studies showing poor prognosis for patients with amplified MET expression in other cancer types, such as non-small cell lung cancer, colorectal, ovarian, and breast cancer(Sierra and Tsao, 2011). Given that a significant proportion of gastric cancers harbor cMET overexpression and/or gene amplification and that such overexpression has been implicated in the progression of gastric carcinoma(Inoue et al., 1997; Nakajima et al., 1999), c-Met has been identified as a potential therapeutic target. Multiple agents targeting this tyrosine kinase receptor have been introduced into clinical trials including monoclonal antibodies such as rilotumumab (monoclonal antibody to HGF) and onartuzumab(monoclonal antibody to cMET), selective small molecule inhibitors (AMG 337tivantinib), and non-selective small molecule inhibitors (crizotinib, foretinib, cabozantinib) (Peters and Adjei, 2012). The randomized phase III clinical trial of mFOLFOX6 combined with either onartuzumab or placebo in patients with metastatic HER2-negative and MET-positive gastric adenocarcinoma (NCT01662869) showed no benefit with the addition of onartuzumab when combined with mFOLFOX6. The phase I trial of AMG 337 in advanced MET-positive solid tumors (NCT01253707) showed treatment response to AMG 337 in the subset of patients with MET-amplified GI tumors. Additionally, a phase III multicenter study is accruing patients with untreated advanced MET-positive gastric adenocarcinoma to compare epiribucin, cisplatin and capecitabine (ECX) with rilotumumabor placebo(NCT01697072). Results are not yet available. In addition, a phase II trial of tivantinib combined with FOLFOX as first-line therapy in metastatic gastric adenocarcinoma (NCT01611857), a phase II trial assessing efficacy of crizotinib in patients (including gastric adenocarcinoma) harboring ALK, MET, or ROS1 alterations (NCT02034981). Notably, phase II studies of foretinib and cabozantinib in patients with advanced gastric adenocarcinoma indicated lack of objective response(Schoffski P, 2010; Shah et al., 2013). If the current trials demonstrate benefit for these agents, then c-Met may serve as a prognostic and predictive biomarker in gastric cancer. PD-1and PD-L1 PD-1, an immune-inhibitory receptor of the CD28 family expressed on the surface of T cells, plays a critical role in tumor immune escape. Under normal physiologic conditions, PD-1/PD-L1 interaction maintains peripheral tolerance of self-antigen. However, in the tumor microenvironment, binding of PD-1 to its ligand, PD-L1 on a tumor antigen-presenting cell, propagates an inhibitory signal with subsequent reduction of T cell proliferation, induction of tumor-specific T cell apoptosis, and resistance of tumor cells to cytotoxic T cell effects(Dong et al., 2002; Keir et al., 2006; Okazaki and Honjo, 2006; Tsushima et al., 2007). PD-1 and PD-L1

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are being evaluated as prognostic markers and predicative biomarkers in several tumor types including gastric cancer. There is currently no consensus on the methodology to evaluate PD-1/PD-L1 pathway. Assays can be divided into serum-based or tissue based assays evaluating PD-L1. Zheng et al compared circulating soluble PD-L1 (sPD-L1) level by enzyme-linked immunosorbent assay (ELISA) in 80 patients with advanced gastric adenocarcinoma with 40 healthy volunteers. It was found that expression of PD-L1 was upregulated in the former group (p=0.006) and that such upregulation was associated with nodal metastasis (p=0.041). Interestingly, in a subanalysis of the 80 patients with gastric adenocarcinoma, higher sPD-L1 levels were associated with better overall survival (p=0.028)(Zheng et al., 2014). These results are contrary to other series evaluating PD-L1 in tissue-based assays. Oki et al reported that PD-L1 overexpression, as assessed by flow cytometry, was associated with tumor invasion (p=0.011) and worse overall 5-year survival (36.7% vs. 48.9%) in patients with gastric carcinoma(Eiji Oki, 2014). Similarly, high levels of PD-L1 in gastric carcinoma tissue detected by IHC correlated with poor overall survival(Wu et al., 2006). The role of PD-L1 as a prognostic biomarker is still investigational at this time. The role for targeting PD-1/PD-L1 in gastric cancer is evolving with promising preliminary data. Muro and colleagues reported findings from the gastric cohort of an international Phase Ib trial, which evaluated tolerability and efficacy of pembrolizumab, an anti-PD-1 antibody that has shown anti-tumor activity in other solid malignancies(Antoni Ribas, 2014; Naiyer A. Rizvi, 2014). Of the 162 patients with advanced gastric cancer screened, 65 were defined as PD-L1 positive by IHC, of which 39 patients enrolled. After treatment with pembrolizumab 10 mg/kg every 2 weeks, patients were reported to have an overall response rate (ORR) of 31% at 6 months with preliminary evidence suggesting an association between PD-L1 expression and ORR (p= 0.071). These findings suggest a role for PD-L1 as a predictive biomarker in gastric cancer. A phase II study, expected to begin enrollment in early 2015(K. Muro, 2014) will be evaluating pembrolizumab in gastric cancer.

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3. Pancreatic Cancer Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related mortality in Western civilization with a 5 year OS of less than 5%(Surveillance, 2014; Yeo and Cameron, 1998). Due to its late clinical presentation and intrinsic resistance to standard of care therapy(Pizzi et al., 2009), PDAC has the worst prognosis of all gastrointestinal malignancies(Surveillance, 2014; Yeo and Cameron, 1998). Identifying accurate biomarkers to detect subtypes of PDAC that may benefit from radiation or specific chemotherapy approaches would be critical in improving the therapeutic outcome of these patients. Challenges in past studies have involved limited availability of tissue as well as the uniformly poor outcome of these patients. At present, the only biomarker commonly used in PDAC is CA 19-9. However, it is limited to monitoring therapy, rather than for predictive purposes(Bunger et al., 2011). SMAD4 SMAD4 (also known as DPC4), a member of the SMAD family, is located on chromosome 18q21.1 and identified as a tumor suppressor gene(Hahn et al., 1996). SMAD4 mediates signal transduction of the transforming growth factor-β (TGF-β) family, which regulates a plethora of biological events, in particular potent inhibition of cell growth by G1 arrest(Blobe et al., 2000; Massague et al., 2000). TGF-β ligand initiates signaling by binding to and activating TGF-β type I receptor (TGFBRI) and TGF-β type II receptor (TGFBR2). TGFBR1 phosphorylates receptor-regulated SMADs (SMAD2 or SMAD3), which in turn heterodimerizes with SMAD4. This prompts translocation of the complex to the nucleus where SMAD4 plays a critical role in transcriptional activation(Liu et al., 1997). In 55% of PDAC cases, there is inactivation of SMAD4, either by homozygous deletion (35%), or by mutation in one allele coupled with loss of the other allele (20%)(Hruban et al., 1998). These abnormalities in SMAD4 usually occur in the advanced stage disease and are associated with subsequent rapid progression (Wilentz et al., 2000). SMAD4 expression is associated with improved clinical outcome in patients with PDAC. Tascilar et al reported that median survival of PDAC patients with intact SMAD4 protein expression appears to be longer than patients without SMAD4 expression (19.2vs 14.7 months, p=0.03), independent of tumor size, metastatic status or pathological stage (Tascilar et al., 2001). Iacobuzio-Donahue et al obtained tissue from 76 pancreatic cancer patients via rapid autopsy and compared the tissues’ histologic features with initial stage at diagnosis, progression of disease, and SMAD4 expression. Findings indicated that SMAD4 expression correlates closely with the development of locally advanced disease while loss of SMAD4 expression predicts progression to metastatic PDAC (Iacobuzio-Donahue et al., 2009). These findings were further supported in subsequent work(Blackford et al., 2009) showing worse median overall survival in surgically resected pancreatic cancer patients with SMAD4 gene inactivation (11.5 months) compared to an improved survival in patients with SMAD4 expression (14.2 months). In the same context, there is an ongoing phase II study of SMAD4 expression as a biomarker in unresectable pancreatic cancer to guide therapy in terms of high dose radiochemotherapy versus systemic therapy based on the likelihood of local or metastatic progression of disease (RTOG 1201). Targeting the TGF-β pathway in the setting of SMAD4 inactivation would be a rational strategy since loss of Smad4 function causes TGF-β to change from a tumor-suppressive to tumorigenic pathway (Chen et al., 2014b; Zhao et al., 2008). In line with these findings, it has been demonstrated that re-activation of SMAD4 restores TGF-β mediated growth inhibition and apoptosis in PDAC cell lines(Dai et al., 1999; Lagna et al., 1996). Furthermore, TGF-β isoforms are found to be overexpressed in PDAC and are 8

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correlated with decreased overall survival(Friess et al., 1993; Lu et al., 1997). Wang et al identified compounds UA62001(Wang et al., 2006) and UA62784(Wang et al., 2009) that specifically target SMAD-4 deficient pancreatic cancer cells, but this has not yet been translated to the clinical setting. BRCA or PALB2 mutations BRCA1 and BRCA2 tumor suppressor genes encode enzymes that regulate the homologous recombination repair double strand breaks (HR-DSBs) pathway(Venkitaraman, 2002) in response to DNA damage. In patients with BRCA mutation(s), this pathway is compromised, allowing for genomic instability, and predisposing cells to malignant transformation(Tutt and Ashworth, 2002). Germline mutations of BRCA1 and BRCA2 are associated with increased risk of developing PDAC (doubled in female BRCA carriers)(Iqbal et al., 2012), breast and ovarian cancer(King et al., 2003). While incidence of BRCA1 and BRCA2 germline mutations in PDAC varies between 5-10% in the general population, it is higher (16%) in patients of Ashkenazi Jewish descent(Lucas et al., 2013). The PALB2 (Partner and Localizer of BRCA2) physically binds BRCA1 and BRCA2 together, forming the BRCA complex, and as a result plays a significant role in tumor suppression(Hofstatter et al., 2011). Studies have shown that familial pancreatic cancer may be closely associated with mutations in the PALB2 gene with a prevalence of approximately 3% (Jones et al., 2009; Slater et al., 2010). Mutation in the BRCA or PALB2 gene could provide a rationale target for anti-tumorigenic agents, including poly-ADP ribose polymerases (PARP) inhibitors, DNA cross-linking agents (mitomycin C and platinum), and irinotecan. PARP are nuclear enzymes that repair DNA single-stranded breaks via the base excisional repair (BER) pathway, a pathway believed to serve as a compensating mechanism of DNA repair in the presence of BRCA mutation(Carden et al., 2010; Plummer and Calvert, 2007; Rouleau et al., 2010). By virtue of synthetic lethality, it is hypothesized that concomitant compromise of both pathways (HR-DSB and BER) via mutation and inhibition, respectively, induces tumor cell death(Polyak and Garber, 2011). A number of preclinical(Chen et al., 2014a; Jacob et al., 2007; Porcelli et al., 2013; Yuan et al., 2013) and clinical (Fogelman et al., 2011; Kaufman et al., 2014; Lowery et al., 2011; Pishvaian MJ, 2013) studies have found that PARP inhibition either as monotherapy or in combination with chemotherapy shows promising efficacy in PDAC patients with BRCA mutation. More recently during ASCO 2014, O’Really et al presented findings from their phase IB study of veliparib (ABT-888) in combination with gemcitabine and cisplatin in PDAC patients with BRCA or PALB2 mutations. They report veliparib 80 mg BID on days 1-12 every 21 days in combination with platinum based regimen to be well tolerated and to induce a partial response in 56% (5/9) of BRCA mutated PDAC patients(O'Reilly EM, 2014). These results have provided the basis for a phase II trial of gemcitabine and cisplatin with or without veliparib (NCT01281150). Investigation of other oral PARP inhibitors such as rucaparib and olaparib are underway. For example, a phase II single arm study aims to assess the response rate of BRCA mutated PDAC patients who receive continuous rucaparib (NCT02042378). Additionally, in a multicenter phase II trial, olaparib (AZD2281) was assessed for single agent efficacy in patients with BRCA1/2 associated malignancies; a tumor response rate of 22% (5/23) was reported in the BRCA mutated PDAC cohort(Kaufman et al., 2014). Furthermore, there is an ongoing multicenter phase I/II trial of ICM (irinotecan, cisplatin, mitomycin C) with or without olaparib in PDAC patients who harbor BRCA mutation (NCT01296763). DNA crosslinking agents such as mitomycin C (MMC) and platinum-based therapy have also been demonstrated to have particular efficacy in patients with BRCA mutated PDAC. In the presence of BRCA mutation, the tumor cell is rendered more susceptible to DNA cross linking 9

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agents given its decreased ability to repair the DNA crosslinks(McHugh et al., 2001). MMC is an anti-tumor antibiotic with potent DNA cross-link activity that interferes with fundamental cellular process such as DNA replication, RNA transcription, cell cycle arrest and apoptosis. Chalasani et al(Chalasani et al., 2008) reports a case of clinical response to MMC in a patient with BRCA mutated PDAC. Of note, a phase II clinical trial that evaluated efficacy of MMC in PDAC patients carrying BRCA2 mutation was proposed, but withdrawn prior to enrollment (NCT00386399). Superior therapeutic response to platinum-based chemotherapy in BRCAassociated PDAC is suggested by a number of studies(Golan et al., 2014; Lowery et al., 2011; Oliver GR, 2010; Sonnenblick et al., 2011). In a recent publication, Golan et al(Golan et al., 2014) performed a retrospective study of patients with BRCA/12-associated PDAC at three institutions. They report superior OS for patients with stage III/IV disease treated with platinum (n=22) compared to patients treated with non-platinum (n= 21) based chemotherapy regimens (22 vs 9 months, p = 0.039). Even though the general patient population with PDAC was not shown to have an OS benefit when treated with a platinum-based regimen(Heinemann et al., 2006; Poplin et al., 2009), the above data indicate that patients with BRCA-associated PDAC may actually experience a therapeutic advantage. Further studies are also needed to elucidate the efficacy of irinotecan, a DNA topoisomerase I inhibitor, in this subset of patients with BRCA-associated PDAC. DNA topoisomerases are nuclear enzymes that regulate basic DNA processes, such as replication, transcription, and recombination(Creemers et al., 1994). Patients with BRCA defect and subsequent defective homologous recombination are suggested to be particularly sensitive to anti-tumor activity of irinotecan. James et al report a case of a 71-year-old patient with BRCA2 mutated PDAC who experienced disease control for 13 months while receiving single agent irinotecan(James et al., 2009). Aside from the previously mentioned phase I/II trial of ICM (irinotecan, cisplatin, mitomycin C) with or without olaparib in BRCA-associated PDAC (NCT01296763), to our knowledge, there is currently no clinical trial dedicated to assessing efficacy of single agent irinotecan in this patient population. KRAS KRAS is a well-established proto-oncogene commonly involved in a wide range of human malignancies. In the presence of KRAS mutation, the KRAS protein is constitutively activated and upregulates downstream proliferative pathways, independent of upstream epidermal growth factor receptor signaling(Vakiani and Solit, 2011). In PDAC, KRAS mutations are the most frequently noted genetic alterations (>90%)(Jones et al., 2008). Patients with PDAC tend to harbor KRAS mutations in codon 12, occasionally in codon 13, and less frequently in codon 61(Garcea et al., 2005; Smit et al., 1988). Sinn et al performed Sanger sequencing of 153 patients and found 68% of cases to harbor KRAS mutations in codon 12 or 13. The presence of KRAS mutation was found to be an independent prognostic factor with an associated decreased OS compared to KRAS wild-type (12.7 vs 20.7 months, p=0.03). Interestingly, there was no significant association between KRAS status and pathologic parameters including tumor grade, tumor stage or lymph node status(Sinn et al., 2014). Targeting the KRAS pathway in PDAC has been relatively discouraging. Given that attempts to directly inhibit KRAS oncogenic activity have been ineffective(Berndt et al., 2011), focus has transitioned from targeting upstream to downstream effectors of KRAS, such as MEK(Bodoky et al., 2012). Altogether, KRAS mutational status holds prognostic value though its role as a predicative marker remains to be determined.

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4. Colon Cancer Colorectal cancer (CRC) is the fourth leading cause of cancer death globally, accounting for 60,000 deaths yearly. While hereditary factors certainly play a key role in the development of CRC, the majority occurs sporadically and progress via the adenoma-carcinoma sequence. The incidence of colorectal cancer is higher in men than women and the median age at diagnosis is 70 years. Geographically, Europe, North America, and Oceania harbor the highest incidence while countries in South/Central Asia and Africa appear to have the lowest incidence(Ferlay et al., 2015). With the advancement of treatment modalities, the prognosis of patients with colorectal cancer has gradually improved in the past decade. The most important prognostic factor is stage at diagnosis. The 5-year survival is greater than 90% in patients with stage I disease, 69% in patients with regional spread, compared to 10% in patients with stage IV disease(Siegel et al., 2012) Treatment options for colorectal cancer consist of multiple modalities. Surgical resection remains the cornerstone of treatment for stage I/II colon cancer while adjuvant chemotherapy is indicated for patients with high-risk stage II or stage III colon cancer(Brenner et al., 2014). Chemotherapy is the main treatment for patients with stage IV disease. Several prognostic and predictive biomarkers are commonly used in the clinical management decisions for patients with stage II, III and IV colorectal cancer. Microsatellilte Instability Testing (MSI) Three distinct genetic/epigenetic pathways have been shown to drive the transformation towards colon cancer: microsatellite instability (MSI), chromosomal instability (CIN) or CpG island hypermethylation (CIMP)(Dutrillaux, 1995). Microsatellites are repetitive DNA di- and tri-nucleotide sequences MSI also known as mismatch repair deficiency (MMR-D), occurs when there are mutations in the DNA mismatch repair genes, such as MLH1,MSH2, PMS2 or MSH6 that result in inability to repair errors in these repetitive sequences. Clinically, the presence of MSI can be detected by immunohistochemistry (IHC) or genotyping. IHC confirmation of MSI involves staining for mismatch repair proteins on formalinfixed paraffin-embedded (FFPE) tumors. Genotype confirmation of MSI involves identifying microsatellite DNA markers from primary tumor via polymerase chain reaction (PCR)(Boland and Goel, 2010). The term MSI-high (MSI-H) is designated to a tumor when 2 or more of the 5 microsatellite sequences recommended by the National Cancer Institute (NCI) are mutated. MSI –Low (MSI-L) refers to tumors having a mutation in only one of the microsatellite sequences. Microsatellite stable (MSS) disease refers to tumors having no mutation in the microsatellite panel(Kurzawski et al., 2004). While MSI may occur in tumors of many organs, it is predominately found in CRC. In sporadic CRC, MSI-H is present in approximately 15% of lesions. This contrasts to its presence in 90% of lesions in patients with the Lynch syndrome or hereditary non-polyposis colorectal carcinoma (HNPCC)(Aaltonen et al., 1994). Studies indicate that the prevalence of MSI differs depending on the CRC stage(Gryfe et al., 2000). More specifically, 20% of stage II CRC harbors MSI while 12% of stage III has MSI in contrast to 4% MSI prevalence in the stage IV CRC population(Koopman et al., 2009). Ribic et al. explored the utility of MSI as a prognostic tool for stage II and stage III colon cancer patients in the setting of adjuvant fluorouracil (5-FU). They analyzed tumor samples from 570 patients enrolled in five randomized trails that compared adjuvant 5FU to surgery alone. In the patients who received adjuvant 5-FU, patients with MSS or MSI-L tumors benefited from adjuvant therapy with improved OS. Patients with MSI-H tumor did not benefit from adjuvant 5FU. In the patients who did not receive adjuvant 5-FU, patients with MSI-H tumors were found 11

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to have superior 5-year OS compared to those with MSI-L or MSS tumors(Ribic et al., 2003). These observations were replicated in a number of papers, confirming that patients with stage II MSI-H CRC would notbenefit from fluorouracil based adjuvant chemotherapy(Sargent et al., 2010; Watanabe et al., 2001)thus providing a molecular basis tostratify patients with stage II colon cancer by MMR status when considering adjuvant FU therapy. The Programmed death-1 (PD-1), found on T cells, causes T cell activation upon ligation with its ligand, PD-L1(Sznol and Chen, 2013). The majority of PD-1/PD-L1 data currently focuses on its prognostic significance as a biomarker rather than its relationship with specific genetic alterations, such as microsatellite stability. Gatalica et al. correlated the presence of PD-1 tumor infiltrating lymphocytes (TIL) and the expression of PD-L1 with microsatellite stability status in colorectal malignancy. Results show that the presence of PD-1 TIL is more closely associated with MSI-H than MSS CRC (77% vs. 39% respectively). Similarly, PD-L1 was also found to be more prevalent in MSI-H CRC population(Gatalica et al., 2014). In contrast, Droeser et al. analyzed tissue specimen (n= 1420) with tissue microarray and found a strong correlation between PD-L1 expression and MSS status as well as early T stage, absence of lymph node metastases, lower tumor grade, absence of vascular invasion and improved survival(Droeser et al., 2013). Further studies are necessary to better understand the relationship between PD-1 and microsatellite status. Given that the status of microsatellite stability may serve as a prognostic biomarker for colon cancer patients, several clinical trials are currently underway. There is an ongoing phase II study of MK-3475 (Anti-PD1, pembrolizumab) in patients with MSI tumors (NCT01876511) as well as a phase II study of nivolumab alone vs. in combination with Ipilimumab in advanced MSI-H CRC (NCT02060188). Front line therapy for stage IV CRC is currently a flurouracil (5-FU) based chemotherapy regimen, frequently with irinotecan(Punt, 2004). While overall survival has significantly improved in the last decade with the development of more effective treatment, there remains a considerable population of patients whom fail to respond to the 5-FU based regimens. In a nonrandomized, prospective study, Liang et al. evaluated the clinical significance of MSI-H in stage IV sporadic CRC patients (n=244). The study population was divided into two cohorts (with or without high-dose 5-FU chemotherapy), with each cohort further assigned to one of two subgroups (MSI-H+, MSI H-). Results show that patients receiving high dose 5-FU with MSI-H disease had superior median overall survival compared to the MSI-L subgroup receiving high dose 5-FU (24 vs 13 months). This suggests that stage IV sporadic CRC patients with MSI-H disease may have increased chemosensitivity(Liang et al., 2002). Other studies explored the association between MSI and irinotecan-based chemotherapy regimen as first line therapy for stage IV CRC. Kim et al (n=297 patients) found that microsatellite stability status did not significantly affect PFS or OS in patients treated with first line irinotecan-based chemotherapy(Kim et al., 2011). These findings contrasts with findings from the Cancer and Leukemia Group B 89803 study; results from this study showed that stage III patients with MSI-H status (n=1,264) had improved 5 year disease free survival after treatment with irinotecan-based regimen as compared to patients with mismatch repair intact tumors(Bertagnolli et al., 2009). Loss of heterozygosity of 18q Loss of heterozygosity (LOH) of the long arm of chromosome 18 (18q) frequently occurs late in the multi-step carcinogenic process. While there is no standard method to detect 18q LOH, the most common approach involves identifying two distinct alleles at a certain chromosomal location. In the process of comparing tumor sample and normal tissue specimen from the same patient, DNA loss is determined if one of the alleles present in the normal specimen is lost in the 12

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tumor. This condition is known as loss of heterozygosity (LOH) (Jen et al., 1994). The concept of 18qLOH is especially significant as 18q harbors multiple tumor suppressor genes associated with colorectal malignancy(Park do et al., 2007; Thiagalingam et al., 1996). Wang et al. obtained pathology from 102 stage II colon cancer patients and detected the frequency of 18qLOH to be approximately 49%. Moreover, it was noted that LOH was more frequently associated with left sided lesions, poorly differentiated, non-mucinous colon cancers(Wang et al., 2010). Multiple studies have explored the relationship between 18qLOH and treatment outcomes in stage II and stage III colon cancer patients. Unfortunately, due to conflicting data and lack of comparability between studies, results are split between results that show tumor 18qLOH to predict poor survival(Choi et al., 2002; Diep et al., 2003; Jen et al., 1994; Kern et al., 1989; Ogunbiyi et al., 1998) and those suggesting lack of association between tumor 18q status and treatment outcome(Lindforss et al., 2000; Ogino et al., 2009a; Zauber et al., 2004). Specifically, Lanza et al. evaluated the prognostic significant of 18qLOH in 118 patients with stage II or III CRC. It was found that 18qLOH was significantly associated with inferior OS and disease-free survival compared to that of patients with no evidence of chromosome 18q allelic loss(Lanza et al., 1998). In contrast, the Cancer and Leukemia Group B (CALGB) conducted separate randomized trials of adjuvant therapy in patients with stages II (CALGB protocol 9581) and III (CALGB protocol 89803) colon malignancy. Each of these trials prospectively evaluated the relationship between tumor 18q status and treatment outcome. It was shown that independent of microsatellite stability status, tumor 18qLOH determination does not have a prognostic significance in stage II and III colon cancer patients(Bertagnolli et al., 2011). As a result, conclusions regarding the prognostic and predictive value of the 18qLOH biomarker remain elusive. More data will be provided by the ECOG 5202 investigational randomized phase III study (NCT00217737) in which stage II patients are stratified as high risk (MSS/18q LOH) or low risk (MSI/no LOH on 18q), and randomized to chemotherapy with or without bevacizumab or observation alone. Prospective studies are needed to conclusively determine the impact of 18qLOH status on the prognosis and clinical outcome of patients with stage II and stage III colon cancer. Oncotype DX Based on results from the MOSAIC trial(Andre et al., 2009), current guidelines recommends adjuvant oxaliplatin-based therapy for stage III and high-risk stage II CRC. However, controversy regarding adjuvant therapy for stage II patients remains since conventional clinical and pathologic markers for “high risk” (T4 stage, high tumor grade, lymphovascular invasion, or inadequate nodal sampling) lack systematic validation and individualized assessment of recurrence risk(Vicuna and Benson, 2007). Thus, standardized, quantitative tools are necessary to provide individualized assessment of disease recurrence risk and treatment benefit in the adjuvant setting. The Oncotype DX Colon Cancer Recurrence Score is a standardized assay that measures the expression of 12 genes (7 recurrence and 5 reference genes) and produces an algorithm that yields a recurrence score of 0 to 100. Specific gene expressions of interest were chosen based on their fundamental role in cell cycle control and stromal response. This reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using tumor gene expression data from 1,851 patients with stage II and III colon cancer enrolled in four large, independent studies(O'Connell et al., 2010). Since its conception, a number of validation studies have been performed to assess the assay’s predictive utility. In the prospective Quick and Simple and Reliable (QUASAR) validation study, 1,436 patients with stage II colon cancer were randomly assigned to either surgery alone or surgery followed 13

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by adjuvant fluorouracil/leucovorin. Gray et al. report that recurrence score was significantly associated with recurrence risk after surgery, independent of attributed high risk factors such as mismatch repair (MMR), T stage, tumor grade, number of nodes examined, and lymphovascular invasion(Gray et al., 2011). Furthermore, two additional prospective studies have confirmed the relationship between the Recurrence Score and recurrence risk(Venook et al., 2013; Yothers et al., 2013). Altogether, available data suggests that the Oncotype DX colon Cancer Recurrence Score accurately predicts recurrence risk in stage II and stage III colon cancer patients. Subsequently, incorporating this tool into routine clinical practice may better inform adjuvant therapy decision-making for this patient population. PI3K The phosphatidylinositide-3-kinases (PI3Ks) belong to a family of lipid kinases that modulate bindings of intracellular signaling proteins, which ultimately regulate cell survival, proliferation and differentiation. The three subtypes of PI3Ks are classified based on their structural characteristics and substrate preferences. Class I PI3Ks, heterodimeric proteins composed of a p85 regulatory subunit and a p110 catalytic subunit, are found to harbor oncogenic mutations, specifically in the alpha isoform of the catalytic subunit, PIK3CA(Ogino et al., 2009b). Upon ligation of growth factors to its receptor tyrosine kinase, such as members of the ERBB-family, platelet-derived growth-factor (PDGFR) and insulin-like growth-factor 1 receptors IGF1R), activated PIK3CA phosphorylates phosphatidyl-inositol-4,5-biphosphate (PIP2) to produce phosphatidyl-inositol-3,4,5-triphosphate (PIP3), subsequently activating the serine threonine kinase Akt, which phosphorylates downstream effectors and amplifies the signaling cascade(De Roock et al., 2011; Ogino et al., 2009b). Inappropriate activation of the PI3K-AKT pathway, primarily by PIK3CA mutations, plays a critical role in the pathogenesis of CRC(Malinowsky et al., 2014; Samuels et al., 2004; Zhang et al., 2011). Frequency of the PIK3CA mutation in CRC approximates 15-25% of which 80% occurs in exon 9 (60-65%) or exon 20 (20-25%)(Dasari and Messersmith, 2010; Samuels et al., 2004). Prognostic role of PIk3CA exon 9 and exon 20 mutations remain unclear. De Roock et al. report data, which suggest the PIK3CA exon 20 mutations to confer cetuximab- resistance in KRAS wild-type tumors. In contrast, PIK3CA exon 9 mutations were found to be associated with KRAS mutations and to not have an independent effect on cetuximab efficacy(De Roock et al., 2011). In general, PIK3CA mutations are commonly associated with negative clinical outcome, but large prospective studies are needed to establish the predictive value of PIK3CA mutations for EGFR inhibitors. Observational and experimental evidence suggest that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2, COX-2) by aspirin or COX-2 inhibitors down-regulates PI3K signaling and reduces risk of developing CRC(Arber et al., 2006; Baron et al., 2003; Bertagnolli et al., 2006; Sandler et al., 2003). Further data suggest that regular aspirin use in patients with established diagnosis of CRC is also associated with improved overall survival(Chia et al., 2012; Rothwell et al., 2010; Rothwell et al., 2012; Zell et al., 2009). The specific mechanism underlying the superior clinical outcome associated with the aspirin use in patients with CRC is poorly understood. Liao et al., in an analysis of 964 patients, found that CRC patients with mutated PIK3CA tumor had superior cancer-specific survival (HR 0.18, p< 0.001) and OS (HR 0.54, p =0.01) compared to that of patients with WT PIK3CA tumor in the setting of regular aspirin therapy(Liao et al., 2012). Similarly, Domingo et al. report CRC tumor harboring the PIK3CA mutation to preferentially benefit from aspirin therapy. Patients with PIK3CA-mutated tumors had reduced rate of CRC recurrence (HR 0.11, p= 0.027) compared to patients with WT PIK3CA tumors (HR 0.92, p = 0.71)(Domingo et al., 2013). In a multi-center international phase III ASCOLT study (NCT00565708), aspirin is hypothesized to improve 14

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overall survival in patients with Dukes C or high risk Dukes B CRC. Findings from these studies suggest utility of PIK3CA mutation as a predictive biomarker for adjuvant aspirin therapy, but the complex relationship between aspirin, PI3K, and other cellular interactions need to be further elucidated. RAS mutations The RAS family of genes encodes guanosine-5’triphosphate (GTP)ase- binding proteins that serve as downstream effectors by coupling epidermal growth factor receptor (EGFR) with intracellular signaling within the mitogen-activated protein kinase (MAPK) pathway. RAS mutations can be detected by a number of molecular techniques including Sanger sequencing, pyrosequencing, BEAMing assays, and real-time PCR with or without melting curve analysis (Tsiatis et al., 2010). Tsiatis et al. assessed KRAS status of 180 colon adenocarcinoma samples by these three techniques, and found pyrosequencing to be superior in sensitivity and objectivity(Tsiatis et al., 2010). Notably, the Scorpion Amplified Refractory Mutation System (ARMS) PCR is, at the present, the only FDA-approved test for clinical use(Atreya et al., 2015) in assessing patient for cetuximab or panitumumab therapy. Due to varying technical performance, expenditure, and feasibility, there is no consensus on a gold standard methodology for RAS mutational analysis(Bellon et al., 2011). Of the RAS genes, Kirsten rat sarcoma viral oncogene (KRAS) has the highest incidence of mutation; approximately 30-40% of CRC harbor somatic single nucleotide point mutations in the KRAS gene with 85-90% of mutations occurring in codons 12 and 13 on exon 2(Neumann et al., 2009). Other mutations have been observed in KRAS exons 3 and 4 and NRAS exons 2, 3, and 4. These mutations lead to constitutive activation of the MAPK pathway and predict resistance to EGFR directed therapy(Allegra et al., 2009; Lievre et al., 2006). Table 1 shows the various trials evaluating the role of EGFR inhibitors in first line setting in colorectal cancer patients. In Table 1, as patients are selected for RAS mutations, the activity of EGFR inhibitors as measured by overall survival or progression free survival improves confirming that RAS is a biomarker for resistance to EGFR inhibitors. Furthermore, these results suggest that this patient population may benefit from extended RAS status analysis rather than exon 2 mutational analysis prior to initiation of treatment of EGFR inhibitors. Translating these findings to clinical practice, investigators propose that patients undergo screening for extended RAS mutations, rather than just exon 2 mutations, prior to treatment with anti-EGFR monoclonal antibodies. The National Comprehensive Cancer Network recently updated guidelines to reflect these suggestions. The guidelines encourage practitioners to test extended RAS mutations “whenever possible” with the understanding that patients who test positive for any RAS mutations would not receive cetuximab or panitumumab(Benson et al., 2014). BRAF BRAF, a member of the RAF family, is a protein kinase downstream of RAS in the RAS/RAF/MEK/ERK signaling cascade responsible for cell growth, differentiation and survival(Vanessa Deschoolmeester, 2012). Pre-clinical data indicates that BRAF mutation leads to constitutive activation of the mitogen activated protein kinase (MAPK) pathway, thereby inducing tumorigenic proliferation(Brunet et al., 1994). Similar to RAS analysis, there is a lack of consensus on a gold standard for BRAF mutation detection with options including gene sequencing, pyrosequencing and PCR. Activating mutations of BRAF are reported to occur in 515% of CRC(Nash et al., 2010). Yokota et al. analyzed genotype of 229 patients with chemotherapy-refractory CRC and found 6.5% of patients to have a BRAF mutation(Yokota et al., 2011). Similarly, Busu et al. reports 8% incidence of BRAF mutation in CRC patients (13.5% 15

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of the KRAS WT cohort)(Gargi Dan Basu, 2014). The most common BRAF mutation is a valineto-glutamic acid substitution at codon 600 (V600E)(Vanessa Deschoolmeester, 2012). Despite its relatively low incidence, BRAF-mutated CRC is a distinct pathologic, phenotypic, and clinical subset, characterized by sporadic microsatellite instability (MSI), CpG island methylatorphyenotype (CIMP) and right-sided colon tumors(Dasari and Messersmith, 2010; De Roock et al., 2011). BRAF-mutated tumors portend a poor prognosis in patients with metastatic CRC(Yokota et al., 2011). In a retrospective review of patients with metastatic CRC (n=524), overall survival of patients with BRAF-mutant tumors was found to be significantly inferior to that of patients with BRAF WT tumors (10.4 m vs 34.7 m)(Tran et al., 2011). Clinical outcomes with BRAF inhibitors have been disappointing in BRAF-mutant CRC. In a phase I study of vemurafenib, Kopetz et al. found only a 5% response among 20 patients with metastatic CRC(S. Kopetz, 2010). Similarly, Corcoran et al. also reports a 5% response rate in 20 BRAF mutated metastatic CRC patients treated with combination dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor)(Corcoran R.B., 2012). Clinical trials of combination therapy are underway. In a multicenter phase Ib/II study, patients with BRAF mutant solid tumors are enrolled to receive BRAF inhibitor, LGX818, in combination with MEK162 (NCT01543698). In an ongoing Phase I/II study, patients with BRAF V600E mutant CRC receive varying combinations of dabrafeinib, trametinib and panitumumab (NCT01750918). Pre-clinical data showed that BRAF-mutant CRC cell lines experienced heterogeneous responses upon treatment with BRAF-inhibitor(Yang et al., 2012), suggestive of both inherent and acquired resistance mechanisms. Prahallad et al. assessed vemurafenib-resistant BRAFmutant CRC cells and found that BRAF inhibition by vemurafenib induced positive feedback activation of EGFR(Prahallad et al., 2012), an indication that EGFR may mediate resistance to BRAF inhibition. Corcoran et al. demonstrated that combination therapy with BRAF inhibitor (vemurafinib) and EGFR inhibitor (erlotinib) yielded effective tumor response in CRC xenografts and reduced Ki-67(Corcoran et al., 2012). Furthermore, Liu et al. compared BRAF/MEK inhibition with and without EGFR inhibition in five BRAF V600E human CRC cell lines and reports triple combination therapy to effectively delay tumor growth compared to doublet therapy(LI Liu, 2014). In addition, Mao et al. found that combination therapy with BRAF and PI3K inhibitors also synergistically improved inhibition of tumor growth(Mao et al., 2013). Taken together, combination strategies with BRAF inhibitors plus agents that inhibit alternative pathways implicated in the development of resistance may yield superior clinical response compared to single agent BRAF inhibitor in patients harboring BRAF mutations. 5. Conclusion This review summarizes the most pertinent biomarkers in GI malignancy. The understanding of their predictive and prognostic value continues to evolve as our comprehension of their clinical application expands. Moreover, the identification of novel biomarkers and elucidation of their clinical significance holds great promise for further development of therapeutic targets, improved survival, and ultimately personalized medicine for our patients.

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REFERENCES

Aaltonen, L.A., Peltomaki, P., Mecklin, J.P., Jarvinen, H., Jass, J.R., Green, J.S., Lynch, H.T., Watson, P., Tallqvist, G., Juhola, M., et al., 1994. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer research 54 (7), 1645-1648. Akiyama, T., Sudo, C., Ogawara, H., Toyoshima, K., Yamamoto, T., 1986. The product of the human cerbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 232 (4758), 1644-1646. Alan P. Venook, D.N., Heinz-Josef Lenz, Federico Innocenti, Michelle R. Mahoney, Bert H. O'Neil, James Edward Shaw, Blase N. Polite, Howard S. Hochster, James Norman Atkins, Richard M. Goldberg, Robert J. Mayer, Richard L. Schilsky, Monica M. Bertagnolli, Charles David Blanke, 2014. CALGB/SWOG 80405: Phase III trial of irinotecan/5-FU/leucovorin (FOLFIRI) or oxaliplatin/5-FU/leucovorin (mFOLFOX6) with bevacizumab (BV) or cetuximab (CET) for patients (pts) with KRAS wild-type (wt) untreated metastatic adenocarcinoma of the colon or rectum (MCRC). Journal of Clinical Oncology 32:5s, 2014 (suppl; abstr LBA3). Allegra, C.J., Jessup, J.M., Somerfield, M.R., Hamilton, S.R., Hammond, E.H., Hayes, D.F., McAllister, P.K., Morton, R.F., Schilsky, R.L., 2009. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (12), 2091-2096. Andre, T., Boni, C., Navarro, M., Tabernero, J., Hickish, T., Topham, C., Bonetti, A., Clingan, P., Bridgewater, J., Rivera, F., de Gramont, A., 2009. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (19), 3109-3116. Antoni Ribas, F.S.H., Richard Kefford, Omid Hamid, Adil Daud, Jedd D. Wolchok, Wen-Jen Hwu, Tara C. Gangadhar, Amita Patnaik, Anthony M. Joshua, Peter Hersey, Jeffrey S. Weber, Roxana Stefania Dronca, Hassane M. Zarour, Kevin Gergich, Xiaoyun (Nicole) Li, Robert Iannone, Soonmo Peter Kang, Scot Ebbinghaus, Caroline Robert; David Geffen, 2014. Efficacy and safety of the anti-PD-1 monoclonal antibody MK-3475 in 411 patients (pts) with melanoma (MEL). Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32:5s (suppl; abstr LBA9000). Arber, N., Eagle, C.J., Spicak, J., Racz, I., Dite, P., Hajer, J., Zavoral, M., Lechuga, M.J., Gerletti, P., Tang, J., Rosenstein, R.B., Macdonald, K., Bhadra, P., Fowler, R., Wittes, J., Zauber, A.G., Solomon, S.D., Levin, B., Pre, S.A.P.T.I., 2006. Celecoxib for the prevention of colorectal adenomatous polyps. The New England journal of medicine 355 (9), 885-895. Atreya, C.E., Corcoran, R.B., Kopetz, S., 2015. Expanded RAS: Refining the Patient Population. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Bang, Y.J., Van Cutsem, E., Feyereislova, A., Chung, H.C., Shen, L., Sawaki, A., Lordick, F., Ohtsu, A., Omuro, Y., Satoh, T., Aprile, G., Kulikov, E., Hill, J., Lehle, M., Ruschoff, J., Kang, Y.K., To, G.A.T.I., 2010. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 376 (9742), 687-697.

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Barok, M., Tanner, M., Koninki, K., Isola, J., 2011. Trastuzumab-DM1 is highly effective in preclinical models of HER2-positive gastric cancer. Cancer letters 306 (2), 171-179. Baron, J.A., Cole, B.F., Sandler, R.S., Haile, R.W., Ahnen, D., Bresalier, R., McKeown-Eyssen, G., Summers, R.W., Rothstein, R., Burke, C.A., Snover, D.C., Church, T.R., Allen, J.I., Beach, M., Beck, G.J., Bond, J.H., Byers, T., Greenberg, E.R., Mandel, J.S., Marcon, N., Mott, L.A., Pearson, L., Saibil, F., van Stolk, R.U., 2003. A randomized trial of aspirin to prevent colorectal adenomas. The New England journal of medicine 348 (10), 891-899. Bellon, E., Ligtenberg, M.J., Tejpar, S., Cox, K., de Hertogh, G., de Stricker, K., Edsjo, A., Gorgoulis, V., Hofler, G., Jung, A., Kotsinas, A., Laurent-Puig, P., Lopez-Rios, F., Hansen, T.P., Rouleau, E., Vandenberghe, P., van Krieken, J.J., Dequeker, E., 2011. External quality assessment for KRAS testing is needed: setup of a European program and report of the first joined regional quality assessment rounds. The oncologist 16 (4), 467-478. Benson, A.B., 3rd, Venook, A.P., Bekaii-Saab, T., Chan, E., Chen, Y.J., Cooper, H.S., Engstrom, P.F., Enzinger, P.C., Fenton, M.J., Fuchs, C.S., Grem, J.L., Hunt, S., Kamel, A., Leong, L.A., Lin, E., Messersmith, W., Mulcahy, M.F., Murphy, J.D., Nurkin, S., Rohren, E., Ryan, D.P., Saltz, L., Sharma, S., Shibata, D., Skibber, J.M., Sofocleous, C.T., Stoffel, E.M., Stotsky-Himelfarb, E., Willett, C.G., Gregory, K.M., Freedman-Cass, D.A., 2014. Colon cancer, version 3.2014. Journal of the National Comprehensive Cancer Network : JNCCN 12 (7), 1028-1059. Berndt, N., Hamilton, A.D., Sebti, S.M., 2011. Targeting protein prenylation for cancer therapy. Nature reviews. Cancer 11 (11), 775-791. Bertagnolli, M.M., Eagle, C.J., Zauber, A.G., Redston, M., Solomon, S.D., Kim, K., Tang, J., Rosenstein, R.B., Wittes, J., Corle, D., Hess, T.M., Woloj, G.M., Boisserie, F., Anderson, W.F., Viner, J.L., Bagheri, D., Burn, J., Chung, D.C., Dewar, T., Foley, T.R., Hoffman, N., Macrae, F., Pruitt, R.E., Saltzman, J.R., Salzberg, B., Sylwestrowicz, T., Gordon, G.B., Hawk, E.T., Investigators, A.P.C.S., 2006. Celecoxib for the prevention of sporadic colorectal adenomas. The New England journal of medicine 355 (9), 873-884. Bertagnolli, M.M., Niedzwiecki, D., Compton, C.C., Hahn, H.P., Hall, M., Damas, B., Jewell, S.D., Mayer, R.J., Goldberg, R.M., Saltz, L.B., Warren, R.S., Redston, M., 2009. Microsatellite instability predicts improved response to adjuvant therapy with irinotecan, fluorouracil, and leucovorin in stage III colon cancer: Cancer and Leukemia Group B Protocol 89803. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (11), 1814-1821. Bertagnolli, M.M., Redston, M., Compton, C.C., Niedzwiecki, D., Mayer, R.J., Goldberg, R.M., Colacchio, T.A., Saltz, L.B., Warren, R.S., 2011. Microsatellite instability and loss of heterozygosity at chromosomal location 18q: prospective evaluation of biomarkers for stages II and III colon cancer--a study of CALGB 9581 and 89803. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29 (23), 3153-3162. Biomarkers Definitions Working, G., 2001. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clinical pharmacology and therapeutics 69 (3), 89-95. Blackford, A., Serrano, O.K., Wolfgang, C.L., Parmigiani, G., Jones, S., Zhang, X., Parsons, D.W., Lin, J.C., Leary, R.J., Eshleman, J.R., Goggins, M., Jaffee, E.M., Iacobuzio-Donahue, C.A., Maitra, A., Cameron, J.L., Olino, K., Schulick, R., Winter, J., Herman, J.M., Laheru, D., Klein, A.P., Vogelstein, B., Kinzler, K.W.,

19

Page 19 of 36

Velculescu, V.E., Hruban, R.H., 2009. SMAD4 gene mutations are associated with poor prognosis in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 15 (14), 4674-4679. Blobe, G.C., Schiemann, W.P., Lodish, H.F., 2000. Role of transforming growth factor beta in human disease. The New England journal of medicine 342 (18), 1350-1358. Bodoky, G., Timcheva, C., Spigel, D.R., La Stella, P.J., Ciuleanu, T.E., Pover, G., Tebbutt, N.C., 2012. A phase II open-label randomized study to assess the efficacy and safety of selumetinib (AZD6244 [ARRY142886]) versus capecitabine in patients with advanced or metastatic pancreatic cancer who have failed first-line gemcitabine therapy. Investigational new drugs 30 (3), 1216-1223. Boland, C.R., Goel, A., 2010. Microsatellite instability in colorectal cancer. Gastroenterology 138 (6), 2073-2087 e2073. Brenner, H., Kloor, M., Pox, C.P., 2014. Colorectal cancer. Lancet 383 (9927), 1490-1502. Brunet, A., Pages, G., Pouyssegur, J., 1994. Constitutively active mutants of MAP kinase kinase (MEK1) induce growth factor-relaxation and oncogenicity when expressed in fibroblasts. Oncogene 9 (11), 33793387. Bunger, S., Laubert, T., Roblick, U.J., Habermann, J.K., 2011. Serum biomarkers for improved diagnostic of pancreatic cancer: a current overview. Journal of cancer research and clinical oncology 137 (3), 375389. Carden, C.P., Yap, T.A., Kaye, S.B., 2010. PARP inhibition: targeting the Achilles' heel of DNA repair to treat germline and sporadic ovarian cancers. Current opinion in oncology 22 (5), 473-480. Chalasani, P., Kurtin, S., Dragovich, T., 2008. Response to a third-line mitomycin C (MMC)-based chemotherapy in a patient with metastatic pancreatic adenocarcinoma carrying germline BRCA2 mutation. JOP : Journal of the pancreas 9 (3), 305-308. Chen, S., Wang, G., Niu, X., Zhao, J., Tan, W., Wang, H., Zhao, L., Ge, Y., 2014a. Combination of AZD2281 (Olaparib) and GX15-070 (Obatoclax) results in synergistic antitumor activities in preclinical models of pancreatic cancer. Cancer letters 348 (1-2), 20-28. Chen, Y.W., Hsiao, P.J., Weng, C.C., Kuo, K.K., Kuo, T.L., Wu, D.C., Hung, W.C., Cheng, K.H., 2014b. SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells. BMC cancer 14, 181. Chia, W.K., Ali, R., Toh, H.C., 2012. Aspirin as adjuvant therapy for colorectal cancer--reinterpreting paradigms. Nature reviews. Clinical oncology 9 (10), 561-570. Choi, S.W., Lee, K.J., Bae, Y.A., Min, K.O., Kwon, M.S., Kim, K.M., Rhyu, M.G., 2002. Genetic classification of colorectal cancer based on chromosomal loss and microsatellite instability predicts survival. Clinical cancer research : an official journal of the American Association for Cancer Research 8 (7), 2311-2322. Corcoran R.B., e.a., 2012. BRAF V600 mutant colorectal cancer (CRC) expansion cohort from the phase I/II clinical trial of BRAF inhibitor dabrafenib (GSK2128436) plus MEK inhibitor trametinib (GSK1120212).

20

Page 20 of 36

Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30(15) suppl 3528. Corcoran, R.B., Ebi, H., Turke, A.B., Coffee, E.M., Nishino, M., Cogdill, A.P., Brown, R.D., Della Pelle, P., Dias-Santagata, D., Hung, K.E., Flaherty, K.T., Piris, A., Wargo, J.A., Settleman, J., Mino-Kenudson, M., Engelman, J.A., 2012. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer discovery 2 (3), 227-235. Creemers, G.J., Lund, B., Verweij, J., 1994. Topoisomerase I inhibitors: topotecan and irenotecan. Cancer treatment reviews 20 (1), 73-96. Dai, J.L., Bansal, R.K., Kern, S.E., 1999. G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation. Proceedings of the National Academy of Sciences of the United States of America 96 (4), 1427-1432. Dasari, A., Messersmith, W.A., 2010. New strategies in colorectal cancer: biomarkers of response to epidermal growth factor receptor monoclonal antibodies and potential therapeutic targets in phosphoinositide 3-kinase and mitogen-activated protein kinase pathways. Clinical cancer research : an official journal of the American Association for Cancer Research 16 (15), 3811-3818. De Roock, W., De Vriendt, V., Normanno, N., Ciardiello, F., Tejpar, S., 2011. KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer. The Lancet. Oncology 12 (6), 594-603. De Vita, F., Giuliani, F., Silvestris, N., Catalano, G., Ciardiello, F., Orditura, M., 2010. Human epidermal growth factor receptor 2 (HER2) in gastric cancer: a new therapeutic target. Cancer treatment reviews 36 Suppl 3, S11-15. Diep, C.B., Thorstensen, L., Meling, G.I., Skovlund, E., Rognum, T.O., Lothe, R.A., 2003. Genetic tumor markers with prognostic impact in Dukes' stages B and C colorectal cancer patients. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21 (5), 820-829. Domingo, E., Church, D.N., Sieber, O., Ramamoorthy, R., Yanagisawa, Y., Johnstone, E., Davidson, B., Kerr, D.J., Tomlinson, I.P., Midgley, R., 2013. Evaluation of PIK3CA mutation as a predictor of benefit from nonsteroidal anti-inflammatory drug therapy in colorectal cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31 (34), 4297-4305. Dong, H., Strome, S.E., Salomao, D.R., Tamura, H., Hirano, F., Flies, D.B., Roche, P.C., Lu, J., Zhu, G., Tamada, K., Lennon, V.A., Celis, E., Chen, L., 2002. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nature medicine 8 (8), 793-800. Douillard, J.Y., Oliner, K.S., Siena, S., Tabernero, J., Burkes, R., Barugel, M., Humblet, Y., Bodoky, G., Cunningham, D., Jassem, J., Rivera, F., Kocakova, I., Ruff, P., Blasinska-Morawiec, M., Smakal, M., Canon, J.L., Rother, M., Williams, R., Rong, A., Wiezorek, J., Sidhu, R., Patterson, S.D., 2013. PanitumumabFOLFOX4 treatment and RAS mutations in colorectal cancer. The New England journal of medicine 369 (11), 1023-1034. Douillard, J.Y., Siena, S., Cassidy, J., Tabernero, J., Burkes, R., Barugel, M., Humblet, Y., Bodoky, G., Cunningham, D., Jassem, J., Rivera, F., Kocakova, I., Ruff, P., Blasinska-Morawiec, M., Smakal, M., Canon,

21

Page 21 of 36

J.L., Rother, M., Oliner, K.S., Wolf, M., Gansert, J., 2010. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28 (31), 4697-4705. Droeser, R.A., Hirt, C., Viehl, C.T., Frey, D.M., Nebiker, C., Huber, X., Zlobec, I., Eppenberger-Castori, S., Tzankov, A., Rosso, R., Zuber, M., Muraro, M.G., Amicarella, F., Cremonesi, E., Heberer, M., Iezzi, G., Lugli, A., Terracciano, L., Sconocchia, G., Oertli, D., Spagnoli, G.C., Tornillo, L., 2013. Clinical impact of programmed cell death ligand 1 expression in colorectal cancer. European journal of cancer 49 (9), 22332242. Dutrillaux, B., 1995. Pathways of chromosome alteration in human epithelial cancers. Advances in cancer research 67, 59-82. Eiji Oki, S.O., Koji Ando, Yukiharu Hiyoshi, Shuhei Ito, Masaru Morita, Yoshihiko Maehara, 2014. HER2 and programmed death-1 ligand-1 (PD-L1) expression in gastric carcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32 (suppl; abstr e15041). Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D., Bray, F., 2015. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer. Journal international du cancer 136 (5), E359-386. Fogelman, D.R., Wolff, R.A., Kopetz, S., Javle, M., Bradley, C., Mok, I., Cabanillas, F., Abbruzzese, J.L., 2011. Evidence for the efficacy of Iniparib, a PARP-1 inhibitor, in BRCA2-associated pancreatic cancer. Anticancer research 31 (4), 1417-1420. Friess, H., Yamanaka, Y., Buchler, M., Ebert, M., Beger, H.G., Gold, L.I., Korc, M., 1993. Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival. Gastroenterology 105 (6), 1846-1856. Fujimoto-Ouchi, K., Sekiguchi, F., Yasuno, H., Moriya, Y., Mori, K., Tanaka, Y., 2007. Antitumor activity of trastuzumab in combination with chemotherapy in human gastric cancer xenograft models. Cancer chemotherapy and pharmacology 59 (6), 795-805. Garcea, G., Neal, C.P., Pattenden, C.J., Steward, W.P., Berry, D.P., 2005. Molecular prognostic markers in pancreatic cancer: a systematic review. European journal of cancer 41 (15), 2213-2236. Gargi Dan Basu, J.X., David Arguello, Rebecca A. Feldman, Sherri Z. Millis, Ryan P. Bender, Zoran Gatalica, Les Paul, Fadi S. Braiteh, 2014. Prevalence of KRAS, BRAF, NRAS, PIK3CA, and PTEN alterations in colorectal cancer: Analysis of a large international cohort of 5,900 patients. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32, 2014 (suppl 3; abstr 399). Gatalica, Z., Snyder, C., Maney, T., Ghazalpour, A., Holterman, D.A., Xiao, N., Overberg, P., Rose, I., Basu, G.D., Vranic, S., Lynch, H.T., Von Hoff, D.D., Hamid, O., 2014. Programmed cell death 1 (PD-1) and its ligand (PD-L1) in common cancers and their correlation with molecular cancer type. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 23 (12), 2965-2970.

22

Page 22 of 36

Giordano, S., Di Renzo, M.F., Narsimhan, R.P., Cooper, C.S., Rosa, C., Comoglio, P.M., 1989. Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene 4 (11), 1383-1388. Golan, T., Kanji, Z.S., Epelbaum, R., Devaud, N., Dagan, E., Holter, S., Aderka, D., Paluch-Shimon, S., Kaufman, B., Gershoni-Baruch, R., Hedley, D., Moore, M.J., Friedman, E., Gallinger, S., 2014. Overall survival and clinical characteristics of pancreatic cancer in BRCA mutation carriers. British journal of cancer 111 (6), 1132-1138. Gomez-Martin, C., Plaza, J.C., Pazo-Cid, R., Salud, A., Pons, F., Fonseca, P., Leon, A., Alsina, M., Visa, L., Rivera, F., Galan, M.C., Del Valle, E., Vilardell, F., Iglesias, M., Fernandez, S., Landolfi, S., Cuatrecasas, M., Mayorga, M., Jose Paules, M., Sanz-Moncasi, P., Montagut, C., Garralda, E., Rojo, F., Hidalgo, M., LopezRios, F., 2013. Level of HER2 gene amplification predicts response and overall survival in HER2-positive advanced gastric cancer treated with trastuzumab. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31 (35), 4445-4452. Gravalos, C., Jimeno, A., 2008. HER2 in gastric cancer: a new prognostic factor and a novel therapeutic target. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 19 (9), 1523-1529. Gray, R.G., Quirke, P., Handley, K., Lopatin, M., Magill, L., Baehner, F.L., Beaumont, C., Clark-Langone, K.M., Yoshizawa, C.N., Lee, M., Watson, D., Shak, S., Kerr, D.J., 2011. Validation study of a quantitative multigene reverse transcriptase-polymerase chain reaction assay for assessment of recurrence risk in patients with stage II colon cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29 (35), 4611-4619. Gryfe, R., Kim, H., Hsieh, E.T., Aronson, M.D., Holowaty, E.J., Bull, S.B., Redston, M., Gallinger, S., 2000. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. The New England journal of medicine 342 (2), 69-77. H. Lenz, D.N., F. Innocenti, C. Blanke, M. Mahony, B. O'Neil, J. Shaw, B. Polite, H. Hochster, J. Atkins, R. Goldberg, R. Mayer, R. Schilsky, M. Bertagnolli, A. Venook 2014. CALGB/SWOG 80405: PHASE III trial of irinotecan/5-FU/leucovorin (FOLFIRI) or oxaliplatin/5-FU/leucovorin (mFOLFOX6) with bevacizumab (BV) or cetuximab (CET) for patients (pts) with expanded ras analyses untreated metastatic adenocarcinoma of the colon or rectum (mCRC). ESMO. Hahn, S.A., Schutte, M., Hoque, A.T., Moskaluk, C.A., da Costa, L.T., Rozenblum, E., Weinstein, C.L., Fischer, A., Yeo, C.J., Hruban, R.H., Kern, S.E., 1996. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271 (5247), 350-353. Hara, T., Ooi, A., Kobayashi, M., Mai, M., Yanagihara, K., Nakanishi, I., 1998. Amplification of c-myc, Ksam, and c-met in gastric cancers: detection by fluorescence in situ hybridization. Laboratory investigation; a journal of technical methods and pathology 78 (9), 1143-1153. Hayes, D.F., Bast, R.C., Desch, C.E., Fritsche, H., Jr., Kemeny, N.E., Jessup, J.M., Locker, G.Y., Macdonald, J.S., Mennel, R.G., Norton, L., Ravdin, P., Taube, S., Winn, R.J., 1996. Tumor marker utility grading

23

Page 23 of 36

system: a framework to evaluate clinical utility of tumor markers. Journal of the National Cancer Institute 88 (20), 1456-1466. Heinemann, V., Quietzsch, D., Gieseler, F., Gonnermann, M., Schonekas, H., Rost, A., Neuhaus, H., Haag, C., Clemens, M., Heinrich, B., Vehling-Kaiser, U., Fuchs, M., Fleckenstein, D., Gesierich, W., Uthgenannt, D., Einsele, H., Holstege, A., Hinke, A., Schalhorn, A., Wilkowski, R., 2006. Randomized phase III trial of gemcitabine plus cisplatin compared with gemcitabine alone in advanced pancreatic cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 24 (24), 3946-3952. Hofstatter, E.W., Domchek, S.M., Miron, A., Garber, J., Wang, M., Componeschi, K., Boghossian, L., Miron, P.L., Nathanson, K.L., Tung, N., 2011. PALB2 mutations in familial breast and pancreatic cancer. Familial cancer 10 (2), 225-231. Hruban, R.H., Offerhaus, G.J., Kern, S.E., Goggins, M., Wilentz, R.E., Yeo, C.J., 1998. Tumor-suppressor genes in pancreatic cancer. Journal of hepato-biliary-pancreatic surgery 5 (4), 383-391. Hsieh, A.C., Moasser, M.M., 2007. Targeting HER proteins in cancer therapy and the role of the nontarget HER3. British journal of cancer 97 (4), 453-457. Iacobuzio-Donahue, C.A., Fu, B., Yachida, S., Luo, M., Abe, H., Henderson, C.M., Vilardell, F., Wang, Z., Keller, J.W., Banerjee, P., Herman, J.M., Cameron, J.L., Yeo, C.J., Halushka, M.K., Eshleman, J.R., Raben, M., Klein, A.P., Hruban, R.H., Hidalgo, M., Laheru, D., 2009. DPC4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (11), 1806-1813. Inoue, T., Chung, Y.S., Yashiro, M., Nishimura, S., Hasuma, T., Otani, S., Sowa, M., 1997. Transforming growth factor-beta and hepatocyte growth factor produced by gastric fibroblasts stimulate the invasiveness of scirrhous gastric cancer cells. Japanese journal of cancer research : Gann 88 (2), 152-159. Iqbal, J., Ragone, A., Lubinski, J., Lynch, H.T., Moller, P., Ghadirian, P., Foulkes, W.D., Armel, S., Eisen, A., Neuhausen, S.L., Senter, L., Singer, C.F., Ainsworth, P., Kim-Sing, C., Tung, N., Friedman, E., Llacuachaqui, M., Ping, S., Narod, S.A., Hereditary Breast Cancer Study, G., 2012. The incidence of pancreatic cancer in BRCA1 and BRCA2 mutation carriers. British journal of cancer 107 (12), 2005-2009. J. Randolph Hecht, Y.-J.B., Shukui Qin, Hyun-Chul Chung, Jian-Ming Xu, Joon Oh Park, Krzysztof Jeziorski, Yaroslav Shparyk, Paulo M. Hoff, Alberto F. Sobrero, Pamela Salman, Jin Li, Svetlana Protsenko, Marc E. Buyse, Karen Afenjar, Tomomi Kaneko, Allison Kemner, Sergio Santillana, Michael F. Press, Dennis J. Slamon; David Geffen, 2013. Lapatinib in combination with capecitabine plus oxaliplatin (CapeOx) in HER2-positive advanced or metastatic gastric, esophageal, or gastroesophageal adenocarcinoma (AC): The TRIO-013/LOGiC Trial. Journal of Clinical Oncology suppl; abstr LBA4001. Jacob, D.A., Bahra, M., Langrehr, J.M., Boas-Knoop, S., Stefaniak, R., Davis, J., Schumacher, G., Lippert, S., Neumann, U.P., 2007. Combination therapy of poly (ADP-ribose) polymerase inhibitor 3aminobenzamide and gemcitabine shows strong antitumor activity in pancreatic cancer cells. Journal of gastroenterology and hepatology 22 (5), 738-748. James, E., Waldron-Lynch, M.G., Saif, M.W., 2009. Prolonged survival in a patient with BRCA2 associated metastatic pancreatic cancer after exposure to camptothecin: a case report and review of literature. Anti-cancer drugs 20 (7), 634-638.

24

Page 24 of 36

Janjigian, Y.Y., Tang, L.H., Coit, D.G., Kelsen, D.P., Francone, T.D., Weiser, M.R., Jhanwar, S.C., Shah, M.A., 2011. MET expression and amplification in patients with localized gastric cancer. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 20 (5), 1021-1027. Jean-Yves Douillard, S.S., Josep Tabernero, Ronald L. Burkes, Mario Edmundo Barugel, Yves Humblet, Gyorgy Bodoky, David Cunningham, Jacek Jassem, Fernando Rivera, Ilona Kocáková, Paul Ruff, Martin Smakal, Jean-Luc Canon, Mark Rother, Kelly S. Oliner, Ying Tian, Roger Sidhu, 2013. Overall survival (OS) analysis from PRIME: Randomized phase III study of panitumumab (pmab) with FOLFOX4 for first-line metastatic colorectal cancer (mCRC). Journal of Clinical Oncology 31, 2013 (suppl; abstr 3620). Jeffers, M., Fiscella, M., Webb, C.P., Anver, M., Koochekpour, S., Vande Woude, G.F., 1998. The mutationally activated Met receptor mediates motility and metastasis. Proceedings of the National Academy of Sciences of the United States of America 95 (24), 14417-14422. Jen, J., Kim, H., Piantadosi, S., Liu, Z.F., Levitt, R.C., Sistonen, P., Kinzler, K.W., Vogelstein, B., Hamilton, S.R., 1994. Allelic loss of chromosome 18q and prognosis in colorectal cancer. The New England journal of medicine 331 (4), 213-221. Jones, S., Hruban, R.H., Kamiyama, M., Borges, M., Zhang, X., Parsons, D.W., Lin, J.C., Palmisano, E., Brune, K., Jaffee, E.M., Iacobuzio-Donahue, C.A., Maitra, A., Parmigiani, G., Kern, S.E., Velculescu, V.E., Kinzler, K.W., Vogelstein, B., Eshleman, J.R., Goggins, M., Klein, A.P., 2009. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science 324 (5924), 217. Jones, S., Zhang, X., Parsons, D.W., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Kamiyama, H., Jimeno, A., Hong, S.M., Fu, B., Lin, M.T., Calhoun, E.S., Kamiyama, M., Walter, K., Nikolskaya, T., Nikolsky, Y., Hartigan, J., Smith, D.R., Hidalgo, M., Leach, S.D., Klein, A.P., Jaffee, E.M., Goggins, M., Maitra, A., Iacobuzio-Donahue, C., Eshleman, J.R., Kern, S.E., Hruban, R.H., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V.E., Kinzler, K.W., 2008. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321 (5897), 1801-1806. K. Muro, Y.B., V. Shankaran, R. Geva, D.V.T. Catenacci, S. Gupta, J.P. Eder, R. Berger, E.J. Gonzalez, J. Pulini, A.B. Ray, M. Dolled-Filhart, K. Emancipator, K. Pathiraja, X. Shu, M.R. Koshiji, J.D. Cheng, H.C. Chung, 2014. A phase 1b study of pembrolizumab (Pembro; MK-3475) in patients with advanced gastric cancer. Annals of Oncology 25 (5): 1-41. Kaufman, B., Shapira-Frommer, R., Schmutzler, R.K., Audeh, M.W., Friedlander, M., Balmana, J., Mitchell, G., Fried, G., Stemmer, S.M., Hubert, A., Rosengarten, O., Steiner, M., Loman, N., Bowen, K., Fielding, A., Domchek, S.M., 2014. Olaparib Monotherapy in Patients With Advanced Cancer and a Germline BRCA1/2 Mutation. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Keir, M.E., Liang, S.C., Guleria, I., Latchman, Y.E., Qipo, A., Albacker, L.A., Koulmanda, M., Freeman, G.J., Sayegh, M.H., Sharpe, A.H., 2006. Tissue expression of PD-L1 mediates peripheral T cell tolerance. The Journal of experimental medicine 203 (4), 883-895. Kern, S.E., Fearon, E.R., Tersmette, K.W., Enterline, J.P., Leppert, M., Nakamura, Y., White, R., Vogelstein, B., Hamilton, S.R., 1989. Clinical and pathological associations with allelic loss in colorectal carcinoma [corrected]. Jama 261 (21), 3099-3103.

25

Page 25 of 36

Kim, J.E., Hong, Y.S., Ryu, M.H., Lee, J.L., Chang, H.M., Lim, S.B., Kim, J.H., Jang, S.J., Kim, M.J., Yu, C.S., Kang, Y.K., Kim, J.C., Kim, T.W., 2011. Association between deficient mismatch repair system and efficacy to irinotecan-containing chemotherapy in metastatic colon cancer. Cancer science 102 (9), 1706-1711. King, M.C., Marks, J.H., Mandell, J.B., New York Breast Cancer Study, G., 2003. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 302 (5645), 643-646. Koopman, M., Kortman, G.A., Mekenkamp, L., Ligtenberg, M.J., Hoogerbrugge, N., Antonini, N.F., Punt, C.J., van Krieken, J.H., 2009. Deficient mismatch repair system in patients with sporadic advanced colorectal cancer. British journal of cancer 100 (2), 266-273. Kuniyasu, H., Yasui, W., Kitadai, Y., Yokozaki, H., Ito, H., Tahara, E., 1992. Frequent amplification of the cmet gene in scirrhous type stomach cancer. Biochemical and biophysical research communications 189 (1), 227-232. Kurzawski, G., Suchy, J., Debniak, T., Kladny, J., Lubinski, J., 2004. Importance of microsatellite instability (MSI) in colorectal cancer: MSI as a diagnostic tool. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 15 Suppl 4, iv283-284. Lagna, G., Hata, A., Hemmati-Brivanlou, A., Massague, J., 1996. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 383 (6603), 832-836. Lanza, G., Matteuzzi, M., Gafa, R., Orvieto, E., Maestri, I., Santini, A., del Senno, L., 1998. Chromosome 18q allelic loss and prognosis in stage II and III colon cancer. International journal of cancer. Journal international du cancer 79 (4), 390-395. LI Liu, H.S., Maureen R Bleam, Vivian Zhang, Jun Zou, Junping Jing, Kurtis E. Bachman, Monica Motwani, Keith W. Orford, Axel Hoos, 2014. Antitumor effects of dabrafenib, trametinib, and panitumumab as single agents and in combination in BRAF-mutant colorectal carcinoma (CRC) models. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32:5s (suppl; abstr 3513). Liang, J.T., Huang, K.C., Lai, H.S., Lee, P.H., Cheng, Y.M., Hsu, H.C., Cheng, A.L., Hsu, C.H., Yeh, K.H., Wang, S.M., Tang, C., Chang, K.J., 2002. High-frequency microsatellite instability predicts better chemosensitivity to high-dose 5-fluorouracil plus leucovorin chemotherapy for stage IV sporadic colorectal cancer after palliative bowel resection. International journal of cancer. Journal international du cancer 101 (6), 519-525. Liao, X., Lochhead, P., Nishihara, R., Morikawa, T., Kuchiba, A., Yamauchi, M., Imamura, Y., Qian, Z.R., Baba, Y., Shima, K., Sun, R., Nosho, K., Meyerhardt, J.A., Giovannucci, E., Fuchs, C.S., Chan, A.T., Ogino, S., 2012. Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival. The New England journal of medicine 367 (17), 1596-1606. Lievre, A., Bachet, J.B., Le Corre, D., Boige, V., Landi, B., Emile, J.F., Cote, J.F., Tomasic, G., Penna, C., Ducreux, M., Rougier, P., Penault-Llorca, F., Laurent-Puig, P., 2006. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer research 66 (8), 3992-3995. Lindforss, U., Fredholm, H., Papadogiannakis, N., Gad, A., Zetterquist, H., Olivecrona, H., 2000. Allelic loss is heterogeneous throughout the tumor in colorectal carcinoma. Cancer 88 (12), 2661-2667.

26

Page 26 of 36

Liu, F., Pouponnot, C., Massague, J., 1997. Dual role of the Smad4/DPC4 tumor suppressor in TGFbetainducible transcriptional complexes. Genes & development 11 (23), 3157-3167. Lorenzen, S., Lordick, F., 2011. How will human epidermal growth factor receptor 2-neu data impact clinical management of gastric cancer? Current opinion in oncology 23 (4), 396-402. Lowery, M.A., Kelsen, D.P., Stadler, Z.K., Yu, K.H., Janjigian, Y.Y., Ludwig, E., D'Adamo, D.R., Salo-Mullen, E., Robson, M.E., Allen, P.J., Kurtz, R.C., O'Reilly, E.M., 2011. An emerging entity: pancreatic adenocarcinoma associated with a known BRCA mutation: clinical descriptors, treatment implications, and future directions. The oncologist 16 (10), 1397-1402. Lu, Z., Friess, H., Graber, H.U., Guo, X., Schilling, M., Zimmermann, A., Korc, M., Buchler, M.W., 1997. Presence of two signaling TGF-beta receptors in human pancreatic cancer correlates with advanced tumor stage. Digestive diseases and sciences 42 (10), 2054-2063. Lucas, A.L., Shakya, R., Lipsyc, M.D., Mitchel, E.B., Kumar, S., Hwang, C., Deng, L., Devoe, C., Chabot, J.A., Szabolcs, M., Ludwig, T., Chung, W.K., Frucht, H., 2013. High prevalence of BRCA1 and BRCA2 germline mutations with loss of heterozygosity in a series of resected pancreatic adenocarcinoma and other neoplastic lesions. Clinical cancer research : an official journal of the American Association for Cancer Research 19 (13), 3396-3403. Malinowsky, K., Nitsche, U., Janssen, K.P., Bader, F.G., Spath, C., Drecoll, E., Keller, G., Hofler, H., SlottaHuspenina, J., Becker, K.F., 2014. Activation of the PI3K/AKT pathway correlates with prognosis in stage II colon cancer. British journal of cancer 110 (8), 2081-2089. Mao, M., Tian, F., Mariadason, J.M., Tsao, C.C., Lemos, R., Jr., Dayyani, F., Gopal, Y.N., Jiang, Z.Q., Wistuba, II, Tang, X.M., Bornman, W.G., Bollag, G., Mills, G.B., Powis, G., Desai, J., Gallick, G.E., Davies, M.A., Kopetz, S., 2013. Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents. Clinical cancer research : an official journal of the American Association for Cancer Research 19 (3), 657-667. Massague, J., Blain, S.W., Lo, R.S., 2000. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 103 (2), 295-309. Matsui, Y., Inomata, M., Tojigamori, M., Sonoda, K., Shiraishi, N., Kitano, S., 2005. Suppression of tumor growth in human gastric cancer with HER2 overexpression by an anti-HER2 antibody in a murine model. International journal of oncology 27 (3), 681-685. Maughan, T.S., Adams, R.A., Smith, C.G., Meade, A.M., Seymour, M.T., Wilson, R.H., Idziaszczyk, S., Harris, R., Fisher, D., Kenny, S.L., Kay, E., Mitchell, J.K., Madi, A., Jasani, B., James, M.D., Bridgewater, J., Kennedy, M.J., Claes, B., Lambrechts, D., Kaplan, R., Cheadle, J.P., Investigators, M.C.T., 2011. Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet 377 (9783), 2103-2114. McHugh, P.J., Spanswick, V.J., Hartley, J.A., 2001. Repair of DNA interstrand crosslinks: molecular mechanisms and clinical relevance. The Lancet. Oncology 2 (8), 483-490. Moses, H., 2007. Committee on Developing Biomarker-Based Tools for Cancer Screening, Diagnosis, and Treatment. US National Academy Press, Institute of Medicine.

27

Page 27 of 36

Naiyer A. Rizvi, E.B.G., Amita Patnaik, Leena Gandhi, Natasha B. Leighl, Ani Sarkis Balmanoukian, Jonathan Wade Goldman, Joseph Paul Eder, Elizabeth Johnson, George R. Blumenschein, Matthew A. Gubens, Kyriakos P. Papadopoulos, Gregory M. Lubiniecki, Jin Zhang, Michelle Niewood, Kenneth Emancipator, Marisa Dolled-Filhart, Mary Elizabeth Hanson, Rina Hui, 2014. Safety and clinical activity of MK-3475 as initial therapy in patients with advanced non-small cell lung cancer (NSCLC). Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32:5s (suppl; abstr 8007). Nakajima, M., Sawada, H., Yamada, Y., Watanabe, A., Tatsumi, M., Yamashita, J., Matsuda, M., Sakaguchi, T., Hirao, T., Nakano, H., 1999. The prognostic significance of amplification and overexpression of c-met and c-erb B-2 in human gastric carcinomas. Cancer 85 (9), 1894-1902. Nash, G.M., Gimbel, M., Shia, J., Nathanson, D.R., Ndubuisi, M.I., Zeng, Z.S., Kemeny, N., Paty, P.B., 2010. KRAS mutation correlates with accelerated metastatic progression in patients with colorectal liver metastases. Annals of surgical oncology 17 (2), 572-578. Neumann, J., Zeindl-Eberhart, E., Kirchner, T., Jung, A., 2009. Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathology, research and practice 205 (12), 858-862. O'Connell, M.J., Lavery, I., Yothers, G., Paik, S., Clark-Langone, K.M., Lopatin, M., Watson, D., Baehner, F.L., Shak, S., Baker, J., Cowens, J.W., Wolmark, N., 2010. Relationship between tumor gene expression and recurrence in four independent studies of patients with stage II/III colon cancer treated with surgery alone or surgery plus adjuvant fluorouracil plus leucovorin. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28 (25), 3937-3944. O'Reilly EM, L.M., Segal MF, Smith SC et al, 2014. Phase IB trial of cisplatin, gemcitabine, and veliparib in patients with known or potential BRCA or PALB2-mutated pancreas adenocarcinoma. Journal of Clinical Oncology 35:5s (suppl; abstr 4023). Ogino, S., Nosho, K., Irahara, N., Shima, K., Baba, Y., Kirkner, G.J., Meyerhardt, J.A., Fuchs, C.S., 2009a. Prognostic significance and molecular associations of 18q loss of heterozygosity: a cohort study of microsatellite stable colorectal cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (27), 4591-4598. Ogino, S., Nosho, K., Kirkner, G.J., Shima, K., Irahara, N., Kure, S., Chan, A.T., Engelman, J.A., Kraft, P., Cantley, L.C., Giovannucci, E.L., Fuchs, C.S., 2009b. PIK3CA mutation is associated with poor prognosis among patients with curatively resected colon cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (9), 1477-1484. Ogunbiyi, O.A., Goodfellow, P.J., Herfarth, K., Gagliardi, G., Swanson, P.E., Birnbaum, E.H., Read, T.E., Fleshman, J.W., Kodner, I.J., Moley, J.F., 1998. Confirmation that chromosome 18q allelic loss in colon cancer is a prognostic indicator. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 16 (2), 427-433. Okazaki, T., Honjo, T., 2006. The PD-1-PD-L pathway in immunological tolerance. Trends in immunology 27 (4), 195-201. Okines, A., Cunningham, D., Chau, I., 2011. Targeting the human EGFR family in esophagogastric cancer. Nature reviews. Clinical oncology 8 (8), 492-503.

28

Page 28 of 36

Oldenhuis, C.N., Oosting, S.F., Gietema, J.A., de Vries, E.G., 2008. Prognostic versus predictive value of biomarkers in oncology. European journal of cancer 44 (7), 946-953. Oliver GR, S.E., Laheru D, Diaz LA, 2010. Family history of cancer and sensitivity to platinum chemotherapy in pancreatic adenocarcinoma. Gastrointestinal Cancers Symposium (Orlando, FL) Abstract 180. Park do, Y., Sakamoto, H., Kirley, S.D., Ogino, S., Kawasaki, T., Kwon, E., Mino-Kenudson, M., Lauwers, G.Y., Chung, D.C., Rueda, B.R., Zukerberg, L.R., 2007. The Cables gene on chromosome 18q is silenced by promoter hypermethylation and allelic loss in human colorectal cancer. The American journal of pathology 171 (5), 1509-1519. Pazdur, R., 2013. FDA Approval for Trastuzumab. National Cancer Institute. Peng, Z., Zhu, Y., Wang, Q., Gao, J., Li, Y., Li, Y., Ge, S., Shen, L., 2014. Prognostic significance of MET amplification and expression in gastric cancer: a systematic review with meta-analysis. PloS one 9 (1), e84502. Peters, S., Adjei, A.A., 2012. MET: a promising anticancer therapeutic target. Nature reviews. Clinical oncology 9 (6), 314-326. Pishvaian MJ, W.H., Zhuang T, He AR et al, 2013. A phase I/II study of ABT-888 in combination with 5fluorouracil (5-FU) and oxaliplatin (Ox) in patients with metastatic pancreatic cancer (MPC). . Journal of Clinical Oncology 31 (suppl 4; abstr 147). Pizzi, S., Porzionato, A., Pasquali, C., Guidolin, D., Sperti, C., Fogar, P., Macchi, V., De Caro, R., Pedrazzoli, S., Parenti, A., 2009. Glucose transporter-1 expression and prognostic significance in pancreatic carcinogenesis. Histology and histopathology 24 (2), 175-185. Plummer, E.R., Calvert, H., 2007. Targeting poly(ADP-ribose) polymerase: a two-armed strategy for cancer therapy. Clinical cancer research : an official journal of the American Association for Cancer Research 13 (21), 6252-6256. Polyak, K., Garber, J., 2011. Targeting the missing links for cancer therapy. Nature medicine 17 (3), 283284. Poplin, E., Feng, Y., Berlin, J., Rothenberg, M.L., Hochster, H., Mitchell, E., Alberts, S., O'Dwyer, P., Haller, D., Catalano, P., Cella, D., Benson, A.B., 3rd, 2009. Phase III, randomized study of gemcitabine and oxaliplatin versus gemcitabine (fixed-dose rate infusion) compared with gemcitabine (30-minute infusion) in patients with pancreatic carcinoma E6201: a trial of the Eastern Cooperative Oncology Group. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27 (23), 3778-3785. Porcelli, L., Quatrale, A.E., Mantuano, P., Leo, M.G., Silvestris, N., Rolland, J.F., Carioggia, E., Lioce, M., Paradiso, A., Azzariti, A., 2013. Optimize radiochemotherapy in pancreatic cancer: PARP inhibitors a new therapeutic opportunity. Molecular oncology 7 (3), 308-322.

29

Page 29 of 36

Prahallad, A., Sun, C., Huang, S., Di Nicolantonio, F., Salazar, R., Zecchin, D., Beijersbergen, R.L., Bardelli, A., Bernards, R., 2012. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 483 (7387), 100-103. Punt, C.J., 2004. New options and old dilemmas in the treatment of patients with advanced colorectal cancer. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 15 (10), 1453-1459. Ribic, C.M., Sargent, D.J., Moore, M.J., Thibodeau, S.N., French, A.J., Goldberg, R.M., Hamilton, S.R., Laurent-Puig, P., Gryfe, R., Shepherd, L.E., Tu, D., Redston, M., Gallinger, S., 2003. Tumor microsatelliteinstability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. The New England journal of medicine 349 (3), 247-257. Rothwell, P.M., Wilson, M., Elwin, C.E., Norrving, B., Algra, A., Warlow, C.P., Meade, T.W., 2010. Longterm effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials. Lancet 376 (9754), 1741-1750. Rothwell, P.M., Wilson, M., Price, J.F., Belch, J.F., Meade, T.W., Mehta, Z., 2012. Effect of daily aspirin on risk of cancer metastasis: a study of incident cancers during randomised controlled trials. Lancet 379 (9826), 1591-1601. Rouleau, M., Patel, A., Hendzel, M.J., Kaufmann, S.H., Poirier, G.G., 2010. PARP inhibition: PARP1 and beyond. Nature reviews. Cancer 10 (4), 293-301. Ruschoff, J., Dietel, M., Baretton, G., Arbogast, S., Walch, A., Monges, G., Chenard, M.P., Penault-Llorca, F., Nagelmeier, I., Schlake, W., Hofler, H., Kreipe, H.H., 2010. HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing. Virchows Archiv : an international journal of pathology 457 (3), 299-307. S. Kopetz, J.D., E. Chan, J. R. Hecht, P. J. O'Dwyer, R. J. Lee, K. B. Nolop, L. Saltz, 2010. PLX4032 in metastatic colorectal cancer patients with mutant BRAF tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28(15) suppl 3534. S. Stintzing, A.J., L. Rossius, D.P. Modest, L. Fischer von Weikersthal, T. Decker, M. Mohler, W. Scheithauer, T. Kirchner, V. Heinemann, 2013. Analysis of KRAS/NRAS and BRAF mutations in FIRE-3: A randomized phase III study of FOLFIRI plus cetuximab or bevacizumab as first-line treatment for wildtype (WT) KRAS (exon 2) metastatic colorectal cancer (mCRC) patients. European journal of cancer 49, supplement 3. Samuels, Y., Wang, Z., Bardelli, A., Silliman, N., Ptak, J., Szabo, S., Yan, H., Gazdar, A., Powell, S.M., Riggins, G.J., Willson, J.K., Markowitz, S., Kinzler, K.W., Vogelstein, B., Velculescu, V.E., 2004. High frequency of mutations of the PIK3CA gene in human cancers. Science 304 (5670), 554. Sandler, R.S., Halabi, S., Baron, J.A., Budinger, S., Paskett, E., Keresztes, R., Petrelli, N., Pipas, J.M., Karp, D.D., Loprinzi, C.L., Steinbach, G., Schilsky, R., 2003. A randomized trial of aspirin to prevent colorectal adenomas in patients with previous colorectal cancer. The New England journal of medicine 348 (10), 883-890.

30

Page 30 of 36

Sargent, D.J., Marsoni, S., Monges, G., Thibodeau, S.N., Labianca, R., Hamilton, S.R., French, A.J., Kabat, B., Foster, N.R., Torri, V., Ribic, C., Grothey, A., Moore, M., Zaniboni, A., Seitz, J.F., Sinicrope, F., Gallinger, S., 2010. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracilbased adjuvant therapy in colon cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28 (20), 3219-3226. Satoh, T., Xu, R.H., Chung, H.C., Sun, G.P., Doi, T., Xu, J.M., Tsuji, A., Omuro, Y., Li, J., Wang, J.W., Miwa, H., Qin, S.K., Chung, I.J., Yeh, K.H., Feng, J.F., Mukaiyama, A., Kobayashi, M., Ohtsu, A., Bang, Y.J., 2014. Lapatinib plus paclitaxel versus paclitaxel alone in the second-line treatment of HER2-amplified advanced gastric cancer in Asian populations: TyTAN--a randomized, phase III study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32 (19), 2039-2049. Schoffski P, S.M., Burris HA, Lutzky J, Rearden T, Sikic B, 2010. Phase 2 randomized discontinuation trial (RDT) of XL184 in patients with advanced solid tumors. EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics EJC Supplements 8: 117. Shah, M.A., Wainberg, Z.A., Catenacci, D.V., Hochster, H.S., Ford, J., Kunz, P., Lee, F.C., Kallender, H., Cecchi, F., Rabe, D.C., Keer, H., Martin, A.M., Liu, Y., Gagnon, R., Bonate, P., Liu, L., Gilmer, T., Bottaro, D.P., 2013. Phase II study evaluating 2 dosing schedules of oral foretinib (GSK1363089), cMET/VEGFR2 inhibitor, in patients with metastatic gastric cancer. PloS one 8 (3), e54014. Siegel, R., DeSantis, C., Virgo, K., Stein, K., Mariotto, A., Smith, T., Cooper, D., Gansler, T., Lerro, C., Fedewa, S., Lin, C., Leach, C., Cannady, R.S., Cho, H., Scoppa, S., Hachey, M., Kirch, R., Jemal, A., Ward, E., 2012. Cancer treatment and survivorship statistics, 2012. CA: a cancer journal for clinicians 62 (4), 220241. Sierra, J.R., Tsao, M.S., 2011. c-MET as a potential therapeutic target and biomarker in cancer. Therapeutic advances in medical oncology 3 (1 Suppl), S21-35. Sinn, B.V., Striefler, J.K., Rudl, M.A., Lehmann, A., Bahra, M., Denkert, C., Sinn, M., Stieler, J., Klauschen, F., Budczies, J., Weichert, W., Stenzinger, A., Kamphues, C., Dietel, M., Riess, H., 2014. KRAS mutations in codon 12 or 13 are associated with worse prognosis in pancreatic ductal adenocarcinoma. Pancreas 43 (4), 578-583. Slater, E.P., Langer, P., Niemczyk, E., Strauch, K., Butler, J., Habbe, N., Neoptolemos, J.P., Greenhalf, W., Bartsch, D.K., 2010. PALB2 mutations in European familial pancreatic cancer families. Clinical genetics 78 (5), 490-494. Smit, V.T., Boot, A.J., Smits, A.M., Fleuren, G.J., Cornelisse, C.J., Bos, J.L., 1988. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic acids research 16 (16), 7773-7782. Sonnenblick, A., Kadouri, L., Appelbaum, L., Peretz, T., Sagi, M., Goldberg, Y., Hubert, A., 2011. Complete remission, in BRCA2 mutation carrier with metastatic pancreatic adenocarcinoma, treated with cisplatin based therapy. Cancer biology & therapy 12 (3), 165-168. Stenzinger, A., von Winterfeld, M., Aulmann, S., Warth, A., Weichert, W., Denkert, C., Ruschoff, J., Dietel, M., Klauschen, F., 2012. Quantitative analysis of diagnostic guidelines for HER2-status assessment. The Journal of molecular diagnostics : JMD 14 (3), 199-205.

31

Page 31 of 36

Surveillance, E., and End Results Program (SEER), 2014. SEER: Stat Fact Sheet. Pancreas Cancer, in: Institute, N.C. (Ed.). Sznol, M., Chen, L., 2013. Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 19 (5), 1021-1034. Tang, Z., Zhao, M., Ji, J., Yang, G., Hu, F., He, J., Shen, H., Gao, Z., Zhao, A., Li, J., Lu, Y., 2004. Overexpression of gastrin and c-met protein involved in human gastric carcinomas and intestinal metaplasia. Oncology reports 11 (2), 333-339. Tanner, M., Hollmen, M., Junttila, T.T., Kapanen, A.I., Tommola, S., Soini, Y., Helin, H., Salo, J., Joensuu, H., Sihvo, E., Elenius, K., Isola, J., 2005. Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 16 (2), 273-278. Tascilar, M., Skinner, H.G., Rosty, C., Sohn, T., Wilentz, R.E., Offerhaus, G.J., Adsay, V., Abrams, R.A., Cameron, J.L., Kern, S.E., Yeo, C.J., Hruban, R.H., Goggins, M., 2001. The SMAD4 protein and prognosis of pancreatic ductal adenocarcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 7 (12), 4115-4121. Thiagalingam, S., Lengauer, C., Leach, F.S., Schutte, M., Hahn, S.A., Overhauser, J., Willson, J.K., Markowitz, S., Hamilton, S.R., Kern, S.E., Kinzler, K.W., Vogelstein, B., 1996. Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nature genetics 13 (3), 343-346. Tran, B., Kopetz, S., Tie, J., Gibbs, P., Jiang, Z.Q., Lieu, C.H., Agarwal, A., Maru, D.M., Sieber, O., Desai, J., 2011. Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer. Cancer 117 (20), 4623-4632. Trusolino, L., Bertotti, A., Comoglio, P.M., 2010. MET signalling: principles and functions in development, organ regeneration and cancer. Nature reviews. Molecular cell biology 11 (12), 834-848. Tsiatis, A.C., Norris-Kirby, A., Rich, R.G., Hafez, M.J., Gocke, C.D., Eshleman, J.R., Murphy, K.M., 2010. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. The Journal of molecular diagnostics : JMD 12 (4), 425432. Tsushima, F., Yao, S., Shin, T., Flies, A., Flies, S., Xu, H., Tamada, K., Pardoll, D.M., Chen, L., 2007. Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy. Blood 110 (1), 180-185. Tutt, A., Ashworth, A., 2002. The relationship between the roles of BRCA genes in DNA repair and cancer predisposition. Trends in molecular medicine 8 (12), 571-576. Vakiani, E., Solit, D.B., 2011. KRAS and BRAF: drug targets and predictive biomarkers. The Journal of pathology 223 (2), 219-229.

32

Page 32 of 36

Van Cutsem, E., Kohne, C.H., Lang, I., Folprecht, G., Nowacki, M.P., Cascinu, S., Shchepotin, I., Maurel, J., Cunningham, D., Tejpar, S., Schlichting, M., Zubel, A., Celik, I., Rougier, P., Ciardiello, F., 2011. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29 (15), 2011-2019. Vanessa Deschoolmeester, F.L., Patrick Pauwels and Marc Peeters, 2012. Biomarkers, in: Khan, P.T. (Ed.), Biomarkers in Gastrointestinal Cancer: Focus on Colon, Pancreatic and Gastric Cancer. INTECH. Venkitaraman, A.R., 2002. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 108 (2), 171182. Venook, A.P., Niedzwiecki, D., Lopatin, M., Ye, X., Lee, M., Friedman, P.N., Frankel, W., Clark-Langone, K., Millward, C., Shak, S., Goldberg, R.M., Mahmoud, N.N., Warren, R.S., Schilsky, R.L., Bertagnolli, M.M., 2013. Biologic determinants of tumor recurrence in stage II colon cancer: validation study of the 12-gene recurrence score in cancer and leukemia group B (CALGB) 9581. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31 (14), 1775-1781. Vicuna, B., Benson, A.B., 3rd, 2007. Adjuvant therapy for stage II colon cancer: prognostic and predictive markers. Journal of the National Comprehensive Cancer Network : JNCCN 5 (9), 927-936. Volker Heinemann, L.F.v.W., Thomas Decker, Alexander Kiani, Ursula Vehling-Kaiser, Salah-Eddin AlBatran, Tobias Heintges, Juergen Lerchenmueller, Christoph Kahl, Gernot Seipelt, Frank Kullmann, Martina Stauch, Werner Scheithauer, Joerg Hielscher, Michael Scholz, Sebastian Mueller, Britta Schaefer, Dominik Paul Modest, Andreas Jung, Sebastian Stintzing, 2013. Randomized comparison of FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment of KRAS wild-type metastatic colorectal cancer: German AIO study KRK-0306 (FIRE-3). Journal of Clinical Oncology 31 (suppl; abstr LBA3506). Wagner, A.D., Moehler, M., 2009. Development of targeted therapies in advanced gastric cancer: promising exploratory steps in a new era. Current opinion in oncology 21 (4), 381-385. Wang, H., Han, H., Von Hoff, D.D., 2006. Identification of an agent selectively targeting DPC4 (deleted in pancreatic cancer locus 4)-deficient pancreatic cancer cells. Cancer research 66 (19), 9722-9730. Wang, H., Stephens, B., Von Hoff, D.D., Han, H., 2009. Identification and characterization of a novel anticancer agent with selectivity against deleted in pancreatic cancer locus 4 (DPC4)-deficient pancreatic and colon cancer cells. Pancreas 38 (5), 551-557. Wang, W., Li, Y.F., Sun, X.W., Chen, G., Zhan, Y.Q., Huang, C.Y., Wan, D.S., Pan, Z.Z., Zhou, Z.W., 2010. Correlation analysis between loss of heterozygosity at chromosome 18q and prognosis in the stage-II colon cancer patients. Chinese journal of cancer 29 (8), 761-767. Watanabe, T., Wu, T.T., Catalano, P.J., Ueki, T., Satriano, R., Haller, D.G., Benson, A.B., 3rd, Hamilton, S.R., 2001. Molecular predictors of survival after adjuvant chemotherapy for colon cancer. The New England journal of medicine 344 (16), 1196-1206.

33

Page 33 of 36

Webb, C.P., Taylor, G.A., Jeffers, M., Fiscella, M., Oskarsson, M., Resau, J.H., Vande Woude, G.F., 1998. Evidence for a role of Met-HGF/SF during Ras-mediated tumorigenesis/metastasis. Oncogene 17 (16), 2019-2025. Wilentz, R.E., Iacobuzio-Donahue, C.A., Argani, P., McCarthy, D.M., Parsons, J.L., Yeo, C.J., Kern, S.E., Hruban, R.H., 2000. Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression. Cancer research 60 (7), 2002-2006. Wu, C., Zhu, Y., Jiang, J., Zhao, J., Zhang, X.G., Xu, N., 2006. Immunohistochemical localization of programmed death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical significance. Acta histochemica 108 (1), 19-24. Wu, C.W., Li, A.F., Chi, C.W., Chung, W.W., Liu, T.Y., Lui, W.Y., P'Eng F, K., 1998. Hepatocyte growth factor and Met/HGF receptors in patients with gastric adenocarcinoma. Oncology reports 5 (4), 817-822. Yamashita-Kashima, Y., Iijima, S., Yorozu, K., Furugaki, K., Kurasawa, M., Ohta, M., Fujimoto-Ouchi, K., 2011. Pertuzumab in combination with trastuzumab shows significantly enhanced antitumor activity in HER2-positive human gastric cancer xenograft models. Clinical cancer research : an official journal of the American Association for Cancer Research 17 (15), 5060-5070. Yan, B., Yau, E.X., Choo, S.N., Ong, C.W., Yong, K.J., Pang, B., Salto-Tellez, M., 2011. Dual-colour HER2/chromosome 17 chromogenic in situ hybridisation assay enables accurate assessment of HER2 genomic status in gastric cancer and has potential utility in HER2 testing of biopsy samples. Journal of clinical pathology 64 (10), 880-883. Yang, H., Higgins, B., Kolinsky, K., Packman, K., Bradley, W.D., Lee, R.J., Schostack, K., Simcox, M.E., Kopetz, S., Heimbrook, D., Lestini, B., Bollag, G., Su, F., 2012. Antitumor activity of BRAF inhibitor vemurafenib in preclinical models of BRAF-mutant colorectal cancer. Cancer research 72 (3), 779-789. Yeo, C.J., Cameron, J.L., 1998. Prognostic factors in ductal pancreatic cancer. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie 383 (2), 129-133. Yokota, T., Ura, T., Shibata, N., Takahari, D., Shitara, K., Nomura, M., Kondo, C., Mizota, A., Utsunomiya, S., Muro, K., Yatabe, Y., 2011. BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer. British journal of cancer 104 (5), 856-862. Yoon, H.H., Shi, Q., Sukov, W.R., Lewis, M.A., Sattler, C.A., Wiktor, A.E., Wu, T.T., Diasio, R.B., Jenkins, R.B., Sinicrope, F.A., 2012. Adverse prognostic impact of intratumor heterogeneous HER2 gene amplification in patients with esophageal adenocarcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30 (32), 3932-3938. Yothers, G., O'Connell, M.J., Lee, M., Lopatin, M., Clark-Langone, K.M., Millward, C., Paik, S., Sharif, S., Shak, S., Wolmark, N., 2013. Validation of the 12-gene colon cancer recurrence score in NSABP C-07 as a predictor of recurrence in patients with stage II and III colon cancer treated with fluorouracil and leucovorin (FU/LV) and FU/LV plus oxaliplatin. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31 (36), 4512-4519. Yu, S., Yu, Y., Zhao, N., Cui, J., Li, W., Liu, T., 2013. C-Met as a prognostic marker in gastric cancer: a systematic review and meta-analysis. PloS one 8 (11), e79137.

34

Page 34 of 36

Yuan, K., Sun, Y., Zhou, T., McDonald, J., Chen, Y., 2013. PARP-1 regulates resistance of pancreatic cancer to TRAIL therapy. Clinical cancer research : an official journal of the American Association for Cancer Research 19 (17), 4750-4759. Zauber, N.P., Wang, C., Lee, P.S., Redondo, T.C., Bishop, D.T., Goel, A., 2004. Ki-ras gene mutations, LOH of the APC and DCC genes, and microsatellite instability in primary colorectal carcinoma are not associated with micrometastases in pericolonic lymph nodes or with patients' survival. Journal of clinical pathology 57 (9), 938-942. Zell, J.A., Ziogas, A., Bernstein, L., Clarke, C.A., Deapen, D., Largent, J.A., Neuhausen, S.L., Stram, D.O., Ursin, G., Anton-Culver, H., 2009. Nonsteroidal anti-inflammatory drugs: effects on mortality after colorectal cancer diagnosis. Cancer 115 (24), 5662-5671. Zhang, J., Roberts, T.M., Shivdasani, R.A., 2011. Targeting PI3K signaling as a therapeutic approach for colorectal cancer. Gastroenterology 141 (1), 50-61. Zhao, S., Venkatasubbarao, K., Lazor, J.W., Sperry, J., Jin, C., Cao, L., Freeman, J.W., 2008. Inhibition of STAT3 Tyr705 phosphorylation by Smad4 suppresses transforming growth factor beta-mediated invasion and metastasis in pancreatic cancer cells. Cancer research 68 (11), 4221-4228. Zheng, Z., Bu, Z., Liu, X., Zhang, L., Li, Z., Wu, A., Wu, X., Cheng, X., Xing, X., Du, H., Wang, X., Hu, Y., Ji, J., 2014. Level of circulating PD-L1 expression in patients with advanced gastric cancer and its clinical implications. Chinese journal of cancer research = Chung-kuo yen cheng yen chiu 26 (1), 104-111.

Trials

N

CRYSTAL

1198

PRIME

1183

COIN

1630

CALGB 80405

1137

FIRE-3

735

Table 1. Clinical trials of EGFR targeted therapy in the colorectal metastatic setting Median PFS (mo) Median OS (mo) KRAS All KRAS All Comparative All All codon RAS codon RAS Regimens patients patients 12 WT WT 12 WT WT FOLFIRI + C 8.9 9.9 10.9 19.9 23.5 25.1 (Van C FOLFIRI 8.0 8.4 8.8 18.6 20.0 21.6 FOLFOX + P 9.6 10.1 23.8 25.8 (Douillard et al., 201 FOLFOX 8.0 7.9 19.4 20.2 FOLFOX/XELOX + C FOLFOX/XELOX FOLFOX/FOLFIRI + B FOLFOX/FOLFIRI + C FOLFIRI + C FOLFIRI + B

-

8.6 8.6 10.8 10.4 10.3 10.4

10.2 10.3 10.5 10.4

-

17.0 17.9 29.0 29.9 28.8 25.0

25.0 33.1 33.1 25.9

(Mau

(Alan P. V

(S. Stin Hei

Abbreviations: B= bevacizumab; C= cetuximab; EGFR= epidermal growth factor receptor; FOLFOX= 5-fluorouracil, leucov FOLFIRI= 5-fluorouracil, leucovorin, irinotecan; mo= months; N= number of patients; OS= overall survival; P= panitumum free survival; Refs= references; WT= wild type; XELOX= capecitabine, oxaliplatin; "+"= plus; "/"= or

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