EGFR targeted therapy

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Review Article

EGFR-targeted therapy
Loredana Vecchione a, b, d, 1 , Bart Jacobs a, b, 1 , Nicola Normanno c , Fortunato Ciardiello d , Sabine Tejpar a, b,?
Digestive Oncology Unit, University Hospital Gasthuisberg, Herestraat 49 bus 602 Be 3000, Leuven, Belgium Center for Human Genetics, Katholieke Universiteit Leuven, Herestraat 49 bus 602 Be 3000, Leuven, Belgium c Cell Biology and Biotherapy Unit, INT-Fondazione Pascale, via Semmola 142, 80131, Naples, Italy d Division of Medical Oncology, Department of Experimental and Clinical Medicine and Surgery “F. Magrassi and A. Lanzara”, Second University of Naples, via Pansini 5, 80131, Naples, Italy
b a

A R T I C L E I N F O R M A T I O N Article Chronology: Received 1 July 2011 Revised version received 30 August 2011 Accepted 30 August 2011

A B S T R A C T Anti-Epidermal Growth Factor Receptor (EGFR) therapies have been recently developed for the treatment of multiple cancer types. At the time when they were introduced in clinical practice, there was little knowledge of the molecular bases of tumor sensitivity and resistance to these novel targeted compounds. By using the framework of anti-EGFR inhibitors as treatment for colorectal cancer patients, we will review the knowledge we have reached until now in improving the development of a personalized cancer therapy and we will try to indicate the future challenges this field will face in the future. ? 2011 Elsevier Inc. All rights reserved.

Keywords: EGFR signaling Oncogenic dependency Colorectal cancer Anti-EGFR inhibitors Molecular subgroups Personalized cancer therapy

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . General background . . . . . . . . . . . . . . . . . . . . . . . KRAS mutations as marker of resistance to anti-EGFR therapies Beyond KRAS mutations . . . . . . . . . . . . . . . . . . . . . Identifying EGFR dependent tumors . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0 0 0 0 0 0

? Corresponding author at: Digestive Oncology Unit, University Hospital Gasthuisberg, Herestraat 49 bus 602 Be 3000, Leuven, Belgium. E-mail addresses: loredana_vecchione@hotmail.com (L. Vecchione), bart.jacobs@med.kuleuven.be (B. Jacobs), nicnorm@yahoo.com (N. Normanno), fortunato.ciardiello@unina2.it (F. Ciardiello), sabine.tejpar@uz.kuleuven.ac.be (S. Tejpar). 1 Loredana Vecchione and Bart Jacobs contributed equally. 0014-4827/$ – see front matter ? 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.08.021

Please cite this article as: L. Vecchione, et al., Exp. Cell. Res. (2011), doi:10.1016/j.yexcr.2011.08.021

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Introduction
This review is intended to give an overview of what the clinical development of Epidermal Growth Factor Receptor (EGFR) antagonists has taught us in colorectal cancer, and to translate biological concepts into future implications of anti-EGFR therapies into the clinic. The review is highly focused on EGFR targeting drugs in colorectal cancer, and is not meant to be comprehensive. Due to the space constraints, we apologize beforehand to researchers whose work could not be cited. Extensive reviews have already focused on ErbB signaling, including in the gastrointestinal tract [1,2], so our aim is to try to highlight the challenges this field will face in the future.

General background
Colorectal cancer (CRC) represents one of the most common cancers worldwide. Approximately 40–50% of newly diagnosed patients are affected by metastatic disease [3]. The prognosis of these patients still remains poor with a median overall survival of 18–21 months [4]. Even though the classical tumor progression model for CRC proposed by Fearon and Vogelstein [5] has been recently slightly reviewed [6], there is still a common agreement on the different histological stages that characterize the development of colorectal tumors: from normal mucosa to invasive adenocarcinoma through dysplastic lesions, adenoma and adenocarcinoma in situ [7]. The multistage process of CRC formation is driven by the activation of dominant growth enhancing genes and by the inactivation of recessive growth inhibitory genes secondary to genetic (mutations) and epigenetic (DNA methylation and histone acetylation) abnormalities. CRCs have a complex genomic landscape, with each tumor containing a median of 76 no silent mutations [8]. However, on average only 15 of these mutations affect candidate cancer genes, i.e. genes that were likely to be involved in tumor pathogenesis and progression. Therefore, most of the mutations identified in CRC may be considered as “passenger mutations”. Nevertheless, it cannot be excluded that infrequent mutations can be drivers and that they function through known pathways. Several different pathways are deregulated in colon carcinogenesis including the WNT-β catenin signaling, the transforming growth factor β (TGF-β) and the EGFR pathways. However, the EGFR pathway is the only for which target based agents have been approved for CRC patients. Although several mutations in genes involved in signaling pathways downstream of EGFR are reported in CRC, there is no clear consensus about the role of EGFR signaling in colorectal cancer development. The EGFR is a member of the human epidermal growth factor receptor (HER)-erbB family of receptor tyrosine kinases. This transmembrane glycoprotein can be selectively activated through the binding with ligands belonging to the EGF family of peptide growth factors [9,10]. Once these ligands bind to the extracellular domain, EGFR forms homo- or heterodimers with its family members ErbB2/Neu, Erbb3/HER3 and Erbb4/HER4, which induces autophosphorylation of the intracellular domain through intrinsic tyrosine kinase activity and subsequent activation of downstream signaling [1]. EGFR activation triggers the activation of a multitude of intracellular signaling pathways, including the Ras/Raf mitogen activated

protein kinase (MAPK), the PI3K/AKT and the Jak2/Stat3 pathways, responsible for cancer cell proliferation, survival, invasion, metastasization and neo-angiogenesis [1,2]. Roberts et al. elegantly demonstrated the involvement of EGFR signaling in a genetic mouse model of intestinal tumorigenesis. By crossing the hypomorphic, 90% inactive EGFR mutant allele wa2 into an APCMin background, they showed that there was no reduction in polyp number measured at 1 month of age, but a 90% reduction in the number of macroadenomas at 3 months of age in these mice compared with the APCMin model [11]. A role for EGFR signaling in establishment of initiated microadenomas was therefore put forward although the mechanism still remains elusive. Although they provide the rationale for EGFR signaling as playing a distinct role in this colon cancer model, EGFR function in normal intestinal development and crypt homeostasis still remains poorly understood. There are however indications that EGFR has a function in intestinal homeostasis. Hypomorphic homozygous Wa2 mice show a delay in intestinal adaptation after a 50% bowel resection [12,13]. More recently, Sato et al. showed that exogenous EGF was required to culture isolated stem cells from mouse intestinal crypts in vitro. The authors state that EGFR signaling is required for propagation of these stem cells and for in-vitro growth of colon crypts and organoids originating from these stem cells [13]. In a follow-up report they have also shown that the Paneth cells, which the authors proposed to be part of the intestinal stem cell niche, highly express EGFR ligands [14]. Whether or not EGFR is required for stem cell maintenance or proliferation and differentiation of stem cell progeny warrants further investigation. On the other hand, there is large evidence of hyperactivation of EGFR signaling in multiple cancer types. Several mechanisms are known to contribute to this phenomenon, including mutations in the kinase domain of EGFR, overexpression of EGFR and its ligands and gene copy number changes. These findings, together with the druggable properties of EGFR, have led to the successful preclinical development first and clinical use then of the class of anti-EGFR agents. Two classes of EGFR antagonists, small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs), have been approved by the Food and Drug administration (FDA) and the European Medicines Evaluation Agency (EMEA) for the treatment of metastatic non small cell lung cancer (mNSCLC), colorectal cancer (mCRC), squamous-cell carcinoma of the head and neck and pancreatic cancer [15–19]. Gefitinib and erlotinib, two reversible TKIs, inhibit the EGFR phosphorylation and its downstream cascade by blocking the ATP pocket located in the intracellular catalytic domain of the receptor. Cetuximab and panitumumab, two anti-EGFR mAbs, target the extracellular domain of the receptor and upon the receptor binding they inhibit its dimerization and subsequent phosphorylation and signal transduction. The introduction of cetuximab and panitumumab in clinical practice, either in combination with chemotherapy or as single agent, has shown to improve the outcome of metastatic CRC (mCRC) patients [20–22]. The first clinical trials of cetuximab and panitumumab were designed for an unselected population of chemorefractory mCRC patients and the objective response rate they recorded in monotherapy was only 10%. Therefore, although the efficacy of EGFR targeting agents represents a major step

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forward in the treatment of this disease, the clinical benefit obtained from the administration of these therapies is still limited to a small percentage of patients. The challenge for these and hopefully more targeted agents in the coming years will be to identify the subset of CRC disease that displays oncogenic dependency on EGFR signaling in order then to improve the patients selection and the optimization of the therapeutic index.

KRAS mutations as marker of resistance to anti-EGFR therapies
Mutations of the KRAS gene occur in approximately 40% of CRC. They represent an early event in the colorectal carcinogenesis [5,7,23] and are maintained during the CRC development, from early stage of the disease to advanced metastatic stages with an approximately 95% concordance rate in paired primary cancers and metastatic samples [24,25]. Mutations in KRAS are single nucleotide point mutations that mostly occur in codons 12 and 13 of exon 2 (codon 12 approximately 70–80% and codon 13 about 15–20% of all Kras mutations). The remaining mutations are mainly located on codons 61, 146 and 154 [26]. These mutations induce the protein to accumulate in a constitutively active GTP-bound status by the impairment of intrinsic and GTPase Activated Protein (GAP) mediated hydrolysis of GTP to GDP [27]. Retrospective analysis of single arm studies in heavily pretreated mCRC patients suggested that KRAS mutations are a marker of resistance to anti-EGFR mAbs [28–33]. Although retrospective and small, these studies were able to strongly support the hypothesis that the Kras mutations were associated with the lack of response to cetuximab and panitumumab in chemorefractory mCRC patients, leading the American and European health authorities to restrict the use of panitumumab monotherapy, as well cetuximab monotherapy or in combination with chemotherapy, only to patients with Kras wildtype tumors [34]. Recently, three prospective trials, the OPUS (Oxaliplatin and Cetuximab in First-Line Treatment of Metastatic Colorectal Cancer) [35], the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) [36], and the PRIME (the Panitumumab Randomized Trial in Combination with Chemotherapy for Metastatic Colorectal Cancer to Determine the Efficacy) [37] confirmed these data: when administered in first line, cetuximab and panitumumab, either in combination with an oxaliplatin-based or in combination with an irinotecan-based chemotherapy, result to be effective only in Kras wild type (WT) mCRC patients. KRAS is a molecular switch that is usually activated by tyrosine kinase receptors. When mutated, it results to be constitutively active leading the cells to become independent from the EGFR signaling activation. This event does not completely explain the mechanisms of resistance of KRAS mutant tumors to anti-EGFR agents but it is intriguingly coming up that the different mutations occurring in the same gene might not have the same effects on the resistance to anti-EGFR drugs. As recently reported by De Roock et al., chemotherapy-refractory mCRC patients which carry a KRAS mutation in codon 13 may have longer progression free survival (median, 4.0 months vs 1.9 months) and longer overall survival (median, 7.6 months vs 5.7 months) compared with patients harboring other KRAS mutations when treated with cetuximab [38]. The molecular mechanism behind this

phenomenon has not yet been identified. Nevertheless, it introduces the concept of different mutations occurring in the same gene as playing different roles in terms of response to treatment and highlights the paradigm that mutations may not always be considered as the main driver of tumor sensitivity to therapy but that other mechanisms may be involved. Clearly, the KRAS biomarker has enriched for a CRC population likely to benefit from anti-EGFR therapy. But since only 20–40% of KRAS wild-type patients will respond to cetuximab, either in monotherapy or in combination, it still does not accurately help to select the subset of CRC patients for which these compounds will be efficacy.

Beyond KRAS mutations
The findings with regards to KRAS, have lightened the search for additional genetic determinants of primary resistance to cetuximab and panitumumab. The idea that mutations in molecules involved in downstream pathways of the EGFR signaling could be responsible for the lack of sensitivity to EGFR targeting agents has directed to the search to BRAF and PIK3CA mutations and on PTEN deregulation. BRAF is one of the primary downstream effectors of KRAS signaling [39]. The V600E is the most common point mutation that involves the BRAF gene and it is present in approximately 10% of mCRC. Since KRAS and BRAF belong to the same pathway downstream of EGFR and mutations in these genes are mutually exclusive, one could speculate that the presence of an active mutation in one of these two molecules is able alone to drive the constitutive activation of the pathway. At least three studies [40–42] have shown that BRAF V600E mCRC tumors do not respond to cetuximab and have lower progression free survival and lower overall survival as compared with BRAF wild type patients. However, these studies investigated the role of BRAF mutations in response to anti-EGFR agents used as monotherapy or in combination with irinotecan-based regimens in chemorefractory patients so we can speculate that these results might be mostly driven by a baseline prognostic effect of BRAF mutations instead of a predictive effect, as also nicely reported by Roth et al. [43]. Recent results from first line randomized studies (OPUS [35] and CRYSTAL [36]) make the relationship between the BRAF mutation and the response to cetuximab not completely clear since they showed a trend in favor to cetuximab treatment for BRAF V600E patients. This highlights, at the moment, the need of further investigations. One of the main pathways activated by EGFR is the PI3K/PTEN/ AKT signaling cascade. Mutations in the PIK3CA gene, which encodes for the p110 catalytic subunit of the PI3K, are not frequent in CRC occurring in about 15% of the tumors [42]. PIK3CA mutations mainly occur in exons 9 and 20, with exon 9 showing the highest incidence (68.5% approximately). These mutations can be found in the same tumor together with KRAS and BRAF mutations, and this makes difficult to evaluate their own role in defining the sensitivity to anti-EGFR mAbs. Patients with mutation in KRAS or BRAF and PIK3CA do not respond to cetuximab while patients wild type for KRAS and BRAF but mutant for PIK3CA may have different sensitivity, depending on the kind of mutation they harbor: while PIK3CA exon 20 mutations are associated with resistance to cetuximab, PIK3CA exon 9 variants have no significant effect on response [42]. Again, this underlines the paradigm of different

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mutations affecting the same gene playing different roles in terms of drug sensitivity. However, as for BRAF V600E, the correlation between PIK3CA mutations and cetuximab response is still not clear since several conflicting data have been reported [44–47]. PTEN inhibits the PI3K and its inactivation, by genetic or epigenetic mechanisms, leads to the constitutive activation of the pathway. Recent reports suggested that PTEN inactivation is associated to resistance to EGFR targeting agents [46,47] However, these findings have not been confirmed yet in randomized clinical trials. Furthermore, the lack of standardized methods for its detection limits the possibility to use this marker in a clinical routine. Finally, the role of NRAS mutations, a member of the RAS oncogene family, has also been investigated. The frequency of these mutations in mCRC is very low (about 2.6%). Preliminary findings suggest that NRAS mutations are associated with resistance to cetuximab and panitumumab [42,48] even if further studies are needed to confirm these results. The above summarized data suggest that molecular alterations that involve BRAF, PIK3CA, PTEN and NRAS might add important information to only KRAS mutations in order to select mCRC patients who should be excluded from EGFR targeted therapies because of their primary resistance to these compounds. In fact, when assessed together, these mutations can improve the objective response rates reached by mCRC under anti-EGFR treatment as suggested by De Roock et al. [42]. Authors reported objective response rate (ORR) in an unselected population of approximately 24.4%; within the KRAS wild type population the ORR increased to 36.3%, while a 41.2% ORR was then observed in patients wild type for KRAS, BRAF, NRAS and PIK3CA-exon 20. These data were obtained in a specific population of patients and need to be confirmed in randomized clinical trials in which chemonaive mCRC patients are treated with anti-EGFR agents in order to clarify the role of these mutations as predictive markers of resistance. Nevertheless, they will not completely be able to define which patients will benefit.

Identifying EGFR dependent tumors
As above mentioned, tumor genotyping of CRC has allowed the identification of markers negatively associated with sensitivity to antiEGFR therapies. Although behaving as good negative predictive factors, these markers still remain unable to selectively identify patients who will experience benefit from therapy. This could be probably due to the fact that tumors carrying constitutively active signaling pathways downstream of the EGFR do not depend on EGFR activation for their growth. Thus, the identification of factors responsible for EGFR dependency might improve the ability to predict the response to anti-EGFR agents. In this context, positive predictors of response would be much more useful than negative predictors in the clinical practice, since they would enable to administer drugs only to a target population, the one which will selectively benefit from it. Several different studies have shown that EGFR expression assessed by immunohistochemistry does not predict clinical outcome in patient treated with anti-EGFR agents [49,50], while EGFR gene copy number has shown a limited evidence of association with objective response and clinical benefit [51–53]. In this regard, gene expression microarrays have enabled the development of transcriptional signatures associated with EGFR

sensitivity. Khambata-Ford et al. were the first to publish a gene signature obtained from snap-frozen liver metastasis which was associated with disease control (DC) in mCRC patients who received cetuximab as monotherapy. In this signature, two EGFR ligands, Amphiregulin (AREG) and Epiregulin (EREG), were found amongst the top genes as predicting the cetuximab response [54]. As follow-up to this discovery study, our group put an effort in the characterization of the predictive value of EREG and AREG expression in primary formalin-fixed-paraffin-embedded (FFPE) tumors for the response of chemorefractory mCRC patients to cetuximab combined with chemotherapy. Here, we confirmed the original findings of Khambata-ford et al. and further enriched for responders to cetuximab in the Kras-wild-type high EREG population [55]. In further agreement with these findings, Tabernero et al. found that in a cohort of 106 patients receiving first line cetuximab combined with an irinotecan based regimen, AREG and EREG expression were elevated in tumors of patients without disease progression, either in the total population or in the KRAS wild type tumor subgroup. Although AREG and EREG expression in mCRC wild type for KRAS strongly correlated with sensitivity to cetuximab, responses to cetuximab independent from the expression of these ligands are still observed [56]. Due to the linear relation between the expression of these ligands and the outcome it is hard to accurately select the patients who will respond. It is therefore necessary to explore additional markers present in sensitivity or resistance signatures to increase the predictive ability of the model. This was exactly the approach followed by Baker et al. who have tested mRNA expression of 110 candidate genes in 144 primary KRAS wild type mCRC patients treated with cetuximab. The authors found most of these genes to be strongly associated with all the clinical outcome variables considered (disease control, ORR and PFS) and a number of them to be compatible with the known biology of CRC: AREG, EREG and VAV3, activators of the EGFR signaling, associate with an increase of clinical benefit while DUSP6, a phosphatase known as a feed-back inhibitor of the MAPK pathway, associates with a decrease of the clinical benefit [57]. Moreover, by performing a multivariate analysis on the entire set of identified genes, a four-gene classifier that includes EREG, AREG, DUSP6 and SCL26A3 was generated. This four-gene classifier, together with the KRAS status, significantly improved the specificity and the predictive positive value of cetuximab benefit (in terms of disease control and ORR) compared to the selection of patients based only on the KRAS status. When applied to the PFS, this model performed better than either AREG/EREG alone or KRAS status alone. An unsupervised hierarchical clustering of gene expression profile of mCRC treated with cetuximab, has lead Rhodes et al. [58] to identify gene modules associated to cetuximab response. In particular, they have been able to find a RAS activation module associated to lack of response to cetuximab (defined as surrogate of RAS mutations), a carcinoma-like proliferating module (including EREG and AREG expression) correlated to cetuximab response and several modules associated to cetuximab resistance, correlated to metastasis/invasion, leukocytes, stromal response, cellular defense and interferon response. The finding of a gene expression module of RAS activation that could serve as surrogate for KRAS mutations and the finding of modules of resistance independent of these mutations demonstrate how the mutational status is not the only marker

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we should consider in stratifying the target population. Even though KRAS mutations are accepted as a dichotomous state in CRC, to be mutated or not, and used as predictive negative marker for cetuximab and panitumumab administration, there are still missing data on how they can really affect the sensitivity to anti-EGFR mAbs and whether this effect is homogenous or not. Recently, Tejpar et al. [59], by profiling RNA extracted from a total of 1378 formalin-fixed tissues of primary CRC, have identified that BRAF V600E CRC can be easily discriminated from the BRAF/KRAS wild-type and from the KRAS mutant since they show a specific gene expression pattern, while the KRAS wild type and KRAS mutant population seem to be heterogeneous underlying the need to better identify CRC subgroups that can be then stratified for selective and personalized treatments. It is clear that the focus in the field is evolving from analyzing dichotomous mutational data, into multi-dimensional expression signatures that are able to catch variability within tumors much more sensitively. The challenge will remain to link these signatures to pathways that are perturbed and contributing to tumor growth.

above described mechanisms to the sensitivity/resistance to anti EGFR compounds will depend on the genetic and molecular background of each individual tumor. However, the baseline gene expression profile of primary CRC has allowed the prediction of responsiveness to anti-EGFR treatment, basically identifying those tumors with autocrine ligand production. It is not unconceivable that this approach will yield results for drugs that are now in preclinical and clinical development. Indeed, gene expression signatures have been able to identify cell lines responsive to inhibitors of the MAPK [61] and PI3K/Akt pathway respectively. The characterization of the molecular subgroups in colon cancer and the functional annotation thereof will allow optimal treatment strategies to be deployed in a tailored fashion.

REFERENCES

Conclusions
The observation that only a subset of mCRC may benefit from cetuximab and panitumumab has driven the scientific community to identify predictive markers of response to optimize the patient selection and to maximize the therapeutic index. The main characteristics for a tumor to respond to anti-EGFR mAbs are to be dependent on EGFR signaling for growth and to have no activations of molecules responsible for the induction of pathways downstream to EGFR. In colorectal tumors that develop in absence of mutations in KRAS or BRAF, it is possible that the effects of these molecular alterations are substituted by autocrine and/or paracrine loops that involve the EGFR and its ligands. These tumors result to be addicted to the production of EGF-like growth factors and display high sensitivity to cetuximab and panitumumab since, by binding the EGFR, these compounds are able to block the functional interaction between ligands and receptor. The clinical application of this phenomenon comes out from studies that have demonstrated that the ligand expression, in particular EREG and AREG, in mCRC wild type for KRAS is correlated with response to cetuximab and panitumumab [54–57,60]. At the same time, when clustered together with other genes, these ligands are able to better define a subset of colon cancer responsive to anti-EGFR mAbs [58]. On the contrary, the activation of molecules involved in intracellular signaling pathways leads to an EGFR-independent tumor phenotype. Mutations in the KRAS oncogene, responsible of its constitutive activation, have represented the first negative predictive factor of response to cetuximab and panitumumab. Mutations and deregulations of other molecules downstream of the EGFR, such as BRAF, NRAS, PI3K and PTEN have also been demonstrated to be associated to lack of response to anti-EGFR mAbs [28–33,40–42,44–46]. Nevertheless, neither the KRAS/BRAF/NRAS/PIK3CA/PTEN molecular alterations nor the ligands levels of expression allow certain identification of patients that will completely benefit from anti-EGFR agents. Cancer is the result of several different molecular alterations and it is conceivable that the relative contribution of the

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