Oncogenic mutations as predictive factors in

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Oncogene (2010) 1–11

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REVIEW

Oncogenic mutations as predictive factors in colorectal cancer A Lie`vre1,2,3, H Blons1,4,5 and P Laurent-Puig1,4,5 1

INSERM UMR-S 775 Molecular Basis of Response to Xenobiotics, Paris, France; 2Assistance-Publique Hoˆpitaux de Paris, Hoˆpital Ambroise Pare´, Service d’He´pato-Gastroente´rologie et Oncologie Digestive, Boulogne-Billancourt, France; 3Universite´ Versailles Saint-Quentin-en-Yvelines, Versailles, France; 4Universite´ Paris Descartes, Paris, France and 5Assistance-Publique Hoˆpitaux de Paris, Hoˆpital Europe´en Georges Pompidou, Service de Biochimie, Paris, France

The anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies cetuximab and panitumumab have been demonstrated to be new therapeutic options for metastatic colorectal cancer (mCRC). Oncogenic activation of intracellular signalling pathways downstream of EGFR has a major role in colorectal carcinogenesis but has also been reported to be an important mechanism of resistance to anti-EGFR antibodies. Among the activating mutations found in colorectal cancers, tumour KRAS mutations, which are found in B40% of the cases, have been widely demonstrated as a major predictive marker of resistance to cetuximab or panitumumab, therefore, opening the way to individualized treatment for patients with mCRC. Other oncogenic mutations, such as BRAF or PIK3CA mutations or loss of PTEN expression, may also be additional interesting predictive markers of response to anti-EGFR monoclonal antibodies but required further evaluation before being incorporated in clinical practice. The identification of these molecular markers involved in the resistance of antiEGFR antibodies will allow the development of new therapies that should target ‘escape mechanisms’ used by tumours to circumvent a pathway that has been pharmacologically blocked by anti-EGFR. Oncogene advance online publication, 12 April 2010; doi:10.1038/onc.2010.89 Keywords: colorectal cancer; EGFR; cetuximab; panitumumab; KRAS; personalized medicine

Introduction The epidermal growth factor receptor (EGFR), a member of the human epidermal growth factor receptor HER-erbB family of receptor tyrosine kinases, is an important therapeutic target in metastatic colorectal cancer (mCRC). The activation of the EGFR pathway is indeed involved in colorectal carcinogenesis through the binding of EGF or other ligands on the extracellular part of the receptor, which results in the initiation of an Correspondence: Professor P Laurent-Puig, INSERM U775 Molecular Basis of Response to Xenobiotics, 45 Rue des Saints Pe`res, 75006 Paris, France. E-mail: [email protected] Received 17 December 2009; revised 29 January 2010; accepted 9 February 2010

oncogenic intracellular signalling cascade involving several pathways, including the RAS-mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, phospholipase C, signal transducer and activator of transcription (STAT) and SRC/FAK pathways. Monoclonal antibodies are one of the major strategies of EGFR inhibition that has been developed in cancer therapy. In the treatment of mCRC, two monoclonal antibodies, cetuximab (Erbitux, Merck-Serono, Darmstadt, Germany) and panitumumab (Vectibix, Amgen, Thousand Oaks, CA, USA) have been approved. Cetuximab, which is the first anti-EGFR monoclonal antibody that was developed, is an IgG1 chimeric antibody. Its efficacy was first demonstrated in combination with irinotecan in irinotecan or oxaliplatin-resistant mCRC expressing EGFR by immunohistochemistry (Cunningham et al., 2004; Saltz et al., 2004; Sobrero et al., 2008), then in monotherapy in chemotherapy-refractory patients (Jonker et al., 2007). More recently, the association of cetuximab plus chemotherapy was proved to be superior to chemotherapy alone in first-line therapy (Bokemeyer et al., 2007; Van Cutsem et al., 2007a). Panitumumab, a fully human monoclonal IgG2 antibody, was registered in chemotherapy-refractory mCRC in which it was shown to improve progression-free survival in patients compared with best supportive care (Van Cutsem et al., 2007b). Even if the efficacy of cetuximab and panitumumab has been clearly demonstrated, it remains modest, as objective response rates are comprised between 8 and 23%. The identification of predictive markers of response or resistance to these anti-EGFR antibodies is therefore of major importance to improve treatment strategy of mCRC by better defining whose patients will benefit from them, avoiding unnecessary and potentially dangerous exposure to these drugs and reducing treatment costs. The most relevant predictive markers of resistance to anti-EGFR antibodies are currently represented by somatic gene mutations, which are both implicated in colorectal carcinogenesis and responsible for a ligand-independent activation of intracellular signalling pathways downstream of EGFR. Signalling pathways and oncogenic mutations in colorectal cancer In the last few years, development of molecular biology has led to a growing knowledge of mechanisms involved

Oncogenic mutations as predictors in colorectal cancer A Lie`vre et al

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in the occurrence of cancers, including colorectal cancer. Among them, an activation of several pathways downstream of EGFR, including the RAS/MAPK and PI3K/ AKT pathways, and also the PLC, STAT and SRC/ FAK pathways, has been shown to be a key event in tumour proliferation, angiogenesis and cell survival. These pathways form an interconnected network, which involves phosphorylation of proteins that activate transcription factors triggering carcinogenesis through deregulation of protein synthesis, cell-cycle progression, apoptosis, angiogenesis and altered metabolism (Shaw and Cantley, 2006). Among genetic alterations that can activate intracellular signalling pathways involved in colorectal carcinogenesis, somatic mutations of genes encoding for effectors of the EGFR pathway are the most common and best known. Although the EGFR gene is not or very rarely mutated in colorectal tumours, activating mutations of KRAS or BRAF, involved in the RAS/MAPK pathway, are present in 32–37% and in 10–17% of the cases in large population-based studies, respectively (Samowitz et al., 2000; Rajagopalan et al., 2002; Brink et al., 2003; Samowitz et al., 2005a; Nosho et al., 2008a; de Vogel et al., 2009; Ogino et al., 2009b), and mutations of PI3KCA that encodes the catalytic subunit of the PI3K protein involved in the PI3K/AKT pathway are observed in 15% of the cases (Bamford et al., 2004; Barault et al., 2008b; Nosho et al., 2008b). MAPK pathway mutations RAS isoforms are small molecules of 21 kD localized at the inner surface of the plasma membrane, which are coded by the three genes KRAS, NRAS and HRAS. They are GDP/GTP-binding proteins that act as intracellular signal transducers (Vetter and Wittinghofer, 2001) and the GTP-active form interact with a variety of downstream effector proteins (Marshall, 1996). RAS genes have long been known as protooncogenes mutated in various types of human cancers, including 35–40% of colorectal cancers. They cause RAS to accumulate in the active GTP-bound state by impairing intrinsic GTPase activity and conferring resistance to GTPase-activating proteins (GAPs) (Bos, 1989). They are thus activating mutations of the RAS/ MAPK pathway. The vast majority of these oncogenic RAS mutations in colorectal cancer affect amino-acid residues G12, G13 and more rarely Q61 of the KRAS gene (Bamford et al., 2004). NRAS mutations are much less frequent (o5%) (Lea et al., 2009) and HRAS mutations are not present (Bamford et al., 2004). After binding and activation by GTP, RAS recruits the serine/threonine kinase RAF, which also acts as an oncogene that phosphorylates MAPK-1 and -2 and initiates MAPK signalling that leads to the expression of proteins having important role in cell proliferation, differentiation and cell survival. A quasi-unique point mutation is observed in the BRAF gene, leading to the substitution of a valine by a glutamic acid in codon 600 (V600E) and is found in 10–15% of colorectal tumours (13%) (Bamford et al., 2004). This mutation is Oncogene

associated with elevated kinase activity and is able to transform NIH3T3 cells (Davies et al., 2002). It is important to note that BRAF and KRAS mutations are mutually exclusive (Rajagopalan et al., 2002; Barault et al., 2008b). The PI3K/AKT pathway mutations The PIK3CA gene codes for the catalytic subunit p110a of PI3K that can be activated directly by phosphorylation of the EGFR tyrosine kinase domain or through the interaction with RAS proteins. This gene is mutated in 15–30% of colorectal cancers, with hotspots identified in exons 1, 2, 9 and 20, and mutated p110a proteins have shown in vitro a gain of enzymatic function by activating the AKT signalling in the absence of growth factors and are thus oncogenic in cell culture and animal models (Samuels et al., 2004; Ikenoue et al., 2005; Kang et al., 2005). In colorectal cancer, PIK3CA mutations occur more frequently in women, and in the proximal part of the colon (Benvenuti et al., 2008; Barault et al., 2008b), a double mutation of the gene is observed in 6–9% of the mutated cases (Samuels et al., 2004; Barault et al., 2008b) and there is a significant concomitant occurrence of KRAS and PIK3CA mutations (Parsons et al., 2005; Velho et al., 2005; Barault et al., 2008b). The tumuor-suppressor gene PTEN encodes a phosphatase that has an activity against lipid and protein substrates. The loss of lipid-phosphatase activity is sufficient to cause cancer phenotype. The PTEN gene may be inactivated in colorectal cancers cells by somatic mutations, which are a relatively rare event (9%), allelic losses and hypermethylation of the enhancer region. The prevalence of inactivating mutation is clearly related to the microsatellite status of the tumour as it varies from 18 to o2% in MSI and MSS tumours, respectively (Wang et al., 1998; Guanti et al., 2000; Shin et al., 2001). In colorectal cancers, PIK3CA mutation and loss of PTEN expression in tumour cells are mutually exclusive (Frattini et al., 2007), as it is observed with KRAS and BRAF mutations, which is consistent with the hypothesis that alteration in only one gene of the same pathway is sufficient to activate this one.

Oncogenic mutations as predictive markers of response to anti-EGFR antibodies As somatic mutations described above are responsible for an ligand-independent activation of signalling pathways downstream of the EGFR, it was hypothesized that they may lead to a resistance to anti-EGFR antibodies that act upstream, at the EGFR level (Figure 1). KRAS somatic mutations After the results of a first study on 30 mCRC patients showing a relationship between the presence of tumour KRAS mutations and lack of response to cetuximab (Lievre et al., 2006), several retrospective studies performed in patients who had progressed under an

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Figure 1 Different EGFR signalling pathways induced by the interaction between ligand and EGF receptor are described. The roles of these pathways in carcinogenesis are given. The frequency of the different mutation observed in colorectal cancers was depicted.

irinotecan-based chemotherapy and who were treated with cetuximab or panitumumab confirmed that KRAS mutations were associated with a resistance to antiEGFR monoclonal antibodies and a shorter progression-free or overall survival (Benvenuti et al., 2007; Di Fiore et al., 2007; Finocchiaro et al., 2007; Frattini et al., 2007; Khambata-Ford et al., 2007; De Roock et al., 2008; Lievre et al., 2008). These results were reinforced by in vitro experiments as the transfection of an activating KRAS mutation (Gly12Val) in the nonmutated and highly sensitive to cetuximab DiFi colorectal cancer cell line was responsible for an acquired resistance to this targeted therapy (Benvenuti et al., 2007). The first large randomized controlled phase III study that clearly demonstrated the negative predictive value of KRAS mutations on response to anti-EGFR monoclonal antibodies compared panitumumab monotherapy with best supportive care in 463 chemoresistant patients (Van Cutsem et al., 2007b). KRAS status was tested in a blinded manner in 427 patients (92%) and was found mutated in 43% of them (Amado et al., 2008). KRAS mutations were significantly associated with lack of

response to panitumumab alone (0% for KRASmutated patients vs 17% for KRAS wild-type patients) and a poorer progression-free survival (7.4 weeks for KRAS-mutated patients vs 12.3 weeks for KRAS wildtype patients) (Table 1). In fact, progression-free survival in KRAS-mutated patients receiving panitumumab and in those with best supportive care was similar (7.4 and 7.3 weeks, respectively) and a significant benefit of panitumumab over best supportive care was found only in KRAS wild-type patients, with a hazard ratio (HR) of 0.45 (95% confidence interval (CI): 0.34–0.59; Po0.0001) vs 0.99 (95% CI: 0.73–1.36) in KRASmutated patients. No difference in overall survival was observed between the control and panitumumab arms among all patients or among KRAS wild-type patients, but it is important to mention that 77% of the 219 KRAS-evaluable patients initially assigned to the control arm crossed over to receive panitumumab treatment (Amado et al., 2008). The results of this first randomized controlled study have also led to the registration, for the first time, of an anti-cancer drug based on the presence of a molecular alteration in an individual colorectal cancer tissue since panitumumab was approved by the Oncogene

Oncogenic mutations as predictors in colorectal cancer A Lie`vre et al

4 Table 1 Randomized controlled trials evaluating tumour KRAS mutations and outcome of anti-epidermal growth factor receptor monoclonal antibodies in metastatic colorectal cancer Reference

Treatment

No. of patients

Objective response, % (n/N) MT

Amado et al. (2008)

Panitumumab

BSC Karapetis et al. (2008) Cetuximab Van Cutsem et al. (2009b) Bokemeyer et al. (2009a); Van Cutsem et al. (2009b)

BSC Cetuximab þ FOLFIRI FOLFIRI

Progression-free survival

WT

Overall survival

MT

WT

MT

WT

7.4 weeks





— 4.5 months 4.6 months 16.2 months

— 9.5 months (Po0.001) 4.8 months 23.5 months

208

0 (0/84)

17 (21/124)

219 198

— 1.2 (1/81)

— 12.8 (15/117)

7.3 weeks 1.8 months

196 630

0 (0/83) 0 (0/113) 31.3 (67/214) 57.3 (181/316)

1.8 months 7.4 months

12.3 weeks (Po0.0001) 7.3 weeks 3.7 months (Po0.001) 1.9 months 9.9 months

533

36.1 (66/183) 39.7 (143/360) (P ¼ 034) (Po00001) 33.8 (27/77) 57.3 (47/82)

7.7 months (P ¼ 0.26) 5.5 months

8.4 months (P ¼ 0.0012) 8.3 months

16.7 months (P ¼ 0.75) 13.4 months

20.0 months (P ¼ 0.0094) 22.8 months

8.6 months (P ¼ 0.015)

7.2 months (P ¼ 0.0064)

17.5 months (P ¼ 0.20)

18.5 months (P ¼ 0.38)

Cetuximab þ FOLFOX

159

FOLFOX

166

52.5 (31/59) (P ¼ 0029)

34 (33/97) (P ¼ 00027)

Abbreviations: BSC, best supportive care; FOLFIRI, 5-fluorouracil/irinotecan; FOLFOX, 5-fluorouracil/oxaliplatin; MT, mutated KRAS; WT, wild-type KRAS.

European Medicines Agency and the Food and Drug Administration in metastatic and chemoresistant mCRC patients without KRAS mutation in their tumour, which represents acknowledgement of the research approach to personalized care. A similarly designed randomized controlled trial performed by the National Cancer Institute of Canada-Clinical Trial Group (NCI-CCTG) included 572 chemorefractory patients to receive cetuximab in monotherapy or best supportive care (Jonker et al., 2007). Data from 394 (69%) KRAS-evaluable patients of this NCI-CCTG trial confirmed that patients with a tumour KRAS mutation in codon 12 or 13 did not benefit from cetuximab monotherapy, contrary to patients with a KRAS wild-type tumour who had a longer progressionfree survival (3.7 vs 1.9 months; HR ¼ 0.40; 95% CI: 0.30–0.54; Po0.001) and overall survival (9.5 vs 4.6 months; HR ¼ 0.55; 95% CI: 0.41–0.74; Po0.001) in the cetuximab arm (Karapetis et al., 2008) (Table 1). The overall survival benefit associated with cetuximab treatment can be explained by the fact that crossover was not allowed in this study unlike in the phase III study with panitumumab. The clinical relevance of KRAS status has not only proved to be important in chemorefractory patients, but also in first-line treatment. Indeed, it was showed in two randomized studies, the CRYSTAL and OPUS studies, that patients with KRAS-mutated tumours do not benefit from the addition of cetuximab to a 5fluorouracil/irinotecan (FOLFIRI)- or 5-fluorouracil/ oxaliplatin (FOLFOX)-based chemotherapy in terms of response and progression-free survival (Van Cutsem et al., 2009a; Bokemeyer et al., 2009b). Updated data have been recently presented at the 34th European Society of Medical Oncology Congress, with a KRAS status available for 88% of the 1198 patients of the CRYSTAL study and 93.5% of the 337 patients of the Oncogene

OPUS study, confirming that KRAS mutations are associated with lack of benefit from the addition of cetuximab to FOLFIRI or FOLFOX (Bokemeyer et al., 2009a; Van Cutsem et al., 2009b). The findings of the OPUS study also showed a deleterious effect of KRAS mutations on the combination of FOLFOX plus cetuximab, as progression-free survival was of 5.5 months with the combined treatment compared with 8.6 months with FOLFOX alone (HR ¼ 1.72 95% CI:1.10–2.68; P ¼ 0.0153) (Bokemeyer et al., 2009a; Bokemeyer et al., 2009b; Van Cutsem et al., 2009b). These data led the European Medicines Agency to restrict the indication of cetuximab to patients with KRAS non-mutated tumours and the Food and Drug Administration not to recommend this drug for the treatment of colorectal cancer harbouring KRAS mutations. From a practical point of view, KRAS status may be determined from either primary colorectal tumour or metastatic tissue as it has been shown a good concordance between metastatic sites and matched primary tumour (Artale et al., 2008; Etienne-Grimaldi et al., 2008), which is not very surprising as KRAS mutations are an early event during colorectal carcinogenesis (Fearon and Vogelstein, 1990). Despite its predictive role, KRAS mutation has not been shown as a prognostic marker in many large-scale studies (Finkelstein et al., 1993; Samowitz et al., 2000; Gnanasampanthan et al., 2001; Ince et al., 2005; Barault et al., 2008b; Zlobec et al., 2009; Ogino et al., 2009a; Ogino et al., 2009b; Roth et al., 2010), except for one large study (Barault et al., 2008a). Overall evidence supports a null prognostic role of KRAS mutation even if a previous meta-analyses (Andreyev et al., 1998; Andreyev et al., 2001) led to wrong conclusions likely due to the well-known positive publication bias.

Oncogenic mutations as predictors in colorectal cancer A Lie`vre et al

5 Table 2 Summary of the studies evaluating the prognostic and predictive value of BRAF mutations on response to cetuximab or panitumumab in colorectal cancer Reference

Total number of patients

Number of KRAS WT patients

Number of BRAF-mutated patients (%)

Objective responsea (%)

Progression-free survivala

Overall survivala

0 0 0

— 1.2 vs 4.6 months Shorter P ¼ 0.001

— 5.4 vs 9.8 months Shorter Po0.0001

2.6 vs 4.4 months HR ¼ 0.59 (0.24–1.07) (P ¼ 0.073) 2 vs 3.9 months HR ¼ 3.6 (1.8–7.4) (P o0.0005) 8 vs 31 weeks (P ¼ 0.0005)

4.1 vs 13.9 months HR ¼ 0.51 (0.18–0.95) (P ¼ 0.037) HR ¼ 4.1 (2.1–8) (P o0.0001) 6.5 vs 14.8 months (P ¼ 0.0004)

Benvenuti et al. (2007) Cappuzzo et al. (2008b) Di Nicolantonio et al. (2008) Loupakis et al. (2009b)

48 85 113

32 38 79

6 (12.5) 4/79 (5.1) 11 (9.7)

138

87

13 (10)

0 (P ¼ 0.016)

Souglakos et al. (2009)

92

60

9 (10)

0

173

116

5/171 (3)

Laurent-Puig et al. (2009)

0 (P ¼ 0.063)

Abbreviations: HR, hazard ratio; WT, wild type. a Objective response, progression-free and overall survival in BRAF-mutated patients (vs BRAF-non-mutated patients) among the KRAS WT subgroup of patients.

BRAF somatic mutations As for KRAS mutations, mutations of the BRAF protein, located downstream of KRAS in the RAS/ MAPK pathway, seems to confer a resistance to antiEGFR therapies (cetuximab or panitumumab), as it was suggested in two small retrospective studies (Benvenuti et al., 2007; Cappuzzo et al., 2008a). This was confirmed in a series of 113 irinotecan-resistant mCRC patients treated by cetuximab or panitumumab (Di Nicolantonio et al., 2008) (Table 2). As BRAF and KRAS mutations are mutually exclusive, the authors analysed the impact of BRAF mutations on response to anti-EGFR therapy in KRAS wild-type patients. The BRAF V600E mutation was detected in 11 of the 79 patients, among whom none was responder to the treatment (P ¼ 0.029). The BRAF mutation was also associated with a significantly shorter progression-free and overall survival. in vitro, the authors also showed that transfection of the BRAF V600E allele in the wild-type DiFi cell line rendered this latter one resistant to either cetuximab or panitumumab compared with non-mutated parental cells (Di Nicolantonio et al., 2008). Two other colorectal cancer cell lines (HT-29 and COLO-205) naturally carrying the BRAF mutation were shown to be highly refractory to antiEGFR antibodies. Treatment with sorafenib, a BRAF inhibitor, restored the sensitivity to cetuximab or panitumumab of these three BRAF-mutated colorectal cancer cell lines. Since then, three independent retrospective studies have also shown lack of response to cetuximab and a shorter survival of patients with BRAF-mutated tumour among the subgroup of KRAS wild-type patients (Laurent-Puig et al., 2009; Souglakos et al., 2009; Loupakis et al., 2009b) (Table 2). These findings clearly show that BRAF mutations are an additional tool for the selection of patients who might be resistant to anti-EGFR antibodies and that they should be considered before considering antiEGFR therapies for mCRC. However, no randomized phase III study with a placebo control arm is currently available to definitively demonstrate the negative predictive value of BRAF mutations in mCRC.

In addition to their predictive value, BRAF mutations seem to be a poor prognostic factor, independently of the administration of an anti-EGFR therapy, as it was observed in the CAIRO-2 study, a phase III randomized trial in which capecitabine, oxaliplatin and bevacizumab was compared with the same combination plus cetuximab in first-line treatment (Tol et al., 2009a; Tol et al., 2009b). In this study, progression-free and overall survival of patients with a BRAF mutation were dramatically reduced compared with that of patients with a KRAS mutation, and of course to that of KRAS/ BRAF wild-type patients, in both arms with and without cetuximab (Tol et al., 2009b). Similar results were observed in the retrospective analysis of both KRAS and BRAF status of patients included in the CRYSTAL study (Kohne et al., 2009). In addition to its predictive role, BRAF mutation is a prognostic marker in colon cancer as shown in most of the large series This prognostic impact is mainly due to the impact of BRAF mutations in the subgroup of microsatellite stable tumours (Samowitz et al., 2005b; French et al., 2008; Kim et al., 2009; Zlobec et al., 2009; Ogino et al., 2009b; Roth et al., 2010), although it was not the case in one large series (Barault et al., 2008a). PIK3CA mutations and loss of PTEN expression Concerning PIK3CA mutations, the first small series that have investigated their clinical impact on resistance to EGFR inhibitors in colorectal cancer were not very convincing as no significant correlation was observed between PIK3CA mutations and response to cetuximab or panitumumab, even if only one responder was reported among the nine mutated patients on the 92 patients included in three studies (Lievre et al., 2006; Moroni et al., 2008; Perrone et al., 2008). In a series of 110 patients treated with cetuximab or panitumumab, Sartore-Bianchi et al. (2009) showed that exons 9 and 20 PIK3CA mutations (found in 13.6% of the cases) were correlated with the lack of response to anti-EGFR antibody in multivariate analysis after adjustment for Oncogene

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KRAS mutation and PTEN loss of expression (odds ratio ¼ 0.11; 95% CI: 0.00–0.85; P ¼ 0.033). This was also confirmed when only KRAS wild-type patients were analysed. Progression-free survival of patients with a PIK3CA mutation was significantly shorter when compared with PIK3CA non-mutated patients both in the overall population and in the KRAS wild-type subgroup of patients, but the overall survival was not statistically different according to PIK3CA status. However, conflicting, even contradictory, results have been reported since the publication of this study as no clear correlation was observed between the presence of a PIK3CA mutation and response to cetuximab in a homogeneous series of 200 chemorefractory mCRC patients treated by this anti-EGFR antibody (Prenen et al., 2009). The results of an European Consortium on the role of PIK3CA, BRAF and KRAS mutations as markers of resistance to cetuximab in chemorefractory mCRC patients reported at the 34th European Society of Medical Oncology Congress were quite similar (Tejpar and De Roock, 2009). These findings do not provide any evidence for a strong role of PIK3CA mutations as a single marker for prediction of resistance to cetuximab in chemorefractory patients. Nevertheless, PIK3CA mutation seems to be a prognostic marker both in colon and rectal cancers (He et al., 2009; Ogino et al., 2009c). Several studies have thus assessed PIK3CA mutations in association with loss of PTEN expression, as PTEN has an important role as a tumour-suppressor protein that antagonizes PI3K function. The loss of PTEN protein expression (30% of colorectal tumours) may therefore be an additional factor of PI3K/AKT pathway activation, which may be responsible for resistance to anti-EGFR antibodies, as it was firstly reported in a small series of 27 patients in which PTEN protein expression was evaluated by immunohistochemistry (Frattini et al., 2007). However, subsequent studies have reported discrepant results on the predictive value of loss of PTEN expression (Razis et al., 2008; LaurentPuig et al., 2009; Sartore-Bianchi et al., 2009; Loupakis et al., 2009a), in part, due to the use of different methods for the determination of PTEN status and to the absence of standardization of immunohistochemistry. These findings show that, as for PIK3CA mutations, loss of PTEN expression cannot be currently used as a single marker for prediction of resistance to cetuximab in mCRC patients. An in vitro study examined the effect of cetuximab on several colon cancer cell lines and found that cell lines with loss of PTEN expression and/or PIK3CA gene mutation were resistant to cetuximab (Jhawer et al., 2008), as it was also recently suggested in a small clinical series of colorectal cancer patients in which the activation of PI3K/AKT pathway by means of PI3KCA mutation and/or PTEN allelic loss was observed in 28% of the cases, all being non-responders to cetuximab (Perrone et al., 2008). Sartore-Bianchi et al. (2009) also showed in a larger series of 110 mCRC patients treated with cetuximab or panitumumab that those with a tumour combining PIK3CA mutation and/or PTEN Oncogene

loss had a significantly shorter progression-free survival than patients with wild-type PI3KCA and normal PTEN (P ¼ 0.0066), which was confirmed in a multivariate analysis (P ¼ 0.009) and was also suggested for overall survival (P ¼ 0.061). Other genetic alterations involved in the prediction of response to anti-EGFR antibodies EGFR gene copy number and expression As EGFR is the target of cetuximab and panitumumab, EGFR genetic alterations have been proposed as potential markers of response to these monoclonal antibodies. Autonomous EGFR activation can arise through mutational events or gene amplification. Although activating mutations in the tyrosine kinase domain of the EGFR gene have been described to have an important role in response to tyrosine kinase inhibitors in lung cancer (Paez et al., 2004; Pao et al., 2004; Taron et al., 2005), such mutations are very rare or absent in colorectal tumours (Barber et al., 2004; De Roock et al., 2008; Moroni et al., 2008). However, an overexpression of EGFR through amplification or increased copy number of the gene is found in 10–15% of colorectal cancers (Sauer et al., 2005; Shia et al., 2005). A first study described an association between an increased EGFR copy number, analysed by fluorescence in situ hybridization (FISH), and tumour response to cetuximab (Moroni et al., 2008). This association was reported both with cetuximab and panitumumab in subsequent studies in which EGFR gene copy number was also evaluated by FISH or chromogenic in situ hybridization (Personeni et al., 2005; Lievre et al., 2006; Frattini et al., 2007; Sartore-Bianchi et al., 2007; Cappuzzo et al., 2008a; Laurent-Puig et al., 2009; Scartozzi et al., 2009). This association between EGFR high polysomy or amplification and a good response to anti-EGFR antibodies was observed independently of the KRAS status and was especially described in the subgroup of KRAS wild-type patients (Personeni et al., 2005; Laurent-Puig et al., 2009; Scartozzi et al., 2009). In a recent series of 173 progressive colorectal cancer patients receiving cetuximab-based regimen, a FISHpositive phenotype (EGFR high polysomy or amplification) was found in 16% of the 138 cases analysed for EGFR status and in 18% in the subgroup of 96 KRAS wild-type tumours in which it was associated with a higher response rate than seen in tumours with normal EGFR copy number (71 vs 37%; P ¼ 0.015) and with a trend towards longer progression-free and overall survival (Laurent-Puig et al., 2009). In this study, patients EGFR FISH positive and wild type for KRAS and BRAF (16% of the entire cohort) were particularly sensitive to cetuximab, with a response rate of about 80%. However, tumour response was observed in colorectal tumours without an increase of EGFR copy number (Italiano et al., 2008) and discrepant results were observed when EGFR copy number was assessed by quantitative PCR and not by FISH (Vallbohmer et al., 2005; Lenz et al., 2006; Khambata-Ford et al., 2007).

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The lack of sensitivity of the PCR technique for detection of an increase in EGFR copy number, partly due to tumour DNA dilution and the lack of reproducibility of FISH data because of the absence of standardized EGFR scoring and heterogeneity of FISH pattern may explain these differences and render this molecular marker difficult to include in clinical practice (Personeni et al., 2005). In conclusion, even if clinical decisions based on EGFR copy number are not warranted as long as FISH technology and scoring are not standardized, EGFR high polysomy or amplification represents a promising marker of high response to cetuximab, which could identify, in addition to KRAS and BRAF mutational status, a small subgroup of patients for whom early and aggressive cetuximab-based therapy may be very beneficial, especially if a surgery of metastases is considered. EGFR ligands expression Contrary to previous studies that had focussed their research on one to several molecular markers, some authors have used a more global genomic approach by analysing on microarrays the gene expression profiles of 95 fresh-frozen liver metastases of colorectal cancer patients treated by cetuximab monotherapy (KhambataFord et al., 2007). This approach showed that the expression of the EGFR ligands epiregulin (EREG) and amphiregulin (AREG) were very discriminant to distinguish patients with a disease control from those with progressing under cetuximab. The EREG and AREG gene expression level was significantly higher in the group of patients with a disease control. Moreover, patients with a high EREG and AREG expression had a longer progression-free survival than those with a low expression (EREG: median of 103.5 vs 57 days; P ¼ 0.0002, and AREG: median of 115.5 vs 57 days; Po0.0001, respectively). These preliminary findings indicate ligand-driven autocrine oncogenic EGFR signalling that could characterize an EGFR-dependent tumour potentially more sensitive to the receptor blockade by cetuximab. Interestingly, among KRAS wild-type patients, those whose tumours express high levels of AREG or EREG had a higher disease control rate and progression-free survival compared with those whose tumours express low levels of these genes, suggesting that expression of these EGFR ligands may bring complementary information to KRAS status in the prediction of response to cetuximab (Harbison et al., 2008).These results have been recently confirmed in a study performed on formalin-fixed paraffin-embedded primary tumour of 220 mCRC patients treated with the combination of cetuximab and irinotecan (Jacobs et al., 2009). In this series, EREG and AREG expression were significantly associated with progressionfree survival (EREG: HR ¼ 0.41; 95% CI: 0.27–0.61; Po0.001, and AREG: HR ¼ 0.43; 95% CI: 0.29–0.64; Po0.001) and overall survival (EREG: HR ¼ 0.42; 95% CI: 0.28–0.63; Po0.0001, and AREG: HR ¼ 0.40; 95% CI: 0.27–0.64; Po0.0001), which was observed only in wild-type KRAS patients. However, the authors showed

that a single cut point of ligands expression that would be applicable in all clinical situations can not be currently determined, which avoid the use of these markers in clinical practice (Jacobs et al., 2009). Conclusions and perspectives Recent advances in the understanding of signalling pathways involved in colorectal carcinogenesis led to the identification of oncogenic mutations that activate these pathways downstream of the EGFR and thus representing markers of resistance to anti-EGFR. Among them, KRAS mutations are the strongest predictive marker and the only one that has been integrated in clinical practice in patients with mCRC. Indeed, the European Medicines Agency and the Food and Drug Administration now recommend the determination of KRAS status before initiating a treatment with anti-EGFR antibodies and restrict these treatments to wild-type KRAS patients (http://www.emea.europa.eu/humandocs/Humans/EPAR/ erbitux/erbitux.htm; http://www.emea.europa.eu/humandocs/ Humans/EPAR/vectibix/vectibix.htm; http://www.fda.gov/ AboutFDA/CentersOffices/CDER/ucm172905.htm). However, only 40–60% of wild-type KRAS patients achieved an objective response to anti-EGFR monoclonal antibodies, suggesting the existence of other molecular markers involved in response to these targeted therapies. In addition to KRAS status, BRAFactivating mutations are probably the most robust predictive marker of resistance, given that oncogenic alterations of PI3K/AKT pathway (PIK3CA mutations and/or loss of PTEN expression) have shown too discrepant results to be incorporated in clinical practice. As for amplification or high polysomy of EGFR gene assessed by FISH or chromogenic in situ hybridization, even if it adds an supplemental information to KRAS and BRAF status by identifying patients who will be high responders, it remains limited for technical reasons. With regard to KRAS status, mutations are currently searched from tumour tissue, which can sometimes be a limitation, as clinicians do not always have an easy access to tumour material from colorectal primary cancer or metastases when patients have been operated in another centre or have never been operated from their colorectal cancer. Some authors have shown that detection of KRAS mutations in the peripheral blood of mCRC patients is possible by the development of combining tests indicating in blood, first the presence of tumour DNA (circulating tumours cells), then KRAS status, with a highly significant correlation to KRAS mutations in tumours (Di Fiore et al., 2008; Yen et al., 2009). Moreover, it was reported in a series of 76 patients that those with KRAS wild-type circulating tumour cells had a better progression-free and overall survival when treated with cetuximab plus chemotherapy (Po0.0001) (Yen et al., 2009). These findings encourage to collect blood samples of mCRC patients to validate the clinical relevance of KRAS mutation detection in blood in future clinical trials with antiEGFR monoclonal antibodies. Oncogene

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8

A new interesting point concerning KRAS mutations is the description of other mutations than those usually described in codons 12 and 13 of the gene, which might be useful to select non-responder patients to anti-EGFR antibodies (Loupakis et al., 2009b). These mutations, located within KRAS codons 61 and 146, even if they are much less frequent than codons 12 and 13 mutations (present in 8 and 1% of the tumours, respectively), have been shown, in addition to BRAF mutations, to correlate with lack of response to cetuximab (0 vs 37%; P ¼ 0.0005) and to a lower progression-free survival (3.3 vs 5.3 months; P ¼ 0.006) and overall survival (9.7 vs 14.8 months; P ¼ 0.027) in the subgroup of 89 wild-type KRAS patients of the study by (Loupakis et al. (2009b), which included a total of 138 irinotecan-resistant colorectal cancers treated with the combination of cetuximab and irinotecan. The identification of strong molecular markers of resistance to anti-EGFR antibodies such as KRAS or BRAF mutations makes necessary the development of new therapies that should target ‘escape mechanisms’ used by mutated tumours to circumvent a pathway that has been pharmacologically blocked by cetuximab or panitumumab. Therapies that may inhibit the activation of MAPK pathway downstream of the RAS protein would be promising. In this perspective, an in vitro study showed that human colon cancer cells harbouring the G12D KRAS mutation were hypersensitive to RAF inhibitors, but surprisingly not to MEK inhibitors (Haigis et al., 2008). On the contrary, it was shown that pharmacological MEK inhibition completely stopped tumour growth in BRAF mutant xenografts, whereas KRAS mutant tumours were only partially inhibited

(Solit et al., 2006). In a more recent study, some authors have shown that PI3K pathway activation (by PIK3CA or PTEN mutation) strongly mediates resistance of KRAS mutant cancer cells to MEK inhibitors (Wee et al., 2009). They also demonstrated that cells with coexisting mutations in KRAS and PIK3CA remain dependent on KRAS signalling, explaining why targeted pharmacological inactivation of both MEK and PI3K pathways is required to achieve a complete tumour growth inhibition of these cancer cells, whereas inhibition of either MEK or PI3K pathway leads only to a partial tumour growth inhibition. These findings bring a strong rationale for the clinical testing of combination MEK- and PI3K-targeted therapies in KRAS-mutated colorectal cancer.

Conflict of interest P Laurent-Puig received compensation as a member of the advisory board of MerckSerono and Amgen company. His research was in part sponsored by MerckSerono and Myriad Genetics. Dr Lievre has consulted for Merck and received compensation. Dr Blons declares no conflict of interest.

Acknowledgements We thank Institut National du Cancer (N12009-1-RT03) and the Region Ile de France for their financial support.

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