Cancer Treatment Reviews

Cancer Treatment Reviews 35 (2009) 262 271 Contents lists available at ScienceDirect Cancer Treatment Reviews journal homepage: www.elsevierhealth.com/journals/ctrv ANTI-TUMOUR TREATMENT Clinical relevance of EGFR- and -status in colorectal cancer patients treated with monoclonal antibodies directed against the EGFR Volker Heinemann *, Sebastian Stintzing, Thomas Kirchner, Stefan Boeck, Andreas Jung Department of Hematology/Oncology, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Marchionini-Street 15, 81377 Munich, Germany article info summary Article history: Received 13 November 2008 Accepted 17 November 2008 Keywords: Epidermal growth factor receptor EGFR mutation Cetuximab Panitumumab Colorectal cancer The epidermal growth factor receptor (EGFR) plays an important role in tumorigenesis and tumor progression of colorectal cancer (CRC). As a result, the EGFR has evolved as a relevant target in the treatment of metastatic CRC. serves as a mediator between extracellular ligand binding and intracellular transduction of signals from the EGFR to the nucleus. The presence of activating mutations has been identified as a potent predictor of resistance to EGFR-directed antibodies such as cetuximab or panitumumab. These agents should therefore be applied only in tumors with a wild-type status of the gene. Further parameters of resistance are lack of EGFR amplification, PTEN loss or BRAF mutation. However, they are less well studied or associated with less consistent data and therefore require prospective analyses before integration into clinical decision making. Future studies need to identify patterns of single or multiple mutations to further increase the power of patient selection for anti-egfr therapy. While molecular parameters help to predict treatment efficacy upfront, skin toxicity has been accepted as an independent predictor of response during exposure to anti-egfr therapy. Ó 2008 Elsevier Ltd. All rights reserved. Introduction The epidermal growth factor receptor (EGFR) has become an important target of cancer therapy. Specifically, the antibodies cetuximab and panitumumab, which are directed against the EGFR, have proven efficacy in the treatment of metastatic colorectal cancer (mcrc). Several reports indicate that an increased gene copy number of EGFR or mutations of genes responsible for downstream signalling are important determinants of response or resistance to anti-egfr antibodies. This review analyses the impact of mutations on the clinical activity of anti-egfr directed treatment. The underlying hypothesis is that most mutations cause a gain of function activating the Ras/MAPK pathway. As signal transduction is activated at the level of proteins, upstream inhibition by EGFR-targeted agents becomes ineffective. EGFR-related signal transduction * Corresponding author. Tel.: +49 89 7095 0; fax: +49 89 7095 2257. E-mail address: Volker.Heinemann@med.uni-muenchen.de (V. Heinemann). The EGFR constitutes the link between the extracellular space and the intracellular signal transduction which regulates nuclear processes involved in cell growth, differentiation, survival, cell cycle progression, angiogenesis, and drug sensitivity. It is a member of the erbb family of receptor tyrosine kinases, which also include erbb2 (HER2/neu), erbb3 (HER3), and erbb4 (HER4). The EGFRtransmembrane protein is composed of three components: an extracellular ligand binding domain, a lipophilic transmembrane domain, and an intracellular tyrosine kinase domain. Apart from erbb2, specific ligands have been identified for each of the erbb receptors. Among these, the epidermal growth factor (EGF) and the transforming growth factor-a (TGF-a) selectively bind to the EGFR. Extracellular ligand binding induces activation of the transmembrane receptors, subsequent homo- or heterodimerization between the different receptors and intracellular autophosphorylation at the tyrosine kinase domain which, in turn, activates downstream signalling pathways. 1,2 Several pathways of signal transduction have been identified. Activation of the Ras-Raf-mitogen-activated protein kinase (MAPK) pathway induces phosphoprotein expression of p-mek and p- ERK1/2. Preclinical and clinical data suggest that MAPK plays a key role in the regulation of cell growth, differentiation, proliferation, apoptosis, and cellular invasiveness. 2,3 EGFR-mediated signalling via PI3 kinase (PI3K) causes activation of AKT, which results in the expression of phosphoproteins such as p-akt, p-gsk3, and p-p70s6k. The PI3K/AKT pathway is regulated by phosphatase protein homologue to tensin (PTEN) the deficiency of which has been associated with an unsuppressed activation of signal transduction. 0305-7372/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctrv.2008.11.005

V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 263 EGFR protein expression The EGFR is the primary target of monoclonal antibodies such as cetuximab or panitumumab. It is, therefore, reasonable to evaluate the predictive potential of EGFR protein expression with regard to sensitivity towards these agents. Analyses performed by immunohistochemistry (IHC) indicate an EGFR protein expression in 60 80% of colorectal tumors. 4 However, only a fraction of IHC-positive tumors also show EGFR gene amplification. Shia et al. investigated 158 tumors and demonstrated that IHC had a low specificity (17% in primary and 23% in metastatic tumors) to predict gene amplification. 5 In agreement with this finding, also clinical analyses have consistently reported no correlation between the extent of EGFR expression as detected by IHC and response to EGFR directed antibodies. 6 8 Moreover, several authors reported that cetuximab was also active in tumors which were EGFR-negative by IHC yielding response rates up to 25%. 9,10 EGFR gene copy number Several methods are used to determine the EGFR gene copy number (GCN). While most studies used fluorescence in situ hybridisation (FISH) or chromogenic in situ hybridisation (CISH), others also employed polymerase chain reaction (PCR)-based methods (Table 1). The comparability of these methods and their differential impact on results still needs to be defined. Also it has to be pointed out that an increased EGFR GCN is not only caused by gene amplification but also by polysomy of chromosome seven. As a reflection of methodological uncertainties, the prevalence of EGFR gene amplification shows a wide variation of 6 51% in different reports. 5,11 16 Evidence from several studies indicates that EGFR gene amplification, as determined by CISH or FISH, is associated with clinical response. 11,13,16 However, methodological problems and discrepancies in data interpretation and reporting so far have prevented this parameter to gain a broad clinical application for decision making. Among others, Cappuzzo et al. reported on 85 chemorefractory patients, where cetuximab-based therapy induced a significantly higher efficacy in EGFR FISH+ compared to EGFR FISH- patients with regard to ORR (32.5% vs. 2.3%, p < 0.0001) and TTP (6.6 vs. 3.5 months, p = 0.02). 16 In EGFR FISH- negative patients sensitivity to cetuximab was not affected by the or BRAF status. According to this study, mutation may confer resistance to cetuximab only in tumors where the EGFR target is present. Using FISH Frattini and coworkers could differentiate cells with an increased EGFR copy number into markedly polysomic cells and cells with an EGFR amplification. 14 Partial response to cetuximab-based therapy was observed in 6 of 8 patients with EGFR gene amplification, 4 of 16 patients with marked polysomy, and 0 of 3 patients with eusomy (p < 0.05). Sartore-Bianchi et al. investigated 58 mcrc patients who were refractory to standard therapies and received panitumumab plus best supportive care within a phase III trial. 17 In the unselected population, the response rate was 10%. 18 Sartore-Bianchi and coworkers employed a FISH method and clearly pointed out that the pattern of EGFR GCN was non-homogeneous in individual tumors displaying various ratios of disomy versus polysomy of chromosome 7 and/or EGFR gene amplification. 17 Selecting P2.47 EGFR copies/nucleus as a cut-off value they were able to establish an optimal discrimination of responders versus non-responders. While 6 of 20 (30%) patients with an increased gene copy number (GCN) responded to panitumumab, no response was observed in 38 patients without an increased EGFR GCN. With a specificity of 98.1% (95% CI, 89.7 99.9%), the negative predictive value was 89.5% (95% CI, 78.5 96.0%). Analysing chromosome 7 polysomy and/or EGFR gene amplification and choosing a cut-off of P43% the specificity rose to 100% (95% CI, 93.2 100%) and the negative predictive value for response was 89.7% (95% CI, 78.8 96.1%). This analysis clearly demonstrates that tumors which lack the target of anti-egfr directed therapy are very unlikely to respond to this treatment approach. It, therefore, may be concluded that the greatest specificity of response prediction may be achieved when EGFR gene amplification as well as chromosome 7 polysomy is taken into account. By contrast, Lenz and coworkers used a PCR-based method to determine the EGFR gene copy number in 34 intensively pretreated mcrc patients exposed to single agent cetuximab. 10 This group did not find a relationship between EGFR copy number and response or PFS, but observed a significant correlation with survival (p = 0.03). In a further analysis of 39 patients, the same group reported no correlation between EGFR gene expression and response to cetuximab, but indicated a significantly longer survival in patients with low gene expression levels of Cox-2, EGFR, and IL-8. 8 In this context, it still needs to be clarified to which extent EGFR gene expression not only is a predictive factor but also has a prognostic importance. EGFR gene mutations Activating mutations of the EGFR gene have been reported in NSCLC and were linked to the clinical efficacy of tyrosine kinase inhibitors such as gefitinib. 19 It appears, however, that EGFR gene mutations are rare in colorectal cancer and have no clinical relevance with regard to the activity of anti-egfr therapy. 20,21 Barber Table 1 Analysis of EGFR gene copy number in colorectal cancer. Reference No tumors Method Cut-off GCN/ nucleus EGFR GCN increased no, (%) ORR (%) EGFR GCN increased ORR (%) EGFR GCN not increased TTP (mo) EGFR GCN increased Moroni 11 30 FISH na 31 89 d 4.8 Sauer 12 48 FISH P1.2 d 7/48 (15) Frattini 14 27 FISH >3 d 30 75 21 a Cappuzzo 16 85 FISH P2.9 43/85 (50.6) 32.5 b 2.3 6.6 c 3.5 Sartore 17 58 FISH P2.5 20/58 (34.5) 30 0 Shia 5 123 CISH >5 15/123 (12.2) Lievre 13 30 CISH P6 10 100 29.6 Khambata- Ford 15 80 PCR 6 EGFR, epidermal growth factor receptor; ORR, objective response rate; GCN, gene copy number; PFS, progression-free survival; OS, overall survival. a p < 0.05. b p < 0.0001. c p = 0.02. d Ratio EGFR gene/cep7. TTP (mo) EGFR GCN not increased

264 V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 et al. investigated a large cohort of 239 CRC patients and found only one mutation. 20 Anti-EGFR antibodies Monoclonal antibodies were designed as effective inhibitors of the EGFR. Cetuximab (Erbitux Ò, Merck KgaA, Darmstadt, Germany) is a chimeric mouse/human antibody targeted against the extracellular domain of the EGFR. Binding of cetuximab to the receptor prevents ligand binding, induces receptor internalization and causes a direct inhibition of the receptor tyrosine kinase activity. 22 This in turn blocks downstream signal transduction via the PI3K/Akt and RASRAF/MAPK pathways inducing pro-apoptotic mechanisms and inhibiting cellular proliferation, angiogenesis and metastasis. 23,24 As an IgG1 antibody cetuximab may also induce antibody-dependent cell-mediated cytotoxicity (ADCC). However, the clinical relevance of ADCC with regard to antitumor efficacy is likely to be rather low. 22 Cetuximab has a single agent activity in mcrc refractory to irinotecan, oxaliplatin and fluoropyrimidines 10 and was shown to restore chemosensitivity in irinotecan-refractory mcrc patients. 6 Response and survival of cetuximab-treated patients strongly relates to the severity of an acneiform skin rash. 10 Panitumumab (Vectibix Ò, Amgen Thousand Oaks, CA, USA), by contrast, is a fully human antibody which is also directed against the EGFR but being an IgG2 MoAb lacks ADCC activity. Other antibodies targeting the EGFR such as matuzumab, zalutumumab and nimotuzumab may have a similar spectrum of activity, but are not discussed in this review since registration has not been performed. Ras Ras constitutes a family of proto-oncogenes encoding small G- proteins with a molecular weight of 21 kda (p21). Three different members contribute to the RAS gene family known as Harvey- Ras (HRAS), Kirsten-Ras (), and N-RAS. These genes are located on different chromosomes. The expression levels of the respective genes vary in different tissues. 25,26 The protein is located at the inner cell membrane and has GTPase activity. 27 Extracellular binding of ligands to transmembrane receptors like the EGFR causes activation of the downstream signal transduction cascade to the nucleus. In the first step the intracellular tyrosine kinase domain of the EGFR is phosphorylated which in turn induces a transient activation of the RAS protein. While in its inactive state, RAS is bound to guanosine diphosphate (GDP), activation occurs by the conversion of GDP to guanosine triphosphate (GTP). Incidence of RAS mutations in solid tumors Different tumor entities are characterized by different frequencies of RAS mutations (Table 2). Also within defined tumor entities, the range of reported RAS mutations appears to be wide which can be explained not only by patient selection, but also by the methodology of analysis. Higher rates of RAS mutations were, for example, recorded when microdissection was used to specifically designate tumor tissue for the mutation analysis. 37 The rate of mutations was 16 38% in non-small-cell cancer, 30 40% in CRC, 45% in intrahepatic cholangiocarcinoma, and >90% in pancreatic cancer. Inconsistent results have been reported for gastric cancer. While Tajima et al. described comparable and low rates of mutation in tumors of the cardia (7.3%) and the distal stomach (6.3%), Lee and coworkers reported a sitedependent rate of mutations for the upper third (3/8, 37.5%), the middle third (4/29, 13.8%), and the lower third (3/99, 3.0%) of the stomach. 29,30 The number of upper third sample was, however, too low to allow firm conclusions. In 152 head-and-neck Table 2 mutations in selected gastrointestinal tumors. Reference Tumor entity No. cases analysed tumor samples, Sathyan et al. did not observe any mutations, but the rate of HRAS mutations was 12.5%. 38 mutation in colorectal cancer Oncogenic mutations of the genes are observed in about 40% (20 50%) of sporadic colorectal cancers. 25,26,39 These are point mutations and are generally observed as somatic mutations. Up to 90% of activating mutations of the RAS gene are detected in codons 12 and 13, but less frequently also in codons 61 and 63. With regard to codon 12- and 13-mutations only, 70% of mutations occur in codon 12% and 30% in codon 13. 14 The most frequent types of mutations in colorectal cancers are G-to-A transitions 40 and G-to-T transversions. 41 The codons 12 and 13 code for two adjacent glycine residues located in the proximity of the catalytic site of RAS. 42 Different mutations result in an exchange of different amino acids at these catalytic sites, and therefore, may be responsible for the different levels of intrinsic GTPase activity reduction. As a consequence, variable RAS mutations may imply variable effects on the biology of disease. For example, the presence of codon 12 glycine-to-valine mutations has been associated with a more aggressive tumor biology in advanced CRC. 42 Recent work from Milano and coworkers indicates that the level of EGFR gene copy number in CRC tumors was independent of the presence of mutations. 43 Detection of mutation Mutations (n) Lord 28 Esophagus cancer 23 7 30.4 Tajima 29 Cardia 52 3 7.3 Distal stomach 82 5 6.3 Mutation rate (%) Lee 30 Stomach 140 11 12.5 Yashiro 31 Stomach 108 20 18.5 Rashid 32 Biliary tract 126 18 14.3 Bile duct 75 2 2.7 Ampulla 18 11 61.1 gallbladder 33 5 15.2 Tannapfel 33 Cholangiocarcinoma 69 31 45 Almoguera 34 Pancreas 22 21 95 Smit 35 Pancreas 30 28 93 Rozenblum 36 Pancreas 42 42 100 Immervoll 37 Pancreas 43 39 91 For the detection of mutations several methods are known. The hotspots and thus the most frequent mutations - given in the recommended genetic nomenclature 44 are g.34g > C (p.g12r), g.35g > C (p.g12c), g.34g > A (p.g12s), g.35g > A (p.g12d), g.35g > C (p.g12a), g.35g > T (p.g12v), g.38g > A (p.g13d) and rarely g.183g > T (p.q13h). 25,26,39 As the mutation in codon 61 is a rather rare event, the molecular-pathologic detection of mutations nowadays focuses only on codons 12 and 13. 45,46 Moreover, as the detection of mutations has become a routine diagnostic method, the methods in use are selected on the basis of velocity, robustness and easiness. Four PCR-based techniques dominate: sequencing methods as the gold standard being either (1) the dideoxy-method 47 51 or (2) the pyrosequencing method. 52,53 Allele specific PCRs like (3) the ARMS-PCR or SnaPshot PCR 45 as well as the employment of four allele specific probes followed by a melting curve analysis 55 are fast working methods. The

V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 265 quality of the DNA does not have to be superior as all the methods are based on the generation of short fragments. Even in case there is no tumor material available, an H&E section from a former histological inspection can be used as a source of DNA after the cover slip has been removed carefully. Moreover, the starting amounts of DNA can be kept quite low as either short amplicons are used 52,53 or nested PCR technology is employed. 51 It should be kept in mind, however, that dideoxy-sequencing needs a high purity of tumor material in the range of 70%, whereas other methods such as pyrosequencing or ARMS-PCR are in need of just 2% or even 1% tumor material in the later DNA template, respectively. Therefore, when using dideoxy-sequencing, microdissection usually becomes an important issue for the preparation of the DNA template. Generally, there is no best method for the detection of mutations. The method used mostly depends on the technical equipment of the laboratory performing the analysis. Of course, specificity and sensitivity and thus the quality of the method must be validated. Close collaboration with a pathologist is clearly recommended. This becomes critical when tumors with low amounts of highly infiltrative growing tumor cells are the subject of investigation. In some European countries programs for interlaboratory quality assurance are in development or are already established as in Germany by the German Society for Pathology. Consistency of mutations in primary tumor and metastases? In a retrospective investigation, Loupakis and coworkers compared PTEN expression and mutations in primary tumors and in related metastatic tissue. 54 In 45 paired samples, they observed a concordance of PTEN analyses in 60% (95% CI, 46 74%), while the concordance of mutation analyses was 95% (95% CI, 84 99%) in 43 primaries and related metastases. This observation indicates that mutation analyses from primary tumors can be used with a great reliability for treatment decisions in the metastatic setting. Role of in carcinogenesis and tumor progression According to the multistep carcinogenesis model of the adenoma-carcinoma sequence, the transition from the adenoma to the colorectal carcinoma occurs by multiple gene mutations. 56 In this process, the accumulation of mutations appears to be of greater importance than the actual sequence. 57 Several studies support the importance of mutational activation of in the progression of CRC. 58 Specific genetic mutations may be responsible for different biological alterations. Codon 12 mutations of the gene were associated with a mucinous phenotype of colorectal cancer. By contrast, codon 13 mutations were rather non-mucinous, but were characterised as more aggressive tumors with a greater metastatic potential. 57 Oliveira and coworkers reported that the frequency of concomitant and BRAF mutations increased along with the depth of wall invasion: T2 2.8%, T3 3.5%, T4 9.4%. 59 Also, a higher frequency of mutations was observed in lymph node metastases as compared to the primary tumors (p = 0.0002). The authors, therefore, suggested that mutations induce a more invasive behaviour of tumor cells. mutations in HNPCC families There is evidence that in colorectal cancer mutations occur independent of microsatellite instability (MSI). Aaltonen et al. were among the first to investigate the frequency of mutations in HNPCC families fulfilling the Amsterdam criteria. 60 They reported that mutations were as frequent in HNPCC families as in sporadic CRC. While these findings were supported by further two studies, 61,62 other reports did not confirm them. 63,64 These studies were criticized for small patient numbers and in most cases for a lack of proof of the hereditary defect. Oliveira and coworkers evaluated 158 HNPCC tumors from patients with germline hmlh1, hmsh2 or hmsh6 mutations and compared these to 166 patients with sporadic MSI-H (high grade microsatellite instability) and 688 patients with sporadic microsatellite stable (MSS) CRC tumors. mutations were detected in 40% of the HNPCC tumors. 65 The mutation frequency varied according to the affected mismatch repair gene and amounted to 48% (29/61) in hmsh2, 32% (29/91) in hmlh1, and 83% (5/6) in hmsh6 patients. 65 While the frequency of mutations was similar in HNPCC patients (40%) and MSS sporadic CRC s (34%), it was significantly smaller in sporadic MSI-H carcinomas (22%). Prognostic relevance of mutations The reports on the prognostic relevance of mutations are inconsistent. While some studies have indicated a negative impact of mutations on survival, 66,67 others did not. 68 71 The first RASCAL study evaluated the mutational status of the gene in 2721 CRC cases from 22 centres. The analyses were performed on DNA extracted from tumor samples by various methods such as PCR amplification followed by SSCP analyses, PCR and direct sequencing, PCR with allele specific primers and hybridisation. 72,73 This study demonstrated that the gene mutation status was important for the progression and outcome of the established CRC. The RASCAL II study investigated 3439 CRC patients from 35 centres with a median follow-up of 55 months. 74 Using the same techniques as in the previous study mutations were detected in 35% of cases (26% in codon 12, 9% in codon 13). A replacement of glycine with valine in codon 12 (g.35g > T, p.g12v) was detected in 9% of overall mutations. A multivariate analysis demonstrated that advanced Dukes stage, age, and the codon 12 (p.g12v) mutations were significantly associated with a poorer prognosis. A significant reduction of the disease-free interval (RR = 1.5, p = 0.0076) and the survival rate (RR = 1.45, p = 0.02) was observed only in Dukes C, but not in Dukes B tumors. 72,73 The RASCAL studies indicate that mutations may imply an unfavourable prognosis in colorectal cancer and that specifically in Dukes C stage patients a p.g12v mutation in codon 12 may lead to a higher risk of relapse and death. Bazan and coworkers evaluated 160 previously untreated patients undergoing surgery for primary operable sporadic CRC. 42 Mutations within the exon 2 of the gene and exons 5 8 of the TP53 gene were detected by PCR SSCP analyses following sequencing. Mutation analyses of the gene showed mutations in 46% (74/160) of cases. Fifty-seven percent of mutations were identified in codon 12, 43% in codon 13. With a frequency of 70%, overall transitions were the predominant alteration. In this analysis, no germline mutations were detected. 42 In a multivariate analysis, mutation in codon 13 was significantly associated with a worse overall survival (HR = 1.86 (95% CI, 1.21 4.21), p < 0.05). In this context, it may be added that germline mutations are not expected to occur in proto-oncogenes. Like in the tumors these mutations are dominant. Thus, the phenotype would be lethal already during the embryogenesis. This is the reason why not a single familial syndrome with a mutation in a dominant acting oncogene is known. BRAF mutation BRAF mutations occur at a lower rate than mutations in CRC (0 12.5%) (Table 3).

266 V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 Table 3 mutation in relation to BRAF mutation or loss of PTEN. Reference In most tumors - and BRAF mutations are mutually exclusive. 47,13 This was corroborated by several studies investigating 113 586 patients who observed combined - and BRAF mutations in only 0 0.4% of tumors. 75 77 The ORR to EGFR-targeted therapy in BRAF mutant patients (1/6, 16.6%) was reported to be lower than that in wild-type patients (26/98, 26.5%). 75 Cappuzzo et al. observed a BRAF mutation in 5% (4/85) of patients who did not respond to cetuximab therapy and showed a lower TTP (1.2 months vs. 4.6 months, p = 0.09) and OS (5.4 months vs. 9.8 months, p = 0.3) than wild-type patients. 16 PI3K-AKT pathway Phosphatidylinositol 3-kinase (PI3K) is a major component of the PI3K-AKT pathway. Mutated PI3K may promote growth and invasion of cancer cells. 78 PI3K is activated by recruitment to the cell surface by activated receptor tyrosine kinases, as well as by binding to activated RAS. Kato and coworkers carried out an analysis of 158 CRC tissue samples and investigated the effect of PIK3CA mutation and mutation on p-akt expression. The frequency of mutations was 11.4% for PIK3CA and 31.6% for the gene. 79 Although 6/158 tumors showed both PIK3CA- and mutations, these mutations were not correlated with each other. In a multivariate analysis, PIK3CA was identified as the only independent and significant prognostic factor for relapse-free survival in stage II/III patients. Neither - nor PIK3CA mutation did, however, correlate with p-akt expression. This observation is in contrast to Cappuzzo et al. who observed a PIK3CA mutation in 17.7% (14/ 85) of cetuximab-treated mcrc patients, but found no difference in ORR, TTP and OS compared to the wild-type comparators. 16 In addition, no AKT mutation was observed in 82 evaluated patients. PTEN Tumors analysed (n) mutation n (%) BRAF mutation n (%) Loss of PTEN expression Ince 76 255 88 (35) 14/250 (5.6) na Lievre 13 30 13 (47) 0 (0) na Benvenuti 47 48 16 (33) 6 (12.5) na Oliveira 59 250 93 (37) 10 (4) na Khambata- 80 30 (38) 0 (0) na Ford 15 Lievre 46 89 24 (27) 2 (2) na De Rook 75 113 46 (41) 6 (5.6) a na Frattini 14 27 10 (37) na 11 (41) Barault 77 586 198 (34) 78 (13.3) na Loupakis 54 88 35 (40) na 36 (42) b Cappuzzo 16 80 42 (53) 4 (5.1) c na a b c BRAF analysis performed in 107 assessable patients. PTEN analysis performed in 85 assessable patients. BRAF analysis performed in 79 assessable patients. Phosphatase homologue to tensin (PTEN) is a tumor suppressor protein that regulates the PI3K/AKT signal transduction. Its loss is associated with an intrinsic activation of the AKT pathway and is known to confer resistance to inhibitors of the HER-family. 80 Thomas et al. demonstrated a loss of PTEN in 30% of sporadic CRC. 81 Frattini et al. analysed 27 cetuximab-treated mcrc patients and documented a loss of PTEN protein in 11/27 (41%). 82 The ORR was 62.5% in patients with intact PTEN, but no response was observed in patients with a loss of PTEN. A loss of PTEN occurring in combination with a mutation was found in 36% (4/11) of patients. Loupakis et al. performed a retrospective analysis of patients receiving cetuximab plus irinotecan after the failure of irinotecanbased therapy. They reported a loss of PTEN in 22 of 55 (40%) patients, 95% of whom were qualified as non-responders. 54 By contrast, in PTEN-positive patients the response rate was 36% (Table 3). Networks of signal transduction The, BRAF, and PI3K genes code for proteins which interact within an intracellular signalling network. Within this network the protein not only binds to protein kinases of the RAF family such as BRAF, but also to PI3K and to a family of exchange factors for the GTPase RAL. 83 Barault and coworkers suggested that an activation of the network may be induced by the mutation of one or more of its various components. On the other hand, disruption of one component would drive signal transduction into the remaining channels of the network. 77 Within a population-based study, Barault at al. evaluated 586 adenocarcinomas from the colon. An activation of the network with a mutation in at least one of the three genes was observed in 56.4% of tumors. Mutations of the gene occurred in 34.4%, of the PIK3CA gene in 17.8%, and of the BRAF gene in 13.3%. Combined mutations of and PIK3CA were identified in 8.2%, of BRAF and PIK3CA in 2.2%, and of and BRAF in 0.2% of patients. In a multivariate analysis, activation of the network was associated with a higher risk of death (HR = 1.48, p = 0.017). 77 Effect of mutation status on response to anti-vegf therapy The vascular endothelial growth factor (VEGF) is regulated downstream of EGFR and preclinical investigations indicate that the inhibition of EGFR by cetuximab may cause a down-regulation of VEGF expression. 84,85 Both pathways are connected via the neuropilin-1 receptor. 86 Ince and coworkers carried out a retrospective analysis of a phase III trial, where the addition of bevacizumab to first-line therapy with irinotecan, 5-fluorouracil and leucovorin (IFL) was investigated. 76,87 Microdissected tumors from 295 patients were available for a determination of mutations in (35%), BRAF (5.6%), and p53 (68%). As for overall survival, - and BRAFwild-type patients had a better prognosis than patients with mutant tumors, but all subgroups had a benefit from treatment with bevacizumab. In patients who were wild-type in both, and BRAF, the hazard ratio in favour of bevacizumab treatment was 0.57 (95% CI, 0.31 1.06), while it was 0.67 (95% CI, 0.37 1.20) in patients with mutant tumors. However, survival was not correlated with p53 mutation or p53 overexpression. Considering the limitations of a retrospective analysis, the authors suggested that the survival benefit induced by bevacizumab was independent of -, BRAF- or TP53 mutation status. 76 More recently, Hurwitz and coworkers also reported on the objective response rate according to the mutation status. In wild-type patients, the addition of bevacizumab to IFL increased ORR from 37.3% to 60%, while it had no effect in mutant patients (41.2% vs. 43.2%). 88 These data need to be verified by prospective studies. Effect of mutation on response to anti-egfr therapy in first-line treatment Several randomised trials were performed which support an increased activity when anti-egfr agents were applied in combination with first-line chemotherapy. Post hoc analyses are now available which evaluate the influence of mutation status on treatment efficacy (Table 4). The CRYSTAL trial compared chemotherapy with 5-fluorouracil, folinic acid and irinotecan (FOLFIRI) plus cetuximab to FOLFIRI

V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 267 Table 4 Effect of mutation status on the efficacy of anti-egfr therapy in first-line therapy. Reference Patients recruited tumors evaluated wt n (%) mut n (%) Treatment ORR (%) PFS months -wt -mut -wt -mut Bokemeyer 92 337 233 134/233 (58) 99/233 (42) FOLFOX + Cetuximab 61 a 33 7.7 b 5.5 c FOLFOX 37 a 49 7.2 b 8.6 c van Cutsem 91 1198 540 348/540 (64.4) 192/540 (35.6) FOLFIRI + Cetuximab 59 d 36 9.9 e 7.6 FOLFIRI 43 d 40 8.7 e 8.1 wt, wild-type; mut, mutant; ORR, objective response rate; PFS, progression-free survival; Cet, cetuximab. a p = 0.011. b p = 0.016. c p = 0.019. d p = 0.0025. e p = 0.017. alone. 89 While the ITT population included 1198 patients, a analysis could be performed in 540 evaluable patients (45.1%) among whom 64% were classified as wild-type. This retrospective analysis revealed that the addition of cetuximab to FOLF- IRI significantly improved PFS in wild-type patients (HR = 0.68, p = 0.017), while this was not the case in mutant patients (HR = 1.07, p = 0.47). Likewise, cetuximab also caused a significant increase of ORR in wild-type patients (43% vs. 59%, p = 0.0025), while no improvement was observed in the mutant population (40% vs. 36%, p = 0.46). The OPUS trial chose the FOLFOX regimen as a chemotherapy backbone and investigated the addition of cetuximab in a randomised trial. 90 Of 337 patients included into this first-line trial, 233 patients could be evaluated for their status and a mutation was found in 42% (Table 4). In wild-type patients, the addition of cetuximab to FOLFOX induced a significant increase of ORR (61% vs. 37%, p = 0.011) and PFS (HR = 0.57, p = 0.016). By contrast, a negative impact on treatment efficacy was noted when cetuximab was applied in mutant patients with regard to ORR (33% vs. 49%, p = 0.106) and PFS (HR = 1.83, p = 0.0192). To this point, the basis for a negative interaction of cetuximab with chemotherapy in mutant patients is not explained. The therapeutic efficacy of double targeting combining anti- VEGF and anti-egfr therapy was investigated in the CAIRO II trial. 91 This study compared capecitabine/oxaliplatin (CapOx) plus bevacizumab to the same regimen plus cetuximab. In contrast to the expectations, the combined use of CapOx plus bevacizumab and cetuximab had a negative impact on PFS (HR = 1.21, p = 0.018) and left ORR and OS unaffected when compared to CapOx plus bevacizumab alone. Interestingly, cetuximab did not affect ORR and PFS in wild-type patients, while in mutant patients it induced a shorter duration of PFS (8.6 months vs. 12.5 months, p = 0.043) and OS (19.2 months vs. 24.9 months). A further trial investigating the efficacy of panitumumab in mcrc was the PACCE trial. In this first-line study patients received irinotecan- or oxaliplatin-based chemotherapy and were randomised to an additional treatment with either bevacizumab or bevacizumab plus panitumumab. 92 A subgroup analysis was made available for the patients with irinotecan-based chemotherapy (n = 200). In wild-type patients (n = 115), the addition of panitumumab to irinotecan/bevacizumab-based chemotherapy induced an ORR of 54% compared to 47% without the EGFR-inhibitor. No improvement of ORR was observed in 85 mutant patients (30% vs. 38%). Effect of mutation on response to anti-egfr-based therapy in pretreated patients Also in pretreated patients the relation between the mutation status and response to anti-egfr directed therapy was intensively investigated (Table 5). Most of these studies were performed as case series or post hoc analyses of clinical trials. It may be criticized that the evidence from prospective studies is largely lacking. However, the concordance between the various analyses is great, and the consistence of results is convincing. Lievre and coworkers analysed 30 patients predominantly treated with cetuximab plus irinotecan after previous exposure to chemotherapy. 13 Only three patients received first-line treatment with cetuximab plus FOLFIRI (irinotecan plus infusional 5-fluorouracil plus folinic acid). This analysis observed no mutation in clinical responders (0/11), while 13 tumors from the 19 non-responders (68.4%) had the mutated gene (p = 0.0003). Also survival was significantly longer in pretreated wild-type- compared to mutant patients (16.3 months vs. 6.9 months, p = 0.016). None of the tumors demonstrated a BRAF mutation which was consistent with the absence of microsatellite instability. All the patients with amplification (10% of tumors) of the EGFR locus were responders. However, no significant correlation was found between PIK3CA mutation and response. In a more recent publication, the same group presented an analysis of 89 mcrc patients and found a mutation rate of 27% which again was associated with resistance to cetuximab (ORR = 0% vs. 40%). 46 mutant patients had a significantly shorter PFS than non-mutant patients (2.5 months vs. 7.9 months, p = 0.0001) and also a significantly shorter overall survival (10.1 months vs. 14.3 months, p = 0.026). In a multivariate analysis, a strong correlation was observed between mutation and both PFS and OS. However, skin toxicity, another predictor of response to cetuximab, was only associated with overall survival. Benvenuti et al. reported 48 colorectal cancer patients receiving panitumumab- or cetuximab- based regimens for first-line to fourth-line therapy. 47 They observed a mutation rate of 33% and a BRAF mutation rate of 12.5%. The presence of - and BRAF mutations was negatively associated with the achievement of a partial response (p = 0.005) and also correlated with a shorter time to tumor progression (p = 0.0259). The group by Frattini and coworkers investigated tumors from 27 patients. 14 The mutation rate was 37%. In addition, they observed an EGFR gene amplification in 30% and a loss of PTEN in 41%. Partial response was noted in six of eight patients with EGFR amplification, while resistance to cetuximab was noted in the majority of patients with mutations and/or a loss of PTEN. Di Fiore et al. studied 59 patients and reported a mutation rate of 37%. 93 No mutations were found in 12 patients responding to cetuximab-based therapy. In addition, mutations were associated with a significantly shorter time to tumor progression (3 months vs. 5.5 months, p = 0.015). One of the largest analyses was performed by De Rook et al.. 75 This group evaluated 113 irinotecan-refractory mcrc patients treated with cetuximab-based therapy. Mutations of the gene

268 V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 Table 5 Effect of mutation status on the efficacy of anti-egfr therapy after failure of irinotecan-based chemotherapy Reference Line of treatment Treatment Tumors evaluated mut n (%) ORR n (%) TTP/PFS months OS months Lievre 13 1st/2nd Cet ± irinotecan or FOLFIRI 30 13 (43) 11/17 (65) 0/13 (0) 16.3 a 6.9 Benvenuti 47 1st 4th Cet ± chemotherapy or Pan monotherapy 48 16 (33) 10/32 (31) 1/16 (6) 4 b 2 b Frattini 14 1st 4th Cet + chemotherapy 27 10 (37) 9/17 (53) 1/10 (10) Di Fiore 93 P2nd Cet+ chemotherapy 59 22 (37) 12/43 (28) 0/22 (0) 5.5 c 3 Lievre 46 P2nd Cet ± chemotherapy 89 24 (27) 26/65 (40) 0/24 (0) d 7.9 d 2.5 14.3 e 10.1 De Rook 75 P2nd Cet ± chemotherapy 113 46 (41) 27/66 (41) 0/42 (0) 6 3 10.8 f 6.8 Cappuzzo 16 P2nd Cet ± chemotherapy g 80 42 (53) 10/38 (26) 4/42 (9.5) 5.4 4.4 10.8 9.5 Khambata-Ford 15 P2nd Cet monotherapy 80 30 (38) 5/50 (10) 0/30 (0) 2.0 2.0 Amado 45 P3rd Pan monotherapy 208 84 (40) 21/124 (17) 0/84 (0) 3.1 1.9 8.1 4.9 ORR, objective remission rate; TTP, time to progression; PFS, progression-free survival; OS, overall survival; wt, wild-type; mut, mutant; Cet, cetuximab; Pan, panitumumab. ORR not assessable in five of 113 patients. a Evaluation of 27 pretreated patients, p = 0.016. b Numbers extrapolated from survival plot: p = 0.044. c p = 0.015. d p < 0.001. e p = 0.026. f p = 0.020. g Personal communication from Dr. Cappuzzo. wt mut wt mut wt mut were observed in 40.7%, mutations of the BRAF gene in 5.6% of patients. The ORR rate was 41% in wild-type patients versus 0% in mutant patients. Only one of six BRAF mutant patients experienced a response to cetuximab-based therapy. mutant patients had a trend towards a shorter PFS (3 months vs. 6 months) and a significantly shorter overall survival (6.8 months vs. 10.8 months, p = 0.020). In a logistic regression analysis using age, sex, mutation status, skin toxicity, number of previous chemotherapy lines and treatment regimens covariates, status and skin toxicity were identified as the predictors of ORR and OS. They also report that early tumor size reduction translating into a longlasting and more profound tumor control only occurred in wild-type patients in whom it caused a significant improvement of survival. Greatest benefit from cetuximab-based therapy may therefore be achieved in wild-type patients and an even greater benefit may be expected in patients with an early radiological response. Effect of on outcome in patients receiving cetuximab or panitumumab as a single agent The study by Khambata-Ford stands out since it is one of the few trials prospectively investigating the relevance of molecular markers on anti-egfr therapy. 15 This group evaluated 110 pretreated mcrc patients that were enrolled onto a cetuximab-monotherapy study. Pretreatment biopsies from metastatic lesions were mandatory for all patients. Gene expression profiles showed that patients with tumors expressing the high levels of mrna for EGFR ligands (epiregulin, amphiregulin) were more likely to have disease control with cetuximab and also had a significantly longer progression-free survival. Also a wild-type was associated with a greater probability of disease control (p = 0.0003). However, the mutation status had no effect on median progression-free survival. Amado and coworkers report on a trial which compared panitumumab plus best supportive care (BSC) to BSC alone in mcrc patients with documented disease progression after the failure of fluoropyrimidines and prespecified exposure to oxaliplatin and irinotecan. 45 A status was obtained in 92% (427/463) of patients randomized within this phase III trial. mutations were observed in 43% of patients. When the panitumumab plus BSC arm (n = 208) was analysed, none of the mutant patients, but 17% of wild-type patients responded to treatment. mutations were associated with a shorter PFS (7.4 weeks vs. 12.3 weeks) and a shorter overall survival (4.9 months vs. 8.1 months) compared to the wild-type status. Clinical relevance of skin toxicity Skin toxicity evolving during the first weeks of treatment has been identified as a potent predictor of response to anti-egfr therapy. Lenz and coworkers investigated 346 mcrc patients refractory to a standard chemotherapy, who received cetuximab in a multicenter phase II trial. 10 Median response rate in the whole patient group was 12.4%. Patients with grade 1, 2, and 3 toxicity had a partial remission rate of 7.2%, 17.0% and 20%, respectively. No response was observed in patients without skin toxicity. Accordingly, the question was raised if an escalation of cetuximab dose would increase skin toxicity and treatment efficacy. The EVEREST trial investigated irinotecan-refractory patients who received cetuximab plus irinotecan. 94 Patients reacting to cetuximab with skin toxicity grades 0 1 on a day 22 evaluation were randomised to either a standard dose or an escalated dose of cetuximab. In the experimental arm, cetuximab was escalated by steps of 50 mg/m 2 q two weeks up to a maximal dose of 500 mg/m 2. As expected, increased doses of cetuximab improved treatment efficacy. However, an analysis of the status revealed that the dose escalation prolonged PFS only in wild-type patients, while it had no effect in mutant patients. Comparably, the escalation of cetuximab did also not affect the remission rate of 0% in mutant patients. Skin toxicity and mutation were identified as independent predictors of treatment outcome. From this it may be concluded that greatest benefit from cetuximab therapy may be expected in wild-type patient reacting with grade P2 skin toxicity. 94 Small molecule tyrosine kinase inhibitors in colorectal cancer Tyrosine kinase inhibitors (TKI) so far have no place in the treatment of colorectal cancer. At present, only few studies are available with conflicting results. 95,96 The effect of the mutation status on TKI activity was studied in a clinical trial performed by Ogino and coworkers. 97 This group investigated 30 mcrc patients receiving gefitinib in combination with chemotherapy. In

V. Heinemann et al. / Cancer Treatment Reviews 35 (2009) 262 271 269 wild-type patients, the response rate was 47% as compared to 33% in mutant patients. Conclusions Determination of the mutation status is required in all mcrc patients who may receive anti-egfr directed antibody therapy. Studies using cetuximab plus chemotherapy for first-line treatment of mcrc could detect an improvement of treatment efficacy only in wild-type patients. By contrast, mutant patients either had no benefit from the addition of cetuximab or even showed a worse outcome than their comparators. At present, it is not clear if additional toxicity impairs treatment intensity in mutant patients or if negative interactions between cetuximab and chemotherapy take place. In studies where anti-egfr antibodies were applied in Psecond-line therapy as single agents or in combination with chemotherapy mostly no remissions were observed in mutant patients. The exact role of the mutation status for patients receiving small molecule tyrosine kinase inhibitors remains to be clarified in larger trials. Clinical implications The EGFR has become an important target in the treatment of metastatic colorectal cancer. Mutations of the gene may activate downstream signal transduction and confer resistance to upstream inhibition of the EGFR by monoclonal antibodies. The mutation status has been identified as a strong predictor of response to anti-egfr antibodies. EGFR directed therapy is not only not effective in mutant mcrc patients, but it may also induce unnecessary toxicity and has been associated with an inferior outcome in some clinical trials. Agents such as cetuximab or panitumumab should, therefore, only be offered to patients with a wild-type. EGFR gene amplification and PTEN expression were also identified as important predictors of tumor sensitivity to anti-egfr therapy, but are less well evaluated in large clinical trials. Mutations of PI3K and BRAF are less frequent and are clinically less relevant. However, they may gain importance in the context of mutational networks affecting the EGFR pathway. Future approaches to predict for sensitivity to anti-egfr treatment strategies may, therefore, involve the analysis of networks of signal transduction rather than single mutations. Conflict of interest statement Prof. Dr. Volker Heinemann, MD: No employment, stock ownership, paid expert testimony or patent applications/registrations Honoraria: Merck, Roche Consultancies: Merck, Roche Grants: Merck, Roche Research support: Merck, Roche Dr. Sebastian Stintzing, MD: No employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Prof. Dr. Thomas Kirchner, MD: No employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Dr. Stefan Boeck, MD: No employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Dr. Andreas Jung, MD: No employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Thomas Kirchner, MD No employment, stock ownership, paid expert testimony or patent applications/registrations Honoraria: Amgen, Merck, Roche Consultancies: Amgen, Merck, Roche Grants: Roche Research Support: Amgen Andreas Jung, MD PhD No employment, stock ownership, paid expert testimony, patent applications/registrations, grants or research support. Honoraria: Amgen, Merck Consultancies: Amgen, Merck, Roche References 1. Lemmon MA, Schlessinger J. Regulation of signal transduction and signal diversity by receptor-oligodimerization. Trends Biochem Sci 1994;19: 459 63. 2. Ono M, Kuwano M. 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