Prognostic and predictive biomarkers for epidermal growth factor receptor-targeted therapy in colorectal cancer: Beyond KRAS mutations

Critical Reviews in Oncology/Hematology 85 (2013) 45–81

Prognostic and predictive biomarkers for epidermal growth factor receptor-targeted therapy in colorectal cancer: Beyond KRAS mutations Ana Custodio ∗ , Jaime Feliu Medical Oncology Department, La Paz Universitary Hospital, IDiPAZ, RTICC (RD06/0020/1022), Spain Accepted 4 May 2012

Contents 1. 2.

3.

4.

5. 6.

7.

8. 9.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EGFR as a target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. EGFR protein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. EGFR gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. EGFR gene copy number (GCN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Alternative EGFR ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Germline polymorphisms within the EGFR signalling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. EGFR polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. EGF and cyclin-D1 polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Polymorphisms in the fragment c gamma (Fc-␥) receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Cyclooxygenase-2 (COX-2) polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. MicroRNAs polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KIRSTEN-RAS (KRAS) status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. KRAS mutations in colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. KRAS mutation status as a predictor of resistance to anti-EGFR mAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Effect of KRAS mutation on response to anti-EGFR therapy in first-line treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Effect of KRAS mutation on response to anti-EGFR therapy in pretreated patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Variation of the treatment effect by KRAS specific mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Prognostic value of KRAS mutation status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroblastoma-RAS (NRAS) status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BRAF status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. BRAF mutation status as a predictor of resistance to anti-EGFR mAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Prognostic value of BRAF mutation status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PIK3CA status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. PIK3CA mutation status as a predictor of resistance to anti-EGFR mAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Prognostic value of PIK3CA mutation status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PTEN status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other potential biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. HER2 gene status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Medical Oncology Department, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046 Madrid, Spain. Tel.: +34 91 2071138; fax: +34 91 7277118. E-mail address: [email protected] (A. Custodio).

1040-8428/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.critrevonc.2012.05.001

46 47 47 47 47 49 50 50 50 51 52 52 53 53 54 55 55 58 59 61 61 62 62 63 64 65 65 65 66 67 67 67

46

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

9.2. c-Met and insulin-like growth factor receptor 1 (IGF1R) pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. TP53 mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Use of multiple biomarkers to predict clinical outcome to ANTI-EGFR mABs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Early response evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1. Early radiological tumour size decrease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2. Skin rash as a biomarker of efficacy of anti-EGFR therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3. Hypomagnesemia as a biomarker of efficacy of anti-EGFR therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 69 69 70 70 70 71 73 74 74 74 81

Abstract The advent of the epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies (mAbs), cetuximab and panitumumab has expanded the range of treatment options for metastatic colorectal cancer (CRC). Despite these agents have paved the way to individualized therapy, our understanding why some patients respond to treatment whereas others do not remain poor. The realization that detection of positive EGFR expression by IHC does not reliably predict clinical outcome of EGFR-targeted treatment has led to an intense search for alternative predictive biomarkers. Data derived from multiple phase III trials have indicated that KRAS mutations can be considered a highly specific negative biomarker of benefit to anti-EGFR mAbs. Oncologists are now facing emerging issues in the treatment of metastatic CRC, including the identification of additional genetic determinants of primary resistance to EGFR-targeted therapy for further improving selection of patients, the explanation of rare cases of patients carrying KRAS-mutated tumours who have been reported to respond to cetuximab and panitumumab and the discovery of mechanisms of secondary resistance to EGFR-targeted therapy. Current data suggest that, together with KRAS mutations, the evaluation of EGFR gene copy number (GCN), BRAF, NRAS, PIK3CA mutations or loss of PTEN expression could also be useful for selecting patients with reduced chance to benefit from anti-EGFR mAbs. This review aims to provide an updated of the most recent data on predictive and prognostic biomarkers within the EGFR pathway, the challenges this emerging field presents and the future role of these molecular markers in CRC treatment. © 2012 Elsevier Ireland Ltd. All rights reserved. Keywords: Prognostic factor; Predictive factor; Molecular marker; Epidermal growth factor receptor (EGFR) inhibitors; KRAS; BRAF; PIK3CA; PTEN

1. Introduction Colorectal cancer (CRC) is the third most common cause of cancer related death in western societies, accounting for approximately 10% of all cancer incidence and mortality [1]. It is widely accepted that CRC is a heterogeneous disease defined by different activating mutations in receptor tyrosine kinases (RTK) or activating loss-of-functions mutations in downstream components of RTK-activated intracellular pathways [2,3]. Therefore, the realization of that treatment should be tailored on an individual case-specific basis is becoming prevalent. Treatment on an “individualized” basis now involves a simultaneous case-specific analysis of clinical and pathological characteristics and analysis of a patientˇıs genetic and tumour biomarker profile [4]. The improvement of our understanding of the biology of CRC has pointed towards the epidermal growth factor receptor (EGFR) pathway as a critical mechanism in CRC tumourigenesis [2,5]. The EGFR (ERB-1 or HER-1) is a member of the human epidermal growth factor receptor (HER)-erbB family of RTKs, that includes three other RTK: HER2/C-neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Since the establishment of the EGFR gene as an oncogene, it has become an important target for cancer treatment because its activation stimulates key processes involved in tumour

growth and progression, including proliferation, angiogenesis, invasion and metastases. The binding of epidermal growth factor (EGF) or other ligands to EGFR initiates a mitogenic signalling cascade through two main axes. On one side, the KRAS–RAF-mitogen-activated protein kinase (MAPK) pathway, responsible for gene transcription, cell-cycle progression and cell proliferation. The other axis involves membrane localization of the lipid kinase phosphatidylinositol 3-kinase (PI3K), which promotes Akt-mammalian target of rapapycin (mTOR) activation, responsible for antiapoptosis and prosurvival signals. Importantly, the two axes (KRAS/BRAF and PIK3CA) are closely related and strictly interconnected, as the p110 subunit of PI3K can also be activated via interaction with RAS proteins. Cetuximab and panitumumab are both monoclonal antibodies (mAbs) that recognize and inactivate the extracellular domain of EGFR, thus leading to inhibition of the downstream signalling pathways [6]. Both agents have been approved for metastatic CRC treatment based on the improvement of progression free survival (PFS) and overall survival (OS) when used either as single agents or in combination with chemotherapy (CT) [7–13]. These costly and potentially toxic treatments are, however, efficient in only a small percentage of patients and it is therefore extremely important to identify specific factors who will lead to a clearer definition of those

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

patients who will benefit from anti-EGFR treatments. KRAS mutation is the first established molecular marker which precludes responsiveness to EGFR-targeted treatment with cetuximab and panitumumab [14–16]. In addition, among CRC tumours carrying wild-type (WT) KRAS, EGFR gene copy number (GCN), mutations of BRAF or PIK3CA or loss of phosphatase and tensin homolog (PTEN) expression may be associated with resistance to anti-EGFR therapy [17–19], although these additional biomarkers require further validation before incorporation into clinical practice. Up until now, each of these markers has been mainly assessed as a single event, but these molecular alterations display overlapping patterns of occurrence, thus adding considerable complexity for drawing an algorithm suitable for clinical decision-making. Therefore, it has been suggested that the future comprehensive analysis of the entire oncogenic pathway triggered by the EGFR should be performed, thus enhancing the prediction ability of the markers individually used. In this review we summarize current progress in the search for molecular markers within the EGFR signalling cascade and further discuss any inconsistent or conflicting findings for these molecular classifiers in early-stage and advanced CRC treatment.

2. EGFR as a target 2.1. EGFR protein expression Positive EGFR protein expression, as determined by immunohistochemistry (IHC), was initially selected as an entry criterion for early studies evaluating EGFR inhibitors on the assumption that sensitivity to such agents was associated with EGFR expression [20]. However, a large body of evidence from patients who were treated with mAbs for metastatic CRC [6,11,21,22] or tyrosine kinase inhibitors (TKI) for other solid tumours [23] indicates that this biomarker is poorly associated with response. Retrospective analysis of multiple series and data from the PRIME trial confirmed that, even in KRAS WT tumours, EGFR expression was not predictive for anti-EGFR therapies efficacy [24–27]. Moreover, several authors reported that cetuximab was also active in tumours which were EGFR-negative by IHC yielding response rates (RR) up to 25% [28,29]. Many technical explanations have been advocated for the lack of association between EGFR protein detection by IHC and response to EGFR-targeted agents [30]. These reasons include disparity between the form of epitope of EGFR detected by IHC and that targeted by anti-EGFR mAbs, as well as issues related to processing and handling of tumour tissue samples, such as prolonged storage, and the fact that IHC could be actually a suboptimal technique for assessing EGFR status. In fact, IHC has shown a low specificity for predicting EGFR gene amplification [30]. EGFR is overexpressed in 60–80% CRC [31], but only a fraction of IHC-positive tumours also

47

show EGFR gene amplification (17% in primary and 23% in metastatic tumours). Copy number variations of EGFR can be caused by polysomy in addition to gene amplification, without a significant increase of the receptor protein [32,33]. IHC is also a semiquantitative method that lacks a standardized scoring system and is subject to interobserver variation. Moreover, EGFR expression differences between primary CRC and their metastases have been reported, indicating that reliance on such marker in the primary tumour to predict treatment response of metastatic growths may be inappropriate [34]. Finally, some tumour specimens contain both low- and high-affinity EGFR binding sites. Because IHC-based methods cannot distinguish between them, these findings may provide further explanation for the lack of correlation between EGFR immunostaining and clinical response to EGFR-targeted treatment [28]. 2.2. EGFR gene mutations Activating mutations, including in-frame deletions and amino acid substitution in exons 18, 19 and 21 in the EGFR catalytic domain, are seen frequently in lung cancer and play an important role in determining responsiveness to TKIs [35]. However, studies on large cohorts of untreated metastatic CRC and on small cohorts of tumour samples taken from treated patients, quickly and unequivocally clarified that EGFR somatic mutations are extremely rare in CRC, and when they do occur, are not associated with response to anti-EGFR mAbs [36,37]. 2.3. EGFR gene copy number (GCN) Contrary to other situations in which the genomic locus corresponding to an oncogene is frankly amplified (>20fold) (MET, PIK3CA), thus resulting in largely increased expression of the corresponding protein, it seems clear that the increase in EGFR GCN is often modest (3–5-fold) and caused by polysomy rather than gene amplification, without a significant increase of the receptor protein, as mentioned before [32,33]. Nevertheless, the association between an increase in EGFR GCN and positive clinical outcome to anti-EGFR agents certainly exists, as confirmed by different series (Table 1) [14,29,30,32,33,37–45]. This molecular alteration can be detected by fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or polymerase chain reaction (PCR)-based methods. The comparability of these methods and their differential impact on results still needs to be defined. Moreover, the cutoffs defining EGFR amplification varied among series between 2- and 6-fold. As a reflection of methodological uncertainties, the prevalence of EGFR gene amplification shows a wide variation of 6–51% in different reports [30,32]. Increased GCN was found in at least 30% of patients when a threshold value of approximately three EGFR copies per nucleus was used, as determined by FISH, compared with only 10% of patients when a threshold of six or more EGFR copies per nucleus

48

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

Table 1 Tumour epidermal growth factor receptor (EGFR) gene copy number (GCN) and outcome of anti-EGFR treatment in advanced CRC. Author (year)

Treatment (type of patients)

GCN cutoff (methodology)

RR (%)

PFS (months)

OS (months)

Frattini (2007) [43]

Cetuximab ± CT (CT naïve or CT refractory)

≥4

22%

NA

NA

<4 (FISH)

14% (p = 0.05)

≥2.92

32.6%

6.6

11.3

<2.92 (FISH)

2.4% (p = 0.0001)

3.5 (p = 0.02)

8.5 (p = 0.8)

30%

8

15

Capuzzo (2007) [33]

Cetuximab ± CT (CT refractory)

Sartore-Bianchi (2007) [39]

Panitumumab (CT refractory)

≥2.47 <2.47 (FISH)

0% (p = 0.0009)

3 (p = 0.039)

10 (p = 0.015)

Lièvre (2006) [14]

Cetuximab ± CT (CT naïve or CT refractory)

≥6

27%

NA

NA

<6 (CISH)

0% (p = 0.04)

≥3

89%

NA

NA

<3 (FISH)

5% (p < 0.001)

≥2.83

NA

5.5

10

4 (p = 0.25)

8.3 (p = 0.037)

Moroni (2005) [32]

Personeni (2008) [42]

Cetuximab/panitumumab ± CT (CT naïve or CT refractory) Cetuximab ± CT (CT refractory)

<2.83 (FISH) Laurent-Puig (2009) [44] (KRAS WT population) Scartozzi (2009) [45] (KRAS WT population)

Lenz (2006) [29]

Cetuximab ± CT (CT refractory)

Cetuximab + irinotecan (CT refractory, KRAS WT)

Cetuximab (CT refractory)

≥2.0

71%

8

16.2

<2.0 (FISH)

37% (p = 0.015)

7 (NS)

11.8 (NS)

≥2.6

60%

7.7

NA

<2.6 (FISH) ≥2.12 <2.12 (CISH)

9% (p = 0.002) 36% 6% (p = 0.03)

2.9 (p = 0.04) 6.4 3.1 (p = 0.02)

NA

[PCR]

NA (NS)

NA (NS)

NA (p = 0.03)

CT = chemotherapy; FISH = fluorescence in situ hybridization; CISH = chromogenic in situ hybridization; PCR = polymerase chain reaction; WT = wild type; RR = response rate; PFS = progression-free survival; OS = overall survival; NA = not available; NS = not statistically significant.

was used, as determined by CISH [38,39]. In addition, poor concordance rates between primary and metastatic tumours have been reported for EGFR GCN [40]. Moroni et al. first demonstrated the association between EGFR GCN, as determined by FISH analysis, and response to anti-EGFR mAbs in unselected populations [32]. Authors described an 89% RR in a subgroup of nine patients with CRC whose tumours had an increased EGFR GCN. These findings were confirmed from a retrospective analysis of a subgroup of patients participating in the pivotal phase III trial of panitumumab monotherapy [39]. The mean EGFR GCN per nucleus and the percentage of tumour cells with chromosome 7 polysomy (≥3 EGFR signals per nucleus) were analyzed by FISH and the association between these parameters and clinical outcome was assessed. None of the patients with a mean of 2.47 or less EGFR gene copies per nucleus, or fewer than 43% of tumour cells with chromosome 7 polysomy, respectively, achieved objective response, compared with six (30%) of the 20 patients

(p = 0.001) and six (32%) of the 19 patients (p = 0.001) who had values above these thresholds. Patients treated with panitumumab with <2.47 EGFR copies/nucleus or <43% of tumour cells displaying chromosome 7 polysomy predicted for shorter PFS (p = 0.039 and p = 0.029, respectively) and OS (p = 0.015 and p = 0.014, respectively). However, EGFR GCN and chromosome 7 polysomy did not draw a parallel with PFS in patients receiving only best supportive care (BSC), suggesting that this parameter is not prognostic in metastatic CRC. The association between EGFR GCN increase and response to cetuximab- or panitumumabbased treatment was confirmed with different cut-offs by subsequent analysis, which are summarized in Table 1 [14,29,32,33,39,42–44]. More recent studies have evaluated the added value of EGFR GCN status in patients with known KRAS WT tumours who received cetuximab-based CT in the second and subsequent treatment lines [44,45]. Laurent-Puig et al. conducted a comprehensive analysis including the evaluation

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

of EGFR amplification/polysomy status by FISH and CISH in 96 KRAS WT tumours [44]. An EGFR FISH-positive phenotype was found in 17 patients (17.7%) who showed a statistically significant higher RR (71%) compared with patients with normal EGFR GCN (37%) (p = 0.015). A trend towards longer PFS and OS was found in patients with FISH-positive phenotype but without reaching a threshold of significance. This methodology was also employed by Scartozzi et al. [45] who evaluated the role of EGFR GCN in 44 irinotecan-refractory KRAS WT CRC patients treated with irinotecan and cetuximab. They reported a statistical significant association between GCN and RR, with a tumour regression in 9 (60%) and 2 (9%) cases with an increased and low FISH EGFR GCN, respectively (p = 0.002) and in 10 (36%) and 1 (6%) cases with an increased and low CISH EGFR GCN, respectively (p = 0.03). Median time to progression(TTP) was 7.7 and 6.4 months in patients showing increased FISH and CISH EGFR GCN, whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02, respectively). These data contrast with earlier findings based on quantitative PCR analysis, showing that when EGFR GCN was assessed by this method, no association was found between this parameter and clinical outcome of cetuximab- or panitumumab-based treatment, probably because of tumour DNA dilution by DNA from normal cells during DNA extraction [23,29]. Lenz et al. did not find a relationship between EGFR GCN and response or PFS in 34 intensively treated patients exposed to single agent cetuximab. However, a significant correlation with survival was observed (p = 0.03) [29]. 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 EGFR, IL-8 and Cox-2 [23]. In this context, it still needs to be clarified to which extent EGFR gene expression is not only a predictive factor of outcome but also has a prognostic importance. Although the most recent data are promising for the use of increased EGFR GCN as a positive predictive factor of clinical outcome to EGFR-targeted mAbs, the reproducibility of data remains the largest obstacle for clinical applicability of this molecular determinant. Methods of tissue processing and EGFR scoring systems need to be standardized before using it for selecting patients for EGFR-targeted therapy [46]. 2.4. Alternative EGFR ligands The overexpression of alternative EGFR ligands, such epiregulin (EREG) and amphiregulin (AREG), is likely to be a key factor in determining sensitivity to anti-EGFR therapies via ligand-driven autocrine oncogenic EGFR signalling [47–49]. The proposed model suggests that high expression of activating ligands may contribute to the cellular addiction to EGFR signalling in KRAS WT CRC.

49

Such tumours demonstrate increased dependence on ligandactivated EGFR signalling and will experience the greatest oncogenic shock following rapid loss of this signalling as a result of ligand withdrawal during EGFR blockade. Retrospective studies have shown the potential predictive value of AREG and EREG expression in KRAS WT tumours. The level of sensitivity to cetuximab was shown to be proportional to the intensity of AREG and EREG tumour mRNA expression [47,48,50,51]. Khambata-Ford et al. compared clinical outcomes for patients with high and low levels of these ligands in fresh-frozen tissues from CRC metastases [47]. Patients with high levels of EREG and AREG expression are more likely to have disease control with cetuximab (EREG, p = 0.000015; AREG, p = 0.000025) and also have significantly longer PFS than patients with low expression (AREG: median, 115.5 vs. 57 days, hazard ratio [HR], 0.44; p = 0.0001; EREG: median, 103.5 vs. 57 days; HR, 0.47; p = 0.0002). The exclusive use of either KRAS status or AREG or EREG gene expression profiles does not result in the selection of identical patient populations who are likely to benefit from treatment with cetuximab: among patients with WT KRAS, those whose tumours expressed high levels of AREG or EREG were likely to experience disease control, whereas patients whose tumours expressed low levels of these genes were not, thus providing important complementary information to KRAS status [48]. Jacobs et al. showed that it is also technically feasible to measure EGFR ligands expression in archival formalin-fixed paraffinembedded (FFPE) tissue of primary CRC [49]. High ligand expression levels were significantly associated with response (AREG, p = 0.0017; EREG, p = 0.0005), PFS (AREG: HR, 0.43; 95% confidence interval [CI], 0.29–0.64; p < 0.001; EREG: HR, 0.41; 95% CI, 0.274–0.609; p < 0.001) and OS (AREG: HR, 0.40; 95% CI, 0.27–0.64; p = 0.0001; EREG: HR, 0.42; 95% CI; 0.28–0.63; p = 0.0001) in KRAS WT metastatic CRC treated with the combination of cetuximab and irinotecan. There was no predictive power of ligand expression in patients with KRAS mutation. In the NCI-CTG 017 phase III trial of cetuximab versus BSC in chemorefractory CRC, high EREG gene expression was also found to be associated with improved median PFS (5.4 vs. 1.9 months) and OS (9.8 vs. 5.1 months) in KRAS WT patients treated with cetuximab [52]. In addition to its predictive value, EREG mRNA expression appeared to be a useful prognostic marker in KRAS WT patients regardless of receiving anti-EGFR therapy [53]. In a cohort of 120 metastatic CRC patients not treated with antiEGFR mAbs, those with KRAS WT status and low EREG mRNA levels showed significantly better OS than those with high levels (p = 0.006). AREG expression showed the same tendency but did not reach significant difference. Similar to EGFR copy number, the lack of standardization of the assays has prevented AREG and EREG expression levels from being used as clinical biomarkers for directing treatment with EGFR-targeted agents.

50

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

3. Germline polymorphisms within the EGFR signalling pathway 3.1. EGFR polymorphisms The regulation of EGFR transcription is not yet completely understood and there is growing interest to explore whether EGFR polymorphisms may influence expression of the receptor and clinical outcome to anti-EGFR mAbs [54–64]. In particular, the EGFR gene contains a highly polymorphic sequence in intron-1, which consists of a variable number tandem repeats (VNTR) of CA dinucleotides ranging from 15 to 26. Due to variable impact on DNA binding, this sequence has been shown to affect the efficiency of gene transcription, mRNA amount and EGFR expression. Preclinical and clinical data have suggested that the longer the CA repeat, the lower the EGFR expression [54]. In experimental models, the transcription of the EGFR gene was found to be inhibited by approximately 80% in (CA)21 -repeat alleles of the EGFR intron-1 variant, whereas decreasing the number of CA pairs down to (CA)12 enhances transcription as much as five-fold [54]. Han et al. [56] and Liu et al. [57] found that a low number of the EGFR intron-1 (CA)n variants was associated with gefitinib responsiveness in non-small-cell lung cancer patients. Relative to the clinical response to anti-EGFR treatment in CRC patients, Amador and coworkers reported that the VNRT of CA dinucleotides in EGFR intron-1 was associated with the pharmacodynamics of gefitinib [54]. Graziano et al. observed a significant association with longer PFS (HR, 0.45; 95% CI, 0.24–0.84; p = 0.01) and OS (HR, 0.41; 95% CI, 0.21–0.78; p = 0.006) for the EGFR intron-1 short/short (S/S) genotype (17 or less CA repeats VNTR) in 110 patients with refractory advanced CRC treated with cetuximab plus irinotecan therapy [58]. EGFR intron-1 S/S carriers also showed more frequent treatment response (p = 0.008) and grade 2–3 skin toxicity (p = 0.001) than EGFR intron-1 long/long (L/L) carriers, whereas no significant association was found between EGFR expression and EGFR intron-1 status. Therefore, a plausible mechanism for explaining the EGFR intron-1 variant influence on cetuximab activity is EGFR upregulation, which in turn is a determinant for the activity of anti-EGFR therapeutics [54,55,59]. The lack of association between EGFR intron-1 variant and EGFR expression is likely to be related to methodological issues regarding the use of poorly validated and non-qualitative IHC methods for EGFR analysis in vivo [60,61]. These findings were confirmed by Pander et al. [62] but are in conflict with results from two previous studies that include 39 patients from a phase II multicenter trial of cetuximab [63,64]. In these studies, Zhang et al. did not find any association between EGFR intron-1 (CA)n and outcome. However, this polymorphism was studied by simply subdividing patients into two groups: 16 carriers of both (CA)n < 20 alleles, and 18 carriers of any (CA)n ≥ 20 alleles, with five missing cases. The sample size of the study by Graziano allowed for a more precise distinction of genotypes [58], with three distinct groups

including two homozygous (S/S and L/L) and the heterozygous S/L genotype. Moreover, Zhang et al. [63,64] may have missed a dose-dependent effect of the EGFR intron-1 (CA)n variant with both S alleles present (CA repeats < 17) in the EGFR intron-1 S/S genotype. EGFR +497G>A is a single nucleotide polymorphism (SNP) affecting exon 13 at residue 521 (previously identified as residue 497, rs11543848), which has been associated with an arginine (Arg) by lysine (Lys) substitution in the extracellular domain of EGFR within subdomain IV. Moriai et al. were able to show that the Lys/Lys (A/A) genotype confers an attenuated function in EGFR ligand binding, growth stimulation, tyrosine kinase activation, and induction of proto-oncogenes [65]. The Arg/Arg genotype has been linked with improved OS in women with metastatic CRC (vs. Lys/Lys and/or Lys/Arg variants), although the reverse pattern was observed in men [66]. This same polymorphism has been linked to cetuximab response in other studies. Lurje et al. showed that EGFR +497 A/A genotype was associated with poor clinical outcome and shorter PFS (median PFS of 1.2 months), compared with other genotypes (median PFS 1.3 months and 1.8 months in patients homozygous and heterozygous for the G-allele, respectively; p = 0.017) [67].

3.2. EGF and cyclin-D1 polymorphisms Modulation of the EGFR ligand, EGF, and of the downstream EGFR signalling, including the cyclin-D1 gene, may also play a role on cetuximab activity [58,62,63]. Functional variants have been described in the EGF 5 -untranslated region (EGF 61 G>A) [68] and in exon 4 of the cyclin-D1 gene (870 A>G) [69,70]. In the analysis by Graziano, EGF 61 G/G homozygous genotype was significantly associated with favourable OS (HR = 0.44; 95% CI, 0.23–0.84; p = 0.01) and a borderline association between EGF 61 A/G heterozygous genotype (HR = 0.58; 95% CI, 0.34–0.99; p = 0.04) and favourable OS was also observed [58]. However, this association was not described by other authors [62,63]. Zhang et al. found that EGF 61 any A allele genotype was correlated with favourable outcome [63]. In addition, a significant association was showed in this study between the cyclin D1 (CCND1) A870G polymorphism and OS. Patients with the A/A homozygous genotype survived for a median of 2.3 months (95% CI 2.1–5.7), whereas those with any G allele (A/G, G/G genotype) survived for a median of 8.7 months (95% CI = 4.4–13.5) (p = 0.019). When the CCND1 and EGF polymorphisms together were analyzed, patients with favourable genotypes (EGF any A allele and CCND1 any G allele) showed a median survival time of 12 months (95% CI = 4.8–15.2), whereas patients with any two unfavourable genotypes (EGF GG or CCND1 A/A) showed a median survival time of 4.4 months (95% CI = 2.1–5.7) (p = 0.004). These results have not been confirmed by Pander et al.: the CCND1 A870G polymorphism was not

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

significantly associated with PFS regardless of receiving cetuximab treatment [62]. 3.3. Polymorphisms in the fragment c gamma (Fc-γ) receptors Modulation of the immune response could be another important mechanism of action to anti-EGFR mAbs. The immunological mechanism antibody-dependent cellular cytotoxicity (ADCC) mediated through the fragment c gamma receptors (Fc-␥R) carried by innate immune cells such as macrophages and natural killers (NK) plays an important role in the antitumour effect of immunoglobulin (Ig) G1 antibodies [71]. In vitro studies have shown that cetuximab was able to induce ADCC [72]. Although it was initially believed that panitumumab, as an IgG2 mAb, would not elicit ADCC, this phenomenon has recently been demonstrated in preclinical studies at concentrations that are analogous to therapeutic doses [73]. Constitutional polymorphisms have been demonstrated on genes encoding for the activating receptors Fc␥RIIa (CD 32, mainly expressed on macrophages) and Fc␥RIIIa (CD16, expressed on NK cells and macrophages), affecting their affinity to human IgG: a histidine (H)/arginine (R) polymorphism at position 131 for Fc␥RIIa (131 H/R) and a valine (V)/phenylalanine (F) polymorphism at position 158 for Fc␥RIIIa (158 V/F) [74]. On the basis of the different binding affinities, patients harbouring Fc␥RIIa-131H/H and Fc␥RIIIa-158V/V genotypes would be expected to mediate a more potent ADCC antitumour response after mAb treatment [75]. Clinical studies have shown that Fc␥RIIa-131H/H and Fc␥RIIIa-158V/V genotypes were associated with better clinical outcome to rituximab as first-line treatment of follicular lymphoma [76,77] and to trastuzumab-based therapy in metastatic breast cancer [78]. Conflicting results have been published in chemorefractory advanced CRC studies, most of them developed in unselected KRAS populations [58,64,79–82]. Zhang et al. reported that Fc␥RIIa-H131R and Fc␥RIIIa-V158F polymorphisms were independently associated with PFS (p = 0.037 and 0.055, respectively) in 39 EGFR-expressing metastatic CRC patients treated with cetuximab monotherapy [64]. Combined analysis of these two polymorphisms showed that patients with the favourable genotypes (FC␥RIIa any H allele, and FC␥RIIIa any F allele) showed a median PFS of 3.7 months (95% CI, 2.4–4.4 months), whereas patients with any unfavourable genotypes (FC␥RIIa R/R or V/V) had a PFS of 1.1 months (95% CI, 1.0–1.4 months; p = 0.004). On the contrary, in the analysis performed by Bibeau et al. [79], Fc␥RIIa-131H and Fc␥RIIIa-158V were favourable alleles, as observed in the studies with rituximab and trastuzumab. Irinotecanrefractory metastatic CRC patients treated with cetuximab plus irinotecan and with combined Fc␥RIIa-131 H/H and/or Fc␥RIIIa-158V/V genotypes had longer PFS than 131R and 158F carriers (5.5 vs. 3.0 months; p = 0.005) for the whole

51

population as well as for the subpopulation of patients with WT KRAS (9.6 vs. 4.6 months; p = 0.015) and mutated KRAS (3.2 vs. 2.8 months; p = 0.015). Fc␥RIIa-131H/H and/or 158V/V polymorphisms also demonstrated significant association with OS for whole group (p = 0.032) and WT KRAS patients (p = 0.027). A pooled analysis of these two polymorphisms in 3 phase III trials of metastatic CRC patients (n = 317) treated with either single agent cetuximab (IMCL0144 and CA225045) or with second-line irinotecan plus cetuximab (EPIC) showed that patients with Fc␥RIIa H/H genotype had shorter PFS (median PFS, 1.3 months; 95% CI, 1.2–2.4) compared to those with H/R or R/R genotypes (median PFS, 2.5 months; 95% CI, 1.5–3.0; p = 0.036). No statistically significant associations were observed between Fc␥RIIIa polymorphisms and efficacy of cetuximab-based treatment [80]. Moreover, other studies have not found a significant association between the Fc␥RIIa or Fc␥RIIIa polymorphisms and the efficacy of cetuximab [58,81]. Finally, in order to provide the statistical power to test the definitive role of these SNPs on cetuximab efficacy, a large international consortium study has been performed including 900 unselected chemorefractory metastatic CRC patients treated with cetuximab alone or in combination [82]. No clinically significant correlation was found between Fc␥R SNPs and cetuximab efficacy in unselected KRAS status patients. However, an increased in disease control rate (DCR) was seen in KRAS mutant patients harbouring the FC␥RIIIa FF genotype (71.3% vs. 47.9% in non-FF; p = 0.049), as well as a trend for increased median PFS (median PFS, 16 vs. 12 weeks for FF and non-FF genotypes, respectively; p = 0.072). In the first-line setting, Pander et al. found that the Fc␥RIIIa V allele (F/V and V/V genotypes combined) was associated with decreased PFS compared with the Fc␥RIIIa F/F genotype (median PFS, 8.2 vs. 12.8 months, respectively; HR, 1.57; 95% CI 1.06–2.34; p = 0.025) in a cohort of WTKRAS metastatic CRC patients treated with capecitabine, oxaliplatin and bevacizumab with cetuximab in the CAIROII study [26], whereas it was not significantly associated with PFS in the arm without cetuximab (p = 0.832) [62]. The Fc␥RIIa polymorphism was not significantly associated with PFS neither in the cetuximab arm nor in the arm without cetuximab. A possible mechanism for the opposite association of theFc␥RIIIa polymorphism with outcome in the studies by Pander and Bibeau could be that the high affinity Vallele [83] results in increased activation of tumour associated macrophages (TAMs) by cetuximab through cross-linking ofthe Fc␥R [84] in the study by Bibeau, instead of increasing ADCC in the study by Pander et al. [62]. As a result of TAM activation, proangiogenic mediators are released in the tumour microenvironment, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) [85]. In the CAIRO-II trial [26], patients had not received palliative CT before, whereas patients in the other studies [58,67,79] had been exposed to irinotecan and/or other lines of CT prior to cetuximab, which could have altered the infiltration of cells of the myeloid lineage such

52

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

as TAMs. However, it must be noted that the FC␥RIIIa F-allele was associated with increased efficacy of the IgG1type mAbs rituximab inlymphoma [76,77] and trastuzumab [78] in advanced breast cancer, as well as cetuximab in the study by Zhang et al. [64]. Therefore, fundamental research should be performed to support this highly speculative hypothesis.

3.4. Cyclooxygenase-2 (COX-2) polymorphisms Another SNP investigated as a predictive factor to pharmacoresistance was found in the COX-2 gene [67]. COX-2 is involved in the regulation of a broad range of cellular processes including tumour onset and progression, metastases, angiogenesis and resistance to CT [86]. The relationship between COX-2 and the EGF/EGFR signalling pathway is still controversial [87]. COX-2 is thought to be a downstream effector of EGFR and was found to be induced by EGF mediated stimulation of EGFR tyrosine kinase in human cell lines [88]. Other studies showed that COX-2 may be an upstream effector of EGFR in human colon cancer cells lines, suggesting that it induces colon cancer carcinogenesis by the activation of EGFR [89]. Furthermore, COX-2 has been reported to be a predictive and an adverse prognostic factor in a variety of malignancies, including CRC [23,86]. COX-2 −765 G>C is a frequent SNP located 765 base pairs upstream of the COX-2 transcription start site. The −765 C allele was shown to be associated with significantly lower COX-2 promoter activity compared with the −765 G variant [90]. Other common variant within the COX-2 gene include the COX-2 +8473 T>C SNP. It locates within the functional region of 3-untranslated region of the gene and, therefore, may have a potential functional relevance in carcinogenesis, perhaps through control of mRNA-stability and degradation [91]. The +8473 C allele is significantly less common in patients with cancer compared with healthy control patients, suggesting a protective effect [91]. Lurje et al. found “low-expression” variants of COX-2 (COX-2 −765 C and COX-2 +8473 C) to be significantly associated with higher PFS in metastatic CRC patients treated with single agent cetuximab (COX-2 −765 G>C C/C genotype: HR, 0.31; 95% CI, 0.12–0.84; p = 0.032; COX-2 +8473 T>C C/C genotype: HR, 0.67; 95% CI, 0.40–1.13; p = 0.003) when compared with other genotypes [67]. In multivariable analysis, COX-2 +8473 T>C (adjusted p = 0.013) remained significantly associated with PFS, independent of skin rash toxicity and KRAS mutation. In summary, data on the predictive and prognostic value of EGFR signalling pathway polymorphisms at the present time are conflicting and need further investigation and validation. Given the retrospective design of all these studies, further larger statistically powered clinical trials are needed to evaluate the significance of these polymorphisms on anti-EGFR mAbs efficacy.

3.5. MicroRNAs polymorphisms MicroRNAs (miRNAs) are small, noncoding RNAs that have revealed a new level of gene regulation in the cell. After being processed by Drosha and Dicer RNase III endonucleases, mature miRNAs can regulate gene expression by degrading and/or suppressing the translation of target messenger mRNA (mRNA) by base pairing in the 3 -untranslated region (UTR) of mRNA [92]. Exerting an effect as either tumour suppressors or oncogenes, miRNAs regulate several genes that have important roles in cancer [93]. Variable levels of miRNA in vivo may affect apoptosis, angiogenesis and specific molecular pathways such as the RAS cascade [94,95]. Recently, miRNA polymorphisms were discovered and are becoming increasingly important on the fast growing field of the personalized medicine. MiRNA polymorphisms could present at or near a miRNA-binding site of functional genes and can affect gene expression by interfering with miRNA function. They have been shown to affect drug response and have the potential to confer drug resistance [96]. The let-7 (Lethal-7) family of miRNA showed RAS regulating activity [97]. Let-7 induced RAS downregulation after binding to specific sites in the 3 -UTR of the human KRAS mRNA [98]. A functional SNP has been described and characterized in the Let-7 complementary site (LCS) in the KRAS 3 -UTR mRNA (LCS6) [99]. The LCS6 SNP (rs61764370) consists in a T-to-G base change and it was found to alter the binding capability of the mature Let-7 to the KRAS mRNA. In experimental models, this variant attenuated the Let-7 control on KRAS with oncogene overexpression [99]. In addition, as a consequence of a possible negative feedback loop, the presence of the LCS6 G variant allele was associated with Let-7 downregulation [99]. The frequency of the LCS6 G- allele in Caucasian population is estimated to be approximately 5–10% in healthy individuals, but it was found to be markedly increased up to 20% in patients with lung cancer [99]. Furthermore, this polymorphism was found to be associated with increased cancer risk in non-small-cell lung cancer patients [99] and reduced OS in squamous cell carcinomas of the head and neck [100], suggesting functional and clinical significance. Moreover, it have been demonstrated that the Let-7 miRNA can also modulate tumour sensitivity to chemotherapeutic agents. Nakajima et al. found the level of expression of Let-7g strongly related to responsiveness to S-1 treatment in 46 patients with recurrent of refractory advanced CRC [101]. The prognostic value of the LCS6 T>G variant in metastatic CRC patients treated with anti-EGFR therapy has been evaluated in two studies [10,103]. Graziano et al. analyzed the KRAS 3 -UTR LCS6 polymorphism, KRAS mutation in codons 12, 13, 61 and the BRAF V600E mutation in the tumour DNA ofpatients with irinotecan-refractory metastatic CRC who underwent salvage irinotecan-cetuximab therapy [102]. In 134 patients there were 100 carriers of the wild-type LCS6 T/T

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

genotype (75%) and 34 carriers of the LCS6 G variant allele (T/G and G/G) genotypes (25%). The distribution of carriers of the LCS6 genotypes was significantly different among carriers of KRAS and BRAF mutation. In particular, all the 13 BRAF V600E mutation carriers were LCS6 T/T WT and Gallele carriers were significantly more frequent in the KRAS mutation group than in patients with KRAS WT (p = 0.004). In the 121 patients without BRAF V600E mutation, OS and PFS times were compared between carriers of the LCS6 G-allele genotypes and carriers of the WT T/T genotype. LCS6 G-allele carriers showed worse OS (p = 0.001) and PFS (p = 0.004) than T/T genotype carriers and these data were confirmed in the multivariate model including the KRAS status. In the exploratory analysis of the 55 unresponsive patients with KRAS mutation, LCS6 G-allele carriers and T/T carriers showed adverse PFS and OS times. Median PFS was 2.5 and 3.4 months (HR = 1.78; 95% CI, 1.1–4.14) and 5.9 and 9.7 months (HR = 1.77; 95% CI, 1.02–3.8) in G-allele carriers and T/T carriers, respectively. The G-allele could also have a role in patients with KRAS/BRAF WT status and its presence could represent an unfavourable predictive marker to the anti-EGFR therapy. However, there were 12 G-allele carriers only among the 63 patients with KRAS/BRAF WT tumours. With this limitation, the log-rank comparison of PFS and OS times showed a trend for unfavourable outcomes in G allele carriers, but the difference was not significant. G allele and T/T carriers showed median OS of 9 and 14.2 months (HR = 1.19; 95% CI, 0.98–5.93) and median PFS of 3.7 and 5.3 months (HR = 1.45; 95% CI, 0.73–3.35), respectively. In general, the worse survival times in G-allele carriers than in WT T/T genotype carriers would suggest that preserved Let7 function may exert some control on the RAS pathway with additive effect to the anti-EGF blockade. If the anti-EGFR blockade does not work, because of the presence of an activating KRAS mutation, the downstream control of Let-7 may still ensure some KRAS downregulation, provided that there is preserved binding between the miRNA and the mRNA of the target gene. In a second study, Zhang et al. have studied the relationships between the KRAS Let-7 LCS6 T>G SNP and outcome in 130 metastatic CRC patients who were refractory to fluoropyrimidines, irinotecan, and oxaliplatin, and treated with cetuximab as monotherapy in the phase II study IMCL0144 [103]. Analysis of KRAS let-7 LCS6 polymorphism was available in 111 patients and 13 of them (12%) were not assessable for tumour response. In 98 patients assessable for tumour response, 67 patients had WT KRAS and 31 patients had mutant KRAS. Fifty-five (82%) of the 67 KRAS WT patients had the KRAS Let-7 LCS6 TT genotype, whereas there are 12 KRAS WT patients harbouring at least one Let-7 LCS6 variant G allele (TG or GG). These 12 patients had a 42% ORR compared with KRAS WT patients with WT TT genotype who had a 9% ORR (p = 0.02). None of the 31 KRAS mutant patients had an objective response to cetuximab regardless of KRAS Let-7 LCS6 polymorphism. KRAS WT patients with TG/GG genotypes had a trend of longer median PFS (3.9 vs. 1.3 months; p = 0.25) and OS

53

(10.7 vs. 6.4 months; p = 0.33) compared with those with T/T genotypes (p = 0.02), but did not reach statistical significance due to the small sample size. Apparently, these two studies [102,103] report conflicting results, but to reach a firm conclusion, it would be of interest to know the percentage of mutated 61 KRAS and the BRAF status in the 55 patients with LCS T/T genotype analyzed by Zhang et al. [103]. In fact, if it is considered that about 18% of WT 12–13 KRAS patients could carry a mutated codon 61 KRAS or BRAF (8% of 61 KRAS plus 10% of BRAF, therefore approximately 9–10/55 cases), the non-responders to cetuximab among the LCS6 T/T patients could be attributed at least in part to the presence of KRAS or BRAF mutations. The same considerations could apply to PFS and OS analyses. In addition, in the abstract presentation of the Zhang et al. work at the 2009 American Society of Clinical Oncology (ASCO) meeting, authors reported an analysis of the LSC6 variant in patients enrolled in the IMCL-0144 trial and in the EPIC trial independent of KRAS mutation status in the tumour [104]. In this report, T/T carriers with mutant KRAS who were treated with irinotecan/cetuximab in the EPIC trial had significantly better PFS of 12 weeks (95% CI, 6.4–18) compared with those harbouring a G-allele with median PFS of 6.4 weeks (95% CI, 5.7–7) (p = 0.037). There was no association between this polymorphism and clinical outcome in patients with WT KRAS enrolled in the EPIC. In a multivariate analysis the polymorphism remained independently associated with PFS in EPIC study. Notably, this finding parallels results of Graziano and colleagues [105]. In summary, these studies support the role of Let-7 LCS6 polymorphisms as a predictive marker of cetuximab efficacy in metastatic CRC patients. However, data were derived retrospectively and involve a relatively small number of patients, and therefore should be considered hypothesis generating and subject to confirmation in prospective and randomized controlled studies.

4. KIRSTEN-RAS (KRAS) status 4.1. Introduction KRAS, a member of the rat sarcoma virus (ras) gene family of oncogenes (including KRAS, HRAS, and NRAS), is located on the short arm of chromosome 12. It is a protooncogene encoding a small 21 kD guanosine diphosphate (GDP)/guanosine triphosphate (GTP) binding protein RAS that acts as a self-inactivating intracellular signal transducer [106]. Following Grb2/SOS mediated activation, GTP-bound KRAS recruits the serine protein BRAF, thus starting a cytoplasmic phosphorylation cascade leading to the activation of transcription factors [CREB, SRF, Fos, nuclear factor ␬B (NF-␬B)] playing important roles in cell growth, differentiation, and survival. The oncogene PIK3CA encodes the p110 subunit of PI3K, which can be also activated via interaction with RAS proteins, as mentioned before [2,107].

54

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

The KRAS WT protein is transiently activated during tightly regulated signal transduction events. The binding of mAbs to EGFR normally induces receptor internalization, causing a direct inhibition of TK activity and the blockage of downstream RAS/RAF/MAPK signalling. However, activating KRAS mutations result in a constitutively active GTP-bound protein which consequently renders the downstream pathway permanently “switched on” irrespective of the activation status of upstream receptors including EGFR. In such an instance, the binding of an anti-EGFR mAb to EGFR and theinhibition of ligand-mediated receptor activation will fail to elicitany pathway suppressive effects. Therefore, this constitutive pathway activation leads tounregulated proliferation, impaired differentiation, and resistance to anti-EGFR therapies [2,107]. 4.2. KRAS mutations in colorectal cancer KRAS is the most commonly mutated gene in the RAS/RAF/MAPK pathway, with approximately 35% to 45% of metastatic CRC patients harbouring KRAS mutations [7,10,26]. Mutations in KRAS or BRAF and PIK3CA may coexist within the same tumour [19,108], but KRAS and BRAF mutations appear to be mutually exclusive [14,17,109]. Up to 90% of activating mutations of the KRAS gene are detected in codons 12 (82–87%) and 13 (13–18%), but less frequently in codons 61, 63 and 146. These are point mutations and are generally observed as somatic mutations. The most common types of KRAS mutations in CRC are G-to-A transitions and G-to-T transversions. The codons 12 and 13 code for two adjacent glycine residues located in the proximity of the catalytic site of RAS [110]. Different KRAS 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 KRAS mutations may be responsible for different biological alterations [110]. Codon 12 mutations of the KRAS gene were associated with a mucinous phenothype of CRC. By contrast, CRCs associated with codon 13 mutations were rather non-mucinous, but were characterized as more aggressive tumours with a greater metastatic potential [111]. KRAS mutation is thought to be an early event in tumourigenesis [112]. Several series have demonstrated a KRAS mutational status concordance ≥95% between primary tumours and paired metastases [40,113–118]. These data have recently been confirmed in a large and homogenous Dutch study, which found a discordance between primary tumours and corresponding liver metastases in only 11 of 305 cases (3.6%) [119], thus suggesting that KRAS mutations analyses from primary tumours can be used with a great reliability for treatment decisions in metastatic setting. However, high rate of heterogeneity of KRAS mutation status (up to 30%) was observed between primary tumours and lymph node metastases in a limited number of studies [116,117,120]. In addition, intratumoural heterogeneity of KRAS mutation

status has also been detected in up to 8% of samples when tumour centers and invasion fronts of primary CRC were compared [116]. Therefore, the site most suitable and reliable for diagnostic mutation analysis has been defined as the tumour center. Interestingly, in a retrospective Spanish report of 230 CRC patients, a higher percentage of KRAS mutations was detected in primary tumours of patients with lung metastases than in those with liver metastases (57% vs. 35%, p = 0.006), suggesting a role for KRAS mutations in the propensity of primary CRC to metastatize to the lung [121]. Despite a general consensus favouring the introduction of KRAS testing in clinical practice as a powerful means to select patients before drug administration, the choice of the most appropriate method for KRAS mutation analysis remains a complex challenge, and it is still not known what level of test sensitivity is required in order to provide useful and predictive information in clinical practice [122–125]. The hotspots and thus the most frequent mutations – given in the recommended genetic nomenclature [122] – 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). Currently, the most widely applied methods for assessing KRAS gene status is direct dideoxy DNA sequencing and the PCR-based assays, which have a relatively low sensitivity because mutant alleles must be present in at least 20% and 10% of cells, respectively, to be reproducibly detected [123,126]. More sensitive methods are available for KRAS analysis. Some are laboratory-made techniques, such as mutant enriched-PCR (ME-PCR), and others are commercial test for diagnostic use, such as matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) technology or amplification refractory mutation system (ARMS). Several studies have compared different methodologies of KRAS analysis but without showing a correlation with the clinical response to anti-EGFR agents in metastatic CRC patients [127,128]. Mollinari et al. retrospectively evaluated objective tumour responses in metastatic CRC patients treated with cetuximab- or panitumumab-based regimen [125]. KRAS codons 12 and 13 mutations were examined by direct sequencing, MALDI-TOF mass spectrometry (MALDI-TOF MS), ME-PCR, and engineered ME-PCR (eMR-PCR), which have a sensitivity of 20%, 10%, 0.1% and 0.1%, respectively. In addition, KRAS codon 61 mutations, BRAF and PIK3CA mutations by direct sequencing and PTEN expression by IHC. Direct sequencing revealed mutations in codons 12 and 13 of KRAS in 43 of the 111 patients considered (39%) and BRAF mutations in 9/111 (8%). Using highly sensitive KRAS analysis methods, additional KRAS mutations were detected in up to 13/68 (19%) patients after directed sequencing had shown them to be WT in codons 12 and 13 of KRAS. With the exception of 2 patients with KRAS G13D mutations and 1 patient with the rare KRAS G60D mutation in exon 3, the patients with KRAS, BRAF, or PIK3CA mutations, as detected by direct sequencing, did

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

not respond to cetuximab- or panitumumab-based therapy. All cases with KRAS mutations detected by MALDI-TOF MS and ME-PCR occurred in non-responders patients, therefore increasing the rate of identified non-responders from 45% (based on the detection of KRAS mutations by direct sequencing alone) to 60% (by adding analysis of KRAS exon 2 by ME-PCR. By adding the evaluation of BRAF, PIK3CA, and PTEN, up to 87% of non-responders patients were identified. The identification of a greater number of KRAS-mutated cases by ME-PCR in primary tumour specimens may be explained by the heterogeneity of the tumours and suggests that clones bearing KRAS mutations might be undetectable when directing sequencing is used [129]. Cells from these clones may display an increased capability to disseminate into peripheral organs where they could predominate (distant metastases). These results seem to support this hypothesis; the 2 cases in which KRAS mutations were identified only by means of M-PCR in the primary tumour and for which a metastatic lesion was available showed the same KRAS mutation in the metastatic specimens simply by using direct sequencing. Santini et al. have assessed KRAS mutacional analysis by pyrosequencing, a new powerful sequencing methodology for SNPs/mutation analysis [124]. The pyrosequencingbased assay detects KRAS mutations in codons 12 and 13 and it is more sensitive than traditional sequencing methods or real-time PCR, being able to detect mutation rate represented in fewer than 20% of analyzed samples [123]. Authors observed a higher percentage, attested on more than 50% of KRAS mutated (codon 12/13) patients, than that usually reported (40–45%). To explore this difference, archival tissue samples from patients previously resulted WT for KRAS codon 12/13 by real-time PCR were reanalyzed by performing real-time PCR and pyrosequencing methods at the same time. Of 29 patients, three (10.3%) were identified as KRAS mutant in codon 12G for 12D mutation by pyrosequencing whereas all of them were reconfirmed KRAS WT by real-time PCR. Moreover, the percentage of mutation rate at pyrosequencing in these three patients resulted of 18.6%, 20% and 18.2%, respectively, which are under the general detection limit of real-time PCR. Unlike that observed in the study performed by Molinari et al. [125], all of these mutated patients showed PR during cetuximab-based CT. Authors suggest that the identification of a very low frequency of KRAS mutations could not impair anti-EGFR therapies activity, as demonstrated by the three mutated responders patients. In conclusion, all these data point the usefulness of increasing the sensitivity of methods to detect mutations in KRAS for enhancing predictions of resistance to cetuximab or panitumumab in metastatic CRC. Therefore, there is an urgent need for the establishment of widely accepted guidelines for KRAS testing, focused on defining the sensitivity threshold that is required for the accurate identification of non-responders patients. In addition, prospective studies are needed to confirm these results in larger number of patients.

55

Finally, recent technological developments have enabled us to develop a highly sensitive method for detection of cellfree DNA (cfDNA) in the peripheral circulation and test this DNA for tumour specific mutations [130,131]. A recent study have investigated the levels of circulating cfDNA in plasma from 108 patients with metastatic CRC in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA KRAS or BRAF mutations in plasma [132]. Authors showed that KRAS analysis in plasma is a feasible alternative to tissue analysis. The majority of KRAS mutations detected in tumours were also found in plasma (32 of 41 [78%]) and there is a clear difference in the outcome between patients with high plasma mutant KRAS (pmKRAS) levels and those with low concentrations. Patients with pmKRAS levels higher than 75% had a DCR of 0% compared with 42% in patients with lower pmKRAS (p = 0.048). In addition, plasma cfDNA and pmKRAS levels were strongly correlated (r = 0.85; p < 10−4 ) and the plasma concentration of baseline cfDNA levels has prognostic value. In the total cohort, the DCR was 77% in patients with low cfDNA (<25% quartile) and 30% in patients with high cfDNA (>75% quartile) (p = 0.009). To summarize, the quantification of cfDNA and KRAS mutations in the peripheral circulation has potential value as a clinical tool for more individualized pretreatment testing and could improve selection of therapy. Further studies along that line seem justified. 4.3. KRAS mutation status as a predictor of resistance to anti-EGFR mAbs KRAS mutations have emerged as a major predictor or resistance to anti-EGFR mAbs. This fact has been consistently shown in small single-arm data sets [14,32,108,133–136] but also in retrospective analysis of large phase III studies and some prospective trials of patients receiving first [8,12,137–139] and subsequent lines of treatment [13–16]. In these studies, patients with metastatic CRC harbouring KRAS mutations did not extract any benefit of treatment with cetuximab of panitumumab either alone or in combination with standard CT. This discovery led to the first practical implementation of personalized medicine in metastatic CRC. 4.3.1. Effect of KRAS mutation on response to anti-EGFR therapy in first-line treatment Several randomized phase II and III trials have been performed to compare CT with standard oxaliplatin and irinotecan-based regimens with or without the addition of cetuximab or panitumumab in the first-line setting [8,9,12,25,26,137–141]. Post hoc analysis are now available which evaluate the influence of KRAS mutation status on treatment efficacy (Table 2). The Cetuximab Combined with Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer (CRYSTAL) study demonstrated that FOLFIRI-cetuximab treatment reduced

56

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

Table 2 KRAS mutation status as a predictive biomarker of response and survival with anti-EGFR monoclonal antibody-based first-line chemotherapy in advanced CRC. Study/year

PFS, mo (HR mAb × no mAb)

OS, mo (HR mAb × no mAb)

39.7% 57.2% 36.1% 31.3%

8.4 9.9 [0.69 (0.55–0.66)] 7.7 7.4 [1.17 (0.88–1.54)]

20 23.5 [0.79 (0.67–0.94)] 16.7 16.2 [1.035 (0.83–1.28)]

FOLFOX (97) FOLFOX + cetuximab (82) FOLFOX (59) FOLFOX + cetuximab (77)

34% 58% 52% 34%

7.2 8.3 [0.567 (0.37–0.85)] 8.6 5.5 [1.72 (1.104–2.67)]

18.5 22.8 [0.85 (0.59–1.21)] 17.5 13.4 [1.29 (0.87–1.90)]

Wild type (656, 60%) Mutated (490, 40%)

FOLFOX (331) FOLFOX + panitumumab (325) FOLFOX (219) FOLFOX + panitumumab (221)

48% 55% 40% 40%

8 9.6 [0.8 (0.7–0.9)] 8.8 7.3 [1.3 (1.04–1.3)]

19.7 23.9 [0.83 (0.67–1.02)] 19.3 15.5 [1.24 (0.98–1.57)]

Wild type (303, 60.84%)

FLOX FLOX cont + cetuximab FLOX int + cetuximab FLOX FLOX cont + cetuximab FLOX int + cetuximab

47% 46% 51% 40% 49% 42%

8.7 7.9 [1.07 (0.79–1.45)] 7.5 7.8 9.2 [0.71 (0.5–1.03)] 7.2

22 20.1 [1.14 (0.80–1.61)] 21.4 [1.08 (0.77–1.52)] 20.4 21.1 [1.03 (0.68–1.57)] 20.5 [1.04 (0.68–1.60)]

FOLFOX (367) FOLFOX/XELOX + cetuximab (362) FOLFOX (268) FOLFOX/XELOX + cetuximab (297) XELOX + BEV (156) XELOX + BEV + cetuximab (158) XELOX + BEV (108) XELOX + BEV + cetuximab (98) Oxaliplatin-based + BEV (203) Oxaliplatinbased + BEV + panitumumab (201) Oxaliplatin-based + BEV (125) Oxaliplatinbased + BEV + panitumumab (135) Irinotecan-based + BEV (58) Irinotecanbased + BEV + panitumumab (57) Irinotecan-based + BEV (39) Irinotecanbased + BEV + panitumumab (47)

57% 64%

8.6 8.6 [0.96 (0.80–1.1)]

17.9 17 [1.04 (0.87–1.23)]

46% 43%

6.6 6.3 [1.08 (0.95–1.2)]

14.8 13.6 [0.98 (0.81–1.17)]

KRAS status (no. patients, %)

Treatment arms (no. patients)

CRYSTAL (2011) [137]

Wild type (666, 62.65%) Mutated (397, 37.34%)

FOLFIRI (350) FOLFIRI + cetuximab (316) FOLFIRI (183) FOLFIRI + cetuximab (214)

OPUS (2011) [138]

Wild type (179, 57%) Mutated (136, 43%)

PRIME (2010) [12]

NORDIC VII (2012) [140]

MRC COIN (2011) [25]

Mutated (195, 39.15%) Wild type (751, 57%) Mutated (565, 43%)

CAIRO-2 (2009) [26]

Wild type (314, 41.5%) Mutated (206, 39.6%) Wild type (404, 61%)

PACCE (2009) [141]

Mutated (260, 39%)

Wild type (115, 57%)

Mutated (86, 43%)

RR

50% 61.4%

10.6 10.5 (NR, p = 0.3)

21.8 22.4 (NR, p = NS)

59.2% 45.9%

12.5 8.1 (NR, p = 0.003)

17.2 24.9 (NR, p = 0.02)

56% 50%

11.5 9.8 [1.36 (1.04–1.77)]

24.5 20.7 [1.89 (1.30–2.75)]

44% 47%

11 10.4 [1.25 (0.91–1.71)]

19.3 19.3 [1.02 (0.67–154)]

48% 54%

38% 30%

12.5 10 [1.5 (0.82–2.76)]

11.9 8.3 [1.19 (0.65–2.21)]

19.8 NE [1.28 (0.50–3.25)]

20.5 17.8 [2.14 (0.82–5.59)]

FOLFOX = infusional 5-FU, leucovorin and oxaliplatin; FOLFIRI = infusional 5-FU, leucovorin and irinotecan; XELOX = oxaliplatin and capecitabine; FLOX = bolus 5-FU, leucovorin and oxaliplatin; BEV = bevacizumab; RR = response rate; PFS = progression-free survival; OS = overall survival; NR = not reported; NE = not estimated; cont = continuous; int = intermittent; mAb = monoclonal antibody; HR = hazard ratio; mo = months.

the risk of progression by 15% as compared with FOLFIRI alone (median PFS, 8.9 vs. 8 months with FOLFIRIcetuximab and FOLFIRI, respectively; HR = 0.85; 95% CI, 0.72–0.99; p = 0.048) [8]. There was no significant difference in OS between the two treatment groups (median OS, 19.9 months in the FOLFIRI-cetuximab and 18.6 months in the FOLFIRI group; HR, 0.93; 95% CI, 0.81–1.07;

p = 0.31). In an updated analysis according to KRAS and BRAF mutation status [137], the rate of patients analyzed for tumour KRAS status was increased from 45% to 89%, with mutations detected in 397 of 1063 patients (37%). This retrospective analysis revealed that only patients with WT KRAS tumours benefited from the addition of cetuximab to FOLFIRI, showing higher RR (57.3% vs. 39.7%; odds

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

ratio (OR), 2.069; p < 0.0001), longer PFS (median, 9.9 vs. 8.4 months; HR, 0.696; 95% CI, 0.558–0.867; p = 0.012) and longer OS (median, 23.5 vs. 20.0 months; HR, 0.796; 95% CI, 0.67–0.94; p = 0.0093). In patients whose tumours carried KRAS mutations, there was no evidence of benefit associated with the addition of cetuximab to FOLFIRI. The Oxaliplatin and Cetuximab in First-Line Treatment of Metastatic Colorectal Cancer (OPUS) trial also showed that the benefit from the addition of cetuximab to FOLFOX4 was restricted to the WT KRAS population [9]. Of 337 patients included, 233 could be evaluated for their KRAS status and a KRAS mutation was found in 42%. In KRAS WT patients, the addition of cetuximab to FOLFOX induced a significant increase in RR (61% vs. 37%; p = 0.011) and PFS (7.7 vs. 7.2 months; HR = 0.57; p = 0.0163) without OS benefit. By contrast, a negative impact on treatment efficacy was noted when cetuximab was added to CT in patients with KRAS mutant metastatic CRC with regard to RR (33 vs.49%; p = 0.106) and PFS (8.6 vs. 5.5 months; HR = 1.83; p = 0.0192) [138]. A meta-analysis of pooled data from the OPUS and CRYSTAL trials showed that patients with KRAS WT tumours (n = 845) had a significant increase in RR (57% vs. 38%; OR, 2.16; p < 0.0001), PFS (9.6 vs. 7.6 months, HR, 0.66; p < 0.0001) and OS (23.5 vs. 19.5 months; HR, 0.81; p = 0.006) with the addition of cetuximab to first-line CT [139]. The Panitumumab Randomized Trial in Combination With Chemotherapy for Metastatic Colorectal Cancer to Determine Efficacy (PRIME) was the first phase III trial to evaluate the addition of panitumumab to FOLFOX4 for the initial treatment of patients with KRAS WT metastatic CRC [12]. Importantly, the results were prospectively analyzed by tumour KRAS status. The study achieved its primary end point by demonstrating a significant longer PFS when panitumumab is added to CT for patients with KRAS WT tumours (9.6 vs. 8 months, respectively; HR, 0.80; 95% CI, 0.66–0.97; p = 0.02). A non-significant increase in OS was also observed for panitumumab-FOLFOX4 versus FOLFOX4 (23.9 vs. 19.7 months, respectively; HR, 0.83; 95% CI, 0.67–1.02; p = 0.072). In the mutantKRASstratum, PFS was significantly reduced in the panitumumab-FOLFOX4 arm versus theFOLFOX4 arm (7.3 vs. 8.8, respectively; HR, 1.29; 95% CI, 1.04–1.62; p = 0.02) and median OS was 15.5 months vs.19.3 months, respectively (HR, 1.24; 95% CI, 0.98–1.57; p = 0.068). In contrast to these results, other phase III trials found that the KRAS mutation status was not predictive of benefit when cetuximab was combined with first-line CT [25,140]. In the NORDIC VII trial, cetuximab combined with the continuous or intermittent FLOX regimen (bolus 5-fluorouracil [5-FU] plus oxaliplatin) did not significantly improve efficacy compared with FLOX alone [140]. Moreover, KRAS mutation status was not predictive for cetuximab effect. The Continuous Chemotherapy Plus Cetuximab or Intermittent Chemotherapy With Oxaliplatin and a Fluoropyrimidine in Metastatic Colorectal Cancer (COIN) trial has neither shown a benefit with the addition of cetuximab to

57

oxaliplatin-based CT [25]. In this study, 1630 patients were randomly assigned to oxaliplatin and fluoropyrimidine CT (n = 815) or the same combination plus cetuximab (n = 815), and the choice of fluoropyrimidine therapy (capecitabine or infused 5-FU plus leucovorin) was decided before randomization. The prognostic value of KRAS status was analyzed prospectively. Although RR increased from 57% with CT alone to 64% with the addition of cetuximab (OR, 1.35; 95% CI, 1–1.82; p = 0.049) in patients with KRAS WT tumours, there was no evidence of a difference in OS between treatment groups (median survival, 17.9 months in the control group and 17 months in the cetuximab group; HR, 1.04; 95% CI, 0.87–1.23; p = 0.67). Improved PFS with cetuximab was seen in patients treated with 5-FU-based therapy (HR, 0.72; 95% CI, 0.53–0.98; p = 0.037), but not in those treated with capecitabine-based therapy (HR, 1.02; 95% CI, 0.82–1.26; p = 0.88; p = 0.10 for interaction). Additionally, patients with no or one metastatic site had improved PFS with cetuximab (HR, 0.73; 95% CI, 0.55–0.97; p = 0.030), whereas those with two or more metastatic sites did not (HR, 1.07; 95% CI, 0.86–1.33; p = 0.56; p = 0.036 for interaction). When an anti-EGFR therapy was combined with bevacizumab-based first-line CT in advanced CRC, no added benefit was observed, even in patients with WT KRAS tumours [26,141]. In the CAIRO-2 (CApecitabine, IRinotecan, and Oxaliplatin trial) study [26], the addition of cetuximab to XELOX plus bevacizumab regimen had no effect on RR (50 vs. 61.4%; p = 0.06) or PFS among those with WT KRAS tumours (median, 10.5 vs. 10.6; p = 0.3). However, this combination had a marked detrimental effect among patients with tumours carrying mutated KRAS: cetuximab-treated patients had significantly shorter PFS (8.1 vs. 12.5 months; p = 0.003) and OS (17.2 vs. 24.9 months; p = 0.03). Similarly, the PACCE (Panitumumab Advanced Colorectal Cancer Evaluation) trial [141] tested the addition of panitumumab to standard first-line oxaliplatin (n = 823) or irinotecan-based therapy (n = 230) plus bevacizumab. Panitumumab was discontinued after a planned interim analysis of 812 oxaliplatin patients showed worse efficacy in this arm. In the final analysis, median PFS was 10 and 11.4 months (HR, 1.27; 95% CI, 1.06–1.52) and median OS was 19.4 months and 24.5 months (HR, 1.43; 95% CI, 1.11–1.83) for the panitumumab and control arms, respectively. KRAS analyses showed adverse outcomes for the panitumumab arm in both WT and mutant groups. These results suggest a detrimental effect with the addition of antiangiogenic agents to anti-EGFR therapies in the first-line setting of advanced CRC. It also raises the possibility of a negative pharmacokinetic and/or pharmacodynamic interaction between these two types of targeted agents in combination with CT, even in the setting when anti-EGFR agents could effectively inhibit EGFR signalling. Another possible explanation that may have contributed to this finding is the increased toxicity of the combination, thus inducing frequent delays and reduction of dose-intensity.

58

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

4.3.2. Effect of KRAS mutation on response to anti-EGFR therapy in pretreated patients The relation of KRAS mutation status and response to antiEGFR directed therapy has also been intensively investigated in pretreated patients (Table 3) [13–16,109,133–135,142]. Some of the studies were performed as case series or post hoc analyses of clinical trials [14,109,133–135,142]. Lièvre et al. first reported the link between KRAS mutations and lack of response to EGFR-targeted therapy [14]. They analyzed 30 patients predominantly treated with cetuximab plus irinotecan after previous exposure to CT. KRAS mutations were observed in 13 of the 30 patients enrolled (43%) and were closely associated with response to treatment. None of the responders (0/11) presented KRAS mutations, whereas they were found in 68.4% (13/19) of non-responders (p = 0.0003). The OS of KRAS WT patients was significantly higher compared with those with mutated KRAS (median OS: 16.3 vs. 6.9 months, respectively, p = 0.016). In a more recent publication, the same group presented an analysis of 89 metastatic CRC patients treated with cetuximab after treatment failure with irinotecan-based CT [133]. A KRAS mutation was present in 27% of patients and was associated with resistance to cetuximab (0% vs. 40% of responders among the 24 mutated and 65 nonmutated patients, respectively; p < 0.001) and poorer survival (median PFS: 10.1 vs. 31.4 weeks in patients without mutation; p = 0.0001; median OS: 10.1 vs. 14.3 months in patients without mutation; p = 0.026). Benvenuti et al. reported 48 CRC patients receiving cetuximab- or panitumumab-based regimens for first-line to fourth-line treatment [109]. They observed a KRAS mutation rate of 33% and a BRAF mutation rate of 12.5%. The presence of KRAS- and BRAF mutations was negatively associated with the achievement of a response (p = 0.005) and also correlated with a shorter time to tumour progression (p = 0.0259). Di Fiore et al. studied 59 patients and reported a KRAS mutation rate of 37%. No KRAS mutations were found in 12 patients responding to cetuximab-based therapy [134]. In addition, KRAS mutations were associated with disease progression (p = 0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs. 5.5 months, p = 0.015). One of the largest analysis was performed by de Roock et al. [135]. A total of 113 patients with irinotecan refractory metastatic CRC treated with cetuximab-based therapy were evaluated and mutations of the KRAS gene were observed in 40.7% of patients. The overall RR was 41% in KRAS WT patients vs. 0% in KRAS mutant ones. KRAS mutant patients had a trend towards a shorter PFS (3 months vs. 6 months) and a significantly longer OS (6.8 months vs. 10.8 months; p = 0.02). In a logistic regression analysis using age, sex, KRAS mutation status, skin toxicity, number of previous CT lines and treatment regimens covariates, KRAS status and skin toxicity were identified as the predictors of ORR and OS. In a retrospective European consortium analysis, de Roock et al. confirmed the negative effect of KRAS mutations on outcome after cetuximab in 649 chemorefractory patients

treated with CT plus cetuximab [142]. The 40% of the tumours harboured a KRAS mutation. KRAS mutants did not derive benefit compared with WT, with a RR of 6.7% vs.35.8% (OR, 0.13; 95% CI, 0.07–0.22; p < 0.0001), a median PFS of 12 vs. 24 weeks (HR, 1.98; 95% CI,1.66–2.36; p < 0.0001) and a median OS of 32 vs. 50 weeks (HR, 1.75; 95% CI,1.47–2.09; p < 0.0001). Phase III trials have shown similar results [13,15,16]. The biomarker analysis of the pivotal phase III trial of panitumumab monotherapy in the relapsed or refractory setting was the first large study (n = 463 patients) to confirm the negative predictive value of KRAS mutations [16]. The relative effect of panitumumab vs. BSC on PFS was significantly greater among patients with KRAS WT (median PFS, 12.3 weeks for panitumumab vs. 7.3 weeks for BSC; HR, 0.45; 95% CI, 0.34–0.59; p < 0.0001) compared with patients with mutant KRAS, in whom no panitumumab benefit was observed (median PFS, 7.4 weeks for panitumumab vs. 7.3 weeks for BSC; HR, 0.99; 95% CI, 0.73–1.36). RR to panitumumab was 17% and 0% for the WT and mutant group, respectively. No statistically significant OS difference was observed between treatment arms among all patients (HR, 0.97; 95% CI, 0.79–1.18) or in either of the KRASgroups; theHR for OS was 1.02 (95% CI, 0.75–1.39) and 0.99 (95% CI, 0.75–1.29) for the mutant and WT KRAS groups, respectively. OS was longer overall in the WT group than in the mutant group adjustingfor stratification factors and randomized treatment (HR, 0.67; 95% CI, 0.55–0.82; both arms combined). Unlike previous single arm uncontrolled studies [14,109,133–135], the presence of a control (BSC) arm in this study enabled the evaluation of the relative effect of panitumumab according to KRAS mutational status regardless of the potential prognostic influences of KRAS mutations of clinical outcomes. Karapetis et al. confirmed the predictive value of KRAS mutations in the NCIC-CTG trial [15], a cetuximab monotherapy study conducted in a similar population of relapsed or refractory patients. Both PFS and OS were similar for the cetuximab and control groups in those patients with tumours carrying KRAS mutations. However, in the subgroup whose tumours carried WT KRAS, cetuximab was associated with statistically significant longer PFS (3.7 vs. 1.9 months; HR, 0.40; 95% CI, 0.30–0.54; p < 0.001) and OS (9.5 vs. 4.8 months; HR, 0.55; 95% CI, 0.41–0.74; p < 0.001) than BSC alone. It should be noted than unlike the pivotal panitumumab phase III study [16], the design of this trial did not allow patients from the control group who had disease progression to cross over to mAb treatment. Another phase III study randomly assigned 1186 patients to FOLFIRI plus panitumumab vs. FOLFIRI alone every 2 weeks [13]. PFS and OS were prospectively analyzed by KRAS status. In the WT KRAS subpopulation, a significant improvement in RR (35% vs. 10%) and PFS was documented when panitumumab was added to CT (median PFS, 5.9 vs. 3.9 months in the FOLFIRI-panitumumab and FOLFIRI arms, respectively; HR, 0.73; 95% CI, 0.59–0.90; p = 0.004). A non-significant trend towards increased OS was

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

59

Table 3 KRAS mutation status as a predictive marker of response and survival with anti-EGFR monoclonal antibody-based chemotherapy in pretreated patients. Study/year

KRAS status (no. patients, %)

Treatment arms (no. patients)

RR

PFS, mo (HR mAb × no mAb)

OS, mo (HR mAb × no mAb)

43%

NA

16.3

0%

NA

6.9 (p = 0.0016)

40%

7.8

14.3

2.5 (p = 0.0001)

10.1 (p = 0.026)

NA

NA

NA (p = 0.0443)

NA

5.5

NA

3 (p = 0.015)

NA

6

10.75

0%

3 (p = 0.074)

6.82 (p = 0.02)

6.7%

6

12.5

Lièvre (2006) [14]

Wild type (17, 56.6%) Mutated (13, 43.3%)

Lièvre (2008) [133]

Wild type (65, 63%) Mutated (24, 27%)

Cetuximab ± CT

Benvenuti (2007) [109]

Wild type (32, 66.6%) Mutated (16, 33.3%)

Cetuximab ± CT or panitumumab

Di Fiore (2007) [134]

Wild type (37, 62.8%) Mutated (22, 37.2%)

Cetuximab ± CT

De Roock (2008) [135]

Wild type (66, 61.1%) Mutated (42, 38.9%)

Cetuximab ± CT

De Roock (2010) [142]

Wild type (448, 60%) Mutated (299, 40%)

Cetuximab ± CT or panitumumab

35.8%

3 (p < 0.001)

8 (p < 0.001)

NCI CTG 017 (2008) [15]

Wild type (230, 58.37%) Mutated (164, 41.62%)

Cetuximab (117) BSC (113) Cetuximab (81) BSC (83)

12.8% 0% 1.2% 0%

3.1 [0.45 (0.34–0.59)] 1.8 1.8 [0.99 (0.73–1.36)] 1.8

8.1 [1.2 (0.75–1.39)] 7.6 4.9 [0.99 (0.75–1.29)] 4.4

Panitumumab Pivotal (2008) [16]

Wild type (243, 56.9%) Mutated (184, 43.4%)

Panitumumab (124) BSC (119) Panitumumab (84) BSC (100)

17% 0% 0% 0%

3.7 [0.4 (0.3–0.54)] 1.9 1.8 [0.99 (0.73–1.35)] 1.8

3.7 [0.4 (0.3–0.54)] 1.9 1.8 [0.99 (0.73–1.35)] 1.8

Wild type (597, 55%)

FOLFIRI (294) FOLFIRI + panitumumab (303) FOLFIRI (248) FOLFIRI + panitumumab (238)

10% 35%

3.9 [0.73 (0.59–0.90)] 5.9

4.9 [0.85 (0.68–1.06)] 5

14% 13%

12.5 [0.85 (0.70–1.04)] 14.5

11.1 [0.94 (0.76–1.15)] 11.8

Peeters (2010) [13]

Mutated (486, 45%)

Cetuximab ± CT

0% 31.25% 6.25% 32.4% 0% 40.9%

CT = chemotherapy; FOLFIRI = infusional 5-FU, leucovorin and irinotecan; BSC = best supportive care; RR = response rate; PFS = progression-free survival; OS = overall survival; NA = not available; HR = hazard ratio; mAb = monoclonal antibody; mo = months.

also observed (median OS, 15.5 vs. 12.5 months, respectively; HR, 0.85; 95% CI, 0.70–1.04; p = 0.12). There was no difference in efficacy in patients with mutant KRAS. Given the results of these clinical trials, the guidelines of the ASCO, the National Comprehensive Cancer Network (NCCN), and the European Society for Medical Oncology (ESMO)/World Congress on Gastrointestinal Cancer recommend that all patients with metastatic CRC who are candidates for anti-EGFR mAb therapy should have their tumour tested for KRAS mutations in an accredited laboratory and subsequently have their treatment tailored based on this information [143–145]. The European Medicines Agency (EMA) and the US Food and Drug Administration

(FDA) have also provided analogous guidelines specifying that panitumumab as well as cetuximab should not be given to patients whose tumours harbour KRAS mutations in codon 12 or 13 [146,147]. 4.4. Variation of the treatment effect by KRAS specific mutations On the basis of the above data, KRAS mutations can be considered a highly specific negative biomarker for benefit of anti-EGFR mAbs. However, it is intriguingly now coming to light that not all KRAS mutations are equal in their biological characteristics and their impact on mediating EGFR

60

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

resistance. First, the pattern of KRAS mutations is tumourtype specific. Although CRC tumours have both codon 12 and 13 mutations, KRAS-mutated pancreatic tumours (75–95%) almost invariably carry codon 12 mutations, and in non-small cell lung cancer, more than 90% of KRAS mutations are located in codon 12 [148]. Second, anecdotal reports indicate that a minority of patients (<10%) with KRAS-mutated tumours can respond to anti-EGFR therapy [32,43,109,149] and that about 15% have long-term disease stabilization [135]. In these patients, codon 13 mutations were overrepresented compared with the overall KRAS-mutated tumour population. Moreover, preclinical data demonstrated that cell lines with KRAS codon 13 glycine (G)-to-aspartate (D) mutations (p.G13D), the most frequent codon 13 mutation in CRC, exhibit weaker in vitro transforming activity than codon 12 mutations [149–151]. In addition, in the presence of cetuximab, G13D KRAS mutant cell lines displayed reduction of proliferation similar to their WT counterparts [125]. A large Japanese study (n = 5887) have suggested that clinicopathological features of tumours with p.G13D mutations were not similar to those with KRAS WT or other mutations [152]. The frequency of KRAS other mutations and p.G13D mutations was 37.6% and 7.4%, respectively. P.G13D mutations were remarkably higher in female. They occurred at a constant rate regardless of age (p = 0.53), while KRAS other mutations increased with age (p = 0.0002) and KRAS WT status decreased with age (p = 0.0001). P.G13D mutations were more common in right-sided colon cancers (9.9%) than in left-sided colon cancers (4.7%), and higher in lung metastasis (9.5%) compared to other metastatic sites. A recently published large retrospective pooled exploratory analysis of 579 patients with chemorefractory CRC treated with cetuximab also suggested that not all the KRAS mutations predict in the same degree refractoriness to anti-EGFR mAbs [151]. Among patients who received any cetuximab-based treatment, OS and PFS were significantly longer in patients with p.G13D-mutated tumours (n = 32) (median OS, 7.6 months; PFS: median, 4 months) than in patients with other KRAS-mutated tumours (OS: median, 5.7 months; median PFS, 1.9 months) in both univariate analysis (OS: HR, 0.52; 95% CI, 0.33–0.80; p = 0.003; PFS: HR, 0.54; 95% CI, 0.36–0.81; p = 0.02) and multivariate analysis adjusting for potential prognostic factors and data set (OS: HR, 0.50; 95% CI, 0.31–0.81; p = 0.005; PFS: HR, 0.51; 95% CI, 0.32–0.81; p = 0.004). No significant difference in OS or PFS was found between patients with p.G13D-mutated and KRAS WT tumours in either univariate (p = 0.98 and p = 0.97, respectively) or multivariate analysis (p = 0.79 and p = 0.66, respectively). RR was not significantly different between patients with p.G13D-mutated and other KRAS-mutated tumours (6.3% vs. 1.6%, respectively; p = 0.15), but patients with KRAS WT tumours had a significantly higher RR than patients with p.G13D-mutated tumours (26.4% vs. 6.3%, p = 0.02). There was a significant interaction between KRAS mutation status (p.G13D vs. other KRAS mutations) and OS benefit

with cetuximab treatment (HR, 0.30; 95% CI, 0.14–0.67; p = 0.003). Even more, those patients with p.G13D mutated tumours who were not treated with cetuximab-based regimen had shorter OS (median, 3.6 months; 95% CI, 2.2–4.8) than those with other KRAS-mutated tumours (median, 4.7 months; 95% CI, 3.6–6.7) (HR, 1.90; 95% CI, 1.03–3.51; p = 0.04) or WT KRAS tumours (median, 5 months; 95% CI, 4.2–5.5) (HR, 1.90; 95% CI, 1.05–3.41; p = 0.03). A benefit from the addition to cetuximab to first-line CT in patients with KRAS p.G13D mutations has also been suggested in a pooled analysis from the CRYSTAL and OPUS studies [153]. There were 689 patients in each treatment arm: 447 vs. 398 patients had KRAS WT tumours, 41 vs. 42 pts had KRAS p.G13D mutant tumours and 201 vs. 249 patients had tumours with other KRAS mutations, in the CT and CT + cetuximab arms, respectively. Heterogeneous treatment effects were seen for all endpoints across the mutation types with significant interaction by KRAS mutation status for response (p < 0.0001), PFS (p < 0.0001) and OS (p = 0.0219). Compared with patients with KRAS WT tumours those with p.G13D mutations had a similar relative treatment effect but at a much lower effect level. However, these results have not been confirmed in the COIN phase III trial [25]. The KRAS p.G13D mutation was identified in 110 patients (6.7%), but was not associated with any difference in outcome with the addition of cetuximab to oxaliplatin-based first-line CT. The association of KRAS codons 61 and 146 mutations with clinical outcomes in metastatic CRC patients treated with cetuximab has also been investigated [142,154]. Among 87 KRAS codons 12 and 13 WT patients treated with the combination of cetuximab and irinotecan, Loupakis et al. found KRAS codons 61 and 146 mutations in 7 (8%) and 1 (1%) cases, respectively [154]. None of mutated patients responded vs. 22 of 68 WT patients (p = 0.096). KRAS 61 and 146 mutations were associated with significantly shorter PFS (median PFS, 3.8 vs. 5.1 months in KRAS 61 and 146 WT; HR, 0.46; p = 0.028), whereas no significant differences were detected in OS (median OS, 9.7 vs. 14.7 months in KRAS 61 and 146 WT; HR, 0.69; p = 0.39). In the European consortium study [142], 40% of patients harboured a KRAS mutation, 2.1% (16/747) in codon 61, and 2% (15/747) in codon 146. Patients with codon 61 mutations had a lower RR than WT ones. By contrast with the previous report based on one patient [154], codon 146 mutations did not affect cetuximab efficacy. The co-occurrence of codon 146 with other KRAS mutations is an additional indication that it might not be an important oncogenic codon. All these analyses suggest that the assessment of KRAS codons 13 and 61/146 mutations might help optimizing the selection of the candidate patients to receive antiEGFR mAbs. However, they represent data on the evidence level of retrospective observational studies and prospectively generated clinical investigations are necessary before conclusions can be made about the potential benefit of cetuximab/panitumumab in subgroups of patients with KRAS mutations.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

4.5. Prognostic value of KRAS mutation status In contrast to the well-accepted KRAS mutation’s negative predictive value for anti-EGFR therapies, the reports on the prognostic value (independent of anti-EGFR mAb treatment) are inconsistent. While some studies have indicated a negative impact of KRAS mutations on survival [111,112,155,156] in patients with stages II and III CRC, others did not [157–162]. The first Kirsten Ras in Colorectal Cancer Collaborative Group study (RASCAL) meta-analysis evaluated the mutational status of the KRAS gene in 2721 CRC cases from 22 groups in 13 countries [155]. Mutations of KRAS codon 12 or codon 13 were detected in 37.7% of the tumours and 80.8% of all the specified mutations occurred in codon 12. Multivariate analysis suggested that the presence of a mutation increased the risk of recurrence (p < 0.001) and death (p = 0.004) in stages II and III CRC patients. This finding was later restricted to the p.G12V mutation (8.6% of all patients), which had a statistically significant impact on treatment failure-free survival (HR, 1.3; p = 0.004) and OS (HR, 1.29; p = 0.008). An updated of this study, the RASCAL II [112], analyzed data on a total of 3439 patients from 35 centers with a median follow-up of 55 months. The multivariate analysis demonstrated that the codon 12 (p.G12V) mutations were significantly associated with poorer OS (p = 0.008, HR, 1.29; 95% CI, 1.08–1.55). When the effects of the p.G12V mutation on the stage II and III patients were analyzed separately, authors observed that its presence in patients with stage III reduced the disease-free survival (DFS) rate significantly (HR, 1.5; 95% CI, 1.13–1.98; p = 0.0076) with a trend towards statistical significance when OS was considered (HR, 1.45; 95% CI, 1.07–1.96; p = 0.02). In contrast, the presence of the p.G12V mutation had no effect on DFS (HR, 1.12; 95% CI, 0.84–1.46; p = 0.46) and OS (HR, 1.15, 95% CI, 0.86–1.53; p = 0.36) in patients with stage II CRC. Hutchins et al. have also analyzed the prognostic role of KRAS mutation status for 1913 predominantly stage II patients randomly assigned between 5-FU and folinic acid CT and no CT in the Quick and Simple and Reliable (QUASAR) trial [156]. Risk of recurrence was significantly higher for KRAS mutant than KRAS WT tumours (28% vs. 21%; HR, 1.40; 95% CI, 1.12–1.74; p = 0.002), thus providing a useful additional risk stratificationto guide use of adjuvant CT. However, translational substudies of large adjuvant trials demonstrated KRAS mutations not to be a prognostic marker for patients treated with adjuvant 5-FU-based CT [160–162]. In the CALGB 89803 study, stage III CRC patients with KRAS mutated tumours did not experience any difference in DFS, relapse-free survival (RFS) and OS rates compared with patients with KRAS WT tumours. The 5-year DFS, RFS and OS rates (KRASmutated vs. KRAS WT patients) were 62% vs. 63% (p = 0.89), 64% vs. 66% (p = 0.84), and 75% vs. 73% (p = 0.56), respectively [160]. In the PETACC-3, KRAS mutation status was not prognostic for RFS or OS in patients with stage II and III combined or when analyzed separately [161]. Finally, the NCCTG N147 study did not find influence

61

of KRAS mutational status on DFS when cetuximab was added to FOLFOX in stage III CRC [162]. In advanced CRC, data on the KRAS prognostic value are also controversial [15,25,26,141,155,163]. In the phase III trial comparing cetuximab vs. BSC in the third line setting, clinical benefit in the WT KRAS subgroup was found in cetuximab treated patients but not in control ones, thus indicating that the benefit was not due to a prognostic effect of KRAS [15]. Two large studies evaluating the addition of cetuximab or panitumumab to CT and bevacizumab in the first-line setting neither found a prognostic value for KRAS mutational status [26,141]. However, other well-conducted large studies argued in favour of its prognostic role [25,155]. In the MRC FOCUS trial [163], KRAS mutations were found to be a poor prognostic factor for OS (HR, 1.24; 95% CI, 1.06–1.46; p = 0.008) but had minimal impact on PFS (HR, 1.14; 95% CI, 0.98–1.36; p = 0.09). Mutation status did not affect the impact of adding irinotecan or oxaliplatin to 5-FU on PFS or OS. The COIN trial also showed that carrying mutant KRAS was a strong negative prognostic factor [25]. Irrespective of treatment received, median PFS and OS were shorter in patients who had KRAS mutations than among those with KRAS WT tumours. The reasons of these discrepancies are unclear and may reflect differences in methodology and datasets and possibly tumour heterogeneity.

5. Neuroblastoma-RAS (NRAS) status NRAS is a member of the KRAS oncogene family and is mapped on chromosome 1 [106]. NRAS and KRAS are highly homologous to one another, sharing a high degree of identity over the first 90% of the protein. The extreme C terminus of the protein constitutes the hypervariable region conferring important differences in trafficking, intracellular localization and function [164]. NRAS mutation rate in CRC is 3–5% and most mutations occur in codon 61, rather than codon 12 or 13. Similarly to BRAF, mutations in NRAS and KRAS are mutually exclusive [142,164]. The presence of NRAS mutations is associated with a lack of response to cetuximab therapy [25,142,165,166] (Table 4). In the study of the European Consortium [142], 2.6% of CTrefractory metastatic CRC harboured NRAS mutations. In KRAS WT patients, NRAS mutantshad a significantly lower RR (7.7% vs. 38.1%; OR, 0.14; p = 0.013) than did WT, and a trend for shorter PFS (median PFS, 14 vs. 26 weeks; HR, 1.82; p = 0.055) and OS (median OS, 38 vs. 50 weeks; HR, 1.89; p = 0.051). The major limitation of this retrospective study was that patients were given heterogeneous cetuximab based regimens. In a randomized clinical trial comparing panitumumab to BSC in chemorefractory CRC, the presence of NRAS mutations (5% of 282 samples) was also associated with lack of response to anti-EGFR treatment [166]. Moreover, in the COIN trial [25], patients with NRAS mutations had worse PFS than those with WT tumours irrespective of the treatment received (p < 0.0088).

62

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

Table 4 NRAS and BRAF mutation status as a predictive marker of response and survival with anti-EGFR mAb-based chemotherapy in advanced CRC. Study/year

KRAS status (no. patients)

NRAS mutation (KRAS wild type) Wild type (289) De Roock (2010) Mutated (13) [142] Oliner (2011) [166]

Wild type (268) Mutated (14)

BRAF V600E mutation (KRAS wild type) Wild type (68) Di Nicolantonio Mutated (11) (2008) [17] De Roock (2010) [142] Laurent-Puig (2009) [44]

Loupakis (2009) [154]

Souglakos (2009) [180]

Wild type (326) Mutated (24) Wild type (111) Mutated (5) Wild type (74) Mutated (13) Wild type (83) Mutated (9) Wild type (397)

Seymour-PICCOLO (2011) [165]

Mutated (63)

Wild type (566) Van Cutsem-CRYSTAL (2011) [137]

Bokemeyer–CRYSTAL –OPUS meta-analysis (2010) [139]

Mutated (59)

Wild type (730) Mutated (70)

Treatment arms

RR

PFS, mo

OS, mo

Cetuximab ± CT or panitumumab (second and further lines)

110/289 (38.1%) 1/13 (7.7%) (p = 0.013)

6.5 3.5 (p = 0.055)

12.5 9.5 (p = 0.051)

Panitumumab (third and further lines)

47/268 (17.5%) 0/9 (0%)

NR NR

NR NR

Cetuximab or panitumumab ± CT (second and further lines)

22/69 (31.8%) 0/11 (0%) (p = 0.029)

NR NR (p = 0.001)

NR NR (p < 0.001)

Cetuximab ± CT or panitumumab (second and further lines)

124/326 (38%) 2/24 (8.3%) (p = 0.0012)

2 6.5 (p < 0.0001)

6.5 13.5 (p < 0.001)

Cetuximab-based CT (second and further lines)

52/111 (46.8%) 0/5 (0%) (p = 0.063)

2 8 (p < 0.001)

7 15 (p < 0.001)

Irinotecan-cetuximab (second and further lines)

24/74 (32%) 0/13 (0%) (p = 0.016)

4.4 2.6 (p = 0.073)

13.9 4.1 (p = 0.037)

CT ± cetuximab (any line)

14/83 (17%) 0/9 (0%) (p = 0.046)

4.3 12.5 (p < 0.0001)

10.9 40.5 (p < 0.0001)

NR

NR

NR

NR [HR pan × no pan = 1.47 (95% CI, 0.85–2.56)]

NR [HR pan × no pan = 2.03 (95% CI, 1.13–3.64)]

123/289 (42.6%) 169/277 (61%) (p < 0.001) 5/33 (15.2%) 5/26 (19.2%) (p = 0.91)

8.8 10.9 (p = 0.0013) 5.6 8 (p = 0.87)

21.6 25.1 (p = 0.0547) 10.3 14.1 (p = 0.74)

156/381 (40.9%) 212/349 (60.7%) (p < 0.001) 5/38 (13.2%) 7/32 (21.9%) (p = 0.46)

7.7 10.9 (p < 0.0001) 3.7 7.1 (p = 0.23)

21.1 24.8 (p = 0.0479) 9.9 14.1 (p = 0.0764)

Irinotecan Irinotecan + panitumumab Irinotecan Irinotecan + panitumumab (second and further lines) FOLFIRI (289) FOLFIRI + cetuximab (277) FOLFIRI (33) FOLFIRI + cetuximab (26) (first line) CT (381) Cetuximab-based CT (349) CT (38) Cetuximab-based CT (32) (first line)

RR = response rate; PFS = progression-free survival; OS = overall survival; NR = not reported; CT = chemotherapy; HR = hazard ratio; mo = months.

The retrospective nature of these data does not allow determining whether NRAS mutations have a true predictive or prognostic effect. However, if these results are confirmed with ongoing studies, then testing for NRAS as a predictor marker for anti-EGFR therapy will be used in patients WT for KRAS and BRAF for treatment selection.

6. BRAF status 6.1. Introduction V-raf murine sarcoma viral oncogene homolog B1 (BRAF), a member of the RAF gene family, encodes a

serine-threonine protein kinase that is a downstream effector of activated KRAS [167]. Activating BRAF mutations are found in approximately 7% of human cancers, specifically in 70% of malignant melanomas, 40% of thyroid cancers and 4–15% of sporadic CRC [17,168]. This frequency is heavily dependent on the patient population studied, since BRAF mutations confer poor prognosis [137,161] and the number of patients with BRAF-mutant tumours declines in the later lines of therapy [44,119,142,161]. Moreover, BRAF mutations occurs in 34–70% of sporadic microsatellite instability-high (MSI-H) CRC, whereas KRAS mutations are more common in microsatellite instability-low(MSI-L) or microsatellite stable(MS-S) tumours [17,161,169–171]. In both stable and unstable cancers, >90% of tumours

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

with BRAF mutations have widespread methylation of CpG islands or what is known as the CpG island methylator phenotype (CIMP) [172,173]. The best described and mostprevalent BRAF mutation in tumours (>95%) is the V600E mutation within the kinase activation domain of the BRAF protein (previously known as V599E), a thymidine to adenine transversion mutation, which results in the substitution of valine with glutamate at codon 600 [174,175]. The signalling changes resulting from a V600E mutation are unclear. In a simple model, there is an increase in MAPK1/3 activation, as seen for mutant KRAS, which results in constitutive activation of the RAF–MAPK–ERK pathway [175]. However, the V600E mutation could have additional functions, since activating mutations in BRAF are only present in CRC that do not carry mutations in the KRAS gene [17,142,170,176,177], suggesting that they occur in different tumour types and might have different outcomes. BRAF mutations have been linked with high grade, right side tumours, female gender, older age and MSI-H tumours [137]. A distinct pattern of metastatic spread has also been observed in BRAF mutant tumours, namely higher rates of peritoneal metastases, distant lymph node metastases and lower rates of lung metastases [178]. These histological and clinical characteristics are different from KRAS-mutant tumours, which also suggests specificity of the mutation for tumour subtypes. Although different studies have demonstrated a high concordance of KRAS mutational status between primary CRC and related metastatic sites [40,113–119], only limited data on BRAF status are available for this concordance [114–116,118,120]. Italiano et al. have reported an identical mutational pattern of BRAF in primary tumours and matching metastases in all but 2 (3%) of 95 patients, mostly with synchronous metastases [115]. Santini et al. have recently investigated in a large series of KRAS WT tumours the grade of concordance in terms of BRAF mutational status between primary tumours and related metastases and, in particular, to exclude the acquisition of a BRAF mutation during metastatic progression [179]. A BRAF V600E mutation was detected in the primary tumour of 13 of the 208 (6.25%) analysed KRAS WT metastatic CRC patients. Then the BRAF V600E mutational status was investigated at metastatic sites in the 195 patients with BRAF WT primary tumours. Five patients were excluded from this analysis for the absence of tumour tissue in the analysed metastatic sample. The majority of the 190 remaining patients had metachronous metastases (125 patients, 65.8%), with the liver as the predominant site. Only 1 of 190 patients (0.5%) showed a BRAF V600E mutation in the metastatic site (liver). In the 13 patients with a BRAF mutation in the primary tumour, however, a BRAF WT in the corresponding metastases was observed in 5 patients (38.5% of concordance). Authors conclude that there is a high concordance in BRAF WT status between primary and metastatic tumours, but the level of concordance in lower when the primary tumour harbours a BRAF mutation.

63

6.2. BRAF mutation status as a predictor of resistance to anti-EGFR mAbs Preclinical data have showed that transfection of the transcript containing the BRAF V600E mutation in WT cell systems confers resistance to both cetuximab and panitumumab [17]. A number of retrospective studies today have shown a significant association between the presence of BRAF mutations and resistance to anti-EGFR mAbs in the CT-refractory setting (Table 4) [17,44,116,142,168,180,181]. Di Nicolantonio et al. [17] first reported that occurrence of oncogenic BRAF alleles negatively interferes with the clinical response in a cohort of 113 patients who received anti-EGFR therapy in a second or subsequent lines of treatment. The BRAF V600E mutation was detected in 11 of 79 (13.9%) metastatic CRC patients who had WT KRAS. None of the BRAF-mutated patients responded to cetuximab or panitumumab, whereas none of the responders carried BRAF mutations (p = 0.029). BRAF-mutated patients had significantly shorter PFS (p = 0.011) and OS (p < 0.0001) than WT patients. Moreover, in the retrospectivepooled study of chemorefractory patients from the European Consortium (n = 761) [142], de Roock et al., reported 4.7% BRAF mutations (35 patient with V600E mutations and 1 patient with D548G mutation). Compared with BRAF WT, BRAF mutants patients had a significantly lower RR (8.3% vs. 38% for WT; OR, 0.15; p = 0.0012), shorter PFS (median, 8 vs. 26 weeks in WT; HR, 3.74; p < 0.0001) and OS (median, 26 vs. 54 weeks in WT; HR, 3.03; p < 0.0001). One of the two BRAF mutants that responded had a p.D594G mutation; the other had a p.V600E mutation present in low copy number. This effect was confirmed in multivariate analysis performed using the mutation status of KRAS, PIK3CA exon 20, PIK3CA exon 9, BRAF and NRAS, and age, sex, number of previous CT lines, and European centre as covariates. Finally, in the PICCOLO phase III trial [165], designed to evaluate the addition of panitumumab to single-agent irinotecan as second-or subsequent-line therapy for prospectively tested KRAS WT advanced CRC, patients with BRAF-mutant tumours (13.6%) have a poor prognosis and panitumumab had an adverse effect on survival in this subgroup (HR, 2.03; 95% CI, 1.13–3.64; p = 0.017). However, as opposed to KRAS mutations, the predictive value of BRAF mutations for lack of benefit with anti-EGFR therapies in the first-line treatment has not been demonstrated [26,137–139]. According to the latest data from the CRYSTAL trial [137], BRAF mutations were identified in 60 patients, which represented 6% of the overall population tested (n = 999) and 9% of those with WTKRAS (n = 625). In the small cohort of patients with KRAS WT/BRAF mutant (n = 59), improvements in PFS (median, 8.0 vs. 5.6 months; HR, 0.934, p = 0.87) and OS (median, 14.1 vs. 10.4 months; HR, 0.908; p = 0.74) associated with the addition of cetuximab to FOLFIRI did not reach statistical significance. However, the magnitude of the difference between treatments in these outcomes was comparable to that for the

64

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

larger subset with KRAS WT/BRAF WT. A similar analysis was attempted in the OPUS trial [138]. Only 11 of the 175 WTKRAS tumours analyzed (6%) had BRAF mutations, including 6 patients allocated to the cetuximab arm. Whether this biomarker is a negative predictor in relation to cetuximab benefit could not be definitively addressed given the relatively small number of patients with BRAF mutations in both CRYSTAL and OPUS trials. A pooled analysis of both studies was conducted to increase the sample size of the KRAS WT/BRAF mutant cohort to a total of 70 patients [139]. Looking at 845 WT KRAS, BRAF mutant patients still derived some benefit from the addition of cetuximab to first-line CT in terms of numerically increased RR (21.9% vs. 13.2%; HR, 1.6; p = 0.46), PFS (7.1 vs. 3.7 months; HR, 0.69; p = 0.23) and OS (14.1 vs. 9.9 months; HR, 0.63; p = 0.07), although it did not reach statistical significance, most likely due to the low BRAF mutation frequency. It is possible that the addition of a biological drug in the first-line setting could modify the particularly unfavourable prognosis of BRAF-mutant patients given CT. However, even if the BRAF mutation had been shown to be predictive, its low prevalence suggests that it may have limited utility in selecting patients for anti-EGFR therapy in clinical practice. 6.3. Prognostic value of BRAF mutation status There is now growing evidence that the presence of a BRAF V600E mutation in the primary tumour identifies patients with poorer prognosis, regardless of the treatment regimen (CT or CT combined with EGFR-targeted therapy) and this prognostic value could override its predictive value [17,25,137,139,140,163,165,173,176,177]. Ogino et al. [173] studied a cohort of 173,200 patients enrolled in the Nurses’ Health Study and the Health Professional Followup study. Among the 649 stages I–IV CRC patients, 105 (17%) had a mutation in BRAF. In both the univariate and multivariate analysis, BRAF mutations were associated with increased colon cancer-specific mortality (multivariate HR, 1.97). In the largest study indicating a predictive role for BRAF in the treatment of advanced CRC with anti-EGFR mAbs done to date, Di Nicolantonio et al. found BRAF mutations in 11 patients (10%) [17]. Although PFS and OS were significantly better in patients with BRAF WT tumours than in those BRAF mutated, it is not possible to judge whether this was due to the negative predictive effect towards treatment with cetuximab/panitumumab, because in this series all patients were treated with these drugs. In the updated analysis of the CRYSTAL trial [137], BRAF V600E mutation indicated poor prognosis in patients with KRAS WT disease in both treatment groups. Patients whose tumours carried BRAF mutations had a worse outcome for all efficacy end points compared with those whose tumours were WT. In the CRYSTAL-OPUS pooled analysis [139], patients with BRAF-mutated tumours treated with CT alone also had

worse outcome compared to patients with WT KRAS/WT BRAF tumours (RR: 13.2% vs. 40.9%; PFS: 3.7 vs. 7.7 months; OS: 9.9 vs. 21.1 months). These data are consistent with the biomarker analysis of the CAIRO-2 trial [176,177]. A BRAF mutation was detected in 45 of 519 tumours (8.7%). In the subgroup of patients treated with irinotecan–bevacizumab–cetuximab, the median PFS and OS were 6.6 and 15.2 months for patients with tumours carrying mutant BRAF vs. 10.4 (p = 0.01) and 21.5 (p = 0.001) months in those with tumours carrying WT BRAF. In the subgroup of patients treated with irinotecan-bevacizumab the median PFS and OS were 5.9 and 15 months for patients with tumours carrying mutant BRAF vs. 12.2 (p = 0.003) and 24.6 (p = 0.002) months in those with tumours carrying WT BRAF. However, the RR in the two treatment groups did not differ significantly. The authors concluded that a BRAF mutation is a negative prognostic marker in patients with metastatic CRC and that this effect, in contrast to KRAS mutations, is not restricted to the outcome of cetuximab treatment [176]. In early-stage CRC, the prognostic value of BRAF mutations is more controversial [156,161,162,182,183]. In the PETACC-3 trial [161], Roth et al. showed no statistically significant differences for RFS between patients with BRAF WT and mutated tumours in the total population (stage and arm corrected) or stratified by stage. In contrast, mutant BRAF was associated with worse OS in patients with stage II and III combined (HR, 1.78; 95% CI, 1.15–2.76; p = 0.010) and in stage III alone, particularly in patients with MSI-L and MS-S tumours (HR, 2.2; 95% CI, 1.4–3.4; p = 0.0003). In the QUASAR study [156], however, the risk of recurrence did not differ significantly between BRAF mutant and WT tumours (19% vs. 24%; OR = 0.84; 95% CI, 0.57–1.23; p = 0.4). Data on OS have not been presented. These apparently contradictory results may be reconciled by new PETACC-3 data showing that BRAF mutation status is strongly associated with survival after relapse (7.5 months for BRAF mutants vs. 25.2 months for BRAF WT; HR, 3.65; p = 1.9e−11 ) [182]. The biological explanation is currently unclear, but may be linked to the MSI or mismatch repair (MMR) status. Previous reports showed that the BRAF V600E mutation in MS-S CRC is associated with significantly poorer survival, but has no effect on the excellent prognosis of microsatellite-unstable tumours [168,171,173]. Because of the strong confounding between BRAF and MMR status (53% of BRAF mutant tumours were deficient MMR), an increased risk of recurrence (OR, 1.32; 95% CI, 0.80–2.16; p = 0.03) was observed in BRAF mutant tumours in analyses stratified by MMR status in the QUASAR trial [156]. In the PETACC-3 study [161,182], the negative prognostic effect of BRAF for OS was only observed in the MSI-L/MS-S population compared with the whole population, and reinforced in stage II disease (HR = 1.84; 95% CI, 1.14–2.97; p = 0.012). No prognostic significance of BRAF mutation status for RFS and OS was found in the MSI-H subpopulation.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

7. PIK3CA status 7.1. Introduction The PI3Ks are a family of lipid kinases grouped into three classes with different structure and substrate preferences. Activation of class IA PI3Ks is initiated when a growth factor binds to its RTK at cytoplasmic surface of the cell membrane. The PI3K class IA is a heterodimeric protein, composed of a regulatory subunit (p85) that mediates anchorage to EGFR-specific docking sites, and a catalytic subunit (p110) generating a second messenger responsible for phosphorylation and activation of AKT. Despite several isoforms being described for both subunits, somatic mutations in cancers are only present in the PIK3CA gene (encoding for the p110␣ subunit) and in PI3KR1 (encoding for p85␣ subunit) [181,183]. Activating mutations in the PIK3CA are described in approximately 10–20% of unselected CRC patients [19,108,184,185] and can co-occur with KRAS and BRAF mutations [142,186]. More than 80% of PIK3CA mutations in CRC are single amino acid substitutions located in “hotspots” in exon 9 (G1624A; E542K) (60–65%) or exon 20 (A3140G; H1047R) (20–25%) [142,185,187]. Exon 9 and 20 hotspots exert different biochemical and oncogenic properties [188,189]. In fact, the gain of function induced by exon 9 (helical-domain) mutations is independent of binding to the p85 regulatory subunit, but requires RAS–GTP interaction for downstream signalling. By contrast, exon 20 (kinasedomain) mutations are active in the absence of RAS–GTP binding but are highly dependent on the interaction with p85. It has been speculated that the contrasting roles of RAS–GTP and p85 in kinase and helical-domain mutant PIK3CA reflect two distinct states of mutated p110␣ and that these mutations should be evaluated separately [188]. 7.2. PIK3CA mutation status as a predictor of resistance to anti-EGFR mAbs In vitro studies in various CRC cell lines have found that activating PIK3CA mutations appear to confer resistance to cetuximab [190,191]. Based on these preclinical data, several groups have retrospectively analyzed the mutational status of PIK3CA as a predictor of resistance to anti-EGFR therapies in the clinical setting (Table 5). Initial reports showed that PIK3CA mutations may render the PI3K/AKT axis constitutively active and render the inhibition of the upstream EGFR superfluous to its oncogenic signalling. So, responses to anti-EGFR therapies are significantly impaired, without any differences with respect to the location of the mutation [14,18,19,33,180,185,190]. The first three studies analyzing PIK3CA mutation status reported together 9 (10%) tumours bearing PIK3CA mutations [14,19,32] and only 1 responded to EGFR-targeted treatment. In a larger patient series, Sartore-Bianchiet al. [18] found activating PIK3CA mutations in 13.6% of the 110 patients treated

65

with cetuximab or panitumumab-based regimens in first- to fourth-line treatment. None of the 15 patients with a PIK3CA mutation achieved an objective response with anti-EGFR mAbs compared with a RR of 23% in the 95 patients with WT PIK3CA (p = 0.0337). The statistical correlation was stronger (p = 0.016) when only KRAS WT patients were analyzed. Patients with PIK3CA mutations displayed a worse clinical outcome also in terms of PFS (p = 0.035). This negative association with PFS was also noted in the study by Souglakos et al. [180], where PIK3CA mutations predicted lower PFS (2.5 vs. 3.9 months; HR, 2.1; 95% CI, 1.2–3.9; p = 0.01) among 92 patients treated with salvage CT and cetuximab. Data conflicting with the aforementioned findings however come from a more recent and larger study by Prenen et al. [185]. Tumours from 200 irinotecan refractory patients treated with cetuximab as monotherapy or in combination with irinotecan were analyzed for activating mutations of PIK3CA. Twenty three (12%) of tumours contained such a mutation, consistent with previous findings. In contrast, five patients with PIK3CA mutations (22%) had an objective response to cetuximab. This means that 5 of 39 responders (13%) and 18 of 160 non-responders (11%) carried a PIK3CA mutation, thus not supporting a significant association between PIK3CA mutations and lack of response to cetuximab (p = 0.781) in CT-refractory metastatic CRC. The median PFS (24 vs. 18 weeks; p = 0.760) and OS (45 vs. 39 weeks; p = 0.698) did also not differ significantly between PIK3CA mutant and WT patients. Finally, the large dataset by the European Consortium [142] showed that among 356 KRAS WT chemorefractory patients treated with cetuximab, those with mutant PIK3CA as a whole had significantly lower RR compared with carriers of WT PIK3CA (17.7% vs. 37.7%; OR, 0.35; 95% CI, 0.13–0.83; p = 0.015). Notably, there was no significant difference in PFS (median PFS, 18 vs. 24 weeks; HR, 1.30; 95% CI, 0.91–1.86; p = 0.17) and OS (median OS, 39 vs. 51 weeks; HR, 1.41; 95% CI, 0.96–2.06; p = 0.09). However, when compared with PIK3CA WT, only PIK3CA exon 20 mutations had a negative effect on RR (0% vs. 36.8%; p = 0.029), DCR (33.3 vs. 76.0%; p = 0.0078), PFS (median, 11.5 vs. 24 weeks; HR, 2.52; 95% CI, 1.33–4.78; p = 0.013) and OS (median, 34 vs. 51 weeks; HR, 3.29; p = 0.0057). By contrast, exon 9 PIK3CA mutations are associated with KRAS mutations and do not have a significant effect on RR (28.6% vs. 36.3% in WT; p = 0.47), DCR (66.7% vs. 75.4% in WT; p = 0.39), median PFS (23.5 vs. 24 weeks; HR, 1.11; 95% CI, 0.72–1.71; p = 0.65) and median OS (46 vs. 51 weeks; HR, 1.30; 95% CI, 0.82–2.05; p = 0.28). Taken together, these data highlight the role of PIK3CA exon 20 mutations in predicting resistance to cetuximab and panitumumab. Because of its low frequency, they should be regarded as hypothesis-generating and require confirmation and further biological studies. Moreover, conflicting results from previous published works could therefore be explained by the heterogeneity of series in terms of the distribution of mutations in the two different exons: the cohort of SartoreBianchi [18] contained more exon 20 mutations (73%) than

66

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

Table 5 PIK3CA mutation and PTEN status as a predictive marker of response and survival with anti-EGFR mAb-based chemotherapy in advanced CRC. Study/year

KRAS status (no. patients)

Treatment arms

RR

PFS, mo

OS, mo

Wild type (94) Mutant exon 9 or 20 (15)

Cetuximab or panitumumab ± CT (any line)

22/94 (23.4%) 0/15 (0%) (p = 0.038)

2 4 (p = 0.0035)

9 11 (p = 0.2516)

Cetuximab-based CT (any line)

NR

3.02 2.85 (p = 0.30)

9.55 11.17 (p = 0.99)

Wild type (177) Mutant exon 9 or 20 (23)

Cetuximab ±irinotecan (second and further lines)

33/177 (18.64%) 5/23 (21.7%) (p = 0.781)

4.4 6 (p = 0.76)

9.75 11.25 (p = 0.69)

Wild type (329) Mutant exon 9 (21) Mutant exon 20 (9)

Cetuximab ± CT or panitumumab (second and further lines)

121/329 (36.8%) 6/21 (28.6%) (p = 0.47) 0/9 (0%) (p = 0.029)

6 5.87 (p = 0.65) 2.87 (p = 0.013)

12.75 11.5 (p = 0.28) 8.5 (p = 0.0057)

Frattini (2007) [43]

PTEN loss (11) No PTEN loss (16) (IHC)

Cetuximab-based CT (any line)

0/11(0%) 10/16 (62.5%) (p < 0.0001)

NR

NR

Loupakis (2009) [197]

PTEN loss (22) No PTEN loss (33) (PTEN at metastatic sites, IHC)

Irinotecan + cetuximab (second and further lines)

1/22 (5%) 12/33 (36%) (p = 0.007)

3.3 4.7 (p = 0.005)

10.6 11.2 (p = 0.37)

Sartore-Bianchi (2009) [18]

PTEN loss (32) No PTEN loss (48) (IHC)

Cetuximab or panitumumab ± CT (any line)

1/32 (3.12%) 17/48 (35.4%) (p = 0.001)

2 4.5 p = 0.0681

7.5 13 p = 0.048

Laurent-Puig (2009) [44]

PTEN loss (89) No PTEN loss (22) (KRAS wild type, IHC)

Cetuximab-based CT (second and further lines)

41/89 (46%) 10/22 (45%) p = NS

NR

12 16.5 p = 0.013

Razis (2008) [201]

PTEN deletion (23) PTEN normal (43) (FISH)

Cetuximab-based CT (any line)

3/23 (13%) 18/43 (41.86%) (p < 0.05)

5.28 7.41 (p = 0.042)

NR

Perrone (2009) [19]

PTEN decreased GCN (4) PTEN increased GCN (11) (FISH)

Cetuximab + irinotecan (second and further lines)

0/4 (0%) 5/11 (45.45%) (p = NS)

NR

NR

PIK3CA mutation Sartore-Bianchi (2009) [18] Souglakos (2009) [180] Prenen (2009) [185] De Roock (2010) [142]

Wild type (142) Mutant exon 9 or 20 (26)

PTEN status

RR = response rate; PFS = progression-free survival; OS = overall survival; CT = chemotherapy; IHC = immunohistochemistry; FISH = fluorescence in situ hybridization; GCN = gain copy number; NR = not reported; mo = months.

exon 9 mutations (27%) compared with the Prenen cohort [185], which included 13% and 78% exons20 and 9 mutations, respectively. 7.3. Prognostic value of PIK3CA mutation status The predictive value of PIK3CA mutation status should be analyzed cautiously, as it also may be a prognostic marker [18,142,180,185]. Some studies have suggested that the presence of PIK3CA mutations conferred a worse outcome in patients who underwent surgical resection [108,192,193]. In a study including 586 patients by Barault et al., decreased rates of 3-year survival were associated with mutations of at least one gene among KRAS, BRAF and PIK3CA [108]. Kato and coworkers carried out analysis of 158 CRC tissues samples and identified PIK3CA mutations as the only independent and significant prognostic factor for worse RFS in stages II/III CRC patients [192]. Ogino et al. reported PIK3CA mutations

in 82 of 450 (18%) patients with stage I-III CRC who underwent curative surgery, as detected by pyrosequencing that specifically targeted exons 9 and 20 [193]. Compared with patients with PIK3CA WT tumours, those with PIK3CAmutated experienced an increase in cancer-specific mortality (HR, 1.64; 95% CI, 0.95–2.86), which was consistent across most strata of clinical and tumoural predictors of patient outcome. Interestingly, this adverse effect seemed to differ according to KRAS mutational status. Among patients with KRAS WT tumours, the presence of PIK3CA mutations was associated with a significant increase in specific mortality (HR, 3.80; 95% CI, 1.56–9.27), but no significant effect on mortality among patients with KRAS-mutated tumours was observed (HR, 1.25; 95% CI, 0.52–2.96). These results are in contrasts with those observed in the metastatic setting. Cappuzzo et al. described a PIK3CA mutation in 17.7% (14/85) of cetuximab-treated metastatic CRC patients, but found no difference in ORR, TTP and OS compared to WT population

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

[181]. Tol et al. did not find PIK3CA mutation to be associated with outcome in KRAS WT tumours treated in the cetuximab arm of the CAIRO 2 study [177]. However, bevacizumab, which was given to all patients, may have confounded its prognostic value. In summary, these emerging data need further analysis in large cohorts of KRAS WT patients treated with anti-EGFR therapies to fully explore both PIK3CA mutations prognostic and predictive value. Moreover, the selection of PIK3CA mutations should be standardized, as these studies did not cover the same mutations within exons 9 and 20.

8. PTEN status PTEN functions as a key tumour suppressor gene involved in the homeostatic maintenance of PI3K/AKT signalling. In nontransformed cells, signal transduction events originating from EGFR activation and directed via PI3K are maintained in equilibrium due to the presence of this negative regulatory molecule. Loss of PTEN function results in increased phosphatidylinositol-3,4,5-triphosphate (PIP-3), the major substrate of PTEN, resulting in PI3K effectors persistent activation [194,195]. PTEN activity can be lost through various mechanisms, including mutations in PTEN (5% on average, with higher frequency in tumours with MSIH), allelic losses at chromosome 10q23 (23%), or epigenetic mechanisms as hypermethylation of the PTEN promoter region (19.9% in CRC with MSI-H vs. 2.2% in MSI-L) [196]. Loss of PTEN protein expression measured by IHC is reported in 19–42% of unselected CRC [20,186] and it has been found to co-occur with KRAS [44,186], BRAF [44,186] and PIK3CA mutations [186] and EGFR polysomy [44]. Interestingly, while KRAS mutational status has been shown to be consistent between primary tumour and distant metastases [40,113–119], PTEN expression shows only approximately 60% concordance [40,197]. Distant metastases often demonstrate loss of PTEN whereas the primary tumoursshow PTEN retention. This lack of concordance could be due to mechanisms of clonal selection that occur naturally in the history of malignancies and it cannot be ruled out that previous medical treatments contribute to this phenomenon. Loss of PTEN expression has been reported to predict resistance to EGFR TKI including erlotinib in glioblastoma [198] and trastuzumab in patients with HER2-overexpressing breast cancer [199,200]. Preclinical data have also demonstrated that PTEN loss appear to confer resistance to cetuximab-induced apoptosis in CRC cell lines [190]. Based on these results, several retrospective studies have analyzed different aspects of the deregulation of PTEN as a potential predictive marker for anti-EGFR mAb responsiveness (Table 5). PTEN protein expression levels were determined using IHC in all studies [18,19,43,44,197,201]. Frattini et al. found that none of 11 patients with lower PTEN expression

67

in tumour tissue responded to a combination of irinotecan and cetuximab, whereas 10 (63%) of 16 patients with intact PTEN protein expression had a PR [43]. Loupakis et al. recently reported that the loss of PTEN expression was not associated with resistance to cetuximab plus irinotecan in the primary tumour (n = 96), but was associated with lack of response in the metastatic lesions (n = 59) [197]. In the PTENpositive group, 12 of 33 patients (36%) were responders, whereas only 1/22 (5%) PTEN-negative patients responded (p = 0.007). Moreover, when PTEN IHC was performed on metastatic samples, the median PFS in patients who had PTEN-positive metastasis was 4.7 months compared with 3.3 months in PTEN-negative patients (HR, 0.49, p = 0.005). More recently, Sartore-Bianchi found that loss of PTEN protein was associated with lack of response to cetuximab and panitumumab (p = 0.001) among 81 evaluated tumours specimens [18]. Loss of PTEN expression was also associated with shorter PFS, that reached statistical significance if this variable was combined with PIK3CA mutations (loss of PTEN and/or PIK3CA mutation; p = 0.0066), and worse OS (p = 0.0048). On the contrary, Laurent-Puig et al. showed no differential effect on RR or PFS according to PTEN protein expression in 162 KRAS WTCRC patients, although the PTEN loss was associated with shorter OS (p = 0.013) [44]. Razis et al. neither found association between PTEN protein expression (IHC) with clinical outcomes although the lack of PTEN gene amplification (FISH) was associated with improved RR to cetuximab and longer TTP (p = 0.042) [201]. The reasons for these discrepant results are unclear. The small sample size of studies, the inclusion of KRAS mutant tumours and the assessment of PTEN expression in primary tumours or metastatic sites make these data difficult to interpret. Moreover, in contrast to the mutation analysis of KRAS, BRAF and PIK3CA, which generate binary “yes–no” results, evaluating PTEN protein expression by IHC and other methods will produce a continuous variable with the challenge to define cut-off and threshold levels for interpretation. In addition, IHC results are known to be affected by significant inter- and intra-observer as well as method-based variability. Therefore, these data should be considered exploratory andthe value of PTEN as a predictive or prognostic marker in CRC cannot be established yet.

9. Other potential biomarkers 9.1. HER2 gene status Some studies conducted in small cohorts of patients have suggested that HER2, the major EGFR partner, could modify the sensitivity to anti-EGFR agents [202]. Recently, data from a large retrospective study have suggested that HER2 gene status evaluated by FISH might represent an additional marker useful for the identification of metastatic CRC patients who might benefit from anti-EGFR-targeted

68

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

therapies [203,204]. A total of 407 chemorefractory metastatic CRC patients treated with cetuximab alone or in combination with irinotecan were collected in an International Consortium effort. HER2 gene status was analyzed using the dual-color FISH assay LSI HER2/neu-CEP17 (PATHVYSION) in one central lab, whereas KRAS and BRAF mutations were investigated locally. HER2 gene status was evaluable in 288 cases (70.8%). RR was observed in 25.3% of the patients. Two different scores were applied for HER2 gene status evaluation: the Colorado (positive vs. negative cases, where positive are 4 copies of the gene in 40% of cells or gene amplification) and the Locarno score (based on the classical cytogenetic criteria, positive case are those with at least low polysomy). With the Colorado score, positive cases (81 cases, 28.8%) experienced response in 34.6% of patients (vs. 15.7% in negative cases, p < 0.001), with an overall median PFS of 5.14 months (vs. 3.0 months in negative cases, p = 0.004) and a median OS of 10.9months (vs. 9.8 months in negative cases, p = 0.44). With the Locarno score, positive cases (81 patients) showed a RR in 30.3% of patients (vs. 11.4% in negative cases, p = 0.027), with a median PFS of 4.1 months (vs. 1.8 months in negative cases, p = 0.002) and a median OS of 11.3 months (vs. 7.8 months in negative cases, p = 0.2). By stratifying cases with KRAS and BRAF mutations, no significant differences in terms of RR, PFS and OS were observed between HER2-positive and negative cases using both cores, although similar trends were found. Authors conclude that the interplay between EGFR and HER2 needs to be further investigated for future best-tailored treatments. 9.2. c-Met and insulin-like growth factor receptor 1 (IGF1R) pathways Several preclinical findings suggest that MET, the hepatocyte growth factor receptor, could interfere with anti-EGFR strategies. MET is a RTK involved in cellular proliferation and apoptosis. Activation of MET may lead to the activation of patways downstream of RAS, such as Raf/MEK/MAPK and the PI3K/protein kinase B pathway (PKB). In addition, MET is able to directly activate PI3K/PKB pathway in a RAS independent manner [205]. Concomitant RTK upregulation including MET is common in human carcinomas with high frequency of KRAS mutations, including CRC [206]. Moreover, studies in lung cancer have shown that MET gene amplification is responsible for acquired resistance to EGFR TKIs [207]. Previous studies have investigated MET in CRC with semiquantitative techniques such as immunoblotting or IHC [208,209]. Inno et al. have recently reported that, compared with low/normal expression, c-Met overexpression significantly correlated with shorter median PFS (3 vs. 5 months; p = 0.018) and median OS (11 vs. 10 months; p = 0.037) among 73 patients with metastatic CRC treated with cetuximab-containing regimens [209]. Cappuzzo et al. have also assessed MET at the genomic level using FISH in 85 EGFR FISH positive metastatic CRC patients treated

with cetuximab [181]. MET increased GCN (MET FISH+) were defined as means ≥5 per cell, which was observed in 7 patients (9.2%), including 2 (2.6%) with gene amplification and 5 (6.6%) with high polysomy. Both patients with MET amplification responded to cetuximab therapy. Although the number of patients was too low for any conclusion, the level of MET gene gain observed in this study population was lower than reported in a previous study conducted on cell lines and patients with acquired resistance to anti EGFR agents [207]. Although obtained in tissues collected before starting cetuximab therapy, these findings suggest that only high level of MET gene gain could be responsible for resistance, levels probably occurring only under therapeutic pressure and rarely present in a general population of patients unexposed to anti-EGFR agents. On the other hand, the IGF1R is also a transmembrane RTK implicated in promoting oncogenic transformation, growth and survival of cancer cells. IGF1R activation triggers a cascade of reactions involving the Raf/MEK/MAPK and the PI3K/PKB pathways [210]. Data on glioblastoma cell lines suggested that IGF1R mediates resistance to anti-EGFR therapy through continued activation of the PI3K-AKT pathway [211]. IGF1R is overexpressed in 50–90% of CRC [212] and preclinical studies suggest that this target results in upregulation in the majority of CRC, most likely contributing to the aggressive growth characteristics of these tumours, poor prognosis and resistance to anti-EGFR strategies [211]. Nevertheless, two retrospective studies have showed that IGF1R overexpression seems a favourable prognostic factor in metastatic CRC [181,209]. Inno et al. found that median OS was significantly longer in metastatic CRC patients treated with cetuximab-based regimens and with high IGF1R expression than in those with low/normal expression (14 vs. 8 months; p = 0.015) [209]. In a second study, metastatic CRC patients treated with cetuximab-based therapy and overexpressing IGF1R (IGF1R IHC+) (74.3%) also had significantly longer survival than patients with low IGF1R expression (16.1 vs. 6.7 months; p = 0.006), although RR and TTP were not significantly different in the two groups [181]. In both studies, IGF1R expression was not associated with cetuximab resistance, probably because the IGF1R pathway did not affect the antiproliferative activity of cetuximab, as recently observed in a lung cancer model [213]. In the study conducted by Morgillo et al, only the treatment with gefitinib, but not cetuximab, induced EGFR-IGF1R heterodimerisation and activation of IGF1R and its downstream signalling mediators, resulting in increased surviving expression in lung cancer cell lines with high levels of IGF1R expression [213]. In addition, in the study performed by Cappuzzo, IGF1R gene amplification was not detected and the level of IGF1R gene gain was very low in the whole analysed population, suggesting that such event is not involved in primary resistance. Moreover, no association was found between IGF1R gene and protein expression, suggesting post-transcriptional events could also interfere with the gene function.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

9.3. TP53 mutations Several data suggest that molecular brakes such as p53, protecting the cells against inappropriate oncogene activation, may also be candidate biomarkers of sensitivity to anti-EGFR therapy, given that their inactivation may be required for tumour progression. Indeed, recent studies have indicated that oncogenic activation of transduction cascades leads to malignant transformation only if p53 is inactivated: alteration of the p53 pathway has been reported to be systematically observed in non-small cell lung cancer with activating EGFR mutations suggesting that p53 inactivation is required to allow expansion of a cell with EGFR pathway activation [214]. Moreover, it has been shown that p53-mediated growth suppression is induced by PIK3CA signalling activation suggesting that p53 acts as a brake for the PIK3CA transduction cascade [215]. Therefore, it is likely to speculate that activation of the EGFR pathway will contribute to cancer and that anti-EGFR antibodies will be efficient on tumour only if p53 is inactivated. Based on these observations, Oden-Gangloff et al. have evaluated the combined impact of KRAS and TP53 status on clinical outcome in 64 metastatic CRC patients treated with cetuximab-based CT, suggesting that p53 mutations are predictive of cetuximab sensitivity [216]. TP53 mutations were found in 41 patients (64%) and were significantly associated with controlled disease (CD), as defined as complete response, PR or SD (p = 0.037) and higher TTP (20 vs. 12 weeks; p = 0.004). Remarkably, in the subgroup of 46 patients without KRAS mutation, TP53 mutation were also associated with CD (p = 0.008) and higher TTP (24 vs. 12 weeks; p = 0.0007). In contrast, in the subgroup of patients with KRAS mutation, the median TTPs were not different between patients with and without TP53 mutation. In conclusion, this data suggest that TP53 genotyping could have and additional value in metastatic CRC patients without KRAS mutation to optimize the selection of patients who should benefit from anti-EGFR therapies. The clinical relevance of these results should be confirmed on larger metastatic CRC series.

10. Use of multiple biomarkers to predict clinical outcome to ANTI-EGFR mABs It is becoming apparent that the key to unraveling the mechanisms of drug response is to adopt the pathway-based approach with simultaneous analysis of multiple parameters required to make an informed treatment decision. This is particular true for anti-EGFR therapies. Current data suggest that the evaluation of not only KRAS mutations [12–16,135–139], but also BRAF [17,44,142,154,166,180,181] and PIK3CA/ PTEN alterations [18,43,180,197] could be useful for selecting patients with metastatic CRC who are unlikely to respond to anti-EGFR-targeted therapy. At present, each of these markers has been mainly assessed as a single event, often in retrospective analysis. As a result, a clinical

69

identification of which biomarkers should be used together with KRAS in the clinical setting remains to be defined. Comprehensive integrated analysis of the entire oncogenic pathway triggered by the EGFR is likely to enhance the prediction ability of the markers used individually [25,108,166,186,217]. In fact, Sartore-Bianchi et al. [186] have demonstrated that when PIK3CA mutations and loss of PTEN expression are combined with KRAS and BRAF mutational analysis, up to 70% of patients unlikely to respond to anti-EGFR mAbs may be identified. Authors have retrospectively analyzed outcome together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumours from cetuximab or panitumumab treated metastatic CRC patients. Among the 106 non-responsive patients, 74 (70%) had tumours with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations in the four markers, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with ≥2 alterations (p < 0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumours harbouring none, 1 or ≥2 molecular alterations (p < 0.001). This observation has led to the idea that CRC may be able to be classified like breast cancers (eg, triplenegative breast cancer) and it has been proposed to define as “quadruple negative” the CRCs lacking alterations in any of these four biomarkers. Laurent-Puig et al. [44] reported a similar comprehensive analysis of molecular biomarkers, including EGFR GCN but not PIK3CA evaluation and focusing on KRAS WT tumours. They retrospectively collected specimens from 173 metastatic CRC patients treated with a cetuximab-based regimen as second or further lines (see previous sections). In patients with KRAS WT tumours (n = 116), BRAF mutations (3%) were weakly associated with lack or response (p = 0.063) but were strongly associated with shorter PFS (p < 0.001) and shorter OS (p < 0.001). A high EGFR polysomy was associated with response (p = 0.013), whereas PTEN null expression was associated with shorter OS (p = 0.013). In multivariate analysis, BRAF mutation and PTEN expression status were associated with OS. A similar study has employed next-generation sequencing technology to investigate whether mutations in multiple genes known to be altered in CRC (AKT1, BRAF, CTNNB1, EGFR, KRAS codon 61, PIK3CA, PTEN and p53) are predictive of response to panitumumab in the pivotal phase III trial. Patients with KRAS, NRAS and BRAF all WT tumours (n = 103) derive the greatest benefit from panitumumab therapy (HR, 0.35; 95% CI, 0.23–0.54) [166]. In addition, the convergence of distinct oncogenic mutations in effector genes downstream of EGFR at the transcriptional level have allowed the discovery of activated profiles that lead to the identification of patients with activating mutations on the EGFR signalling, which may not benefit from drugs that inhibit the EGFR receptor. In a recently reported study, three activating mutation signatures for KRAS (75 genes), BRAF (58 genes) and PIK3CA (49 genes) have been developed using whole genome microarray

70

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

data [218]. Combined into an integrative 3-way classification model, tumours can be classified as “mutation-like” or “nonmutation-like” with a sensitivity of 90.3% and a specificity of 61.7%. The three mutation signatures and the combined model are associated with response to cetuximab treatment in patients with metastatic CRC (p = 0.004 for KRAS signature, p = 0.018 for BRAF signature, p = 0.003 for PIK3CA signature and p = 0.0009 for the combination model). Importantly, approximately 20% of metastatic CRC patients with no response to anti-EGFR targeted therapies do not harbour mutations of KRAS, BRAF, PIK3CA nor loss of PTEN expression [186]. The lack of response in “quadruple negative” patients may be due to multiple reasons, including the limited sensitivity of current sequencing methods in detecting point mutations in DNA extracted from FFPE tumours [219], the oncogenic deregulation of the same four genes by mechanisms other than mutations (such as amplification as reported for PIK3CA), the occurrence of alterations in other key elements of the EGFR-dependent signal cascade (such as for example AKT or MAPK) and the presence of genetic alterations in RTK other than EGFR, providing an alternate pathway of survival and/or proliferation. In summary, each one of the molecular biomarkers evaluated in this review demonstrated to affect clinical outcome to EGFR-targeted mAbs, although, by itself, only KRAS reached the clinical practice. Probably the best strategy has been the evaluation of these biomarkers in the context of a multi-determinants analysis including both the KRAS–RAF–MAPK and the PI3K–PTEN–AKT signalling pathways, providing predictive algorithms that are ready for validation in prospective trials. Moreover, in a near future, the therapeutic armamentarium for metastatic CRC will be further expanded by introduction of novel targeted agents, and current data from comprehensive integrated analysis of different effectors along the EGFR pathway will support a rational among different available options.

11. Early response evaluation 11.1. Early radiological tumour size decrease Early on-treatment changes may help to identify those patients in whom continuation of therapy is worthwhile. Recent correlations between biomarkers and radiological data have shown that KRAS WT tumours clearly differ from KRAS mutant in terms of tumour size changes [135,220–222]. Specifically, an early radiological tumour size decrease has been associated with clinical benefit in irinotecan refractory metastatic CRC patients treated with cetuximab [135]. Within KRAS WT patients, OS was significantly better in patients with an initial relative decrease of tumour size >9.66% at week 6 comparedwith those without (median OS, 74.9 vs. 30.6 weeks; p = 0.00000012). These data have been validated in a large and independent series of 289 patients treated with cetuximab with or without

irinotecan in the BOND trial [220]. Median TTP was 6.1 vs. 1.5 months in patients with (34.3%) or without (65.7%) tumour shrinkage, respectively, at week 6 (HR, 0.23; 95% CI 0.17–0.32). The median OS was 13.7 vs. 6.9 months (HR, 0.21; 95% CI 0.14–0.32), respectively. In a multivariate model, early tumour decrease outperformed skin toxicity as a predictor of long-term outcome. More recently, the same authors have used the CRYSTAL and OPUS studies to quantify the predictive power of tumour size changes at week 8 for long-term outcome, investigating heterogeneity across treatment arms (CT or CT+cetuximab) [221]. A strong relationship between changes in tumour size at week 8 and PFS in KRAS WT metastatic CRC patients receiving CT + cetuximab was found in both studies. This was weaker in the CT arm. Data were similar for OS. Treatment interaction was confirmed by significant interaction terms for PFS (p = 0.020 and 0.004 in the CRYSTAL and OPUS studies, respectively) in the Cox regression models. In the CO.17 trial, the change in tumour size at 8 weeks following commencement of cetuximab using a waterfall plot analysis was a better predictor of OS than standard RECIST categories for the overall population. Results were similar for WT patients, whereas neither change in longest tumour diameters nor RECIST response was associated with OS in KRAS mutated patients [222]. In conclusion, these results suggest that early tumour shrinkage is a reliable and easily accessible tool to predict efficacy of cetuximab for metastatic CRC. Its prognostic impact is probably far more important than with treatment regimens without cetuximab. These findings can facilitate trial design and end points measured as early as at 6 weeks can be used to assess efficacy and adapt further treatments accordingly [223,224]. 11.2. Skin rash as a biomarker of efficacy of anti-EGFR therapy Skin toxicity develops at an early stage in treatment with EGFR inhibitors and has been studied extensively as a potential early marker of response. The incidence of the characteristic “acneiform” skin rash is around 80% in most series. It is usually apparent after approximately 1 week of treatment and reaches maximum severity after 2–3 weeks. The presence and severity of the acne-like rash anti-EGFR agents-related has been consistently linked with higher RR and longer survival among patients with metastatic CRC who have been treated with cetuximab or panitumumab, both in non-selected populations [7,10,11,21,29,225] and subgroup analysis based on KRAS mutational status [27,162,226,227] (Table 6). Subgroup analysis of phase III trials with cetuximab [162,227] and panitumumab [27,226] demonstrated that grade 2 or higher skin toxicity was associated with increased RR and survival outcomes in patients with WT KRAS tumours, but not in patients with mutant KRAS tumours. It is known that EGFR is highly expressed in normal tissues including the skin; therefore, cutaneous toxicity might

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

71

Table 6 Skin rash as a predictive biomarker of efficacy of anti-EGFR therapy in metastatic colorectal cancer. First author (year)

Non selected population

Lenz (2006) [29] Cunningham (2004) [7]

Jonker (2007) [10] Van Cutsem (2007) [11] Subgroup analysis based on KRAS status

O’Callagham (2011) [227] Peeters (2009) [226]

Cunningham (2009) [27]

Treatment (type of study, type of patients)

No. patients

Rash prevalence

Response rate

Survival

Cetuximab monotherapy (phase II, CT refractory) Cetuximab vs. cetuximab + irinotecan (phase III, irinotecan refractory)

346

89.9% (39.8% G1, 44.2% G2, 5.7% G3) 80% (7.95% G3–4)

No rash: 0%. Rash: 12.9% (7% G1, 17% G2, 20% G3) Rash vs. no rash: monotherapy 13% vs.0%, combination 25.8% vs. 6.3%

Cetuximab vs. BSC (phase III, CT refractory) Panitumumab vs. BSC (phase II, CT refractory) Cetuximab vs. BSC (phase III, CT refractory) Panitumumab vs. BSC (phase III, CT refractory)

572 (287 cetuximab, 285 BSC) 463 (231 cetuximab, 232 BSC) 572 (287 cetuximab, 285 BSC) 463 (231 panitumumab) KRAS: 110 WT/72 mutant

88.6% (39.6% G1, 37.2% G2, 11.8% G3) 90% (8% ≥G3)

No data% G1, 37.2% G2, 11.8% G3)

Median OS. No rash: 1.7 mo, G1: 4.9 mo, G2–3: 9.4 mo. Median OS (rash vs. no rash): monotherapy: 8.1 vs. 2.5 mo; combination: 9.1 vs.3 mo. Median OS. No rash: 2.6 mo, G1:4.8 mo, G2:8.4 mo. Median OS. G1: 5.5 mo. G ≥ 2: 8.9 mo.

90%

No data

91% (5% ≥G3)

86% responders had rash ≥G2

FOLFOX4 + panitumumab vs. FOLFOX4 (phase III, 1st line)

1183 (311 KRAS WT treated with panitumumab)

Rash G ≥ 2: 79%

G0-1: 39% G ≥ 2: 62%

329 (111 monotherapy, 218 combination)

No data

Median OS. No rash: 2.6 mo. G1: 4.8 mo. G ≥ 2:8.4 mo. OS (rash G ≥ 2 vs. G 0–1). WT KRAS: HR 0.75 (95% CI, 0.49–1.17). KRAS mut: No difference. Median PFS. G0-1: 6 mo., G≥2: 11.1 mo. Median OS: G0-1:11.5 mo., G≥2: 28.3 mo.

PFS = progression-free survival; OS = overall survival; BSC = best supportive care; WT = wild type; mut = mutant; mo = months; G = grade.

indicate local receptor saturation. So, “dose-to-rash” strategies are being studied with the aim of optimizing response to anti-EGFR treatment. The randomized phases I and II EVEREST (Evaluation of Various Erbitux Regimens by Means of Skin Tumour Biopsies) study has evaluated whether dose escalation of cetuximab in irinotecan-refractory metastatic CRC patients with no rash or a slight rash in the first weeks of treatment may induce a more pronounced rash and subsequently higher activity of the agent [228]. Eightynine patients with grade 0 or 1 skin toxicity on a day 22 evaluation were randomized to the standard dose arm or the arm with “dosing-to-rash” (escalating the dose of cetuximab by 50 mg/m2 every 2 weeks until grade 2 skin toxicity, tumour response or dose = 500 mg/m2 ) in combination with standardregimen irinotecan (180 mg/m2 every 2 weeks). Escalation of the cetuximab dose resulted in a non-significant trend towards higher RR in WT KRAS patients (21.1% vs. 46% for the standard dose; p = 0.396), suggesting that skin toxicity and KRAS status were independent predictors of outcome. However, it has to be kept in mind that patients tolerating high doses of cetuximab might represent a group with an overall better performance status and thus a better PFS. In addition, it is important to note that whereas EGFR rash and KRAS status are independent, not associated markers, patients with KRAS mutated tumours will not benefit from EGFR mAb, even if they develop a severe rash.

There are several limitations to the use of rash as an early physical marker of efficacy. There are no criteria for adverse effects involving skin that are specifically tailored to the activity of EGFR-targeted treatment [135]. Rash often occurs in patients without apparent benefit from anti-EGFR treatment, and conversely, clinical benefit has also been seen in patients without rash [229]. The mechanism of both the rash and its correlation with tumour response is still poorly understood [230]. Although skin rash may indicate local receptor saturation, other factors, such as their immune response and EGFR polymorphisms might alter the individual susceptibility to develop skin rash and also obtain a response [58]. Further prospective studies are underway to clarify the predictive value of skin toxicity in advanced CRC patients treated with EGFR-targeted therapy. 11.3. Hypomagnesemia as a biomarker of efficacy of anti-EGFR therapy Magnesium is a critical cofactor in many enzymatic reactions involved in physiological functions, such as nucleic acid metabolism, protein synthesis and energy production. In addition, it seems to play an important role in tumour biology, such as the regulation of oxidative stress, carcinogenesis, tumour progression and angiogenesis [231,232]. According to the “membrane magnesium mitosis” model of cell growth

72

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

defined by Rubin and colleagues, magnesium plays a pivotal role in regulating cell proliferation and hypomagnesemia induces both growth arrest by affecting cell cycle regulatory proteins, protein synthesis, DNA duplication and increased apoptosis [232,233]. Because EGFR is strongly expressed in the kidney, particularly in the ascending limb of the loop of Henle where 70% of filtered magnesium is reabsorbed, EGFR blockade may interfere with magnesium transport [234]. Groenestege defined EGF as an autocrine/paracrine magnesiotropic hormone that regulates renal magnesium reabsorption by regulating the activity of the magnesium-permeable channel TRPM6 (transient receptor potential cation channel, subfamily M, member 6). A point mutation in pro-EGF that disrupts sorting of the protein to the basolateral membrane of distal convoluted tubule cells in kidney nephrons has been identified. As a consequence, inhibition of the EGFR by anti-EGFR mAbs might lead to suppressed activity of TRPM6 and renal magnesium wasting [235]. Some studies have reported that EGFR-inhibiting antibodies compromised the renal magnesium retention capacity, leading to inappropriate urinary excretion and hypomagnesemia [236–238]. Tejpar et al. reported that 95 (97%) of 98 patients with CRC treated with EGFR-targeting mAbs with or without combined CT had decreasing serum magnesium concentrations during EGFR-targeting treatment compared with baseline measurements [238]. The mean serum magnesium slope during EGFR-targeting treatment was significantly lower compared with CT alone (−0.00157 mmol/L/day vs. 0.00014 mmol/L/day; p < 0.0001). Furthermore, the incidence of severe hypomagnesemia was associated with treatment duration [237]. A meta-analysis has recently been performed to determine the risk of hypomagnesemia in patients treated with cetuximab or panitumumab in randomized clinical trials [239]. Fourteen prospective randomized phase III studies were included, in which patients were randomly assigned to receive standard anti-neoplastic treatmentcombined with cetuximab or panitumumab vs. standard treatment alone. The overall incidence of hypomagnesemia was 17% among the patients who received anti-EGFR therapies. The addition of anti-EGFR mAbs to standard anticancer therapy showed a significantly increased risk of hypomagnesemia compared with patients treated with control medications, with an overall relative risk of 5.83 (p < 0.00001), where 3.87 refers to cetuximab and 12.55 to panitumumab. The risk seems to be even higher for panitumumab, probably correlated with the increased risk of other adverse events (e.g., diarrhea and dehydration). Incidence of severe hypomagnesemia was 3.4% and 0.27%, respectively (p < 0.0001), for treatment and control arms. Overall risk ratio for severe hypomagnesemia was 8.3 (7.48 for cetuximab and 10 for panitumumab). The real clinicalsignificance of this adverse event is unknown, although no deaths or severe complications havebeen reported. Two recently published studies have suggested that the reduction in serum magnesium levels might potentially

provide an early marker of clinical response and outcome during cetuximab treatment in advanced CRC patients [240,241]. In the first trial [240], 68 metastatic CRC patients receiving weekly irinotecan+cetuximab as third line treatment were evaluated for magnesium serum levels at the following time points: just before the beginning of the first day and 1, 21, 50 and 92 days after cetuximab + irinotecan infusion. Patients have been not selected for KRAS mutational status. The median magnesium basal levels were significantly decreased just 7 days after the first anticancer infusion and progressively decreased from the seventh day onward, reaching the highest significance at the last time point (92 days after the first cetuximab infusion), with a median value of 1.75 mmol/L vs. 2.12 mmol/L when compared with the basal time (p < 0.0001). At any time of the programmed time points, 65 of 68 patients showed a reduction of magnesium serum levels; 25 of them showed at least 20% reduction with respect to the basal level, but only 3 patients developed a hypomagnesemia grade 1 according to the Common Toxicity Criteria (CTC). No cases of hypomagnesemia higher than grade 1 were recorded. Patients who developed a decreased of magnesium serum levels of at least 20% between the first and the third week after the first infusion showed a higher RR compared with patients who did not show an equal reduction (64% vs. 25.6%; p = 0.004). Moreover, the median TTP was longer in the group with reduced magnesium than in the other group (6 vs. 3.9 months; p < 0.0001), as well as OS (10.7 vs. 8.9 months; p = 0.021). A second study showed similar results in 143 KRAS WTCRC patients treated with cetuximab+irinotecan as third-line therapy [241]. The median magnesium basal value showed a statistically significant decrease 7 days after the start of cetuximab-based therapy (p = 0.04), with the highest statistical significance recorded 28 days after the start of treatment (p < 0.0001). Eight patients during any of the programmed time points developed a hypomagnesemia grade 1 according to the CTC criteria and only one patient a grade 2 hypomagnesemia. Patients with an early decreased (within the first 4 weeks from the start of treatment) of magnesium levels >50% showed a higher tumour RR (55.8% vs.16.7%, p < 0.0001), a longer TTP (6.3 vs. 3.6 months, p < 0.0001) and longer median OS (11 vs. 8.1 months, p = 0.002). Multivariate analysis was carried out taking into account skin toxicity and magnesium reduction levels; the early magnesium reduction preserved the statistical significance while the acne-like skin rash lost its significance. These results suggest that magnesium reduction seems to act as a stronger prognostic factor than skin toxicity in this population. However, in contrast to prior reports, grade ≥1 or ≥20% reduction from baseline cetuximab-hypomagnesemia at the end of month 1 was associated with poor OS in the NCI-CTG CO.17 clinical trial in pretreated CRC patients (HR, 1.61; 95% CI, 1.12–2.33, p = 0.01 and HR, 2.08; 95% CI, 1.32–3.29, p = 0.002, respectively), even after adjustment for grade of rash [242] To summarize, magnesium circulating level is an easy and economically inexpensive biomarker to routinely and

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

73

Table 7 Summary of prognostic and predictive biomarkers for anti-EGFR therapy. Biomarker

Incidence (%)

Detection method

Positive result

Prognostic value

Predictive value

EGFR protein expression EGFR protein

80–85%

IHC

No prognostic role [24–29]

6–51%

FISH/CISH/PCR-based method

Membranous and cytoplasmic staining ≥2–6 GCN

AREG/EREG expression

NR

Ab enzyme linked immunosorbent assay

Established cut points for mRNA expression levels for each ligand

Prognostic marker in KRAS WT patients [53]

KRAS mutations

35–45%

DNA direct sequencing, pyrosequencing, PCR-based assays

Mutations in codons 12 (82–87%), 13 (13–18%) and 61

BRAF mutations

4–15%

DNA sequencing, PCR-based assays

V600E point mutation (>95%) in exon 15

PIK3CA mutations

10–20%

DNA direct sequencing, pyrosequencing, PCR-based assays

Point mutations (80%) in exon 9 (60–65%) or 20 (20–25%), missense and frame shift mutations

PTEN status

19–42%

IHQ

Loss of immunoreactivity

Data on the KRAS prognostic value are controversial [15,25,26,111,112,141, 155–162] The presence of a BRAF V600E mutation identifies patients with poor prognosis [17,25,137,139,140,163, 165,173,176,177] Conflicting results regarding the PIK3CA prognostic and predictive role [14,17–19,26,32,33,44,116, 137–139,142,165,168,180, 181,185,190] Conflicting results regarding the PTEN status prognostic and predictive role [18,19,43,44,197,201]

No predictive for anti-EGFR therapies efficacy [24–29] Significant association between EGFR GCN and benefit to anti-EGFR therapies both in unselected and KRAS populations [14,29,30,32,33,37–45] Significant association between AREG/EREG expression levels and clinical benefit to anti-EGFR therapies [47–51] KRAS mutations is a major predictor of resistance to anti-EGFR mAbs [8,12–16,133–135,137–139, 142] The predictive value of BRAF mutation for lack of benefit with anti-EGFR therapies is controversial

No prognostic role

EGFR: epidermal growth factor receptor; FISH: fluorescence in situ hybridization; CISH: chromogenic in situ hybridization; PCR: polymerase chain reaction; GCN: gene copy number; AREG: epiregulin; AREG: amphiregulin; Ab: antibody; WT: wild-type; NR: not reported.

serially be detected in patients treated with anti-EGFR mAbs. Magnesium levels also enable a useful prediction of patient benefit from these therapies. However, further studies are clearly required to deeply understand the biological reasons of the contradictory findings reported and to apply a comprehensive assessment of genetic alterations in EGFR signalling pathways.

12. Conclusions and future directions Selection of patients for targeted therapy based on molecular predictors of individual tumours is regarded as the treatment strategy for the future. The introduction of KRAS testing as a diagnostic tool to select patients for EGFRtargeted treatment has been validated and is considered as one of the most important recent advances in the field of personalized CRC therapy. Although KRAS mutations can be considered a highly specific negative biomarker for response to anti-EGFR mAbs (specificity of 93%), it is also

poorly sensitive (sensitivity of 47%), since WT KRAS does not guarantee benefit from these agents [8,12–16,137–139]. This had led to investigation of additional markers of primary resistance to cetuximab and panitumumab in KRAS WT tumours, thus focusing on the analysis of molecules involved in the downstream of EGFR signalling [243]. According to the presently available data, it seems clear that BRAF mutations define a genetically distinct subset of CRCs characterized by an extremely poor prognosis [17,25,137,139,163,173]. Whether BRAF mutational status should be used as a selection factor for treatment with EGFRtargeted agents in patients with WT KRAS tumours is still controversial [17,137–139,142]. The investigation of other biomarkers such as EGFR GCN [33,43–45,136], expression levels of EGFR ligands [47–52], EGFR gene polymorphisms [58,62–64], NRAS [25,142,166] or PIK3CA (exon 20) mutations [18,142,180,185] or loss of PTEN expression [18,43,197] also seem relevant to positively identify responders. Table 7 summarizes potential prognostic and predictive biomarkers for EGFR-targeted therapy in CRC.

74

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

It is becoming increasingly apparent that disease progression is largely driven by complex pathways and analysis of one single marker is unlikely to predict progression of disease with a high degree of accuracy and reproducibility. Therefore, concomitant analysis of multiple genetic and epigenetic events involved in theEGFR-initiated oncogenic signalling cascadeis likely to enhance the prediction ability of the biomarkers used individually [186]. Important future challenges for translational research studies are the standardization of biomarkers assessment and theimplementation and validation of promising molecular markers in large prospective clinical trial. Further research on the mechanisms of secondary acquired resistance, such as alterations in other key elements of the EGFR-dependent signal cascade (MAPK or AKT1), or the presence of genetic alterations in RTK other than EGFR is also warranted to define additional signalling pathways to be targeted. For instance, Montagut and coworkers have recently identified an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to cetuximab [244]. Cells with this mutation, however, retain binding and are growth inhibited by panitumumab. Two of ten subjects studied with disease progression after cetuximab treatment acquired this mutation. Moreover, a patient with cetuximab resistance harbouring the S492R mutation responded to panitumumab treatment. This mutation may provide a molecular explanation for the clinical benefit of panitumumab in a subset of metastatic CRC patients who do not respond to treatment with cetuximab [245]. The specificity of the S492R mutation is expected to facilitate reliable testing to guide the clinical use of panitumumab after cetuximab failure and justifies prospective independent validation of the S492R EGFR mutation. Moreover, pharmacological inhibition of effectors downstream of EGFR is a strategy currently under investigation. Although there are no drugs available for the specific and direct inhibition of KRAS, a number of agents designed to inhibit the kinase activity of BRAF are either already clinically approved or are progressing through the pipeline of phases I and II studies [246,247]. Given the frequency and role of oncogenic PIK3CA mutations in metastatic CRC above described [19,108,184,185], it would be rationale to target this pathway in the KRAS WT population [248]. Finally, when cancer cells are treated with drugs that block a single molecular target, they are often able to activate alternative pathways as escape mechanisms to overcome the blockade and therefore the effectiveness of these drugs. Rational combinations of targeted treatments to circumvent, reverse, or even preclude resistance are therefore necessary for optimum use of molecular therapies in cancer [249,250]. In summary, in the next years research efforts for KRAS WT patients will focus on the evaluation of additional markers of primary resistance to cetuximab or panitumumab by the analysis of molecules involved in the downstream of the EGFR signalling. Other interesting areas of research are the

standardization of biomarkers evaluation in order to increase the sensitivity of methods to detect KRAS mutations and better define the sensitivity threshold that is required for the accurate identification of non-responder patients as well as the assessment of clinical and radiological data as potential early markers of response. Important future challenges are also the potential mechanisms and pathways involved in acquired resistance to anti-EGFR therapies and the development of new strategies for the pharmacological inhibition of effectors downstream of EGFR. For KRAS mutated patients, an important point for further research is the assessment of the impact of KRAS mutations in different codons on mediating EGFR resistance in order to optimize the selection of patients to receive anti-EGFR treatment. Finally, we feel that the best strategy has been the evaluation of all these biomarkers in the context of a multi-determinant analysis of the entire oncogenic pathway triggered by the EGFR, providing algorithms that need to be validated in large, prospective, clinical trials. A better understanding of the functional interactions within RTK-activated intracellular pathways is essential to efficiently target the individual tumour and to select appropriate patients for therapy thereby maximizing drug efficacy and minimizing toxicity.

Conflict of interest The authors declare that they have no conflict of interest relating to the publication of this.

Reviewers Daniele Santini, M.D., Ph.D., Assistant Professor, University Campus Bio-Medico, Medical Oncology, Via Alvaro del Portillo 200, I-00128 Rome, Italy. Milo Frattini, Ph.D., Institute of Pathology, Laboratory of Molecular Diagnostic, via in Selva 24, CH-6600 Locarno, Switzerland.

References [1] Jemal A, Siegel R, Yu J, et al. Cancer statistics 2010. CA: A Cancer Journal for Clinicians 2010;60:277–300. [2] Scaltriti M, Baselga J. The epidermal growth factor receptor pathway: a model for targeted therapy. Clinical Cancer Research 2006;12:5268–72. [3] Labianca R, Beretta GD, Kildani B, et al. Colon cancer. Critical Reviews in Oncology/Hematology 2010;74:106–33. [4] Comella P, Casaretti R, Avallone A, Franco L. Optimizing the management of metastatic colorectal cancer. Critical Reviews in Oncology/Hematology 2010;75:15–26. [5] Winer E, Gralow J, Diller L, et al. American Society of Clinical Oncology, Clinical cancer advances 2008: major research advances in cancer treatment, prevention, and screening—a report from the American Society of Clinical Oncology. Journal of Clinical Oncology 2008;27:812–26.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81 [6] Rodríguez J, Viúdez A, Ponz-Sarvisé M, et al. Improving disease control in advanced colorectal cancer: panitumumab and cetuximab. Critical Reviews in Oncology/Hematology 2010;74:193–202. [7] Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. New England Journal of Medicine 2004;351:337–45. [8] Van Cutsem E, Kohne CH, Hitre E, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. New England Journal of Medicine 2009;360:1408–17. [9] Bokemeyer C, Bondarenko I, Makhson A, et al. Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. Journal of Clinical Oncology 2009;27:663–71. [10] Jonker DJ, O’Callaghan CJ, Karapetis CS, et al. Cetuximab for the treatment of colorectal cancer. New England Journal of Medicine 2007;357:2040–8. [11] Van Cutsem, Peeters M, Siena S, et al. Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. Journal of Clinical Oncology 2007;25:1658–64. [12] Douillard JY, Siena S, Cassidy J, et al. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. Journal of Clinical Oncology 2010;28:4697–705. [13] Peeters M, Price TJ, Cervantes A, et al. Randomized phase III study of panitumumab with fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone as second-line treatment in patients with metastatic colorectal cancer. Journal of Clinical Oncology 2010;28:4706–13. [14] Lièvre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Research 2006;66:3992–5. [15] Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. New England Journal of Medicine 2008;359:1757–65. [16] Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. Journal of Clinical Oncology 2008;26:1626–34. [17] Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. Journal of Clinical Oncology 2008;26:5705–12. [18] Sartore-Bianchi A, Martini M, Molinari F, et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFRmonoclonal antibodies. Cancer Research 2009;69:1851–7. [19] Perrone F, Lampis A, Orsenigo M, et al. PIK3CA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients. Annals of Oncology 2009;20:84–90. [20] Siena S, Sartore-Bianchi A, Di Nicolantonio F, Balfour J, Bardelli A. Biomarkers predicting clinical outcome of epidermal growth factor receptor-targeted therapy in metastatic colorectal cancer. Journal of the National Cancer Institute 2009;101:1308–24. [21] Saltz LB, Meropol NJ, Loehrer Sr PJ, et al. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. Journal of Clinical Oncology 2004;22:1201–8. [22] Mitchell EP, Hecht JR, Baranda J, et al. Panitumumab activity in metastatic colorectal cancer (mCRC) patients (pts) with low or negative tumor epidermal growth factor receptor (EGFR) levels: an updated analysis. Journal of Clinical Oncology 2007;25(Suppl.) [abstract 4082]. [23] Vallböhmer D, Zhang W, Gordon M, et al. Molecular determinants of cetuximab efficacy. Journal of Clinical Oncology 2005;23: 3536–44. [24] Pérez-Soler R, Chachoua A, Hammond LA, et al. Determinants of tumor response and survival with erlotinib in patients with non-smallcell lung cancer. Journal of Clinical Oncology 2004;22:3238–47.

75

[25] Maughan TS, Adams RA, Smith CG, et al. Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet 2011;377:2103–14. [26] Tol J, Koopman M, Cats A, et al. Chemotherapy, bevacizumab and cetuximab in metastatic colorectal cancer. New England Journal of Medicine 2009;360:563–72. [27] Cunningham D, Ruff P, Rother M, et al. Randomised open-label phase 3 study (PRIME) of panitumumab plus FOLFOX4 compared with FOLFOX4 alone as first-line treatment for metastatic colorectal cancer: analysis of efficacy by epidermal growth factor receptor status and severity of skin toxicity. In: Abstract presented at 12th Australasian gastro-intestinal trials group annual meeting. 2010. [28] Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumours that do not express the epidermal growth factor receptor by immunohistochemistry. Journal of Clinical Oncology 2005;23:1803–10. [29] Lenz H-J, Van Cutsem E, Khambata-Ford S, et al. Multicenter phase II and translational study of cetuximab in metastatic colorectal carcinoma refractory to irinotecan, oxaliplatin, and fluoropyrimidines. Journal of Clinical Oncology 2006;24:4914–21. [30] Shia J, Klimstra DS, Li AR, et al. Epidermal growth factor receptor expression and gene amplification in colorectal carcinoma: an immunohistochemical and chromogenic in situ hybridization study. Modern Pathology 2005;18:1350–6. [31] Goldstein NS, Armin M. Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system. Cancer 2001;92: 1331–46. [32] Moroni M, Veronese S, Benvenuti S, et al. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to anti-EGFR treatment in colorectal cancer: a cohort study. Lancet Oncology 2005;6:279–86. [33] Cappuzzo F, Finochiaro G, Rossi E, et al. EGFR FISH assay predicts for response to cetuximab in chemotherapy refractory colorectal cancer patients. Annals of Oncology 2008;19:717–23. [34] Scartozzi M, Bearzi I, Berardi R, et al. Epidermal growth factor receptor (EGFR) status in primary colorectal tumors does not correlate with EGFR expression in related metastatic sites: implications for treatment with EGFR-monoclonal antibodies. Journal of Clinical Oncology 2004;22:4772–8. [35] Taron M, Ichinose Y, Rosell R, et al. Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinoma. Clinical Cancer Research 2005;11:5878–85. [36] Barber TD, Vogelstein B, Kinzler KW, Velculescu VE. Somatic mutations of EGFR in colorectal cancers and glioblastoma. New England Journal of Medicine 2004;351:2883. [37] Tsuchihashi Z, Khambata-Ford S, Hanna N, Jänne PA. Responsiveness to cetuximab without mutations in EGFR. New England Journal of Medicine 2005;353:208–9. [38] Ooi A, Takehana T, Li X, et al. Protein overexpression and gene amplification of HER-2 and EGFR in colorectal cancers: an immunohistochemical and fluorescent in situ hybridization study. Modern Pathology 2004;17:895–904. [39] Sartore-Bianchi A, Moroni M, Veronese S, et al. Epidermal growth factor receptor gene number and clinical outcome of metastatic colorectal cancer treated with panitumumab. Journal of Clinical Oncology 2007;17:895–904. [40] Molinari F, Martin V, Saletti P, et al. Differing deregulation of EGFR and downstream proteins in primary colorectal cancer and related metastatic sites may be clinically relevant. British Journal of Cancer 2009;100:1087–94. [41] Sauer T, Guren MG, Noren T, Dueland S. Demonstration of EGFR copy loss in colorectal carcinomas by fluorescence in situ

76

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81 hybridization (FISH): a surrogate marker for the sensitivity to specific anti-EGFR therapy? Histopathology 2005;47:560–4. Personeni N, Fieuws S, Piessevaux H, et al. Clinical usefulness of EGFR gene copy number as a predictive marker in colorectal cancer patients treated with cetuximab: a fluorescent in situ hybridization study. Clinical Cancer Research 2008;14:5869–76. Frattini M, Saletti P, Romagnani E, et al. PTEN loss of expression predicts cetuximab efficacy in metastatic colorectal cancer. British Journal of Cancer 2007;97:1139–45. Laurent-Puig P, Cayre A, Manceau G, et al. Analysis of PTEN, BRAF and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer. Journal of Clinical Oncology 2009;27:5924–30. Scartozzi M, Bearzi I, Mandolesi A, et al. Epidermal growth factor receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis. BMC Cancer 2009;9:303. Sartore-Bianchi A, Fieuws S, Veronese S, et al. Standardization of EGFR FISH in colorectal cancer: Results of an international, interlaboratory reproducibility ring study. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3597]. Khambata-Ford S, Garrett CR, Meropol NJ, et al. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. Journal of Clinical Oncology 2007;25:3230–7. Harbison CT, Mauro DJ, Clark EA, et al. In reply to correspondence: KRAS mutation signature in colorectal tumors significantly overlaps with the cetuximab response signature. Journal of Clinical Oncology 2008;26:2230–1. Jacobs B, de Roock W, Piessevaux H, et al. Amphiregulin and epiregulin mRNA expression in primary tumors predicts outcome in metastatic colorectal cancer treated with cetuximab. Journal of Clinical Oncology 2009;27:5068–74. Tabernero J, Cervantes A, Rivera F, et al. Pharmacogenomic and pharmacoproteomic studies of cetuximab in metastatic colorectal cancer: biomarker analysis of a phase I dose-scalation study. Journal of Clinical Oncology 2010;28:1181–9. Saridaki Z, Tzardi M, Papadaki C, et al. Impact of KRAS, BRAF, PIK3CA mutations, PTEN, AREG, EREG expression and skin rash in ≥2 line cetuximab-based therapy of colorectal cancer patients. PLoS One 2011;6:e15980. Jonker DJ, Karapetis C, Harbison C, et al. High epiregulin (EREG) gene expression plus K-ras wild-type (WT) status as predictors of cetuximab benefit in the treatment of advanced colorectal cancer (ACRC): results from NCIC CTG CO.17—a phase III trial of cetuximab versus best supportive care (BSC). Journal of Clinical Oncology 2009;27(Suppl.) [abstract 4016]. Kuramochi H, Nakajima G, Kaneko Y, et al. Amphiregulin and epiregulin mRNA expression in primary colorectal cancer and corresponding liver metastases. Journal of Clinical Oncology 2011;29(Suppl.) [abstract e14023]. Amador ML, Oppenheimer D, Perea S, et al. An epidermal growth factor receptor intron-1 polymorphism mediates response to epidermal growth factor receptor inhibitors. Cancer Research 2004;64:9139–43. Gebhardt F, Zanker KS, Brandt B. Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron-1. Journal of Biological Chemistry 1999;274:13176–80. Han SW, Jeon YK, Lee KH, et al. Intron-1 CA dinucleotide repeat polymorphism and mutations of epidermal growth factor receptor and gefitinib responsiveness in non-small-cell lung cancer. Pharmacogenetics and Genomics 2007;17:313–9. Liu G, Gurubhagavatula S, Zhou W, et al. Epidermal growth factor receptor polymorphisms and clinical outcomes in non-small-cell lung cancer patients treated with gefitinib. The Pharmacogenomics Journal 2008;8:129–38.

[58] Graziano F, Ruzzo A, Loupakis F, et al. Pharmacogenomic profiling for cetuximab plus irinotecan therapy in patients with refractory advanced colorectal cancer. Journal of Clinical Oncology 2008;26:1427–34. [59] Bishop PC, Myers T, Robey R, et al. Differential sensitivity of cancer cells to inhibitors of the epidermal growth factor receptor family. Oncogene 2002;21:119–27. [60] McKay JA, Murray LJ, Curran S, et al. Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumours and lymph node metastases. European Journal of Cancer 2002;38: 2258–64. [61] Atkins D, Reiffen KA, Tegtmeier CL, et al. Immunohistochemical detection of EGFR in paraffined embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. Journal of Histochemistry and Cytochemistry 2004;52:893–901. [62] Pander J, Gelderblom H, Antonini NF. Correlation of FCGR3A and EGFR germline polymorphisms with the efficacy of cetuximab in KRAS wild-type metastatic colorectal cancer. European Journal of Cancer 2010;46:1829–34. [63] Zhang W, Gordon M, Press OA, et al. Cyclin D1 and epidermal growth factor polymorphism associated with survival in patients with advanced colorectal cancer treated with cetuximab. Pharmacogenetics and Genomics 2006;16:475–83. [64] Zhang W, Gordon M, Schultheis AM, et al. FCGR2A and FCGR3A polymorphisms associated with clinical outcome of epidermal growth factor receptor-expressing metastatic colorectal cancer patients treated with single-agent cetuximab. Journal of Clinical Oncology 2007;25:3712–8. [65] Moriai T, Kobrin MS, Hope C, Speck L, Korc M. A variant epidermal growth factor receptor exhibits altered type ␣ transforming growth factor binding and transmembrane signaling. Proceedings of the National Academy of Sciences of the United States of America 1994;91:10217–21. [66] Press OA, Zhang W, Gordon MA, et al. Gender-related survival differences associated with EGFR polymorphisms in metastatic colon cancer. Cancer Research 2008;15:3037–42. [67] Lurje G, Nagashima F, Zhang W, et al. Polymorphisms in cyclooxygenase-2 and epidermal growth factor receptor are associated with progression-free survival independent of K-ras in metastatic colorectal cancer patients treated with single-agent cetuximab. Clinical Cancer Research 2008;14:7884–95. [68] Shahbazi M, Pravica V, Nasreen N, et al. Association between functional polymorphism in EGF gene and malignant melanoma. Lancet 2002;359:397–401. [69] Kobayashi S, Shimamura T, Monti S, et al. Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signalling. Cancer Research 2006;66:11389–98. [70] Bélanger H, Beaulieu P, Moreau C, Labuda D, Hudson TJ, Sinnett D. Functional promoter SNPs in cell cycle checkpoint genes. Human Molecular Genetics 2005;14:2641–8. [71] Weiner GJ. Monoclonal antibody mechanisms of action in cancer. Immunologic Research 2007;39:271–8. [72] Kurai J, Chimuki H, Hashimoto K, et al. Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines. Clinical Cancer Research 2007;13:1552–61. [73] Lopez-Albaitero A, Ferris RL. Immune activation by epidermal growth factor receptor specific monoclonal antibody therapy for head and neck cancer. Archives of Otolaryngology – Head and Neck Surgery 2007;133:1277–81. [74] Van Sorge NM, van der Pol WL, van de Winkel JGJ. Fc␥R polymorphisms: implications for function, disease susceptibility and immunotherapy. Tissue Antigens 2003;61:189–202. [75] Strome SE, Sausville EA, Mann D. A mechanistic perspective of monoclonal antibodies in cancer therapy beyond target-related effect. Oncologist 2007;12:1084–95.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81 [76] Weng W-K, Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. Journal of Clinical Oncology 2003;21:3940–7. [77] Cartron G, Dacheux L, Salles G, et al. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphisms in IgG Fc receptor Fc␥RIIIa gene. Blood 2002;99:754–8. [78] Musolino A, Naldi N, Bortesi B, et al. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. Journal of Clinical Oncology 2008;26:1789–96. [79] Bibeau F, López-Crapèz E, Di Fiore F, et al. Impact of Fc-␥-RIIa–Fc␥-RIIIa polymorphisms and KRAS mutations on the clinical outcome of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan. Journal of Clinical Oncology 2009;27:1122–9. [80] Zhang W, Yang D, Harbison C, et al. FCGR2A H131R and FCGR3A V158F polymorphism status in mCRC patients treated with singleagent cetuximab (IMCL0144 and CA225045) or with second-line irinotecan plus cetuximab (EPIC): a pooled statistical analysis. Journal of Clinical Oncology 2011;29(Suppl.) [abstract e14025]. [81] Páez D, Paré L, Espinosa I, et al. Immunoglobulin G fragment C receptor polymorphisms and KRAS mutation: are they useful biomarkers of clinical outcome in advanced colorectal cancer treated with antiEGFR-based therapy? Cancer Science 2010;101:2048–53. [82] Geva R, Jensen BV, Fountzilas G, et al. An international consortium study in chemorefractory metastatic colorectal cancer (mCRC) patients (pts) to assess the impact of FCGR polymorphisms on cetuximab efficacy. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3528]. [83] Pander J, Gelderblom H, Guchelaar HJ. Pharmacogenomics of EGFR and VEGF inhibition. Drug Discovery Today 2007;12:1054–60. [84] Nimmerjahn F, Ravetch JV. Fc gamma receptors as regulators of immune response. Nature Reviews Immunology 2008;8:34–47. [85] Chen JJ, Lin YC, Yao PL, et al. Tumor-associated macrophages: the double-edged sword in cancer progression. Journal of Clinical Oncology 2005;23:953–64. [86] Dandekar DS, Lokeshwar BL. Inhibition of cyclooxygenase (COX)-2 expression by Tet-inducible COX-2 antisense cDNA in hormonerefractory prostate cancer significantly slows tumor growth and improves efficacy of chemotherapeutic drugs. Clinical Cancer Research 2004;10:8037–47. [87] Dannenberg AJ, Lippman SM, Mann JR, Subbaramaiah K, DuBois RN. Cyclooxygenase-2 and epidermal growth factor receptor: pharmacologic targets for chemoprevention. Journal of Clinical Oncology 2005;23:254–66. [88] Xu K, Shu HK. EGFR activation results in enhanced cyclooxygenase2 expression through p38 mitogen activated protein kinase dependent activation of the Sp1/Sp3 transcription factors in human gliomas. Cancer Research 2007;67:6121–9. [89] Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nature Medicine 2002;8:289–93. [90] Papafili A, Hill MR, Brull DJ, et al. Common promoter variant in cyclooxygenase-2 represses gene expression: evidence of role in acute-phase inflammatory response. Arteriosclerosis, Thrombosis, and Vascular Biology 2002;22:1631–6. [91] Hu Z, Miao X, Ma H, et al. A common polymorphism in the 3 UTR of cyclooxygenase 2/prostaglandin synthase 2 gene and risk of lung cancer in a Chinese population. Lung Cancer 2005;48:11–7. [92] Ambros V. The evolution of our thinking about microRNAs. Nature Medicine 2008;14:1036–40. [93] Visone R, Croce CM. MiRNAs and cancer. American Journal of Pathology 2009;174:1131–8. [94] Schickel R, Boyerinas B, Park SM, Peter ME. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene 2008;27:5959–74.

77

[95] Lee YH, Dutta A. MicroRNAs: small but potent oncogenes or tumor suppressors. Current Opinion in Investigational Drugs 2006;7:560–4. [96] Mishra PJ, Bertino JR. MicroRNA polymorphisms: the future of pharmacogenomics, molecular epidemiology and individualized medicine. Pharmacogenomics 2009;10:399–416. [97] Roush S, Slack FJ. The let-7 family of microRNAs. Trends in Cell Biology 2008;18:505–16. [98] Johnson SM, Grosshans H, Shingara J, et al. RAS is regulated by the let-7 microRNA family. Cell 2005;120:635–47. [99] Chin LJ, Ratner E, Leng S, et al. A SNP in a let-7 microRNA complementary site in the KRAS 3 untranslated region increases non-small cell lung cancer risk. Cancer Research 2008;68:8535–40. [100] Christensen BC, Moyer BJ, Avissar M, et al. A let-7 microRNA binding site polymorphism in the KRAS 3 UTR is associated with reduced survival in oral cancers. Carcinogenesis 2009;30:1003–7. [101] Nakajima G, Hayashi K, Xi Y, et al. Non-coding microRNAs hsa-let7g and hsa-miR-181b are associated with chemoresponse to S-1 in colon cancer. Cancer Genomics Proteomics 2006;3:317–24. [102] Graziano F, Canestrari E, Loupakis F, et al. Genetic modulation of the Let-7 microRNA binding to KRAS 3 -untranslated region and survival of metastatic colorectal cancer patients treated with salvage cetuximab-irinotecan. The Pharmacogenomics Journal 2010;10:458–64. [103] Zhang W, Winder T, Ning Y, et al. A let-7 microRNA-binding site polymorphism in 3 -untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy. Annals of Oncology 2011;22:104–9. [104] Winder T, Zhang A, El-Khoueiry D, et al. Association of a germline variant in the K-ras 3 untranslated region with response and progression-free survival in patients with mCRC treated with singleagent cetuximab (IMCL-0144) or in combination with cetuximab (EPIC) independent of K-ras mutation status. Journal of Clinical Oncology 2009;27(Suppl.) [abstract 4061]. [105] Ruzzo A, Canestrari E, Galluccio N, et al. Role of KRAS let-7 LCS6 SNP in metastatic colorectal cancer patients. Annals of Oncology 2011;22:234–5. [106] Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nature Reviews Cancer 2003;3:459–65. [107] Harari PM. Epidermal growth factor receptor inhibition strategies in oncology. Endocrine-related Cancer 2004;11:689–708. [108] Barault L, Veyrie N, Jooste V, et al. Mutations in the RAS-MAPK, PI(3)K (phosphatidylinositol-3-OH kinase) signaling network correlate with poor survival in a population-based series of colon cancers. International Journal of Cancer 2008;122:2255–9. [109] Benvenuti S, Sartore-Bianchi A, Di Nicolantonio F, et al. Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies. Cancer Research 2007;67:2643–8. [110] Bazan V, Migliavacca M, Zanna I, et al. Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 K-ras mutations are associated with mucinous phenotype. Annals of Oncology 2002;13:1438–46. [111] Bazan V, Agnese V, Corsale S, et al. Specific TP53 and/or Ki-ras mutations as independent predictors of clinical outcome in sporadic colorectal adenocarcinomas: results of a 5-year gruppo oncologico dell’Italia meridionale (GOIM) prospective study. Annals of Oncology 2005;16(Suppl. 4):iv50–5. [112] Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study. British Journal of Cancer 2001;85:692–6. [113] Santini D, Loupakis F, Vincenzi B, et al. High concordance of KRAS status between primary colorectal tumors and related metastatic sites: implications for clinical practice. The Oncologist 2008;13:1270–5. [114] Artale S, Sartore Bianchi A, Veronese SM, et al. Mutations of KRAS and BRAF in primary and matched metastatic sites of colorectal cancer. Journal of Clinical Oncology 2008;26:4217–9.

78

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81

[115] Italiano A, Hostein I, Soubeyran I, et al. KRAS and BRAF mutational status in primary colorectal tumors and related metastatic sites: biological and clinical implications. Annals of Surgical Oncology 2010;17:1429–34. [116] 117-Baldus SE, Scahefer KL, Engers R, et al. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clinical Cancer Research 2010;16:790–9. [117] Kaneko Y, Kuramochi H, Nakajima G, et al. Heterogeneity of Kras status in primary colorectal cancer and corresponding liver metastases. Journal of Clinical Oncology 2011;29(Suppl.) [abstract e14037]. [118] Cejas P, López-Gómez M, Aguayo C, et al. Analysis of the concordance in the EGFR pathway status between primary tumors and related metastases of colorectal cancer patients: implications for cancer therapy. Current Cancer Drug Targets 2012;12:124–31. [119] Knijn N, Mekenkamp LJM, Klomp M, et al. KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients. British Journal of Cancer 2011;104:1020–6. [120] Oliveira C, Velho S, Moutinho C, et al. KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression. Oncogene 2007;26:158–63. [121] Cejas P, López Gómez M, Aguayo C, et al. KRAS mutations in primary colorectal cancer tumors and related metastases: a potential role in prediction of lung metastasis. PLos One 2009;18(4): e8199. [122] Den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Human Mutation 2000;15:7–12. [123] Tsiatis AC, Norris-Kirby A, Rich RG, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. Journal of Molecular Diagnostics 2010;12:425–32. [124] Santini D, Galluzzo S, Gaeta L, et al. Should oncologists be aware in their clinical practice of KRAS molecular analysis? Journal of Clinical Oncology 2011;29:e206–7. [125] Molinari F, Felicioni L, Buscarino M, et al. Increased detection sensitivity for KRAS mutations enhances the prediction of anti-EGFR monoclonal antibody resistance in metastatic colorectal cancer. Clinical Cancer Research 2011;17:4901–14. [126] Jimeno A, Messersmith WA, Hirsch FR, Franklin WA, Eckhardt SG. KRAS mutations and sensitivity to epidermal growth factor receptor inhibitors in colorectal cancer: practical application of patient selection. Journal of Clinical Oncology 2009;27:1130–6. [127] Franhlin WA, Haney J, Sugita M, Bemis L, Jimeno A, Messersmith WA. KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer. Journal of Molecular Diagnostics 2010;12:43–50. [128] Oliner K, Juan T, Suggs S, et al. A comparability study of 5 commercial KRAS tests. Diagnostic Pathology 2010;5:23. [129] Marchetti A, Milella M, Felicioni L, et al. Clinical implications of KRAS mutations in lung cancer patients treated with tyrosine kinase inhibitors: an important role for mutations in minor clones. Neoplasia 2009;11:1084–92. [130] Jung K, Fleischhacker M, Rabien A. Cell-free DNA in the blood as a solid tumor biomarker—a critical appraisal of the literature. Clinica Chimica Acta 2010;411:1611–24. [131] Lecomte T, Ceze N, Dorval E, Laurent-Puig P. Circulating free tumor DNA and colorectal cancer. Gastroenterologie Clinique et Biologique 2010;34:662–81. [132] Spindler K, Pallisgaard N, Vogelius I, Jakobsen A. Quantitative cellfree DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clinical Cancer Research 2012;18:1177–85. [133] Lièvre A, Bachet JB, Boige V, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. Journal of Clinical Oncology 2008;26:374–9.

[134] Di Fiore F, Blanchard F, Charbonnier F, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. British Journal of Cancer 2007;96:1166–9. [135] De Roock, Piessevaux H, De Schutter J, et al. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Annals of Oncology 2008;19:508–15. [136] Sastre J, Aranda E, Grávalos C, et al. First-line single-agent cetuximab in elderly patients with metastatic colorectal cancer. A phase II clinical and molecular study of the Spanish group of digestive tumor therapy (TTD). Critical Reviews in Oncology/Hematology 2011;77: 78–84. [137] Van Cutsem E, Köhne CH, Láng I, et al. Cetuximab plus irinotecan, flourouracil and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. Journal of Clinical Oncology 2011;29:2011–9. [138] Bokemeyer C, Bondarenko I, Hartmann JT, et al. Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study. Annals of Oncology 2011;22:1535–46. [139] Bokemeyer C, Kohne C, Rougier P, et al. Cetuximab with chemotherapy (CT) as first-line treatment for metastatic colorectal cancer (mCRC): analysis of the CRYSTAL and OPUS studies according to KRAS and BRAF mutation status. Journal of Clinical Oncology 2010;28(Suppl.) [abstract 3506]. [140] Tveit K, Guren T, Glimelius B, et al. Phase III trial of cetuximab with continuous or intermittent fluorouracil, leucovorin, and oxaliplatin (Nordic FLOX) versus FLOX alone in first-line treatment of metastatic colorectal cancer: the NORDIC-VII study. Journal of Clinical Oncology 2012. April 02 [Epub ahead of print]. [141] Hecht JR, Mitchell E, Chidiac T, et al. A randomized phase IIIB trial of chemotherapy, bevacizumab, and panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer. Journal of Clinical Oncology 2009;27:672–80. [142] De Roock W, Claes B, Bernasconi D, et al. Effects of KRAS BRAF NRAS and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncology 2010;11:753–62. [143] Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clinical Oncology Provisional Clinical Opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. Journal of Clinical Oncology 2009;27:2091–6. [144] National Comprehensive Cancer Network. Clinical practice guidelines in oncology. http://www.nccn.org/professionals/physician gls/ pdf/colon.pdf. [145] Van Cutsem E, Dicato M, Arber N, et al. Molecular markers and biological targeted therapies in metastatic colorectal cancer: expert opinion and recommendations derived from the 11th ESMO/World Congress on Gastrointestinal Cancer, Barcelona, 2009. Annals of Oncology 2010;21(Suppl. 6), vi1–10. [146] European Medicines Agency: Committee for Medicinal Products for Human Use. May 2008 plenary meeting monthly report. http://www.emea.europa.eu/pdfs/human/press/pr/27923508en.pdf. [147] GenomeWeb Daily News. FDA adds KRAS testing info to Vectibix, Erbitux Labels (Press Release, Genome Web News). http://www.genomeweb.com/dxpgx/fda-adds-kras-testing-infovectibix-erbituxlabels. [148] Wellcome Trust Sanger Institute. Catalogue of somatic mutations in cancer. http://www.sanger.ac.uk/genetics/CGP/cosmic/. [149] Lee CN, Chen HY, Liu HE. Favorable response to erlotinib in a lung adenocarcinoma with both epidermal growth factor receptor exon 19 deletion and K-ras G13D mutations. Journal of Clinical Oncology 2010;28:e111–2.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81 [150] Guerrero S, Casanova I, Farré L, Mazo A, Capellà G, Mangues R. K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogen overexpression. Cancer Research 2000;60:67750–76756. [151] De Roock W, Jonker DJ, Di Nicolantonio F, et al. Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 2010;27:1812–20. [152] Uetake H, Watanabe T, Yoshino T, et al. Clinicopathological features of patients with colorectal cancer among KRAS wild type p.G13D and other mutations: results from a multicenter, cross-sectional study by the Japan Study Group of KRAS mutation in colorectal cancer. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3605]. [153] Tejpar S, Bokemeyer C, Celik I, Schlichting M, Sartorius U, Van Cutsem E. Influence of KRAS G13D mutations on outcome in patients with metastatic colorectal cancer (mCRC) treated with firstline chemotherapy with or without cetuximab. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3511]. [154] Loupakis F, Ruzzo A, Cremolini C, et al. KRAS codon 61,146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. British Journal of Cancer 2009;101:715–21. [155] Andreyev HJN, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten Ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study. Journal of the National Cancer Institute 1998;90:675–84. [156] Hutchins G, Southward K, Handley K, et al. Value of mismatch repair, KRAS and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. Journal of Clinical Oncology 2011;29:1261–70. [157] Esteller M, Gonzáles S, Risques RA, et al. K-ras and p16 aberrations confer poor prognosis in human colorectal cancer. Journal of Clinical Oncology 2001;19:286–8. [158] Ahnen DJ, Feigl P, Quan G, et al. Ki-ras mutation and p53 overexpression predict the clinical behavior of colorectal cancer: a Southwest Oncology Group study. Cancer Research 1998;58:1149–58. [159] Belly RT, Rosenblatt JD, Steinmann M, et al. Detection of mutated K12-ras in histologically negative lymph nodes as an indicator of poor prognosis in stage II colorectal cancer. Clinical Colorectal Cancer 2001;1:110–6. [160] Ogino S, Meyerhardt JA, Irahara N, et al. KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803. Clinical Cancer Research 2009;15:7322–9. [161] Roth A, Tejpar S, Delorenzi M, et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. Journal of Clinical Oncology 2009;28:466–74. [162] Alberts SR, Thibodeau SN, Sargent DJ, et al. Influence of KRAS and BRAF mutational status and rash on disease-free survival (DFS) in patients with resected stage III colon cancer receiving cetuximab (Cmab): Results from NCCTG N0147. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3607]. [163] Richman SD, Seymour MT, Chambers P, et al. KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. Journal of Clinical Oncology 2009;27:5931–7. [164] Haigis KM, Kendall KR, Wang Y, et al. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nature Genetics 2008;40:600–8. [165] Seymour MT, Brown SR, Richman S, et al. Addition of panitumumab to irinotecan: results of PICCOLO, a randomized controlled trial in advanced colorectal cancer (aCRC). Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3523]. [166] Oliner K, Peeters M, Siena S, et al. Evaluation of the gene mutations beyond KRAS as predictive biomarkers or response to panitumumab

[167] [168]

[169]

[170]

[171]

[172]

[173]

[174]

[175]

[176] [177]

[178]

[179]

[180]

[181]

[182]

[183]

[184]

[185]

[186]

79

in a randomized, phase III monotherapy study of metastatic colorectal cancer (mCRC). Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3530]. Sridhar SS, Hedley D, Siu LL. Raf kinase as a target for anticancer therapeutics. Molecular Cancer Therapeutics 2005;4:677–85. Samowitz WS, Sweeney C, Herrick C, et al. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Research 2005;65:6063–9. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatchrepair status. Nature 2002;418:934. Minoo P, Moyer MP, Jass JR. Role of BRAF-V600E in the serrated pathway of colorectal tumourigenesis. Journal of Pathology 2007;212:124–33. French AJ, Sargent DJ, Burgart LJ, et al. Prognostic significance of defective mismatch repair and BRAF V600E in patients with colon cancer. Clinical Cancer Research 2008;14:3408–15. Curtin K, Slattery ML, Samowitz WS. CpG island methylation in colorectal cancer: past, present and future. Pathology Research International 2011;12:902674. Ogino S, Nosho K, Kirkner GJ, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 2009;58:90–6. Ikenoue T, Hikiba Y, Kansi F, et al. Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Research 2003;63:8132–7. Fransén K, Klintenäs M, Osterström A, et al. Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas. Carcinogenesis 2004;25:527–33. Tol J, Nategaal ID, Punt CJ. BRAF mutation in metastatic colorectal cancer. New England Journal of Medicine 2009;361:98–9. Tol J, Dijkstra JR, Klomp M, et al. Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab. European Journal of Cancer 2010;46:1997–2009. Tran B, Kopetz S, Tie J, et al. Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer. Cancer 2011 [Epub ahead of print]. Santini D, Spoto C, Loupakis F, et al. High concordance of BRAF status between primary colorectal tumours and related metastatic sites: implications for clinical practice. Annals of Oncology 2010;21: 1565. Souglakos J, Philips J, Wang R, et al. Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. British Journal of Cancer 2009;101:465–72. Cappuzzo F, Varella-Garcia M, Finocchiaro G, et al. Primary resistance to cetuximab therapy in EGFR FISH positive colorectal cancer patients. British Journal of Cancer 2008;99:83–9. Roth A, Klingbiel D, Yan P, et al. Molecular and clinical determinants of survival following relapse after curative treatment of stage II–III colon cancer (CC): results of the translational study of PETACC 3-EORTC 40993-SAKK 60-00 trial. Journal of Clinical Oncology 2010;28(Suppl.) [abstract 3504]. Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. The emerging mechanisms of isoform-specific PI3K signalling. Nature Reviews Molecular Cell Biology 2010;11:329–41. Nosho K, Kawasaki T, Ohnishi M, et al. PIK3CA mutation in colorectal cancer: relationship with genetic and epigenetic alterations. Neoplasia 2008;10:534–41. Prenen H, De Schutter J, Jacobs B, et al. PIK3CA mutations are not a major determinant of resistance to the epidermal growth factor receptor inhibitor cetuximab in metastatic colorectal cancer. Clinical Cancer Research 2009;15:3184–8. Sartore-Bianchi A, Di Nicolantonio F, Nichelatti M, et al. Multideterminants analysis of molecular alterations for predicting clinical

80

[187] [188]

[189]

[190]

[191]

[192]

[193]

[194]

[195] [196]

[197]

[198]

[199]

[200]

[201]

[202]

[203]

[204]

[205]

[206]

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81 benefit to EGFR-targeted monoclonal antibodies in colorectal cancer. PLoS One 2009;4:7287. Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004;304:554. Zhao L, Vogt PK. Hot-spot mutations in p110alpha of phosphatidylinositol 3-kinase (PI3K): differential interactions with the regulatory subunit p85 and with RAS. Cell Cycle 2010;9:596–600. Zhao L, Vogt PK. Helical domain and kinase domain mutations in p110alpha of phosphatidylinositol 3-kinase induce gain of function by different mechanisms. Proceedings of the National Academy of Sciences of the United States of America 2008;105:2652–7. Jhawer M, Goel S, Wilson AJ, et al. PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to epidermal growth factor receptor inhibitor cetuximab. Cancer Research 2008;68:1953–61. Samuels Y, Díaz LA, Schmidt-Kittler O, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005;7:561–73. Kato S, Iida S, Higuchi T, et al. PIK3CA mutation is predictive of poor survival in patients with colorectal cancer. International Journal of Cancer 2007;121:1771–8. Ogino S, Katsuhiko N, Gregory J, et al. PIK3CA mutation is associated with poor prognosis among patients with curatively resected colon cancer. Journal of Clinical Oncology 2009;27:1477–84. Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nature Reviews Cancer 2006;6:184–92. Di Cristofano A, Pandolfi PP. The multiple roles of PTEN in tumor suppression. Cell 2000;100:387–90. Goel A, Arnold CN, Niedwiecki D, et al. Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instabilityhigh sporadic colorectal cancers. Cancer Research 2004;64: 3014–21. Loupakis F, Pollina L, Stasi I, et al. PTEN expression and KRAS mutations on primary tumours and metastases in the prediction of benefit from cetuximab plus irinotecan for patients with metastatic colorectal cancer. Journal of Clinical Oncology 2009;27:2622–9. Mellinghoff IK, Wang MY, Vivanco I, et al. Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. New England Journal of Medicine 2005;353:2012–24. Nagata Y, Lan KH, Zhou X, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:117–27. Wang L, Zhang Q, Zhang J, et al. PI3K pathway activation results in low efficacy of both trastuzumab and lapatinib. BMC Cancer 2011;11:248. Razis E, Briasoulis E, Vrettou E, et al. Potential value of PTEN in predicting cetuximab response in colorectal cancer: an exploratory study. BMC Cancer 2008;13(8):234. Finocchiaro G, Cappuzzo F, Janne PA, et al. EGFR, HER2 and Kras as predictive factors for cetuximab sensitivity in colorectal cancer. Journal of Clinical Oncology 2007;25(Suppl.) [abstract 4021]. Martin V, Sacconi A, Landi L, et al. HER2 gene copy number gain in chemorefractory metastatic colorectal cancer (mCRC) patients treated with cetuximab: results of an International Consortium. Tumori 2011;11(Suppl. i):S2–3. Martin V, Sacconi A, Landi L, et al. An International Consortium in chemo-refractory metastatic colorectal cancer patients shows cetuximab efficacy in patients harboring HER2 gene copy number. Eur J Cancer 2011;47(Suppl. 1):S392 [abstr 6015]. Jiang W, Hiscox S, Matsumoto K, Nakamura T. Hepatocyte growth factor/Scatter factor, its molecular, cellular and clinical implications in cancer. Critical Reviews in Oncology/Hematology 1999;29: 209–48. Long IS, Han K, Li M, et al. Met receptor overexpression and oncogenic Ki-ras mutation cooperate to enhance tumorigenicity of colon cancer cells in vivo. Molecular Cancer Research 2003;1:393–401.

[207] Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007;316:1039–43. [208] Fukuura T, Miki C, Inoue T, Matsumoto K, Suzuki H. Serum hepatocyte growth factor as an index of disease status of patients with colorectal carcinoma. British Journal of Cancer 1998;78:454–9. [209] Inno A, Di Salvatore M, Cenci T, et al. Is there a role for IGF1R and c-Met pathways in resistance to cetuximab in metastatic colorectal cancer? Clinical Colorectal Cancer 2011;10:325–32. [210] Khandwala HM, Mc Cutcheon IE, Flybjerg A, Friend KE. The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocrine Reviews 2000;21:215–44. [211] Chakravarti A, Loeffler JS, Dyson NJ. Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation on phosphoinositide 3-kinase signaling. Cancer Research 2002;62:200–7. [212] Koda M, Reszec J, Sulkowska M, Kanczuga-Koda L, Sulkowski S. Expression of the insulin-like growth factor-I receptor and proapoptotic Bax and Bak proteins in human colorectal cancer. Annals of the New York Academy of Sciences 2004;1030:377–83. [213] Morgillo F, Kim WY, Kim ES, Ciardiello F, Hong WK, Lee HY. Implication of the insulin-like growth factor-1R pathway in the resistance of non-small cell lung cancer cells to treatment with gefitinib. Clinical Cancer Research 2007;13:2795–803. [214] Mounawar M, Mukeria A, Le Calvez F, et al. Patterns of EGFR, HER2, TP53, and KRAS mutations of p14arf expression in non-small cell lung cancers in relation to smoking history. Cancer Research 2007;67:5667–72. [215] Kim JS, Lee C, Bonifant CL, Ressom H, Waldan T. Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA. Molecular and Cellular Biology 2007;27:662–77. [216] Oden-Gangloff A, Di Fiore F, Bibeau F, et al. TP53 mutations predict disease control in metastatic colorectal cancer treated with cetuximabbased chemotherapy. British Journal of Cancer 2009;100:1330–5. [217] Passardi A, Ulivi P, Valgiusti M, et al. The role of KRAS, BRAF, and PI3K mutations as markers of resistance to cetuximab in metastatic colorectal cancer. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3603]. [218] Simon I, Tian S, Moreno V, et al. The role of activating mutations of KRAS, BRAF, and PIK3CA pathway convergence at the transcriptional level and prediction of treatment response to cetuximab in colorectal cancer. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3534]. [219] Marchetti A, Gasparini G. K-ras mutations and cetuximab in colorectal cancer. New England Journal of Medicine 2009;360:833–4. [220] Piessevaux H, Buyse M, de Roock W, et al. Radiological tumor size decrease at week 6 is a potent predictor of outcome in chemorefractory metastatic colorectal cancer treated with cetuximab (BOND trial). Annals of Oncology 2008;19:508–15. [221] Piessevaux H, Van Cutsem E, Bokemeyer C, et al. Early tumor shrinkage and long-term outcome in metastatic colorectal cancer (mCRC): assessment of predictive utility across treatment arms in the CRYSTAL and OPUS studies. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3572]. [222] Van Hazel GA, Tu D, Tebbutt NC, et al. Early change in tumor size from waterfall plot analysis and RECIST response as predictor of overall survival (OS) in advanced, chemotherapy-refractory colorectal cancer (ACRC): NCIC CTG/AGIT CO.17 study. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3602]. [223] Karrison TG, Maitland ML, Stadler WM, et al. Design of phase II cancer trials using a continuous endpoint of change in tumor size: application to a study of sorafenib and erlotinib in non small-cell lung cancer. Journal of the National Cancer Institute 2007;99:1455–61. [224] Rubinstein L, Dancey J, Korn E, et al. Early average change in tumor size in a phase 2 trial: efficient endpoint or false promise? Journal of the National Cancer Institute 2007;99:1422–3.

A. Custodio, J. Feliu / Critical Reviews in Oncology/Hematology 85 (2013) 45–81 [225] Berlin J, Van Cutsem E, Peeters M, et al. Predictive value of skin toxicity severity for response to panitumumab in patients with metastatic colorectal cancer (mCRC): a pooled analysis of five clinical trials. Journal of Clinical Oncology 2007;25(Suppl.) [abstract 4134]. [226] Peeters M, Siena S, Van Cutsem E, et al. Association of progressionfree survival, overall survival, and patient-reported outcomes by skin toxicity and KRAS status in patients receiving panitumumab monotherapy. Cancer 2009;115:1544–54. [227] O’Callaghan CJ, Tu D, Karapetis CS, et al. The relationship between the development of rash and clinical and health-related quality of life outcomes by KRAS mutation status in patients with colorectal cancer treated with cetuximab in NCIC CTG CO.17. Journal of Clinical Oncology 2011;29(Suppl.) [abstract 3588]. [228] Tejpar S, Humblet Y, Vermorken JB, et al. Relationship of efficacy with KRAS status (wild type versus mutant) in patients with irinotecan-refractory metastatic colorectal cancer (mCRC), treated with irinotecan (q2w) and scalating doses of cetuximab (q1w): the EVEREST experience (preliminary data). Journal of Clinical Oncology 2008;26(Suppl.) [abstract 4001]. [229] Pérez-Soler R, Saltz L. Cutaneous adverse effects with HER1/EGFRtargeted agents: is there a silver lining? Journal of Clinical Oncology 2005;23:5235–46. [230] Personeni N, Hendlisz A, Gallez J, et al. Correlation between the response to cetuximab alone or in combination with irinotecan and the activated/phosphorylated epidermal growth factor receptor in metastatic colorectal cancer. Seminars in Oncology 2005;32(Suppl. 9):S59–62. [231] Wolf FI, Maier JA, Nasulewicz A, et al. Magnesium and neoplasia: from carcinogenesis to tumor growth and progression or treatment. Archives of Biochemistry and Biophysics 2007;458: 24–32. [232] Rubin H. Central roles of Mg2+ and MgATP2− in the regulation of protein synthesis and cell proliferation: significance for neoplastic transformation. Advances in Cancer Research 2005;93:1–58. [233] Rubin H. The logic of the membrane, magnesium, mitosis (MMM) model for the regulation of animal cell proliferation. Archives of Biochemistry and Biophysics 2007;458:16–23. [234] Whang R, Hampton EM, Whang DD. Magnesium homeostasis and clinical disorders of magnesium deficiency. Annals of Pharmacotherapy 1994;28:220–6. [235] Groenestege WM, Thebault S, van der Wijst J, et al. Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia. Journal of Clinical Investigation 2007;117:2260–7. [236] Schrag D, Chung KY, Flombaum C, Saltz L. Cetuximab therapy and symptomatic hypogmagnesemia. Journal of the National Cancer Institute 2005;97:1221–4. [237] Fakih MG, Wilding G, Lombardo J. Cetuximab-induced hypomagnesemia in patients with colorectal cancer. Clinical Colorectal Cancer 2006;6:152–6. [238] Tejpar S, Piessevaux H, Claes K, et al. Magnesium wasting associated with epidermal-growth-factor receptor-targeting antibodies in colorectal cancer: a prospective study. Lancet Oncology 2007;8:387–94. [239] Petrelli F, Borgonovo K, Cabiddu M, Ghilardi M, Barni S. Risk of anti-EGFR monoclonal antibody-related hypomagnesemia: systematic review and pooled analysis of randomized studies. Expert Opinion on Drug Safety 2012;11:S9–19. [240] Vincenzi B, Santini D, Galluzzo S, et al. Early magnesium reduction in advanced colorectal cancer patients treated with cetuximab plus irinotecan as predictive factor of efficacy and outcome. Clinical Cancer Research 2008;14:4219–24. [241] Vincenzi B, Galluzzo S, Santini S, et al. Early magnesium modifications as a surrogate marker of efficacy of cetuximab-based anticancer treatment in KRAS wild-type advanced colorectal cancer patients. Annals of Oncology 2011;22:1141–6. [242] Vickers MM, Karapetis CS, Tu D, et al. The influence of hypomagnesemia (hMg) on overall survival (OS) in a phase III randomized study of cetuximab (CET) plus best supportive care (BSC) versus

[243]

[244]

[245]

[246]

[247]

[248]

[249]

[250]

81

BSC: NCIC CTG/AGITG CO.17. Journal of Clinical Oncology 2011;(Suppl.) [abstract 3601]. Rizzo S, Bronte G, Fanale D, et al. Prognostic vs predictive molecular biomarkers in colorectal cancer: is KRAS and BRAF wild type status required for anti-EGFR therapy? Cancer Treatment Reviews 2010;36S3:S56–61. Montagut C, Dalmases A, Bellosino B, et al. Identification of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer. Nature Medicine 2012;18:221–3. Saif MW, Kaley K, Chu E, Copur MS. Safety and efficacy of panitumumab therapy after progression with cetuximab: experiences at two institutions. Clinical Colorectal Cancer 2010;9:315–8. Kopetz S, Desai J, Chan E, et al. PLX4032 in metastatic colorectal cancer patients with mutant BRAF tumors. Journal of Clinical Oncology 2010;28(Suppl.) [abstract 3534]. Hoeflich KP, Herter S, Tien J, et al. Antitumor efficacy of the novel RAF inhibitor GDC-08798 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogenactivated protein kinase pathway suppression. Cancer Research 2009;69:3042–51. Markman B, Atzori F, Pérez-García J, Tabernero J, Baselga J. Status of PI3K inhibition and biomarkers development in cancer therapeutics. Annals of Oncology 2010;21:683–91. Di Nicolantonio F, Arena S, Tabernero J, et al. Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus. Journal of Clinical Investigation 2010;120:2858–66. Zhang YJ, Tian XQ, Sun DF, Zhao SL, Xiong H, Fang JY. Combined inhibition of MEK and mTOR signaling inhibits initiation and progression of colorectal cancer. Cancer Investigation 2009;27:273–85.

Biographies Ana Custodio M.D. is currently a staff physician of the Medical Oncology Department at La Paz University Hospital in Madrid, Spain. Dr. Custodioˇıs interests include research in gastrointestinal cancer, lung cancer and neuroendocrine tumours, focusing on molecular biology and translational oncology. She is author and co-author of several papers and book chapters in these fields. Dr. Custodio is a member of several scientific societies including the Spanish Society of Medical Oncology (SEOM), the Spanish Lung Cancer Group (GECP) and the Spanish Group of Neuroendocrine Tumours (GETNE). Ana Custodio is now also a member of the European Society of Medical Oncology (ESMO). Jaime Feliu M.D., Ph.D. is currently Head of the Medical Oncology Department at La Paz University Hospital in Madrid. He is also Lecturer at the School of Medicine in the Autonoma University in Madrid and Director of the Palliative and Supportive Care Master in the Autonoma University in Madrid. Dr. Feliuˇıs main research interests include basic and clinical research in gastrointestinal cancer, focusing on molecular-targeted therapies and molecular biology. He is author of more than 250 national and international papers, books and book chapters in this field. Dr. Feliu is also a member of several scientific societies including the American Society of Clinical Oncology (ASCO), the Spanish Society of Medical Oncology (SEOM) and the Multidisciplinary Spanish Group of Gastrointestinal Cancer (GEMCAD).