Introduction
Despite the introduction of new treatments, the 5-year survival rate for metastatic colorectal cancer (mCRC) remains below 10% [
1]. Cetuximab, an IgG1 monoclonal antibody (MoAb) targeting epidermal growth factor receptor (EGFR), has proven to be effective in providing clinical benefit in approximately 10% to 20% of patients [
2‐
4]. EGFR is a transmembrane tyrosine kinase receptor that, following ligand binding, triggers two main signaling pathways: the RAS-RAF-MAPK pathway which is involved in cell proliferation, and the PI3K-PTEN-AKT pathway which controls cell survival and motility [
5].
While the presence of a
KRAS mutation permits identification of tumors that are insensitive to these treatments, only less than half of patients with a
KRAS wild type (wt) tumor will benefit from treatments, suggesting a role for additional mechanisms of resistance [
6‐
10]. It thus appears necessary to better define the subpopulation of patients who truly benefit from cetuximab. One approach to resolving this question may be the application of pharmacogenetics, as recently reviewed by Coate and co-workers [
11]. Yet, gene polymorphisms may affect pharmacodynamics of anti-EGFR therapies such as cetuximab, by introducing inter-patient variability at the level of the EGFR target itself, the EGF ligand, as well as in the immunological mechanism called antibody-dependent cellular cytotoxicity (ADCC).
Four functional
EGFR variants have been associated with
EGFR regulation [
12‐
14]: a (CA)n repeat polymorphism in
EGFR intron 1, a G > A single nucleotide polymorphism (SNP) at codon 497, and two SNPs -216 G > T and -191C > A located in the promoter region. Modulation of the EGFR ligand EGF and of the downstream EGFR signaling, including the cyclin-D1 gene (
CCND1), may also play a role in modulating cetuximab activity. Functional variants have been described in the
EGF 5'-untranslated region (
EGF 61 G > A) [
15,
16], and in the exon 4 of the
CCND1 gene (870A > G) [
17,
18]. The ADCC, mediated through Fc receptors (FcγR) carried by immune cells such as macrophages and natural killer cells, plays an important role in the antitumor effect of IgG1 antibodies, such as cetuximab [
19,
20]. The effectiveness of ADCC may depend on the degree of activation of FcγR and constitutional polymorphisms have been demonstrated on genes encoding for these receptors: a histidine (H)/arginine (R) polymorphism at position 131 for
FCGR2A and a valine (V)/phenylalanine (F) polymorphism at position 158 for
FCGR3A [
21].
In the present study, we investigated possible associations between these genetic variants and clinical outcomes of advanced CRC patients treated with cetuximab. Clinical end points were skin toxicity, clinical response, time to progression (TTP) and overall survival (OS).
Discussion
Cetuximab has shown efficacy in patients with metastatic colorectal cancer in several phase II trials leading, in 2004, to FDA approval for the treatment of irinotecan-refractory metastatic colorectal cancer. Several retrospective and prospective studies have clearly demonstrated that
KRAS mutation confers resistance to these patients [
6‐
10,
23,
24] but the complete mechanism of cetuximab sensitivity remains only partially understood. The present study was conducted in patients receiving cetuximab before KRas-mutation testing was introduced as a requirement. As expected, no response rate was observed in mutated
KRAS patients
vs 15.2% in wt tumors, even though the difference did not reach significance. We presently analyzed 8 gene polymorphisms involving 5 relevant candidate genes potentially related to the pharmacodynamics of cetuximab, namely
EGFR,
EGF,
CCND1,
FCGR2A and
FCGR3A, on 58 CRC patients receiving cetuximab-based therapy. Statistical analyses were conducted in the whole set of patients, as well as in the sub-group of 34 patients with wt KRas tumors, so as to reflect the current cetuximab-treated population.
Numerous studies have reported a relationship between favorable outcome of cetuximab-treated patients and related skin toxicity [
2,
3,
25]. Accordingly, present data show a higher response rate in patients developing grade 2-3 cutaneous toxicity as compared to patients with grade 0-1 (14%
vs 5%, respectively), even though not significant. Present results also show a tendency for an association between intron 1
EGFR polymorphism and cetuximab-related skin toxicity: the incidence of grade 2-3 toxicity was 1.5-fold greater in patients bearing short CA-repeats in intron 1 of
EGFR gene (CA sum ≤ 35) as compared to others (p = 0.058, Figure
1). This observation concords well with previous studies by Amador et al. [
26] and Graziani et al. [
27] reporting that patients developing cutaneous rash after anti-EGFR therapies presented shorter CA-repeats in intron 1 of
EGFR gene as compared to patients who did not develop rash. Experimental studies have reported an inverse correlation between the number of CA-repeats in the intron 1 of the
EGFR gene and
EGFR gene transcription [
28‐
30]. It can thus be hypothesized that elevated ubiquitous EGFR expression (including skin and tumor) renders the cells more susceptible to anti-EGFR effects.
In addition to the influence of intron 1 polymorphism on
EGFR gene transcription,
EGFR gene presents two functional polymorphisms in the promoter region: the -216G/T polymorphism located in a Sp1 binding site [
31,
32], and the -191C/A polymorphism located 4 bp upstream of a transcription initiation site [
31]. These two SNPs may thus have an impact on
EGFR gene regulation. Present data obtained on patients with wt KRas tumors show a significantly longer TTP in homozygous
EGFR -191CC patients relative to other patients (p = 0.030, univariate analysis). However, this genotype was not retained in a multivariate analysis.
Cyclin D1 is a downstream effector of EGFR signaling that regulates cell cycle. The
CCND1 A870G gene polymorphism affects the splice donor site at the exon 4/intron 4 boundary, resulting in two different mRNA transcripts (a and b) [
33]. Both the A allele and the G allele can encode these two transcripts. However, the A allele preferentially encodes transcript b, which results in a longer half-life cyclin D1 protein [
33]. The impact of
CCND1 A870G polymorphism on cancer progression has been studied in head and neck cancer patients, with conflicting results [
34,
35]. In our study, patients homozygous for the
CCDN1 870AA genotype had a significantly greater response rate than AG or GG patients, both in the whole population and in patients with a wt KRas tumor (75.0%
vs 7.1%, respectively in wt KRas patients). In addition, patients with the
CCND1 870AA genotype had a significantly longer median TTP than GG patients, with AG patients having an intermediary TTP, both in the whole population and in patients with a wt KRas tumor (median TTP were 7.9, 3.0 and 2.6 months, in AA, AG and GG wt KRas patients, respectively). Of note, in patients with wt KRas tumors,
CCND1 polymorphism also influenced specific survival, with a significantly shorter survival in GG patients. The positive influences of
CCND1 870A allele are thus consistent with one another, even though they do not concord with the sole published study having analyzed the impact of
CCND1 A870G polymorphism on the outcome of advanced colorectal cancer patients receiving cetuximab therapy [
36]. In this latter study, conducted on a limited sample of 39 patients, the 870 G allele had a favorable impact on survival [
36].
In addition to direct anti-EGFR effect, IgG1 mAbs such as cetuximab mediate anti-tumor effects by the ADCC mechanism. Fragment C of the mAb binds to the Fc receptors (FcR) carried by immune cells, thus triggering tumor cell lysis. Functional polymorphisms on two FcR genes (
FCGR2A, FCGR3A) affecting the affinity of FcR for fragment C have been identified [
37,
38]. These polymorphisms may thus influence ADCC efficiency [
39,
40]. Even though some studies have reported significant associations between these polymorphisms and clinical efficacy of rituximab [
41], trastuzumab [
42] or cetuximab [
43,
44], data conflict regarding which alleles are linked to favorable patient outcome. In the present study, we report a significant influence of
FCGR3A F158V polymorphism on survival both in the whole population and in patients with a wt KRas tumor, with VV patients presenting a dramatically shorter survival. The favorable influence of the
FCGR3A 158F allele was also reported in a study by Zhang et al. [
43] and a study by Pander et al. [
45], but not in the study from Bibeau et al. [
44]. These discrepancies related to the impact of
FCGR3A 158F/V polymorphism on cetuximab efficacy are difficult to account for but could be due to the relatively limited sample size of these studies.
We observed that none of the 13 patients pre-treated with bevacizumab had a response to cetuximab and that this subgroup had a significantly decreased specific survival as compared with non-pretreated patients (9.8 months vs 4.9 months, p = 0.018). This difference remains statistically significant in a multivariate analysis adjusted for age, sex, PS status and KRas status (data not shown). This negative influence of bevacizumab pre-treatment cannot be imputed to patient characteristics which were not significantly different between bevacizumab pretreated patients and non-pretreated patients, although it must be noted that 42% of bevacizumab pretreated patients carried KRAS mutated tumors vs 29% in non-pretreated patients (p = 0.48). Importantly, a multivariate analysis including bevacizumab pretreatment revealed that CCND1 A870G and FCGR3A F158V polymorphisms both remained significant independent predictors of patient survival (whole population and KRas wt tumors).
The retrospective design of this study, conducted on a relatively small number of patients, may place intrinsic limitations on the present original data. However, results obtained in the sub-group of wt KRas patients, corresponding to the current cetuximab-treated population, clearly suggest that CCND1 A870G polymorphism may be used as an additional marker for predicting cetuximab efficacy, TTP and overall survival. Of note, FCGR3A F158V polymorphism and CCND1 A870G polymorphism were significant independent predictors of overall survival in patients with wt KRas tumors. Such promising observations deserve further confirmation in a prospective study conducted on a larger population of CRC patients receiving cetuximab-based therapy.
Authors' contributions
Manuscript writing: LD, GM and MCEG
Review: LD, MCEG, GM
Investigators: LD, EN, JFS, MG
All authors read and approved the final manuscript