Clinical data support the use of EGFR mAbs in patients with metastatic colorectal cancer (mCRC) with wild-type RAS status. This notion, hypothesized in the review article by Camp, Ellis, and colleagues in the January 1, 2005, issue of Clinical Cancer Research, serves as an example of the successful application of basic science principles to clinical practice. The exclusion of patients with mCRC with Ras-mutated tumors from therapy with EGFR mAbs has led to improved outcomes while sparing patients unnecessary and potentially harmful therapy. Clin Cancer Res; 21(16); 3578–80. ©2015 AACR.

See related article by Camp et al., Clin Cancer Res 2005;11(1): January 1, 2005;397–405

The therapeutic potential of mAbs targeting the EGFR was established by promising preclinical studies and subsequent early-phase clinical trials in patients with metastatic colorectal cancer (mCRC; ref. 1). Ultimately, in 2004, the FDA approved the human–mouse chimeric EGFR mAb, cetuximab, for use in chemotherapy-refractory patients with mCRC. Similarly, panitumumab, a fully human EGFR mAb, was FDA approved for similar indications in 2006, and ultimately both drugs were subsequently approved in the first-line setting in combination with chemotherapy. However, the earliest clinical trials investigating EGFR mAbs in mCRC demonstrated that these agents only provided modest benefit when used in combination with chemotherapy. Around this time, a concerted effort to identify predictive markers for EGFR-targeted therapies was undertaken simultaneous to advances in genomic sequencing.

As principal investigator of a laboratory, the senior author of this article (L.M. Ellis) learned early on in his career that the best way to educate a trainee on a topic was to have this individual write a review article. This strategy exposes trainees to the entire field of the topic of interest, requiring a critical evaluation of the literature, and identifying knowledge gaps. In 2004, the first author of this article (E.R. Camp) entered the laboratory and was given the task of writing a review on resistance to EGFR-targeted therapies. At this time, EGFR mutations in lung cancer were being identified as markers of sensitivity to EGFR tyrosine kinase receptor inhibitors (2). Our laboratory had been interested in growth factor receptors and downstream intracellular signaling, with a focus on Src in collaboration with G. Gallick at The University of Texas MD Anderson Cancer Center (Houston, TX). In reviewing the literature and applying our own knowledge of cell signaling, the general topics of this review in Clinical Cancer Research (CCR) focused on the following as resistance factors to EGFR-targeted therapies: (i) the presence of redundant tyrosine kinase receptors; (ii) increased angiogenic signaling; (iii) existence of specific EGFR mutations; and (iv) constitutive activation of downstream mediators (3). This last proposed mechanism of resistance was not based on actual data, but a simple hypothesis: If an important intracellular pathway is constitutively activated by a mutation, then blocking signaling at the cell surface would be ineffective.

In our original schematic drawing describing this hypothesis, we listed several activated downstream intermediates as potential resistance pathways. One, in particular, has demonstrated clinical utility in selecting patients with mCRC for EGFR mAb therapy. The therapeutic relevance of downstream RAS mutations on EGFR mAb efficacy in patients with mCRC highlights the importance of understanding the molecular basis of malignant disease to improve personalized medicine. We recognize that this concept is the basis of many preclinical and clinical studies, but at the time of writing this review, there were no publications that had discussed or hypothesized that mutated RAS or other constitutively activated signaling intermediates could serve as markers of resistance (or even detriment) in patients with mCRC. However, at that time, several studies were examining the role of mutated RAS as a resistance marker for EGFR tyrosine kinase inhibitors in lung cancer (4, 5); other investigators had hypothesized that RAS could be a resistance marker for EGFR-targeted therapies, and this work occurred simultaneous to, or shortly after, our publication. Soon after the publication of our review in January 2005, several studies were examining the role of KRAS in EGFR mAb resistance, suggesting that others had this idea and had already begun studies. However, we have not identified any publication prior to ours in CCR addressing this topic. Of course, since then we have learned that additional RAS mutations may render patients resistant to EGFR mAb therapy (6).

In 2006, a retrospective study of 30 patients with mCRC treated with cetuximab (mostly combined with chemotherapy) was the first clinical study to correlate KRAS mutational status with resistance to EGFR mAb therapy (7). In this small study, patients with tumors harboring a KRAS mutation showed a 0% response rate. Overall survival (OS) in cetuximab-treated patients was significantly shorter in those with KRAS mutations in comparison with patients with wild-type (WT) KRAS (6.9 vs. 16.3 months; P = 0.016). A subsequent investigation confirmed the clinical significance of KRAS mutations as predictive markers for cetuximab or panitumumab therapy for patients with mCRC (8). Furthermore, this study was the first to demonstrate a causal relationship between activating KRAS mutation (Gly12Val) and decreased in vitro response to cetuximab therapy in human colon cancer cell lines.

A landmark retrospective analysis of a prospective randomized trial in 392 chemotherapy-refractory patients with mCRC comparing cetuximab treatment with best supportive care (BSC) solidified the significance of KRAS mutational status in clinical practice (9). OS in patients with WT KRAS tumors was significantly longer in those treated with cetuximab than in those who received BSC (9.5 vs. 4.8 months; P < 0.001). In contrast, patients with mutated KRAS tumors experienced an equivalent OS between cetuximab therapy and BSC. A larger concern was that results from the randomized phase II OPUS trial comparing first-line FOLFOX4 with or without cetuximab in patients with mCRC suggested that the addition of an EGFR mAb might actually have a detrimental effect on outcomes in mCRC patients with mutated KRAS tumors (10) and other studies have confirmed these findings (6). These initial translational investigations laid the foundation for personalized molecular medicine in mCRC by demonstrating the predictive capability of KRAS mutational status for improving patient selection and outcomes for EGFR mAb therapy. In response to the growing evidence supporting the predictive implications of KRAS status for EGFR mAb therapy, the FDA restricted the use of cetuximab and panitumumab to patients with WT KRAS mCRC in 2009.

The initial studies investigating the role of KRAS mutation status on the efficacy of cetuximab in patients with mCRC focused on the most prevalent KRAS mutations involving codons 12 and 13 in exon 2, which are present in approximately 40% of patients with mCRC. Even though multiple studies confirmed that exon 2 mutated KRAS rendered patients with mCRC resistant to EGFR mAb therapy, EGFR mAbs were found to be effective in some, but not all, patients with KRAS WT tumors; this suggested that alternative molecular abnormalities might be influencing therapeutic response. Extending this line of investigation further, an expanded or “all-RAS” mutation analysis including all KRAS and NRAS mutations has been proposed for identifying patients with mCRC who would benefit from EGFR mAb therapy. The first study to identify the negative predictive value of driver mutations, including NRAS, reported on a large retrospective cohort of chemotherapy-refractory patients with mCRC who were subsequently treated with irinotecan plus cetuximab (11). In this study, assessment of data from patients with tumors harboring driver mutations beyond KRAS identified even more patients unlikely to respond to EGFR mAbs, thus improving response rates in those patients with tumors without these mutations. Subsequently, a retrospective analysis of 1,060 patient tumor samples from the randomized PRIME trial in patients with mCRC treated with first-line FOLFOX4 with or without panitumumab highlighted the significance of an expanded RAS assessment to predict response for EGFR mAb-containing regimens (6). In this trial, 52% of the analyzed tumors were identified as RAS mutated, including any KRAS or NRAS mutations in exon 2, 3, or 4. Of the original 641 patients categorized as having exon 2 WT KRAS tumors, an additional 108 (17%) had an alternative RAS mutation. Patients with tumors expressing any RAS mutation who received panitumumab with FOLFOX4 experienced a significantly decreased progression-free survival (PFS) as well as OS compared with patients with WT RAS tumors. The survival outcomes were similar between exon 2 KRAS mutations and tumors with alternative RAS mutations, suggesting that any RAS mutation equally negates the therapeutic benefit achieved with EGFR mAbs. However, the numbers of patients with these less frequent RAS mutations limit the ability to firmly state that they are indeed markers of resistance. Similar to the findings from the OPUS trial, patients with any RAS mutation actually experienced a significantly decreased PFS and OS with the addition of panitumumab to FOLFOX4, reinforcing the concern that EGFR mAbs may actually be detrimental in this setting. Similarly, a recent meta-analysis of nine randomized, controlled trials with EGFR mAbs for mCRC patients evaluating RAS mutational status further solidified the predictive impact of an expanded RAS mutational profile on PFS as well as OS (12). In general, expanded RAS analysis may increase the percentage of patients ineligible for EGFR mAb therapy by approximately 15% compared with KRAS exon 2 mutations alone. Based on the potential detrimental impact of any RAS mutation in patients receiving EGFR mAb therapy, the FDA endorsed an extended RAS assessment for EGFR mAb therapy in 2013 and, subsequently, the National Comprehensive Cancer Network (NCCN) published clinical guidelines recommending prospective expanded RAS mutational assessment for all mCRC patients considered candidates for EGFR mAb therapy.

The failure of efficacy with EGFR mAbs in a statistically significant number of patients with WT KRAS tumors fueled further investigations evaluating alternative downstream mediators of EGFR. The first report to extend the molecular analysis beyond RAS in patients with mCRC investigated the role of mutated BRAFv600e as a marker of cetuximab resistance in WT KRAS tumors (13). BRAF is a downstream mediator in the RAS pathway, but, of course, can be constitutively activated as it is mutated in approximately 5% to 10% of colorectal cancers. In this study, patients with tumors expressing the BRAFv600e mutation experienced a shorter PFS and OS in response to cetuximab compared with WT BRAF tumors. Although initial reports suggested that BRAF might be a predictive marker for EGFR mAbs, subsequent studies have not uniformly confirmed this association. For example, in the retrospective analysis of the PRIME trial, patients with tumors expressing the BRAFv600e mutation showed an equally poor PFS and OS in response to FOLFOX4 alone compared with the addition of panitumumab (6). In contrast, the randomized PICCOLO trial investigating the role of irinotecan plus panitumumab in fluorouracil-refractory patients with mCRC demonstrated a significantly worse OS if patients' tumors harbored a BRAF mutation. Despite the inconsistent results regarding BRAF as an EGFR mAb predictive marker, BRAF mutational status appears to have strong negative prognostic significance, as highlighted by results from several trials. Compared with patients with WT RAS/BRAF tumors, BRAFv600e mutations conferred an approximately 50% reduced PFS and OS in both arms of the trial. Given the aggressive biology associated with BRAFv600e mutation, the actual impact on the therapeutic efficacy of EGFR mAbs may be difficult to determine. Similar to BRAF, PIK3CA mutations and loss of PTEN have shown an inconsistent impact on the therapeutic efficacy of EGFR mAbs. Currently, testing for BRAF and PIK3CA/PTEN is not recommended for EGFR mAb mCRC patient selection, although these mutations/alterations may be beneficial in determining prognosis and eligibility for clinical trials.

The improved objective response with the addition of EGFR mAbs observed in “all-RAS” WT tumors highlights the potential of molecularly driven personalized cancer therapy. The role for prospective RAS mutational analysis for cetuximab selection has been explored in recent cooperative group clinical trials that analyzed a comprehensive (“all-RAS”) molecular profile to assess the benefit of using cetuximab in combination with FOLFIRI or mFOLFOX6 (14). The CALGB/SWOG 80405 trial evaluated response to cetuximab-containing regimens in 526 “all-RAS” WT colorectal cancer patients. In the “all-RAS” WT cohort, patients receiving cetuximab plus chemotherapy achieved an improved median PFS and OS of 11.4 and 32 months, respectively, compared with historical controls.

Taken together, the evidence is growing that mutations in RAS can predict for resistance to EGFR mAbs in patients with mCRC. NCCN clinical guidelines, the FDA, and the European Medicines Agency mandate restricted use of EGFR mAbs for patients with RAS WT mCRC. The foundation of this clinical practice change can be traced back to the fundamentals learned from preclinical studies of signaling pathways. This dramatic change in clinical practice highlights the continued need for adequate funding for translational research to improve oncologic outcomes for our patients.

L.M. Ellis is a consultant/advisory board member for Celgene, Eli Lilly, and Genentech/Roche. No potential conflicts of interest were disclosed by the other author.

Conception and design: E.R. Camp, L.M. Ellis

Writing, review, and/or revision of the manuscript: E.R. Camp, L.M. Ellis

E.R. Camp was supported by NIH grant K08CA142904. L.M. Ellis was supported by NIH grant R01CA157880, Department of Defense grant CA100879, and the William C. Liedtke Jr Chair in Cancer Research.

1.
Goldstein
NI
,
Prewett
M
,
Zuklys
K
,
Rockwell
P
,
Mendelsohn
J
. 
Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model
.
Clin Cancer Res
1995
;
1
:
1311
8
.
2.
Lynch
TJ
,
Bell
DW
,
Sordella
R
,
Gurubhagavatula
S
,
Okimoto
RA
,
Brannigan
BW
, et al
Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib
.
N Engl J Med
2004
;
350
:
2129
39
.
3.
Camp
ER
,
Summy
J
,
Bauer
TW
,
Liu
W
,
Gallick
GE
,
Ellis
LM
. 
Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor
.
Clin Cancer Res
2005
;
11
:
397
405
.
4.
Pao
W
,
Wang
TY
,
Riely
GJ
,
Miller
VA
,
Pan
Q
,
Ladanyi
M
, et al
KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib
.
PLoS Med
2005
;
2
:
e17
.
5.
Eberhard
DA
,
Johnson
BE
,
Amler
LC
,
Goddard
AD
,
Heldens
SL
,
Herbst
RS
, et al
Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non–small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib
.
J Clin Oncol
2005
;
23
:
5900
9
.
6.
Douillard
JY
,
Oliner
KS
,
Siena
S
,
Tabernero
J
,
Burkes
R
,
Barugel
M
, et al
Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer
.
N Engl J Med
2013
;
369
:
1023
34
.
7.
Lievre
A
,
Bachet
JB
,
Le Corre
D
,
Boige
V
,
Landi
B
,
Emile
JF
, et al
KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer
.
Cancer Res
2006
;
66
:
3992
5
.
8.
Benvenuti
S
,
Sartore-Bianchi
A
,
Di Nicolantonio
F
,
Zanon
C
,
Moroni
M
,
Veronese
S
, 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 Res
2007
;
67
:
2643
8
.
9.
Karapetis
CS
,
Khambata-Ford
S
,
Jonker
DJ
,
O'Callaghan
CJ
,
Tu
D
,
Tebbutt
NC
, et al
K-ras mutations and benefit from cetuximab in advanced colorectal cancer
.
N Engl J Med
2008
;
359
:
1757
65
.
10.
Bokemeyer
C
,
Bondarenko
I
,
Makhson
A
,
Hartmann
JT
,
Aparicio
J
,
de Braud
F
, et al
Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer
.
J Clin Oncol
2009
;
27
:
663
71
.
11.
De Roock
W
,
Claes
B
,
Bernasconi
D
,
De Schutter
J
,
Biesmans
B
,
Fountzilas
G
, 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 Oncol
2010
;
11
:
753
62
.
12.
Sorich
MJ
,
Wiese
MD
,
Rowland
A
,
Kichenadasse
G
,
McKinnon
RA
,
Karapetis
CS
. 
Extended RAS mutations and anti-EGFR monoclonal antibody survival benefit in metastatic colorectal cancer: a meta-analysis of randomized, controlled trials
.
Ann Oncol
2015
;
26
:
13
21
.
13.
Di Nicolantonio
F
,
Martini
M
,
Molinari
F
,
Sartore-Bianchi
A
,
Arena
S
,
Saletti
P
, et al
Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer
.
J Clin Oncol
2008
;
26
:
5705
12
.
14.
Lenz
H
,
Niedzwiecki
D
,
Innocenti
F
,
Blanke
C
,
Mahony
MR
,
O'Neil
BH
, et al
CALGB/SWOG 80405: Phase III trial of irinotecan/5-FU/leucovorin (FOLFIRI) or oxaliplatin/5-FY/leucovorin (mFOLFOX6) with bevacizumab or cetuximab for patients with expanded ras analyses untreated metastatic adenocarcinoma of the colon or rectum [abstract]
. In:
Proceedings of the ESMO 2014 Congress, 2014 Sep 26–30
,
Madrid, Spain. Lugano (Switzerland)
:
European Society for Medical Oncology
; 
2014
.
Abstract nr 501O
.