1308 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
The epidermal growth factor receptor (EGFR), a member of the
human epidermal growth factor receptor (HER) – erbB family of
receptor tyrosine kinases, represents an important target for cancer
treatment because its activation stimulates key processes involved in
tumor growth and progression, including proliferation, angiogene-
sis, invasion, and metastasis. The binding of EGF or other ligands to
EGFR initiates a mitogenic signaling cascade via several pathways,
including the RAS – RAF – mitogen-activated protein kinase (MAPK),
phosphatidylinositol 3-kinase (PI3K) – Akt, and phospholipase C ?
pathways ( 1 , 2 ). Overexpression of EGFR is found in a range of solid
tumor types and has been linked to poorer outcomes ( 3 , 4 ).
EGFR inhibitors — monoclonal antibodies targeting the extracel-
lular domain and small-molecule tyrosine kinase inhibitors — have
expanded the range of treatment options for various solid tumors.
EGFR-targeted monoclonal antibodies have been extensively stud-
ied in metastatic colorectal cancer ( Table 1 ), whereas tyrosine kinase
inhibitors have thus far shown little activity in this setting ( 5 , 6 ).
Cetuximab (ER-K0034, Erbitux, Merck-Serono KgaA, Darmstadt,
Germany; ImClone Systems Inc, New York, NY), the fi rst anti-
EGFR monoclonal antibody to be approved for clinical use for meta-
static colorectal cancer, is a chimeric mouse – human monoclonal
antibody that has been evaluated primarily in combination with che-
motherapy ( 7 – 10 ) but also as monotherapy ( 7 , 11 , 12 ). Panitumumab
(ABX-EGF, Vectibix; Amgen Inc, Thousand Oaks, CA), a fully
human monoclonal antibody, has shown effi cacy as monotherapy in
chemotherapy-refractory patients with metastatic colorectal cancer
( 13 ), and ongoing chemotherapy combination trials in earlier lines of
Biomarkers Predicting Clinical Outcome of Epidermal Growth
Factor Receptor – Targeted Therapy in Metastatic Colorectal
Salvatore Siena , Andrea Sartore-Bianchi , Federica Di Nicolantonio , Julia Balfour , Alberto Bardelli
The monoclonal antibodies panitumumab and cetuximab that target the epidermal growth factor receptor (EGFR) have expanded
the range of treatment options for metastatic colorectal cancer. Initial evaluation of these agents as monotherapy in patients
with EGFR-expressing chemotherapy-refractory tumors yielded response rates of approximately 10%. The realization that detec-
tion of positive EGFR expression by immunostaining does not reliably predict clinical outcome of EGFR-targeted treatment has
led to an intense search for alternative predictive biomarkers. Oncogenic activation of signaling pathways downstream of the
EGFR, such as mutation of KRAS , BRAF , or PIK3CA oncogenes, or inactivation of the PTEN tumor suppressor gene is central to
the progression of colorectal cancer. Tumor KRAS mutations, which may be present in 35% – 45% of patients with colorectal
cancer, have emerged as an important predictive marker of resistance to panitumumab or cetuximab treatment. In addition,
among colorectal tumors carrying wild-type KRAS , mutation of BRAF or PIK3CA or loss of PTEN expression may be associated
with resistance to EGFR-targeted monoclonal antibody treatment, although these additional biomarkers require further valida-
tion before incorporation into clinical practice. Additional knowledge of the molecular basis for sensitivity or resistance to EGFR-
targeted monoclonal antibodies will allow the development of new treatment algorithms to identify patients who are most likely
to respond to treatment and could also provide rationale for combining therapies to overcome primary resistance. The use of
KRAS mutations as a selection biomarker for anti-EGFR monoclonal antibody (eg, panitumumab or cetuximab) treatment is the
first major step toward individualized treatment for patients with metastatic colorectal cancer.
J Natl Cancer Inst 2009;101:1308–1324
treatment have reported acceptable interim safety data ( 14 , 15 ). In
addition, cetuximab and panitumumab have both been evaluated in
combination with bevacizumab, a monoclonal antibody targeting the
vascular endothelial growth factor (VEGF), plus standard fi rst-line
chemotherapy ( 16 , 17 ). However, increased toxicity and a shorter
progression-free interval were observed in the experimental groups
compared with the control groups. Thus, the strategy of combining
both an EGFR inhibitor and a VEGF inhibitor with chemotherapy
appears to be detrimental and is not being pursued further.
Affiliations of authors: The Falck Division of Medical Oncology, Department
of Oncology, Ospedale Niguarda Ca ’ Granda, Milan, Italy (SS, AS-B);
Laboratory of Molecular Genetics, Institute for Cancer Research and
Treatment, University of Torino Medical School, Turin, Italy (FDN, AB);
Kilconquhar, Fife , Scotland (JB); Fondazione Italiana Ricerca Cancro Institute
of Molecular Oncology , Milan, Italy (AB) .
Correspondence to: Salvatore Siena, MD, The Falck Division of Medical
Oncology, Ospedale Niguarda Ca ’ Granda, Piazza Ospedale Maggiore 3, 20162
Milan, Italy (e-mail: email@example.com ); Alberto Bardelli,
PhD, Laboratory of Molecular Genetics, Institute for Cancer Research and
Treatment, University of Torino Medical School, Strada Provinciale 142, Km
3.95, 10060 Candiolo, Turin, Italy (e-mail: firstname.lastname@example.org) .
See “Funding” and “Notes” following “References.”
© The Author 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Advance Access publication on September 8, 2009.
JNCI | Review 1309
Cetuximab and panitumumab appear to have similar effi cacy,
achieving fairly modest but clinically meaningful objective
response rates of approximately 10% when used as monotherapy
for chemotherapy-refractory EGFR-expressing metastatic col-
orectal cancers ( 7 , 11 – 13 , 18 ). However, panitumumab is likely to
be less immunogenic than cetuximab because of its fully human
composition and, indeed, panitumumab seldom gives rise to
severe infusion reactions ( 13 ). Such events may occur in up to
22% of cetuximab-treated patients, depending on geographical
region ( 19 , 20 ), and appear to be commonly associated with pre-
existing specifi c IgE antibodies against the oligosaccharide com-
ponent of the cetuximab molecule, galactose- ? -1,3-galactose ( 21 ).
Positive EGFR protein expression, as determined by immuno-
histochemistry, was initially selected as an entry criterion for studies
evaluating EGFR inhibitors on the assumption that sensitivity to
such agents was associated with EGFR expression. However, a large
body of evidence from patients who were treated with monoclonal
antibodies for metastatic colorectal cancer ( 7 , 11 , 13 , 22 , 23 ) or tyrosine
kinase inhibitors for other solid tumors ( 24 , 25 ) indicates that this
biomarker is poorly associated with response to EGFR inhibitors in
the clinical setting. Objective responses have been observed in
patients with low or negative, as well as high, EGFR protein expres-
sion, as determined by immunohistochemistry. These fi ndings have
led to intense research to identify alternative predictive molecular
biomarkers that can be used to identify patients who are most likely
to benefi t from EGFR-targeted treatment. This review discusses
progress made toward these ends with a focus on treatment of meta-
static colorectal cancer with anti-EGFR monoclonal antibodies.
Literature was identifi ed in PubMed and oncology conference
databases by use of the search terms “colorectal cancer” and
“molecular markers” and retrieved articles were evaluated by the
authors. All fully published clinical data relating to clinical
response to treatment with monoclonal antibodies in metastatic
colorectal cancer were considered for inclusion, as well as key
conference abstracts. Additional searches of the same databases
were performed to identify suitable background information.
Predicting Response: Molecular Biomarkers
Early work exploring molecular biomarkers of response to EGFR-
targeted monoclonal antibodies (ie, cetuximab or panitumumab) as
alternatives to EGFR protein determined by immunohistochemistry
started in 2005 ( 26 ) and was based on retrospective analyses of
archived tumor tissue from subsets of patients participating in clini-
cal trials. However, more recently studies have been designed to
incorporate biomarker analysis (eg, the pivotal phase III panitu-
mumab study) ( 13 , 27 ). In general, primary tumor tissue was ana-
lyzed, although metastatic tissue was evaluated in some instances. In
the majority of studies, cetuximab was given in combination with
chemotherapy, which could make interpretation difficult, whereas
to date, panitumumab has been administered almost exclusively as
monotherapy. Although most biomarker datasets are from chemo-
therapy-refractory or relapsed patients who had received multiple
previous lines of treatment, first-line data have recently been pre-
sented ( 28 , 29 ). Finally, it should also be mentioned that many analy-
ses were based on objective responses alone, without taking disease
stabilization into account.
Markers Downstream of EGFR
A rapidly growing body of knowledge has indicated that growth of
many tumors is driven by constitutive activation of signaling path-
ways downstream of the EGFR, as will be discussed below. Figure 1
shows the interactions between various signaling pathways involved
in tumor proliferation and progression. Such close interactions
between these pathways may provide “escape mechanisms” that
allow tumors to circumvent a pathway that has been pharmacologi-
The interlinked RAS – MAPK and PI3K signaling pathways
( Figure 1 ) play an important role in tumorigenesis via phosphory-
lation of various proteins and transcription factors that directly
control cell growth, differentiation, and apoptosis ( 1 , 2 , 30 ). KRAS ,
a member of the rat sarcoma virus ( ras ) gene family of oncogenes
(including KRAS , HRAS , and NRAS ), encodes the guanosine
diphosphate (GDP) – and guanosine triphosphate (GTP) – binding
protein RAS that acts as a self-inactivating intracellular signal
transducer ( 31 ). After binding and activation by GTP, RAS
recruits the oncogene RAF , which phosphorylates MAP2K (mito-
gen-activated protein kinase kinase)-1 and MAP2K-2, thus initiat-
ing MAPK signaling that ultimately leads to expression of proteins
playing important roles in cell growth, differentiation, and sur-
vival. The oncogene PIK3CA encodes the p110 subunit of PI3K,
which can be activated via interaction with RAS proteins ( 1 , 2 , 30 ).
Table 1 . Anti – epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) used for treatment of metastatic colorectal
AgentDescriptionCompanyApproved indicationsInvestigational indications
Cetuximab (Erbitux)Chimeric mAbMerck-Serono KGaA,
ImClone Systems Inc,
New York, NY
Treatment of patients with
EGFR-expressing, KRAS wild-type
mCRC in combination with
chemotherapy (EU) or irinotecan
in irinotecan-refractory disease (US)
or as a single agent in patients who
have failed oxaliplatin- and irinotecan-
based therapy or who are intolerant
to irinotecan (EU, US)
Monotherapy for fluoropyrimidine-,
oxaliplatin-, and irinotecan-resistant
EGFR-expressing mCRC with wild-
In combination with other
Panitumumab (Vectibix)Fully human mAbAmgen Inc, Thousand
In combination with
other targeted agents
1310 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
Mutation in KRAS , BRAF , or PIK3CA results in continuous acti-
vation of the downstream RAS – MAPK or PI3K pathways, regardless
of whether the EGFR is activated or pharmacologically blocked.
Such activation in turn enhances transcription of various oncogenes,
including MYC , CREB , and the gene for nuclear factor ? B ( 1 , 2 , 30 ).
A recent population-based study of 586 patients with colon
adenocarcinomas found mutations in KRAS , BRAF , and/or PIK3CA
in 316 (56%) of the 586 tumors studied ( 32 ). KRAS is the most
commonly mutated gene in this pathway, with mutations in
35% – 45% of colorectal adenocarcinomas; mutations in PIK3CA
( ≤ 20%) and BRAF (<15%) are less common ( 32 – 37 ). Mutations in
PIK3CA and KRAS or BRAF may coexist within the same tumor
( 32 , 36 – 38 ), but KRAS and BRAF mutations appear to be mutually
exclusive ( 33 , 34 , 39 – 41 ). KRAS mutation is thought to be an early
event in tumorigenesis ( 42 , 43 ), and, in general, metastatic and
primary sites have been concordant with regard to KRAS status
( 44 – 46 ), with only small differences having been reported ( 47 , 48 ).
KRAS mutations have been explored as prognostic biomarkers
(independent of anti-EGFR monoclonal antibody treatment), but
data are confl icting, refl ecting differences in datasets and method-
Figure 1 . Overview of interlinked cellular signaling pathways involved in
the proliferation and progression of colorectal cancer. Agents targeting
signaling proteins that have been evaluated or are currently being evalu-
ated in phase II, III, or IV clinical trials for colorectal cancer are shown. The
epidermal growth factor receptor (EGFR) – related family of receptor
tyrosine kinases includes human epidermal growth factor receptor
(HER1), EGFR, or c-erbB1; HER2 or c-erbB2; HER3 or c-erbB3; and HER4
or c-erbB4. C-MET = mesenchymal – epithelial transition factor; EGF =
epidermal growth factor; HDAC = histone deacetylases; HGF = hepatocyte
growth factor; IGF-1 = insulin-like growth factor-I; IGF-1R = insulin-like
growth factor-I receptor; IR = insulin receptor; VEGF = vascular endothe-
lial growth factor; VEGF-R = vascular endothelial growth factor receptor.
ologies and possibly tumor heterogeneity ( 32 , 43 , 49 – 54 ).
Retrospective data from 2721 patients with colorectal cancer from
the RASCAL (ie, the Kirsten ras in Colorectal Cancer Collaborative
Group) study ( 43 ) indicated that KRAS mutations may be associ-
ated with increased risk of death ( P = .002). However, in phase III
monotherapy studies of cetuximab ( 55 ) or panitumumab ( 13 , 27 ),
KRAS mutations did not appear to affect outcome among patients
receiving only best supportive care. Furthermore, KRAS mutations
do not appear to have a stage-specifi c prognostic value: No asso-
ciation between tumor KRAS mutations and relapse-free survival
was observed among patients with stage II and stage III colorectal
cancer in the Pan-European Trials in Adjuvant Colon Cancer
(PETACC) 3 study ( 54 ).
KRAS Mutations. A number of groups undertook retrospective
testing of KRAS status of tumors from patients with metastatic
colorectal cancer who were treated with cetuximab or panitu-
mumab (with or without chemotherapy) ( 26 , 33 , 34 ). Lievre et al.
( 34 ) first reported the link between KRAS mutations and lack of
response to EGFR-targeted monoclonal antibodies, a concept
JNCI | Review 1311
previously proposed by Moroni et al. ( 26 ), based on their cohort
study (n = 30 patients). These findings were confirmed and
extended to BRAF in a series of 48 patients by Benvenuti et al. ( 33 ),
who also found that transfection of mutated KRAS (G12V) into
wild-type DiFi colorectal cancer cells confers resistance to cetux-
imab. KRAS mutations have since emerged as a major predictor of
resistance to panitumumab or cetuximab in the clinical setting.
Studies ( 27 , 34 , 36 , 55 – 60 ) of patients receiving first and subsequent
lines of treatment have found that those with tumors carrying
KRAS mutations do not respond to EGFR-targeted monoclonal
antibodies or experience any survival benefit from such treatment.
Indeed, the progression-free interval in patients with tumors car-
rying mutant KRAS generally appears to be approximately half that
of those patients whose tumors carry wild-type KRAS ( Table 2 ).
The pivotal randomized phase III study of panitumumab
monotherapy in the relapsed or refractory setting ( 13 ) was the fi rst
large study (n = 463 patients) to confi rm the negative predictive
value of KRAS mutations ( 27 ). Biomarker analysis of primary
tumor tissue was planned in the protocol and KRAS analysis was
performed in a blinded manner at a central laboratory by use of a
KRAS testing kit (DxS Ltd, Manchester, UK) ( 27 ). Among the 463
patients enrolled in this study, 427 (92%) were included in the
KRAS analysis. Of these 427, 184 (43%) were found to have
tumors harboring mutant KRAS : 84 (40%) of the 208 patients
randomly assigned to panitumumab plus best supportive care and
100 (46%) of the 219 patients assigned to best supportive care
alone. Among the 208 patients assigned to panitumumab, 21
(17%) of the 124 patients in the wild-type KRAS subgroup
achieved objective response, whereas none of the 84 patients in the
mutant KRAS subgroup responded to this treatment. Median
progression-free interval among those treated with panitumumab
was 12.3 weeks among those in the wild-type KRAS subgroup and
7.4 weeks among those in the mutant KRAS subgroup. The hazard
ratio (HR) for disease progression or death (panitumumab vs con-
trol group) was 0.45 (95% confi dence interval [CI] = 0.34 to 0.59)
for panitumumab in the wild-type KRAS subgroup, but there was
no benefi t of panitumumab in the mutant KRAS subgroup (HR =
0.99, 95% CI = 0.73 to 1.36) ( Figure 2 ). A sensitivity analysis that
adjusted for potential bias from unscheduled assessment found
similar results. A total of 168 (77%) of the 219 KRAS -evaluable
patients initially assigned to the control group crossed over to
receive panitumumab after disease progression, at a median time of
7.1 weeks; this crossover confounded analysis of overall survival.
Among these 168 patients, 20 (22%) of the 91 in the wild-type
KRAS subgroup, compared with none in the mutant KRAS sub-
group, responded to panitumumab treatment; it is important to
note that these results were based on local review, whereas tumor
response in the main study was based on central review ( 27 ).
KRAS data from three large randomized phase II – III cetuximab
studies have recently been published, including the fi rst-line
OPUS (ie, Oxaliplatin and Cetuximab in First-Line Treatment of
metastatic colorectal cancer) ( 28 ) and CRYSTAL (ie, Cetuximab
Combined With Irinotecan in First-Line Therapy for Metastatic
Colorectal Cancer) ( 29 ) studies and the NCIC-CTG (ie, National
Cancer Institute of Canada-Clinical Trials Group) monotherapy
study conducted in relapsed or refractory patients or those with
contraindications to chemotherapy ( 55 ). Data from 394 (69%) of
the 572 KRAS -evaluable patients participating in the phase III
NCIC – CTG trial confi rmed that patients with tumors carrying
KRAS mutations do not benefi t from cetuximab monotherapy.
Both progression-free and overall survival were similar for the
cetuximab and control groups in those patients with tumors carry-
ing KRAS mutations (progression-free interval = 1.8 vs 1.8
months [HR = 0.99, 95% CI = 0.73 to 1.35, P = .96]; overall
survival = 4.6 vs 4.5 months [HR = 0.98, 95% CI = 0.70 to 1.37, P =
.89]). However, in the subgroup whose tumors carried wild-type
KRAS , cetuximab treatment was associated with statistically
signifi cantly ( P < .001) longer survival than control treatment
(progression-free interval = 3.7 vs 1.9 months [HR = 0.40, 95%
CI = 0.30 to 0.54]; overall survival = 9.5 vs 4.8 months [HR = 0.55,
95% CI = 0.41 to 0.74, P < .001 ]) ( 55 ). It should be noted that
unlike the pivotal panitumumab phase III study ( 13 ), the design of
this study did not allow patients from the control group who had
disease progression to cross over to monoclonal antibody treat-
ment. Final retrospective data from the OPUS and CRYSTAL
studies indicate that the addition of cetuximab to fi rst-line
FOLFOX (folinic acid, fl uorouracil, and oxaliplatin) ( 28 ) or
FOLFIRI (folinic acid, fl uorouracil, and irinotecan) ( 29 ) chemo-
therapy does not benefi t patients with tumors carrying KRAS
mutations, although those patients can benefi t from chemotherapy
alone ( Table 2 ). Indeed, fi ndings of the OPUS study indicate that
addition of EGFR-targeted treatment to chemotherapy may even
be detrimental in such patients ( 28 ) ( Table 2 ).
In the PACCE (ie, Panitumumab Advanced Colorectal Cancer
Evaluation) study ( 16 ), adding panitumumab to bevacizumab and
chemotherapy was associated with shortening of the progression-
free interval among patients with tumors carrying wild-type KRAS
(11.5 months in the chemotherapy – bevacizumab arm vs 9.8
months in the panitumumab-chemotherapy – bevacizumab arm). In
the CAIRO (ie, Capecitabine, Irinotecan, and Oxaliplatin trial) – 2
study ( 17 ), addition of cetuximab to capecitabine, oxaliplatin, and
bevacizumab as fi rst-line treatment in patients with metastatic
colorectal cancer had no effect on progression-free interval among
those with tumors carrying wild-type KRAS (10.6 months in the
chemotherapy – bevacizumab arm vs 10.5 months in the combined
cetuximab arm). However, this combination had a marked detri-
mental effect among patients with tumors carrying mutated KRAS
(12.5 vs 8.1 months) ( 17 ).
The proportion of patients bearing wild-type KRAS tumors
who fail to achieve either objective response or disease stabilization
with panitumumab or cetuximab varies considerably between
studies ( Table 2 ). Among a total of 124 patients with such tumors
who were treated with panitumumab in the pivotal phase III study,
45 (36%) had a best response of progressive disease ( 61 ). These
patients had a median progression-free interval of 7.3 weeks, as
shown by a post hoc subanalysis ( Figure 3 ). However, among
patients with stable disease or a partial response, median progression-
free interval was 23.9 and 27.0 weeks, respectively ( 61 ).
It is interesting to note that in other solid tumor settings,
EGFR inhibitors (tyrosine kinase inhibitors or cetuximab) have
shown minimal activity in patients with pancreatic cancer ( 62 , 63 ),
which is associated with a very high prevalence of KRAS mutations
(approximately 90%) ( 64 ), and in patients with lung cancer whose
tumors carry KRAS mutations ( 65 – 67 ).
1312 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
Table 2 . Tumor KRAS mutations and outcome of panitumumab- or cetuximab-based treatment in patients with metastatic colorectal cancer *
Treatment [type of study;
type of patients]
No. of patients
No. of patients (%)
Outcome by KRAS status, No. of patients (%) †
Association of KRAS mutation
with response and survival
Complete or partial
Monotherapy Amado ( 27 ) ‡ *
Panitumumab, [phase III,
KRAS mutation associated with
shorter PFS vs wild type (7.4 vs 12.3 wk): no benefit of panitumumab vs BSC in this subgroup
Amado ( 27 ) §
[phase III extension, chemotherapy refractory]
Freeman ( 36 )
Panitumumab [patient cohort,
Median PFS = 7.4 wk for MT KRAS
vs 16.2 wk for WT. Median OS = 22.2 wk for MT KRAS vs 42.9 wk
Karapetis ( 55 )
Cetuximab [phase III,
KRAS mutation associated with
shorter PFS and OS vs WT ( P < .001 for both)
Khambata-Ford ( 59 ) *
Cetuximab [NA ,
KRAS mutations found in three
(11%) DC vs 27 (51%) NR ( P < .001) and associated with
lower DC rate (10% vs 48%; P < .001) but similar PFS
(59 vs 61 d)
Mainly combination therapy Benvenuti ( 33 )
Cetuximab ± CT or
panitumumab [patient cohort, chemotherapy naïve and refractory]
KRAS/BRAF mutation negatively
associated with PR ( P < .01).
KRAS mutation associated with
shorter TTP ( P = .04)
De Roock ( 57 )
Cetuximab ± CT [patient cohort,
KRAS mutation found in 0% of OR
vs 52% of NR ( P < .001) and
associated with shorter OS (27.3 vs 43.0 wk ( P = .02)
Di Fiore ( 56 )
Cetuximab + CT [patient cohort,
KRAS mutation was associated
with PD ( P < .001) and shorter
TTP (3 vs 5.5 mo; P < .01)
Lievre ( 34 )
Cetuximab ± CT [patient cohort,
chemotherapy naïve and refractory]
KRAS mutation associated with
shorter OS (6.9 vs 16.3 mo; P = .02)
Lievre ( 60 )
Cetuximab + CT [patient cohort,
KRAS mutation associated with
shorter PFS (10.1 vs 31.4 wk; P < .001) and OS (10.1 vs 14.3
mo; P = .03)
JNCI | Review 1313
Treatment [type of study;
type of patients]
No. of patients
No. of patients (%)
Outcome by KRAS status, No. of patients (%) †
Association of KRAS mutation
with response and survival
Complete or partial
Combination with chemotherapy (first-line setting) Bokemeyer ( 28 ) FOLFOX [phase II,
Median PFS = 8.6 vs 7.2 mo in MT
and WT patients, respectively
FOLFOX + cetuximab [phase II, chemotherapy naïve]
No benefit of adding cetuximab
to FOLFOX in KRAS MT patients,
median PFS = 5.5 vs 7.7 mo in MT and WT patients, respectively
Van Cutsem ( 29 ) FOLFIRI [phase III,
Median PFS = 8.1 vs 8.7 mo in MT
and WT patients, respectively; median OS = 17.7 vs 21.0 mo in MT and WT patients, respectively
FOLFIRI + cetuximab [phase III, chemotherapy naïve]
No benefit of adding cetuximab
to FOLFIRI in KRAS MT patients,
median PFS = 7.6 vs 9.9 mo in MT and WT patients, respectively; median OS = 17.5 vs 24.9 mo in MT and WT patients, respectively
* Studies that prospectively evaluated biomarkers. BSC = best supportive care; CR = complete response; CT = chemotherapy; DC = disease control (PR or SD); FOLFIRI = folinic acid, fluorouracil, and irinotecan;
FOLFOX = folinic acid, fluorouracil, and oxaliplatin; MT = mutant; NA = not available; NR = nonresponse or nonresponder; OR = objective response or responder; OS = overall survival; PD = progressive disease;
PFS = progression-free interval; PR = partial response or responder; SD = stable disease; TTP = time to disease progression; WT = wild type.
† Expressed as a percentage of patients within the MT and WT subgroup. (the denominator is also shown in the first two columns). ‡ Phase III comparison of panitumumab vs BSC (data for panitumumab recipients only are shown).
§ Patients initially assigned to BSC who crossed over to panitumumab treatment after disease progression in the phase III study.
Table 2 (continued).
1314 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
BRAF Mutations. Recently published retrospective analysis ( 40 )
of 113 tumors from patients who received panitumumab or cetux-
imab in second or subsequent lines of treatment showed that those
with tumors that carried BRAF V600E mutations (n = 11, 10%) did
not respond to EGFR inhibition and had statistically significantly
shorter progression-free interval ( P = .001) and overall survival ( P <
.001) than patients whose tumors carried wild-type BRAF (n = 34).
A similar pattern was observed in an analysis of 231 tumors from
patients treated with first-line cetuximab plus capecitabine, oxalipa-
tin , and bevacizumab in the CAIRO-2 study ( 41 ). The median
progression-free interval and overall survival were 6.6 and 15.2
months for patients with tumors carrying mutant BRAF (n = 28), vs
10.4 ( P = .01) and 21.5 ( P = .001) months in those with tumors car-
rying wild-type BRAF (n = 231). However, the response rate did not
differ between these two patient subgroups (39% vs 48%; P = .04).
Di Nicolantonio et al. ( 40 ) also demonstrated that introduc-
tion of the BRAF V600E allele could confer resistance to either
cetuximab or panitumumab in wild-type BRAF colorectal cancer
cells. Furthermore, they showed that the multikinase inhibitor
sorafenib may restore sensitivity to EGFR inhibitors in BRAF -
mutated colorectal cancer cell lines. Consequently, combined
sorafenib and cetuximab therapy is undergoing clinical evaluation
in metastatic colorectal cancer in a National Cancer Institute –
sponsored trial (NCT00343772; http :// clinicaltrials . gov / ct2 / show /
NCT00343772 ). Evaluation of EGFR-targeted antibodies in
combination with other novel and more selective BRAF inhibi-
tors, such as PLX-4032 and XL-281, is also warranted.
PTEN and PI3K. Loss of expression of the tumor suppressor
PTEN protein, which regulates the PI3K – Akt signaling path-
way, has been reported to confer tumor resistance to EGFR
tyrosine kinase inhibitors in vitro ( 68 ) and has been linked to
erlotinib resistance in patients with glioblastoma ( 69 ) and to
trastuzumab resistance in patients with breast cancer ( 70 ). In
vitro studies in various colon cancer cell lines have found that
activating PIK3CA mutations or loss of PTEN expression
Figure 2 . Progression-free interval and KRAS
mutation status of tumor in patients with meta-
static colorectal cancer who were randomly
assigned to treatment with best supportive care
(BSC) alone or panitumumab (Panit) plus BSC in a
phase III study ( 27 ). A ) Tumors with mutant KRAS
status. B ) Tumors with wild-type KRAS status ( 27 )
(with permission from the American Society of
Clinical Oncology). CI = confi dence interval; HR =
JNCI | Review 1315
appeared to confer resistance to cetuximab. Cell lines that had
mutations in PIK3CA , or were PTEN null, and also had muta-
tions in RAS or BRAF exhibited the greatest resistance to cetux-
imab ( 71 ). Similarly, in the clinical setting, deregulation of either
PIK3CA or PTEN gene (via mutation or loss of expression; P = .02)
( 38 ) or PTEN protein expression loss alone ( P < .001) ( 58 ) statis-
tically significantly impaired response to cetuximab-based treat-
ment in colorectal cancer patients. Frattini et al. ( 58 ) reported
that none of 11 patients with tumor PTEN loss responded to
cetuximab-based treatment, whereas 10 (63%) of 16 patients
with intact PTEN protein expression were partial responders.
Perrone et al. ( 38 ) also noted that none of three patients with
PTEN mutation responded to treatment with cetuximab and
irinotecan. In a total of 92 patients with metastatic colorectal
cancer who were included in three biomarker analyses ( 26 , 34 , 38 ),
nine (10%) had tumors bearing PIK3CA mutations and only one
responded to EGFR-targeted treatment. In a larger patient
series (n = 110), Sartore-Bianchi et al. ( 72 ) found that PIK3CA
mutations and PTEN loss in colorectal tumors were statistically
significantly associated with lack of response to panitumumab
(zero of 15 patients, P = .038) or cetuximab (one of 32 patients,
P = .001) treatment. In the same study, PIK3CA mutations and/
or loss of PTEN expression were negatively associated with
progression-free interval, and loss of PTEN expression was also
linked with poorer overall survival ( P = .005). These investiga-
tors suggested that combining mutation analysis for KRAS and
PIK3CA (loss of PTEN and/or PIK3CA mutation) could identify
up to 70% of patients with metastatic colorectal cancer who are
unlikely to respond to treatment with an EGFR-targeted
monoclonal antibody ( 72 ). Contradictory evidence was recently
reported by Prenen et al. ( 73 ) who found no strong rationale for
using PIK3CA mutations as a single marker for sensitivity to
cetuximab in chemotherapy-refractory metastatic colorectal
cancer. Razis et al. ( 74 ) reported that normal PTEN protein
expression was associated with a higher response rate and longer
time to progression in patients treated with cetuximab-based
therapy, despite a 50% response rate observed in patients who
had lost PTEN protein expression ( 74 ). Because this study
included patients treated with first-line cetuximab and chemo-
therapy, these findings are difficult to interpret. Further inves-
tigation and prospective data from large randomized clinical
trials are required to confirm these findings before they can be
integrated into clinical practice.
Because tumors with oncogenic PIK3CA mutations are likely to
be driven by PI3K as the primary source of growth, proliferation,
and survival signaling ( Figure 1 ), the use of selective PI3K inhibi-
tors is being tested in ongoing trials. Indeed, several PI3K inhibi-
tors are progressing from preclinical development to phase I
clinical studies. This family of compounds includes XL147, GDC-
0941, BGT226, and the pan-PI3K – mammalian target of rapamycin
(mTOR) inhibitors XL765 and NVP-BEZ235 ( 75 ). The combina-
tion of cetuximab and the mTOR inhibitor everolimus has been
shown to be effective in restoring growth inhibition in cetuximab-
resistant colorectal cancer cells ( 76 ). It remains to be evaluated in
clinical trials whether concomitant inhibition of the EGFR and
PIK3CA pathways ( Figure 1 ) will convey clinical benefi t.
In addition, in colorectal cancer, the incidence of PIK3CA and
BRAF mutations (but not mutations of KRAS or TP53 ) displays a
gender bias with a higher frequency occurring in women ( 32 , 77 ).
Thus, it could be hypothesized that female patients with metastatic
colorectal cancer might be less likely to benefi t from treatment
with EGFR-targeted monoclonal antibodies. However, available
clinical data do not support this hypothesis ( 17 , 27 , 29 ).
EGFR as a Target
Many technical reasons have been advocated for the lack of asso-
ciation between EGFR detection by immunohistochemistry and
response to EGFR-targeted treatment, as discussed by Shia et al.
( 78 ). These reasons include disparity between the form or epitope
of EGFR detected by immunohistochemistry and that targeted by
Figure 3 . Progression-free interval by
response to panitumumab in a subgroup of
patients with metastatic colorectal cancer
whose tumors carry wild-type KRAS. Data
are from a phase III study ( 27 , 61 ).
1316 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
anti-EGFR monoclonal antibodies, as well as issues related to
processing and handling of tumor tissue samples, such as pro-
longed storage. Immunohistochemistry is also a semiquantitative
method that lacks a standardized scoring system and is subject to
interobserver variation. Moreover, differences between primary
colorectal tumors and their metastases with regard to EGFR
expression have been reported ( 79 ), indicating that reliance on
such biomarkers in the primary tumor to predict treatment
response of metastatic growths may be inappropriate.
EGFR Affinity and Phosphorylation. Using a specific ligand-
binding assay, Francoual et al. ( 80 ) found that many tumors con-
tain both low- and high-affinity EGFRs: 64 (78%) of 82 tumor
specimens contained only high-affinity binding sites (median dis-
sociation constant = 0.75 nM) and 18 (22%) had both low- and
high-affinity sites. Because immunohistochemistry-based methods
cannot distinguish between low- and high-affinity EGFR, these
findings may provide further explanation for the lack of correlation
between EGFR immunostaining and clinical response to EGFR-
EGFR phosphorylation status may refl ect the level of receptor
utilization by the tumor. This parameter (as determined by immu-
nohistochemistry) was associated with clinical response in patients
treated with cetuximab-based therapy. Patients with an activated
or phosphorylated EGFR score, as indicated by an immunohis-
tochemistry-based visual score of 7 or greater, were almost twice as
likely to have disease control (objective response or stable disease)
than those with a score of less than 7 (100% vs 54%; P = .05) ( 81 ).
EGFR Gene Status. Activating mutations, including in-frame
deletions and amino acid substitutions in exons 18, 19, and 21 in
the EGFR catalytic domain, play an important role in determining
responsiveness to tyrosine kinase inhibitors in the lung cancer set-
ting ( 82 , 83 ). Such mutations are rare ( 26 , 84 ) or absent ( 57 , 85 ) in
colorectal cancer tumors. Moroni et al. ( 26 ) detected one mutation
(3.2%) among 31 patients with metastatic colorectal cancer, occur-
ring in a patient who achieved stable disease for 24 weeks with
cetuximab and chemotherapy treatment. This missense heterozy-
gous mutation in exon 21 (Gly857Arg) affected a residue located
within the activation loop of the EGFR catalytic domain and was
one amino acid away from the Leu858Arg-activating mutation that
has been identified in patients with lung cancer who respond to
gefitinib or erlotinib ( 86 ). At disease progression, the patient
whose tumor had this mutation was treated with gefitinib; this
molecular alteration in EGFR was not associated with clinical
response because the disease progressed after 4 weeks of treatment.
Notably, a specific polymorphism of EGFR affecting exon 13 at
residue 521 Arg/Arg (previously identified as residue 497,
rs11543848) has been linked with improved overall survival in
women with metastatic colorectal cancer (vs Lys/Lys and/or Lys/
Arg variants), although the reverse pattern was observed in men
with this disease ( 87 ). This same polymorphism has been linked to
cetuximab response in other studies ( 88 – 90 ). Conflicting evidence
also exists for a polymorphism affecting the ligand of EGFR, EGF,
at position 61 (rs4444903) ( 89 , 91 , 92 ).
A small proportion of colorectal tumors overexpress EGFR via
amplifi cation of the gene, which can be detected by fl uorescence in
situ hybridization (FISH) ( 93 ) or chromogenic in situ hybridization
( 78 ). Although the intensity of protein expression was associated
with the likelihood of gene amplifi cation, immunohistochemistry
had a low specifi city (17% in primary tumors and 23% in meta-
static tumors) for predicting gene amplifi cation ( 78 ).
When EGFR gene copy number was evaluated by polymerase
chain reaction, no association was found between this parameter
and clinical outcome of panitumumab- or cetuximab-based treat-
ment ( 26 , 57 , 85 ), probably because of tumor DNA dilution by
DNA from normal cells during DNA extraction. However, EGFR
gene copy number as analyzed by FISH or chromogenic in situ
hybridization appears to be a promising biomarker of response to
such treatment ( Table 3 ), and present technical limitations are
being addressed in pathology studies ( 97 ). Methods of tissue pro-
cessing and EGFR scoring systems differed between studies.
Moreover, FISH pattern for EGFR expression is often nonhomo-
geneous in colorectal cancer tumors, with variable ratios of disomy
vs polysomy or amplifi cation being observed ( 93 , 94 ). Increased
gene copy number 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
was used, as determined by chromogenic in situ hybridization
( Table 3 ). Statistically signifi cant concordance between primary
colorectal tumors and their metastases with regard to EGFR gene
copy number has been found, as identifi ed by FISH ( 95 , 96 ).
Available data suggest that patients with less than three EGFR
gene copies per nucleus have a relatively low likelihood of respond-
ing to EGFR-targeted monoclonal antibody treatment ( 34 , 56 , 94 –
96 ). In a retrospective analysis of a subgroup of patients participating
in the pivotal phase III trial of panitumumab monotherapy ( 94 ),
the mean EGFR gene copy number per nucleus and the percentage
of tumor cells with chromosome 7 polysomy (three or more 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 EGFR gene copies per nucleus
or fewer than 43% of tumor cells with chromosome 7 polysomy,
respectively, achieved objective response compared with six (30%)
of the 20 patients ( P = .001) and six (32%) of the 19 patients ( P =
.001) who had values above these thresholds. A mean EGFR gene
copy number threshold of less than 2.5 copies per nucleus or fewer
than 40% of tumor cells with chromosome 7 polysomy discrimi-
nated patients with shorter progression-free ( P = .039 and P = .029,
respectively) and overall survival ( P = .015 and P = .014, respec-
tively). EGFR gene copy number and chromosome 7 polysomy
status did not draw a parallel with progression-free interval in
patients receiving only supportive care in this study, suggesting
that this parameter is not prognostic in metastatic colorectal can-
cer ( 94 ). These data contrast with earlier fi ndings that were based
on quantitative polymerase chain reaction analysis ( 85 ) showing
that EGFR gene copy number, as assessed by this method, related
to neither clinical response nor progression-free interval.
Homogeneous (ie, 100%) chromosome 7 disomy was the most
common pattern found in 58 colorectal tumors with nonincreased
gene copy number (n = 26; 45%) ( 94 ). Chromosome 7 disomy is
easier to detect than an increase in EGFR gene copy number and,
therefore, might enable a more reproducible FISH assay. However,
JNCI | Review 1317
Table 3 . Tumor epidermal growth factor receptor gene copy number and outcome of panitumumab- or cetuximab-based treatment in patients with metastatic colorectal cancer *
Treatment [type of
study; type of patients]
No of patients with
No. of patients (%)
[cutoff † ; methodology]
Outcome by GCN status, No. of patients (%) ‡
Association of increased EGFR GCN
with response and survival parameters
Complete or partial
Monotherapy Sartore- Bianchi ( 94 )
cohort, chemotherapy refractory]
20/58 (34) [ ≥ 2.47; FISH]
No OR if mean EGFR GCN of <2.47
copies per nucleus or <43% of tumor cells with chromosome 7 polysomy vs 6/20 (30%; P < .001) and 6/19
(32%; P < .001) among those with
higher values. Mean EGFR GCN of <2.5
copies per nucleus or <40% of tumor cells with chromosome 7 polysomy associated with shorter PFS ( P =.0153
and .0386, respectively) and OS ( P = .0145 and .0290, respectively)
Other Cappuzzo ( 95 ) Cetuximab ± CT [patient
cohort, chemotherapy refractory]
43/85 (51) [2.92; FISH]
Increased EGFR GCN associated with
higher OR ( P < .001) and longer TTP
(6.6 vs 3.5 mo, P = .02)
Personeni ( 96 ) Cetuximab ± CT [patient
cohort, chemotherapy refractory]
[ ≥ 2.83; FISH]
Longer PFS (5.5 vs 4.0 mo, P = .25)
and OS (10 vs 8.3 mo, P = .037) in
patients with mean GCN ≥ 2.83
Frattini ( 58 )
Cetuximab ± CT [patient
cohort, chemotherapy naïve and refractory]
8/27(30) [ ≥ 3 EGFR /
CEP7; FISH] or 16/27 (59) [ ≥ 4.00
6/8 (75) or
0/8 (0) or
2/8 (25) or
Two patients with increased EGFR GCN
had PD, possibly due to concomitant KRAS mutations. All NR with EGFR
gene amplification or increased GCN also showed concomitant KRAS
mutations and/or absent PTEN expression
Lievre ( 34 )
Cetuximab ± CT [patient
cohort, chemotherapy naïve and refractory]
3/30 (10)] [ ≥ 6; CISH] §
Increased EGFR GCN in 27% of OR
vs 0% of NR ( P = .04)
Moroni ( 26 )
cetuximab ± CT [patient cohort, chemotherapy naïve and refractory]
9/29 (31) [ ≥ 3; FISH]
8/9 || (89)
Increased EGFR GCN in 8/9 (89%)
OR vs 1/20 (5%) NR ( P < .001)
* CEP7 = chromosome 7 control; CISH = chromogenic in situ hybridization; CT = chemotherapy; EGFR = epidermal growth factor receptor; FISH = fluorescence in situ hybridization; GCN = gene copy number;
NA = data not available; NR = nonresponders; OR = objective response or responder (ie, complete or partial response); OS = overall survival; PD = progressive disease; PFS = progression-free interval;
PTEN = phosphatases and tensin homolog; TTP = time to disease progression.
† Expressed as number per nucleus. ‡ Expressed as a percentage of patients with increased or normal GCN (the denominator is also shown in the first two columns). § In more than 50% of cancer cells or presence of large gene copy cluster.
|| High overall response rate in this study was due to a clinical enrichment strategy.
1318 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
methods need to be further standardized for better reproducibility
and optimum sensitivity ( 96 , 97 ).
In comparison with patients with normal EGFR gene copy num-
ber, patients with an increased EGFR gene copy number exhibit
higher response rates to EGFR-targeted monoclonal antibodies,
with a longer progression-free interval or time to progression
( 34 , 58 , 94 – 96 ) ( Table 3 ). Two studies found that six (30%) of
20 patients and 14 (33%) of 43 patients with increased EGFR gene
copy number had an objective response to panitumumab ( 94 ) or
cetuximab with or without chemotherapy ( 95 ). Such response rates
compare favorably with historical response rates reported for popu-
lations selected by EGFR immunostaining alone. Higher response
rates were seen in other studies ( Table 3 ). For instance, Moroni et al.
( 26 ) found an 89% response rate in a subgroup of nine patients with
colorectal cancer whose tumors had an increased EGFR gene copy
number, but these investigators included a relatively high propor-
tion of responders (nine of 29 patients; 31%) in their analysis. In
vitro studies by these investigators also showed that the proliferation
of various colorectal cancer cell lines with amplifi ed EGFR was
completely inhibited by cetuximab at concentrations that did not
affect proliferation of cells with unamplifi ed EGFR ( 26 ). Logistic
regression analysis indicated that the odds ratio for response to pani-
tumumab was 5.62 (95% CI = 1.51 to 21.0) for increased vs normal
mean EGFR gene copy number ( 94 ).
Overexpression of Other EGFR Ligands. Elevated gene expres-
sion of alternative EGFR ligands, such as epiregulin and amphi-
regulin, may promote tumor growth and survival via an autocrine
loop ( 59 , 98 ). Expression of high levels of mRNA for either epi-
regulin or amphiregulin has been associated with sensitivity to
cetuximab monotherapy ( 59 , 98 ). Comparison of clinical outcomes
for patients with high and low levels of these ligands showed a
statistically significantly improved disease control rate ( P < .001)
and longer progression-free interval among patients with high
expression of epiregulin (median = 103.5 vs 57 days, P < .001;
HR = 0.47 [95% CI = 0.24 to 0.64]) or amphiregulin (median =
115.5 vs 57 days, P < .001; HR = 0.44 [95% CI = 0.21 to 0.57]) ( 59 ).
The exclusive use of either KRAS status or amphiregulin or epi-
regulin gene expression profiles ( 59 ) does not result in the selec-
tion of identical patient populations who are likely to benefit from
treatment with cetuximab: Among patients with wild-type KRAS ,
patients whose tumors expressed high levels of amphiregulin or
epiregulin were likely to experience disease control, whereas
patients whose tumors expressed low levels of these genes were
not, thus providing important complementary information to
KRAS status ( 99 ). Increased gene copy number of HER2 (the pre-
ferred heterodimer of EGFR) was linked to a statistically signifi-
cantly shorter overall survival ( P = .03), with a trend toward a
shorter time to progression ( P = .09), in 85 patients receiving
cetuximab with or without chemotherapy ( 100 ).
Other Potential Biomarkers
Markers of angiogenesis and cell cycle regulation appear to be
promising areas for further research. Angiogenesis is a prerequisite
for growth and progression of tumors ( 101 , 102 ). Although driven
by separate mechanisms, EGFR and the key angiogenic factor
VEGF-1 share common downstream pathways ( Figure 1 ). Findings
of preclinical studies (conducted in human tumor cell lines xeno-
grafted into murine models that evaluated the combined pharma-
cological targeting of EGFR-dependent and VEGF-dependent
pathways) indicate direct or indirect angiogenic effects of EGFR
signaling ( 103 – 106 ). Furthermore, expression of VEGF-1 or its
receptor has been linked to resistance to EGFR-targeted agents in
various cancer cell lines ( 107 ) and in patients with metastatic col-
orectal cancer ( 108 ). Markers that have been positively linked to
outcome in patients with metastatic colorectal cancer who were
treated with EGFR-targeted monoclonal antibodies include
expression or gene polymorphisms of cyclooxygenase-2 ( 108 , 109 ),
interleukin-8 ( 108 , 109 ), and the cell cycle regulator cyclin D1
( 93 , 108 , 109 ). For instance, Vallböhmer et al. ( 108 ) concluded that
a combination of low gene expression levels of cyclooxygenase-2,
EGFR, and interleukin-8 was statistically significantly related to
the overall survival of patients who were treated with cetuximab
monotherapy (13.5 vs 2.3 months in those with high gene expres-
sion levels of these three genes; P = .028). Feedback mechanisms
and complex cellular circuits further link expression of VEGF,
interleukin-8, and cyclooxygenase-2 to the oncogenic activation of
KRAS and BRAF genes. For example, expression of cyclooxyge-
nase-2, an upstream regulator of EGFR activity, is driven via the
EGFR cascade ( Figure 1 ), and in particular oncogenic KRAS has
been shown to induce cyclooxygenase-2 expression ( 110 , 111 ). In
addition, increased expression of cyclooxygenase-2 may result in
increased production of prostaglandin E2, which in turn can trans-
activate EGFR ( 112 ). Expression of the transcription factor nuclear
factor ? B has also been linked with resistance to cetuximab ( 113 ).
Polymorphisms in fragment c gamma receptors, surface recep-
tors for immunoglobulin G located on immune effector cells (such
as natural killer lymphocytes and macrophages), are also of interest
as potential markers of response to EGFR-targeted monoclonal
antibodies, although data are confl icting at present ( 109 , 114 – 116 ).
Fragment c gamma receptors are thought to play a role in antibody-
dependent cell-mediated cytotoxicity, which has been postulated as
an additional mechanism of action for the IgG1 type of monoclonal
antibodies, such as cetuximab, rituximab, and trastuzumab ( 117 ).
Although it was initially believed that panitumumab, as an IgG2
monoclonal antibody, would not elicit antibody-dependent cell-
mediated cytotoxicity, this phenomenon has recently been demon-
strated in squamous cell head and neck carcinomas in vitro at
concentrations that are analogous to therapeutic doses ( 117 ).
Molecular brakes that protect against inappropriate oncogene
activation (such as TP53 ) may also be candidate biomarkers of
sensitivity to anti-EGFR therapy, given that their inactivation may
be required for tumor progression. Indeed, Oden-Gangloff et al.
( 118 ) suggest that TP53 mutations may be predictive of increased
likelihood of response to cetuximab treatment, particularly in
patients with wild-type KRAS status ( 118 ).
Early Response Evaluation
The characteristic “acneiform” skin rash observed in most patients
who are treated with EGFR inhibitors has been studied as a poten-
tial marker of efficacy. This adverse effect is usually apparent after
approximately 1 week of treatment and reaches maximum severity
after 2 – 3 weeks. As with the tyrosine kinase inhibitor erlotinib
JNCI | Review 1319
in patients with lung cancer ( 24 , 119 ), skin toxicity has been con-
sistently linked with higher response rates and longer survival
among patients with metastatic colorectal cancer who have
been treated with panitumumab ( 13 , 120 ) or cetuximab ( 7 , 11 , 12 ),
whereas patients without rash appear to have a poor outcome.
Figure 4 illustrates the relationship between survival and worst
grade of rash among patients treated with panitumumab ( 13 ) or
cetuximab ( 12 ) monotherapy in two phase III studies. A landmark
analysis (including only patients who were progression free for
≥ 28 days to allow time for onset) of progression-free interval data
from the panitumumab study (n = 231) showed a statistically sig-
nificant benefit for patients with grade 2 – 4 skin toxicity compared
with those with grade 1 skin toxicity (HR = 0.62, 95% CI = 0.44
to 0.88) ( 13 ).
Rash might indicate receptor saturation, and “dose-to-rash”
strategies are being studied with the aim of optimizing response to
EGFR-targeted treatment. Preliminary data from the phase I – II
Evaluation of Various Erbitux Regimens by means of Skin and
Tumour biopsies (ie, EVEREST) study suggest that among patients
receiving cetuximab-based treatment, cetuximab dose escalation to
500 mg/m 2 per week may improve response rates in those with no or
slight skin reactions ( 121 ), but the difference was not statistically
signifi cant and results require confi rmation in a larger study.
Subsequent analysis of results by KRAS status showed that cetux-
imab dose escalation did not increase response in patients with
tumors carrying a mutant KRAS gene. However, among those with
tumors carrying wild-type KRAS , this strategy improved the
response rate from four (21%) of 19 patients to 13 (46%) of 28
patients ( 122 ). It is important to note that the panitumumab regimen
of 6 mg/kg every 2 weeks, which is approved for treatment of meta-
static colorectal cancer, achieves similar drug exposure to a regimen
of 2.5 mg/kg per week, which was studied in early phase trials and
was found to be associated with a 100% incidence of rash ( 123 ).
There are several limitations to the use of rash as an early physi-
cal marker of effi cacy. As highlighted by De Roock et al. ( 57 ), there
are no criteria for toxic effects involving skin that are specifi cally
tailored to the activity of EGFR-targeted treatment. Rash often
occurs in patients without apparent benefi t from anti-EGFR treat-
ment, and conversely, clinical benefi t has also been seen in patients
without rash ( 124 ). Because EGFR is expressed in the skin, skin
rash may indicate local receptor saturation, but other factors, such
as their immune status, might alter an individual ’ s susceptibility to
rash. An association has been observed between tumor and normal
tissue with regard to high-affi nity EGFR. This fi nding might pro-
vide an explanation for the link observed between skin toxicity and
clinical outcome of patients treated with EGFR-targeted treat-
ment ( 81 ). Moreover, the relationship between rash and clinical
benefi t is inconsistent for the tyrosine kinase inhibitor gefi tinib
( 124 ) and, intriguingly, rash is not observed in patients treated
with the humanized anti-EGFR monoclonal antibody nimotu-
zumab ( 125 , 126 ), although it should be noted that effi cacy data for
this drug are currently limited.
Anti-EGFR monoclonal antibody treatment may compromise
renal magnesium retention capacity, leading to hypomagnesemia
in some patients with colorectal cancer ( 127 ). Vincenzi et al. ( 128 )
recently suggested that reduction in serum magnesium levels
might potentially provide an early marker of effi cacy of combined
treatment with cetuximab and irinotecan. It has also been sug-
gested that treatment with cetuximab may induce a sudden and
lasting modulation of circulating VEGF levels ( 129 ), although
the association between this fi nding and clinical effi cacy was not
Discussion and Future Perspectives
Although the link between clinical benefit and overexpression of
the molecular target is clear for trastuzumab and imatinib and their
respective targets (HER2 and the BCR-ABL tyrosine kinase),
experience has shown that positive expression of EGFR as shown
by immunostaining is not predictive of response to EGFR inhibi-
tors. It is now clear that tumor growth can be driven by constitutive
activation of signaling pathways downstream of the EGFR, such as
the RAS – MAPK – PI3K pathway. Oncogenic activation of compo-
nents in these pathways can bypass the EGFR-driven signaling
cascade and impair the clinical efficacy of anti-EGFR monoclonal
antibodies. Such activation can occur via mutations in oncogenes
such as KRAS or BRAF on one side of the EGFR-mediated path-
way or by PIK3CA mutation or loss of tumor suppressor genes
No. at risk
50 44 30 21 13
149 138 118 96 69 47 34 21
7 3 2 1
Grade 2–4 121/14 (84)
HR = 0.59
(95% CI, 0.42 to 0.85)
Grade 1 53/56 (95)
Probability of survival (%)
2 4 6 8 10 12 14 16 18 20 22 24
Time since random assignment (months)
Probability of survival (%)
10 8 6 4 2 0 12 14 16 18 20 22 24
Time of survival (months)
No. at risk
Grade 2 or higher
Figure 4 . Probability of survival by worst grade of skin toxicity in
patients with metastatic colorectal cancer who were treated with EGFR-
targeted monoclonal antibodies in two randomized phase III studies. A )
Patients treated with panitumumab. Data are from a landmark analysis
that was limited to patients with progression-free interval of at least 28
days ( 13 ) (with permission from the American Society of Clinical
Oncology. B ) Patients treated with cetuximab ( 12 ). Reproduced with
permission from the New England Journal of Medicine . Copyright 2007
Massachusetts Medical Society. All rights reserved. CI = confi dence
interval; HR = hazard ratio.
1320 Review | JNCI Vol. 101, Issue 19 | October 7, 2009
such as PTEN on the opposite side of the cascade ( 72 ) ( Figure 1 ).
These findings may provide some explanation for the rather
modest objective response rates that have been achieved with clini-
cal trials of EGFR inhibitors to date, as well as the disparities
observed between clinical and preclinical findings. It should also
be noted that preclinical models are based on particular tumor
subtypes that may not be representative of most tumors encoun-
tered in clinical practice.
Table 4 summarizes potential biomarkers that may be related to
primary response to the anti-EGFR monoclonal antibodies, pani-
tumumab and cetuximab. Overall, presently available data reviewed
in this article provide convincing evidence that activating muta-
tions of KRAS , which are present in a substantial proportion of
patients with metastatic colorectal cancer, predict lack of response
to anti-EGFR monoclonal antibody treatment ( 27 – 29 , 33 , 34 , 55 , 60 ).
This fi nding is consistent with observations from use of EGFR
tyrosine kinase inhibitors in the treatment of non – small cell lung
cancer, although KRAS mutations are less common in lung tumors
( 66 , 67 ).
KRAS testing is now being integrated into clinical practice. The
European Medicines Agency’s conditional approval of panitumumab
monotherapy in the setting of chemorefractory metastatic colorectal
cancer specifi ed wild-type KRAS as a selection marker (134).
Current data indicate that objective response rates of up to 22% can
be expected in such patients (Table 2). It should be noted that clini-
cal benefi t is not confi ned to objective responders because delaying
disease progression can improve clinical symptoms and the patient’s
quality of life (135). Wild-type KRAS was more recently identifi ed
as a selection marker for cetuximab monotherapy or combination
therapy (136). The European Medicines Agency (20, 130), the US
Food and Drug Administration (137), and the American Society of
Oncology (131) now recommend determining tumor KRAS status
before initiating treatment with an anti-EGFR monoclonal anti-
body and restricting such treatment to patients with tumors bearing
wild-type KRAS .
Because of the complexity of the EGFR signaling system, it is
likely that predictive algorithms will be developed for metastatic
colorectal cancer that incorporate several molecular biomarkers.
For instance, combining analysis of KRAS status with determina-
tion of BRAF and PIK3CA status and PTEN expression may iden-
tify additional patients with metastatic colorectal cancer who are
unlikely to respond to treatment with an EGFR-targeted mono-
clonal antibody ( 133 ). However, these additional markers ( Table 4 )
require further validation before they can be incorporated into
Tumors with an increased EGFR gene copy number as assessed
by FISH or chromogenic in situ hybridization may be dependent
on the EGFR pathway for their survival and growth. There is
evidence that normal diploid EGFR gene copy number may predict
tumor resistance to EGFR-targeted treatment ( Table 3 ). Again,
further research is required to validate this biomarker in larger
patient series ( 97 ).
There are clearly a number of technical issues to be overcome,
particularly standardization of analytical methods and scoring sys-
tems. Lack of standardization may well explain some of the discordant
results that have been reported. Certainly, some biomarkers may not
prove suitable for translation into clinical practice. However, DNA
sequencing of formalin-fi xed paraffi n-embedded tumor samples is a
relatively straightforward method for identifying KRAS mutations.
Because KRAS mutation is an early event in colorectal cancer tumori-
genesis ( 42 , 43 ), archived primary tumor tissue can be used to identify
patients who are unlikely to respond to EGFR-targeted monoclonal
antibodies, even after multiple lines of treatment. A KRAS testing kit
from DxS Ltd can identify the following seven somatic mutations in
KRAS codons 12 and 13: Gly12Ala, Gly12Arg, Gly12Asp, Gly12Ser,
Gly12Cys, Gly12 Val, and Gly13Asp. The polymerase chain reac-
tion – based technique used is highly sensitive but does not detect less
frequent changes that can be detected with direct sequencing (eg,
Gly13Val, Gly13Ala, and Gly13Cys). Although direct sequencing has
the capability to detect all changes at the nucleotide level, it is less
Table 4 . Summary of potential predictive molecular biomarkers for response to the epidermal growth factor receptor (EGFR) – targeted
monoclonal antibodies cetuximab and panitumumab in metastatic colorectal cancer *
Relationship to responseBiomarkerFirst author (reference)
Predicts lack of response and now
incorporated into clinical practice †
Very likely to predict lack of response
KRAS mutationAmado ( 27 ); Bokemeyer ( 28 ); Van Cutsem
( 29 ); Table 2 , this article; ( 20 , 130 , 131 )
Frattini ( 58 ); Perrone ( 38 ); Benvenuti ( 33 );
Di Nicolantonio ( 40 ); Sartore-Bianchi ( 72 );
Di Nicolantonio ( 132 )
Finocchiaro ( 100 )
Table 3 , this article
Personeni ( 81 )
Khambata-Ford ( 59 )
Mutation or lack of expression of PTEN; mutation
of BRAF or PIK3CA
May predict lack of response ‡
May predict increased likelihood of response
Increased HER2 gene copy number
Increased EGFR gene copy number §
Increased EGFR phosphorylation ‡
Overexpression of alternative EGFR ligands
(amphiregulin and/or epiregulin) ‡
pAKt overexpression ‡
Markers of angiogenesis and cell cycle regulation;
transcription factors (VEGF, IL-8, COX-2, cyclin D,
NF ? B) ‡
Razis ( 133 )
Vallböhmer ( 108 ); Nagashima ( 109 );
Zhang ( 92 )
Other potential markers
* Data are based on analysis of tumor tissue from patients participating in clinical trials. COX-2 = cyclooxygenase-2; HER2 = human epidermal growth factor-2;
IL-8 = interleukin-8; NF ? B = Nuclear factor kappa B; pAkt = phosphorylated Akt; VEGF = vascular endothelial growth factor.
† Based on data from a study that prospectively defined biomarker analysis and included a large number of patients ( 13 ).
‡ Limited preliminary data.
§ Data need to be confirmed in large patient datasets, preferably with prospective study design.
JNCI | Review 1321
sensitive and may occasionally miss mutations, especially if the frac-
tion of tumor vs normal cells is low ( 57 ). Testing will be facilitated by
gene panel microarray technology and gene panels that incorporate
KRAS testing are being evaluated ( 138 ).
Further work is also required to explore potential early markers
of response (in patients already receiving EGFR-targeted treat-
ment) that can be incorporated into the design of future prospec-
tive clinical trials and guide therapeutic decisions regarding
continuation of treatment in individual patients. Skin toxicity
develops at an early stage in treatment with EGFR inhibitors and
has been studied extensively as a potential early marker of response,
although further work is required.
Virtually all responding tumors eventually “escape” from
EGFR-targeted treatment (ie, develop acquired resistance). In the
lung cancer setting, acquired resistance to erlotinib or gefi tinib has
been attributed to development of a secondary mutation within the
EGFR catalytic domain ( 139 ). Other potential escape mechanisms
include activation of alternative signaling pathways contributing to
proliferation and survival, such as those involving activation of
HER2, HER3, mesenchymal – epithelial transition factor (C-MET),
insulin-like growth factor-I receptor , MAPK, and Akt ( 140 ).
Consequently, identifi cation of the molecular basis of acquired
resistance to anti-EGFR monoclonal antibodies in metastatic col-
orectal cancer should be a priority for future research.
In conclusion, the quest for predictive biomarkers of response to
EGFR inhibitor therapy has resulted in a rapidly accumulating body
of knowledge that has paved the way for more targeted use of these
agents. More rational use of EGFR-targeted agents should provide
benefi ts for patients and health-care providers alike by sparing
patients unnecessary treatment and allowing better use of health-
care resources. Prospective biomarker-driven studies are now under
way, but in the meantime, the identifi cation of wild-type KRAS
as a selection biomarker for panitumumab or cetuximab therapy
represents an important step toward fulfi lling the promise of
individualized treatment for metastatic colorectal cancer.
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