Prognostic vs predictive molecular biomarkers in colorectal cancer: is KRAS and BRAF wild type status required for anti-EGFR therapy?
ABSTRACT An important molecular target for metastatic CRC treatment is the epidermal growth factor receptor (EGFR). Many potential biomarkers predictive of response to anti-EGFR monoclonal antibodies (cetuximab and panitumumab) have been retrospectively evaluated, including EGFR activation markers and EGFR ligands activation markers. With regard to the "negative predictive factors" responsible for primary or intrinsic resistance to anti-EGFR antibodies a lot of data are now available. Among these, KRAS mutations have emerged as a major predictor of resistance to panitumumab or cetuximab in the clinical setting and several studies of patients receiving first and subsequent lines of treatment have shown that those with tumors carrying KRAS mutations do not respond to EGFR-targeted monoclonal antibodies or show any survival benefit from such treatments. The role of B-RAF mutations, mutually exclusive with KRAS mutations, in predicting resistance to anti-EGFR mAbs is not yet consolidated. It therefore appears that BRAF mutations may play a strong negative prognostic role and only a slight role in resistance to anti-EGFR Abs.
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ABSTRACT: Colorectal cancer (CRC) is one of the most common human malignant diseases and the second leading cause of cancer-related deaths worldwide. The treatment of advanced CRC has improved significantly in recent years. With the emergence of two targeted antibodies, cetuximab (Erbitux), an anti-epidermal growth factor receptor monoclonal antibody and bevacizumab (Avastin), a vascular endothelial growth factor monoclonal antibody, the treatment of metastatic CRC has entered the era of personalized therapy. Predictive and prognostic biomarkers have, and will continue to, facilitate the selection of suitable patients and the personalization of treatment for metastatic CRC (mCRC). In this review, we will focus primarily on the important progresses made in the personalized treatment of mCRC and discuss the potentially novel predictive and prognostic biomarkers for improved selection of patients for anti-cancer treatment in the future.World Journal of Gastroenterology 04/2014; 20(14):3858-3874. · 2.43 Impact Factor
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ABSTRACT: Knowledge about the clinical significance of V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutations in colorectal cancer (CRC) is growing. BRAF encodes a protein kinase involved with intracellular signaling and cell division. The gene product is a downstream effector of Kirsten Ras 1(KRAS) within the RAS/RAF/MAPK cellular signaling pathway. Evidence suggests that BRAF mutations, like KRAS mutations, result in uncontrolled, non-growth factor-dependent cellular proliferation. Similar to the rationale that KRAS mutation precludes effective treatment with anti-EGFR drugs. Recently, BRAF mutation testing has been introduced into routine clinical laboratories because its significance has become clearer in terms of effect on pathogenesis of CRC, utility in differentiating sporadic CRC from Lynch syndrome (LS), prognosis, and potential for predicting patient outcome in response to targeted drug therapy. In this review we describe the impact of BRAF mutations for these aspects.Gastroenterology and hepatology from bed to bench. 01/2013; 6(1):6-13.
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ABSTRACT: Colorectal cancer (CRC) is the second leading cause of cancer-related death. Despite the progress that has been made towards the identification of the molecular mechanisms involved in CRC, currently there are many unclear points. The current opinion is that microsatellite instability (MSI), CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) seem to play a significant role. MSI is related to point mutations in defect mismatch repair system of DNA. There are two well-established MSI phenotypes: MSI-high (MSI-H) and MSI-low (MSI-L or MSS). CIN refers to a different cellular event which originates from the presence of an abnormal chromosome complement or number. CIMP is the third most commonly involved event, and is defined by widespread methylation of CpG islands of suppressor promoters, with two phenotypes: CIMP-high and CIMP-low which interact with MSI or CIN status V-raf murine sarcoma viral oncogene homolog B (BRAF) is a serine-threonine protein kinase that acts as a downstream effector of the Kirsten rat sarcoma viral oncogene homolog (KRAS) pathway. Various studies have revealed that BRAF V600E mutations appear to be a valid indicator of poor prognosis. KRAS is a proto- oncogene which encodes a GTP-ase involved in cellular response to extracellular stimuli. Its prognostic value is still controversial. However, wild-type KRAS is associated with better response to Endothelial Growth Factor Receptor inhibitors combined with standard chemotherapy. Loss of Heterozygosity, especially involving 18q, is a well-known potential mechanism for tumorigenesis that has been studied in CRC. Vascular endothelial growth factor is a pro-angiogenic factor linked with the aggressiveness of CRC. Emerging data show that cycloxygenase 2 overexpression is significantly associated with worse outcomes in CRC. Recent studies highlight mi-croRNAs as promising prognostic biomarkers. More specifically, the down-regulation of miR-451, miR-625, miR-29c, miR-126, miR-129 and miR133 is purported to be a poor prognostic factor, while miR-224 was overexpressed in CRC.Anticancer research 05/2014; 34(5):2061-8. · 1.87 Impact Factor
Cancer Treatment Reviews 36S3 (2010) S56–S61
Contents lists available at ScienceDirect
Cancer Treatment Reviews
journal homepage: www.elsevierhealth.com/journals/ctrv
Prognostic vs predictive molecular biomarkers in colorectal cancer: is KRAS and
BRAF wild type status required for anti-EGFR therapy?
Sergio Rizzoa‡, Giuseppe Brontea‡, Daniele Fanalea, Lidia Corsinia, Nicola Silvestrisb, Daniele Santinic,
Gaspare Gulottad, Viviana Bazana, Nicola Gebbiaa, Fabio Fulfaroa, Antonio Russoa,*
aDepartment of Surgical and Oncological Sciences, Section of Medical Oncology, University of Palermo, Palermo, Italy
bMedical and Experimental Oncology Unit, Cancer Institute “Giovanni Paolo II”, Bari, Italy
cMedical Oncology, Campus Bio-Medico University, Rome, Italy
dDepartment of General Surgery, Urgency, and Organ Transplantation, University of Palermo, Italy
a r t i c l e i n f o
s u m m a r y
An important molecular target for metastatic CRC treatment is the epidermal growth factor receptor
(EGFR). Many potential biomarkers predictive of response to anti-EGFR monoclonal antibodies
(cetuximab and panitumumab) have been retrospectively evaluated, including EGFR activation
markers and EGFR ligands activation markers. With regard to the “negative predictive factors”
responsible for primary or intrinsic resistance to anti-EGFR antibodies a lot of data are now
available. Among these, KRAS mutations have emerged as a major predictor of resistance to
panitumumab or cetuximab in the clinical setting and several studies of patients receiving first
and subsequent lines of treatment have shown that those with tumors carrying KRAS mutations
do not respond to EGFR-targeted monoclonal antibodies or show any survival benefit from such
treatments. The role of B-RAF mutations, mutually exclusive with KRAS mutations, in predicting
resistance to anti-EGFR mAbs is not yet consolidated. It therefore appears that BRAF mutations
may play a strong negative prognostic role and only a slight role in resistance to anti-EGFR Abs.
© 2010 Elsevier Ltd. All rights reserved.
Colorectal cancer (CRC) is the third most common cause of cancer-
related death, with an incidence of almost a million cases annually
in both males and females.1Despite the fact that recent progress in
diagnosis and treatment has increased the number of patients who
have been completely cured at an early stage of the disease, the
prognosis for advanced forms of this cancer is still very poor, with
treatment limited to palliation for the vast majority of patients.2
The development of colorectal cancer (CRC) is a multistep
process brought about by the accumulation of several genetic
alterations, including chromosomal abnormality, gene mutations
and epigenetic modifications involving several genes regulating
proliferation, differentiation, apoptosis and angiogenesis.3,4
Of the various genetic alterations, only a few are involved
in cell growth and will lead to cancer development. This
phenomenon therefore, known as ‘oncogene addiction’ might
represent a rationale for molecular target therapy,5possibly helping
*Corresponding author. Antonio Russo, MD, PhD. Department of
Surgical and Oncological Sciences, Section of Medical Oncology,
Universit` a di Palermo, Palermo, Italy; Via del Vespro 127,
90127 Palermo, Italy. Tel.: +390916552500; fax: +390916554529.
E-mail address: email@example.com (A. Russo).
to develop new and targeted treatment options in patients with
An important molecular target for metastatic CRC treatment is
the epidermal growth factor receptor (EGFR). EGFR is a member
of the HER (ErbB) family of receptor tyrosine kinases involved in a
variety of signal transduction pathways which are able to promote
tumor cell proliferation, angiogenesis, invasion and metastasis in
different epithelial malignancies.7
In more specific terms, the binding of the epidermal growth factor
(EGF) to the extracellular binding site of EGFR activates three major
signal transduction pathways, including the RAS–RAF mitogen-
activated protein kinase (MAPK), phosphatidylinositol 3-kinase
(PI3K) and phospholipase C pathways, causing downstream change
in the gene expression profile leading to cancer development.8
EGFR is expressed on normal human cells but higher levels
of expression of the receptor have also been correlated with
malignancy in a variety of cancers, including CRC.9
developed including monoclonal antibodies (mAbs), which target
the extracellular domain of EGFR, such as cetuximab, and small
molecule tyrosine kinase inhibitors (TKIs), which target the receptor
catalytic domain of EGFR, such as gefitinib and erlotinib.10Although
both classes of agents show clear antitumor activity, only anti-EGFR
‡These authors contributed equally to this work.
0305-7372/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
S. Rizzo et al./Cancer Treatment Reviews 36S3 (2010) S56–S61S57
monoclonal antibodies such as Cetuximab and Panitumumab have
been approved for clinical use in metastatic CRC.11
These costly and potentially toxic treatments are, however,
efficient in only a small percentage of patients, and it is therefore
extremely important to identify specific factors which will lead to a
clearer definition of those patients who will benefit from anti-EGFR
Potential positive predictive biomarkers
The major potential predictive factors of response to cetuximab
and/or panitumumab evaluated up till now in literature are
molecular factors involved more or less directly in the EGFR
signaling pathway. Among these, EGFR protein expression, EGFR
gene copy number, EGFR gene mutations, and overexpression of
EGFR ligands (such as epiregulin and amphiregulin) have been
evaluated in order to select patients who may benefit from EGFR-
Several clinical trials have been performed in patients with
metastatic CRC to relate the level of EGFR protein expression, as
determined by immunohistochemistry with sensitivity to anti-EGFR
antibodies. The results have demonstrated a lack of association
between EGFR detection by immunohistochemistry, and response to
EGFR-targeted treatment. Objective responses have been observed
in patients with low or high EGFR level expression and since
this biomarker has proved to be poorly associated with sensitivity
to anti-EGFR antibody, it cannot therefore be considered as
an inclusion criterion for patients undergoing treatment with
Activating mutations in the EGFR catalytic domain play an
treatment in lung cancer; these alterations are however, rare
or absent in CRC and are not significantly associated with
clinical response of metastatic CRC to the anti-EGFR monoclonal
In a small fraction of CRCs, the overexpression of EGFR is
frequently associated with amplification of the gene. The evaluation
of the EGFR gene copy number evaluated by quantitative PCR
does not seem to correlate with the clinical outcome of patients,
whereas the analysis by fluorescence in situ hybridation (FISH)
appears to be associated with an increase of treatment response.15
The predictive value is, however, uncertain and further studies are
therefore required to assess the increase of EGFR gene copy number
as a predictive marker of response to anti-EGFR treatment.
The overexpression of alternative EGFR ligands, such epiregulin
and amphireguline may promote tumor growth and survival by
an autocrine loop.16Several studies have correlated the expression
of these ligands with sensitivity to cetuximab monotherapy. The
results showed a statistically longer progression free survival (PFS)
among patients with high expression of epiregulin. The exclusive
use of amphiregulin or epiregulin gene expression profile does not,
however, result in the selection of patient populations benefiting
from cetuximab treatment.17
In order to increase the power of patient selection for anti-EGFR
therapy, several studies involving the identification of alternative
predictive molecular biomarkers have been conducted.
Biomarkers downstream to EGFR
EGFR-mediated signaling involves two main intracellular cascades:
KRAS, which, activating BRAF, triggers in its turn the mitogen-
activated protein kinases (MAPKs) and the membrane localization
of the lipid kinase PIK3CA, which counteracts with PTEN and
promotes AKT1 phosphorylation, thereby activating a parallel
KRAS is a proto-oncogene encoding a small 21kD guanosine
triphosphate (GTP)/guanosine diphosphate (GDP) binding protein
involved in the regulation of cellular response to many extracellular
stimuli.19After binding and activation by GTP, RAS recruits the
oncogene RAF, which phosphorylates MAP2K (mitogen-activated
protein kinase kinase), initiating the MAPK signaling leading
to the expression of the protein involved in cell proliferation,
differentiation and survival.20PIK3CA is an oncogene encoding for
the p110 subunit of PI3K, which can be activated via interaction by
the RAS protein.21
The constitutive activation of signaling pathways downstream of
the EGFR by mutations in KRAS, BRAF and PI3KCA and the interaction
between these pathways, drive the growth and progression of
CRC and provide an escape mechanism which allows the tumors
to overcome the pharmacological blockade induced by anti-EGFR
KRAS is the mostly commonly mutated gene in this pathway.
It is mutated in 35–45% of colorectal adenocarcinomas and this
alteration is an early event in colon tumorigenesis.23Up to 90% of
activating KRAS gene mutations are detected in codons 12 (70%)
and 13 (30%), and less in codon 61. Frequently these mutations
result in an exchange of different amino acids at the catalytic sites
which induce the glicine-to-valine substitution associated with a
more aggressive tumor growth.24
Negative predictive role of KRAS gene mutations
KRAS mutations have emerged as a major predictor of resistance
to panitumumab or cetuximab in the clinical setting and several
studies of patients receiving first and subsequent lines of treatment
have shown that those with tumors carrying KRAS mutations do
not respond to EGFR-targeted monoclonal antibodies or show any
survival benefit from such treatments.
Several studies have been conducted in order to explore the
role of KRAS mutations as a predictive biomarker of tumors from
patients with metastatic CRC treated with anti-EGFR monoclonal
antibody (with or without chemotherapy).25
The first study evaluating the correlation between K-RAS
mutational status in primary tumors and absence of response to
treatment with cetuximab or panitumumab, was that of Lievre et al.,
which involved a cohort study of 30 patients, and reported a link
between KRAS mutations and lack of response of metastatic patients
to EGFR-targeted monoclonal antibodies.
In this study K-RAS mutations were observed in 13 of the
30 patients enrolled in the study and these mutations were
closely associated with response to treatment; none of the mutated
tumors responded to cetuximab. Among responders none (0/11)
presented KRAS mutations, while a mutational status of KRAS
was found in 68.4% (13/19) of patients who were non-responders
(p=0.0003). The overall survival of K-RAS wild type patients (WT)
was significantly higher compared with those with mutated KRAS
(median OS: 16.3 vs 6.9 months, p=0.016).26
Amado et al., confirmed the negative predictive value of KRAS
mutations in a randomized phase III study, comparing the effect
of panitumumab monotherapy with best supportive care (BSC) in
patients with chemotherapy-refractory metastatic CRC.
The treatment effect on PFS in the WT KRAS group was
significantly greater (P 0.0001) than in the mutant group. Median
PFS in the WT KRAS group was 12.3 weeks for panitumumab and
7.3 weeks for BSC. The Authors therefore concluded their study
confining the panitumumab monotherapy efficacy in metastatic
CRC to patients with WT KRAS tumors.27
study of 572 patients, Karapetis et al. examined the role of
KRAS mutations in treatment response, comparing the effect of
cetuximab monotherapy with best supportive care (BSC) in patients
with chemo-refractory metastatic CRC.
In a randomized
S58S. Rizzo et al./Cancer Treatment Reviews 36S3 (2010) S56–S61
Disease-Free Survival Overall Survival
HR95% CLP HR95% CLP
Ki-RAS mut. codon 12
Valine1.301.09-1.54< 0.011.29 1.08-1.55< 0.01
Cysteine1.110.85-1.46 NS1.26 0.93-1.62NS
Alanine 1.210.89-1.66 NS1.35 0.98-1.87NS
K-RAS mut. codon 13
Aspartate0.94 0.79-1.12NS 0.930.78-1.12NS
Fig. 1. The RASCAL study: results of the multivariate analysis.
Cetuximab treatment in WT KRAS patients compared to BSC
resulted in a significant increase of the OS (median OS: 9.5 vs
4.8 months; HR=0.55, 95%CI: 0.41–0.74, P<0.001) and PFS (median
PFS: 3.7 vs 1.9 months; HR=0.40, 95%CI: 0:30 to 0:54, p<0.001)
while among KRAS mutated patients the differences were not
significant in either PFS or OS (p=0.96 p=0.89, respectively).28
Reports regarding KRAS data from large randomized trials have
recently been published, including the first-line phase II study OPUS
(Oxaliplatin and Cetuximab in First-Line Treatment of metastatic
CRC) and the first-line phase III study CRYSTAL (Cetuximab
Combined With Irinotecan in First-Line Therapy for Metastatic
Colorectal Cancer).29These results show that the KRAS mutated
patients do not benefit from addition of cetuximab to conventional
chemotherapy. Both PFS and OS were similar for cetuximab and
control groups in patients carrying tumors with KRAS mutations
(progression-free interval = 1.8 vs 1.8 months [HR=0.99, 95%CI =
0.73 to 1.35, P=0.96]; overall survival = 4.6 vs 4.5 months [HR=0.98,
95%CI = 0.70 to 1.37, P=0.89]). In the wild-type KRAS patients,
however, cetuximab treatment was associated with statistically
significantly (P<0.001) 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<0.001].29,30
Retrospective data from the OPUS and CRYSTAL studies indicate
that from the addition of cetuximab to first-line FOLFOX (folinic
acid, fluorouracil, and oxaliplatin)30
fluorouracil and irinotecan)29
chemotherapy does not benefit
patients with KRAS mutations. In fact, the OPUS study indicates that
addition of EGFR-targeted treatment to chemotherapy may even
be detrimental in such patients.30In some cases the addition of
cetuximab or panitumumab to standard chemotherapy may be not
useful even in KRAS wt patients.
In the PACCE (Panitumumab Advanced Colorectal Cancer Eval-
uation) study, the addition of panitumumab to bevacizumab and
chemotherapy was associated with shortening of the progression
free interval among patients with tumors carrying WT KRAS
(11.5 months in the chemotherapy–bevacizumab arm vs 9.8 months
in the panitumumab–chemotherapy–bevacizumab arm).31
In the CAIRO-2 (CApecitabine, IRinotecan, and Oxaliplatin trial)
study, the addition of cetuximab to capecitabine, oxaliplatin, and
bevacizumab as first-line treatment in patients with metastatic CRC
had no effect on progression-free interval among those with tumors
carrying WT KRAS (10.6 months in the chemotherapy–bevacizumab
arm vs 10.5 months in the combined cetuximab arm).32
In the large COIN trial the addition of cetuximab to Oxaliplatin-
based CT did not improve OS or PFS with increased nonhematologi-
cal toxicity in KRAS wt patients, even if the very advanced disease in
the COIN population may be the reason for the negative results..33
or FOLFIRI (folinic acid,
Prognostic role of KRAS gene mutations
The collaborative RASCAL II studies have been conducted with the
aim of investigating the prognostic role of KRAS mutations in CRC
To explore the effect of KRAS mutations at different stages of
CRC, 3493 patients were recruited in this multivariate analysis.
The results obtained suggest that of the 12 possible mutations on
codons 12 and 13 of KRAS, only the substitution glycine to valine on
codon 12, found in 8.6% of all patients, had a statistically significant
impact on PFS (P=0.0004, HR 1.3) and OS (P=0.008, HR 1.29)
(Fig. 1). Furthermore, these mutations have a greater impact on
outcome in Dukes’C cancers (failure-free survival, P=0.008, HR 1.5;
OS P=0.02 HR 1.45) than in Dukes’B cancers (failure-free survival,
P=0.46, HR 1.12; OS P=0.36 HR 1.15).
The RASCAL studies therefore show that tumors carrying mutated
KRAS might have an effect on the survival rate of CRC patients, and
that the specific codon 12 glycine/valine mutation not only might
play a role in the tumor progression, but this alteration might also
predispose to more aggressive biological behavior in patients with
Not all identified mutations of KRAS, however, necessarily have
the same biological, biochemical and functional role.34
The prognostic role of KRAS mutations in CRC progression is still
controversial. In a recent work Roth el al. in accordance with data
from smaller retrospective studies (PETACC-3, EORTC 40993, SAKK
60-00) sustain the lack of prognostic value for KRAS mutation status
for PFS and OS in patients with CRC. The prognostic significance of
KRAS mutations observed in the multivariate analysis of the RASCAL
studies might have been overestimated as a result of the number
of subset analyses. Larger studies are therefore required in order to
confirm whether a specific KRAS mutation might lead to a clinically
relevant prognostic effect in patients with CRC.35
Predictive and prognostic role of BRAF gene mutations
The most frequent BRAF mutation observed is a DNA missense
mutation leading to a valine to glutamic acid amino acid
substitution (V600E). It is functionally the most important
mutation involved in the receptor-independent aberrant activation
of the MEK-ERK pathway and CRC carcinogenesis. Mutations of
BRAF, kinase located downstream of K-RAS in the EGFR signal
transduction pathway, are found in colorectal tumors with a
relatively low frequency (approximately 10%). These alterations
have been studied in recent years in order to reach a better
understanding of its possible role in predicting response to anti-
EGFR drugs. Assuming that BRAF mutations may have, in K-RAS
wild type tumors, a predictive/prognostic role, Di Nicolantonio
et al. have retrospectively analyzed RR, TTP (time to progression),
OS and mutational status of K-RAS and BRAF in 113 patients
S. Rizzo et al./Cancer Treatment Reviews 36S3 (2010) S56–S61S59
KRAS wt/BRAF wt (n=730) KRAS wt/BRAF mut (n=70)
Cetuximab + CT
Cetuximab + CT
Mod. from Bokemeyer - ASCO 2010
Fig. 2. B-RAF mutational status in K-RAS Wt patients: pooled analysis of CRYSTAL and OPUS studies.
with mCRC treated with cetuximab or panitumumab. The BRAF
mutation V600E was the only one present in the cohort of analyzed
patients. Its presence appears inversely related to the activity
of the treatment, especially since some of the mutated patients
(11 out of 79 K-RAS w.t.) responded to the drug administration
and conversely none of the responders presented the mutation in
question (p=0.029). In addition, BRAF-mutated patients compared
with w.t obtained a significantly shorter PFS (p=0.011) and OS
(p<0.0001). The authors concluded by supporting the need to
have BRAF w.t. in order to obtain response from treatment with
EGFR inhibitors.36Furthermore, in BRAF-mutated colorectal cancer
cell lines, the sensitivity to EGFR inhibitors may be restored by
means of the multikinase inhibitor sorafenib. Several studies have
confirmed the negative prognostic role of BRAF mutations. In K-RAS
wild-type patients, BRAF-mutated individuals have shown a worse
outcome in terms of PFS and OS. Furthermore, BRAF is prognostic
for OS, especially in patients with microsatellite instability (MSI)
low (MSI-L) and stable (MSI-S) tumors. In the MSI-H (high)
subpopulation no prognostic value of K-RAS and BRAF mutation
status was found for RFS and OS.35The relatively low frequency of
this genetic alteration in colorectal cancer makes it rather difficult
to draw absolute conclusions also based on post-hoc analysis of
the Phase II and III studies recently published. The retrospective
analysis of the B-RAF mutational status has been performed in pts
in the CRYSTAL and OPUS studies (Fig. 2). The analysis of the OS and
PFS in pts with wt KRAS/wt BRAF showed a significant difference
between the two treatment arms. This difference was not significant
in the mutated BRAF/KRAS wt patients. Nevertheless, these patients
seem to benefit from the addition of Cetuximab, with an increase of
OS and a doubling of PFS rates. Furthermore, there is clearly a worse
outcome in mutated BRAF patients independently of treatment with
Cetuximab, which supports the hypothesis of a possible negative
prognostic role of BRAF mutations.37In the CAIRO-2 study, a similar
pattern was observed in a large series of mCRC patients treated with
chemotherapy and Bevacizumab with or without Cetuximab. It was
seen that the BRAF mutation is associated with a worse outcome,
both in terms of PFS and of OS, independently of the addition
of Cetuximab to the treatment..38To date, therefore, the negative
value of mutations of BRAF is only suggested by some reports,39
while the significant negative prognostic value seems to be now
PTEN-PI3K-AKT-mTOR pathway alterations
In addition to KRAS and BRAF, the HER family of receptors
also activates the PI3K signaling pathway, which in turn can be
oncogenically deregulated either by activating mutations in the
PIK3CA p110 subunit or by inactivation of the PTEN phosphatase.
The role of deregulated PIK3CA/PTEN signaling on the response to
targeted therapy has therefore been investigated in breast cancer,41
glioblastoma42and also mCRC. Mutation constitutive activation of
the PI3K signaling pathway has been reported to occur in ~30%
of colon tumors, primarily due to activating mutations in exons 9
and 20 of the PIK3CA gene43,44and, to a lesser extent, due to
inactivating PTEN mutations or PTEN promoter methylation.45PTEN
is a tumor suppressor that acts as a negative regulator of PI3K
signaling by converting PIP3 to PIP2, and truncating mutations
which result in loss of PTEN expression, reported in ~20% of MSI
The molecular alteration of PTEN is often caused by epigenetic
mechanisms,45supporting the detection of the intact protein by IHC
as a better diagnostic tool than gene sequencing, as it potentially
covers more mechanisms of alteration. PIK3CA mutation and PTEN
expression status predicts response of colon cancer cells to the EGFR
inhibitor cetuximab distinguishing drug sensitive and resistant cell
lines. Colon cancer cell lines with activating PIK3CA mutations
or loss of PTEN expression (PTEN null) were more resistant to
cetuximab than PIK3CA wild type (WT)/PTEN expressing cell lines.
Furthermore, cell lines that were PIK3CA mutant/PTEN null and
Ras/BRAF mutant were highly resistant to cetuximab compared
with those without dual mutations/PTEN loss, indicating that
constitutive and simultaneous activation of the Ras and PIK3CA
pathways confer maximal resistance to this agent. On the other
hand, these patients may be suitable candidates for treatment
with newer targeted drugs currently involved in clinical trials,
which inhibit signaling mediators further downstream, including
PI3K, AKT, or mTOR inhibitors and Ras, Raf, or MEK inhibitors. A
possible mechanism of resistance to cetuximab of these cell lines
may be the existence of alternate mutations in the Ras/BRAF and
or PIK3CA/PTEN pathway. Increased sensitivity to cetuximab was
observed in PIK3CA WT lines. Likewise, breast cancers with either
activating mutations in PIK3CA or with loss of PTEN expression
respond poorly to treatment with the Her2/Neu targeting antibody,
Consistent with the present findings, Frattini
et al. recently reported that colon tumors with loss of PTEN
expression have significantly reduced response to cetuximab.48
Furthermore, PIK3CA mutations and PTEN loss in colorectal tumors
are statistically and significantly associated with lack of response to
panitumumab or cetuximab treatment.18PIK3CA mutations and/or
loss of PTEN expression are negatively associated with PFS, and
loss of PTEN expression is also linked with poorer OS. A priori
screening of colon tumors for PTEN expression status and PIK3CA
and Ras/BRAF mutation status could help stratify patients likely
to benefit from this therapy.53Razis et al.54reported 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. Loupakis et al. performed
a retrospective analysis on the status of PTEN in a cohort of
S60S. Rizzo et al./Cancer Treatment Reviews 36S3 (2010) S56–S61
Non-EGFR-dep. growth → PD
in K-RAS wt
100 unselected mCRC pts
K-RAS wt 40%
Fig. 3. Activity of anti EGFR-Ab monotherapy in chemorefractory mCRC patients.
55 metastases from patients with irinotecan refractatory mCRC
treated with irinotecan and cetuximab: 12 (36%) of 33 patients with
PTEN-positive metastases were responders compared with one (5%)
of 22 who had PTEN-negative metastases.50Patients with PTEN-
positive metastases and KRAS wild type had longer PFS compared
with other patients.
Collectively, these findings show that colon cancer cell lines with
constitutively active PI3K signaling are refractory to cetuximab.
These data imply that colon cancer cell lines which acquire
mutations that result in constitutive activation of the PI3K pathway
have a diminished dependence on canonical EGFR ligand-induced
signaling for their growth and are, therefore, more resistant
to EGFR targeted therapies. Collectively, these studies provide
additional clinical evidence that the mutation status of the PI3K
signaling pathway should be considered before treatment with
EGFR family antagonists. Colon cancer cell lines mutant for
PIK3CA/PTEN null are significantly more resistant to cetuximab
compared with PIK3CA/PTEN WT lines. Furthermore, cell lines
with both constitutively active PIK3CA and Ras/BRAF signaling
were highly refractory to cetuximab. These studies suggest that
combining mutation analysis for K-RAS 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.18
EGFR and its downstream K-RAS/B-RAF and PTEN-PI3K-AKT-mTOR
pathways play an important role in tumorigenesis and tumor
progression of CRC.
Only in a fraction of patients with mCRC (10% according to
RECIST criteria that are not adequate for the evaluation of tumor
response to new molecular agents) anti-EGFR mAbs (cetuximab
and panitumumab) have shown remarkable efficacy (Fig. 3).
Forty percent of patients with mCRC obtain clinical benefit from
monotherapy with anti-EGFR antibodies, which means that they
present EGFR-dependent tumoral growth. The remaining 60% of
the patients are Non Responders, that is, they present tumoral
growth which does not depend on the EGFR block mediated by the
anti-EGFR Abs. This limitation seems linked mainly to oncogenic
KRAS mutations in codon 12 and 13, which implies its continuous
activation and signal transduction to the nucleus, even when the
EGFR is blocked. KRAS mutation seems to be responsible in 35–45%
cases of resistance to anti-EGFR Abs. The potential bias associated
with a retrospective evaluation of the mutational status of KRAS
in the CRYSTAL and OPUS studies seems not relevant today, since
in both studies the difference between the ITT population and the
population assessable for KRAS is negligible.
Another group of patients, representing 10% of the total number
of patients, carry the BRAF mutation that is mutually exclusive with
KRAS mutations. It therefore appears that BRAF mutations may play
a strong negative prognostic role and only a slight role in resistance
to anti-EGFR Abs, since even pts with mCRCs treated without
Cetuximab have significantly reduced PFS and OS when the tumor
presents a BRAF mutation. The investigation of other biomarkers
such as EGFR copy number and expression levels of EGFR ligands,
phosphatase and tensin homolog (PTEN) loss or NRAS mutation may
be useful to further refine the responder population. Nevertheless,
up till now, clinical evidence all points towards the identification
of the KRAS mutation as the only evaluated and reproducible
predictive factor of resistance to anti-EGFR antibodies.
Conflict of interests
All authors have no conflict of interest to declare.
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