Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma

Abstract

Background:
As epidermal growth factor receptor (EGFR) is involved in the pathogenesis of malignant pleural mesotheliomas (MPMs), the anti-EGFR drugs may be effective in treating MPM patients. Mutations of the EGFR gene or its downstream effectors may cause constitutive activation leading to cell proliferation, and the inhibition of apoptosis and metastases. Consequently, molecular profiling is essential for select patients with MPM who may respond to anti-EGFR therapies.

Methods:
After manual macrodissection, genomic DNA was extracted from 77 histological samples of MPM: 59 epithelioid, 10 biphasic, and 8 sarcomatoid. Epidermal growth factor receptor gene mutations were sought by means of real-time polymerase chain reaction (PCR) and direct sequencing, KRAS gene mutations by mutant-enriched PCR, and PIK3CA and BRAF gene mutations by direct sequencing.

Results:
Gene mutations were identified in nine cases (12%): five KRAS, three BRAF, and one PI3KCA mutation; no EGFR gene mutations were detected. There was no difference in disease-specific survival between the patients with or without gene mutations (P=0.552).

Conclusions:
Mutations in EGFR downstream pathways are not rare in MPM. Although none of those found in this study seemed to be prognostically significant, they may support a more specific selection of patients for future trials.

Full text

Mutation analysis of the EGFR gene and
downstream signalling pathway in histologic
samples of malignant pleural mesothelioma
R Mezzapelle
1,2
, U Miglio
1
, O Rena
3
, A Paganotti
4
, S Allegrini
1
, J Antona
1
, F Molinari
5
, M Frattini
5
, G Monga
1
,
O Alabiso
6
and R Boldorini*
,1,4
1
Unit of Pathology, Department of Health Sciences, University of Eastern Piedmont ‘Amedeo Avogadro’, Via Solaroli 17, Novara
28100, Italy;
2
Division of Cancer Genomics, ‘Edo ed Elvo Tempia Valenta’ Foundation, Via Malta 2, Biella 13900, Italy;
3
Unit of
Thoracic Surgery and ‘Maggiore della Carita
´’ Hospital, Corso Mazzini 18, Novara 28100, Italy;
4
Unit of Pathology, ‘Maggiore della
Carita
´’ Hospital, Corso Mazzini 18, Novara 28100, Italy;
5
Institute of Pathology, Via Selva 24, Locarno 6600, Switzerland and
6
Unit of
Oncology, Department of Translational Medicine, University of Eastern Piedmont ’Amedeo Avogadro‘, Via Solaroli 17, Novara
28100, Italy
Background: As epidermal growth factor receptor (EGFR) is involved in the pathogenesis of malignant pleural mesotheliomas
(MPMs), the anti-EGFR drugs may be effective in treating MPM patients. Mutations of the EGFR gene or its downstream effectors
may cause constitutive activation leading to cell proliferation, and the inhibition of apoptosis and metastases. Consequently,
molecular profiling is essential for select patients with MPM who may respond to anti-EGFR therapies.
Methods: After manual macrodissection, genomic DNA was extracted from 77 histological samples of MPM: 59 epithelioid, 10
biphasic, and 8 sarcomatoid. Epidermal growth factor receptor gene mutations were sought by means of real-time polymerase
chain reaction (PCR) and direct sequencing, KRAS gene mutations by mutant-enriched PCR, and PIK3CA and BRAF gene
mutations by direct sequencing.
Results: Gene mutations were identified in nine cases (12%): five KRAS, three BRAF, and one PI3KCA mutation; no EGFR gene
mutations were detected. There was no difference in disease-specific survival between the patients with or without gene
mutations (P¼0.552).
Conclusions: Mutations in EGFR downstream pathways are not rare in MPM. Although none of those found in this study seemed
to be prognostically significant, they may support a more specific selection of patients for future trials.
Malignant mesotheliomas are relatively rare, highly malignant
tumours that arise from the mesothelial cells lining the serosal
cavities of the body. The most frequent are malignant pleural
mesotheliomas (MPMs) (Boutin et al, 1998), whose estimated
annual incidence in Europe (15–33 cases per million inhabitants) is
expected to increase further over the next 20 years because of their
long period of latency (Peto et al, 1999). The main carcinogen
associated with MPMs is asbestos, and occupational exposure to it
has been considered the main risk factor (Wagner et al, 1960;
Lanphear and Buncher, 1992; Carbone et al, 2002); however, other
potential carcinogenic agents include infection by Simian Virus 40
and radiation exposure (Yang et al, 2008).
The median survival of patients with MPM is currently 12
months from the time of diagnosis despite treatment (Vogelzang
et al, 2003). The European Organisation for Research and
Treatment of Cancer has indicated that the main predictors of a
negative prognosis are a poor performance status (PS), high white
blood cell counts, male gender, a sarcomatoid subtype, anaemia,
*Correspondence: Professor R Boldorini; E-mail: renzo.boldorini@med.unipmn.it
Received 1 February 2013; accepted 4 March 2013; published online 4 April 2013
&2013 Cancer Research UK. All rights reserved 0007 – 0920/13
FULL PAPER
Keywords: malignant mesothelioma; EGFR; mutation analysis; target therapy
British Journal of Cancer (2013) 108, 1743–1749 | doi: 10.1038/bjc.2013.130
www.bjcancer.com | DOI:10.1038/bjc.2013.130 1743

and thrombocytosis (Curran et al, 1998), but it has been recently
suggested that short survival may also be related to a high nuclear
tumour grade, a high MIB-1 labelling index (Comin et al, 2000),
and high epidermal growth factor receptor (EGFR) expression in
tumour cells (Rena et al, 2011).
There is no standard of care for MPM, but systemic
chemotherapy is the only possible treatment option for most
patients at the time of diagnosis. It has been found that pemetrexed
combined with cisplatin is more effective than platinum alone in
terms of overall survival (12.1 vs 9.3 months), time to progression
(5.7 vs 3.9 months), and objective responses (41.3% vs 16.7% of
tumour shrinkage) (Vogelzang et al, 2003), but, as most patients
progress during or shortly after these first-line treatments, there is
a clear need to develop more effective antitumour agents.
Epidermal growth factor receptor is a receptor tyrosine
kinase that is overexpressed in 60–70% of MPM tissue specimens
(mainly of the epithelial subtype), but not in the normal
mesothelium (Dazzi et al, 1990; Destro et al, 2006; Okuda et al,
2008). Exposure to asbestos fibres causes EGFR aggregation, and
the subsequent autophosphorylation and activation of EGFR
activates both the RAS/RAF/MAPK pathway (which induces cell
proliferation, metastasis, and invasion) (Pache et al, 1998) and the
PI3KCA/AKT/mTOR pathway, which leads to the inhibition of
apoptosis (Suzuki et al, 2009). Consequently, inhibiting EGFR
pathways should have an antitumoral effect. Two classes of EGFR
inhibitors have been developed for cancer therapy: tyrosine kinase
inhibitors (TKIs), which block EGFR autophosphorylation by
competing with ATP binding, and anti-EGFR monoclonal
antibodies (mABs), which compete with the ligand binding
the extracellular domain of EGFR. It has been reported that
mutation analysis of the EGFR gene and of some of its down-
stream signal-transduction proteins predicts the response of
lung adenocarcinomas to TKIs (Shepherd et al, 2005) and the
response of colorectal cancer to anti-EGFR mABs (cetuximab,
panitumumab) (Jonker et al, 2007).
Preclinical studies have shown that EGFR TKIs are highly
efficacious (Barbieri et al, 2011), but two phase II studies of
gefitinib and erlotinib used alone to treat malignant pleural and
peritoneal mesotheliomas failed to demonstrate their clinical
efficacy, although it needs to be pointed out that the patients in
both trials were not selected on the basis of any molecular criteria
(Govindan et al, 2005; Garland et al, 2007). One recent study has
shown that cetuximab effectively blocks the growth of MPM cells
in cell cultures and mouse models (Kurai et al, 2012) and, as in the
case of colorectal cancer and lung adenocarcinomas, the potential
efficacy of these TKIs in MPM may depend on the mutation status
of EGFR gene and its downstream effectors (Lie
`vre et al, 2006;
Pirker et al, 2011).
To the best of our knowledge, only a few low-powered studies
have investigated the presence and frequency of EGFR gene
mutations in MPM (Cortese et al, 2006; Enomoto et al, 2012), and
none has searched for mutations in the KRAS,BRAF, and PI3KCA
downstream effectors. We searched a large series of histological
MPM samples for mutations in EGFR gene and its main
downstream signalling effectors to evaluate their frequency and
possible prognostic significance, and their possible use as
predictors of the response of MPMs to targeted therapies.
MATERIALS AND METHODS
Patients and samples. The study involved 77 consecutive MPM
patients admitted to the Thoracic Unit of the University Hospital
of Novara between January 2008 and December 2010, all of whom
were diagnosed as having MPM on the basis of multiple pleural
biopsies taken by means of video-assisted thoracoscopy. The
tumour samples were immediately fixed in formalin for 24 h,
embedded in paraffin, and routinely processed for histology and
immunohistochemistry, and the diagnosis of MPM was based on
standard histological and immunohistochemical criteria, including
positivity to calretinin, vimentin, and cytokeratins 5 and 6, and
negativity to carcinoembryonic antigen, thyroid transcription
factor 1, and Ber Epy 4. The MPMs were classified on the basis
of the WHO classification of pleural tumours (Travis et al, 2004),
and clinically and pathologically staged on the basis of the TNM
staging system (Sobin et al, 2009). The patients’ PS at the time of
diagnosis was graded using the Eastern Cooperative Oncology
Group (ECOG) scale (Oken et al, 1982), and the patients with a PS
of 0–2 underwent therapeutic protocols indicated by the referring
oncologist.
Haematoxylin/eosin-stained slides of the pleural biopsies and
corresponding formalin-fixed, paraffin-embedded blocks were
reviewed by a pathologist (RB) to select the area with 450% of
tumour cells.
DNA extraction. The tumoral areas of the formalin-fixed,
paraffin-embedded sections were macrodissected manually, and
then five 5-mm-thick sections were prepared and collected in a
1.5 ml tube. Genomic DNA was extracted using EDTA-SDS/
proteinase K followed by phenol–chloroform, and resuspended
with 30 ml of DEPC-treated and RNAse-free water (Promega,
Madison, WI, USA).
Mutational analysis
Epidermal growth factor receptor gene. All of the samples were
analysed using the TheraScreen EGFR29 Mutation Kit (Qiagen,
Manchester, UK), which combines the two technologies of ARMS
and Scorpion chemistry to detect mutations in a real-time
polymerase chain reaction (PCR). This kit allows the detection of
in-frame deletions on exon 19, insertions on exon 20, and G719X,
S768I, T790M, L858R, and L861Q mutations against a background
of WT genomic DNA with a sensitivity of 1%. Starting from 2 mlof
genomic DNA, the analyses were made in accordance with the
manufacturer’s instruction using RotorGene Q (Qiagen), and the
results were interpreted following the datasheet.
To determine the presence of other less common mutations, the
samples underwent further PCRs to amplify the whole sequence of
exons 18–21 of the EGFR gene as described previously by Paez et al
(2004). Table 1 shows the primers and PCR conditions.
KRAS gene. The KRAS gene was analysed by means of a mutant-
enriched PCR (ME-PCR) to detect the hotspots in codons 12 and
13 of exon 2 that include more than 95% of the known gene
mutations. The ME-PCR consisted of two amplification steps
(seminested PCR) in which artificial restriction sites were
introduced into the wild-type amplicon using mismatched primers
(Table 1). The restriction sites (BstNI for codon 12 and BglI for
codon 13) introduced during the first PCR step were localised
immediately next to the KRAS codon in the analysis to distinguish
wild-type and mutant sequences. The wild-type amplicons were
then digested by BstNI or BglI restriction enzymes, and the mutant
products were enriched for a second round of amplification. The
ME-PCR and digestion conditions have been described previously
(Molinari et al, 2011). Mutant-enriched PCR has a sensitivity of up
to 0.01%. All of the samples underwent automated sequencing by
using an ABI PRISM 3130 (Applied Biosystems, Foster City; CA,
USA) and reverse primers.
BRAF gene. Exon 15 of the BRAF gene (which contains the
hotspot codon 600, where more than 90% of gene mutations occur)
was analysed by means of direct sequencing in accordance with
previously published protocols (Di Nicolantonio et al., 2008),
BRITISH JOURNAL OF CANCER Analysis of EGFR gene and pathway in malignant mesothelioma
1744 www.bjcancer.com | DOI:10.1038/bjc.2013.130

starting from 50 ng of genomic DNA. The primers and PCR
conditions are shown in Table 1.
PI3KCA gene. The analysis of the PIK3CA gene was concentrated
on exons 9 and 20, which include all of the hotspot codons, using
previously published protocols (Sartore-Bianchi et al., 2009). The
primers and PCR conditions are shown in Table 1.
Sequence analysis. All of the PCR products and KRAS second
enzymatic digestions were analysed by means of 3% agarose gel
electrophoresis, and then purified using NucleoSpin Gel and the
PCR clean-up kit (Macherey-Nagel, Du¨ren, Germany). The
sequence of each gene was analysed using an ABIPrism 3130
Genetic Analyzer (Applied Biosystems), and all of the mutated
cases were confirmed two times starting from independent PCR
reactions.
Statistical analysis. This examined the correlations between the
presence of gene mutations and other demographic, clinical and
pathological variables. The associations between categorical vari-
ables were determined using the w
2
or Fisher’s exact test. The
statistical differences of the average values were tested using a
Student’s t-test and analysis of variance, followed by Bonferroni’s
test.
The impact of the different variables on long-term outcomes
was analysed using the Kaplan–Meier method of analysing disease-
specific survival (DSS); the survival data were compared using the
log-rank test.
P-values of o0.05, with a 95% confidence interval, were
considered statistically significant.
RESULTS
In all, 57 patients were male (74%) and 20 were female (26%); their
average age at the time of diagnosis was 68 years (range 43–90,
median 64.5 years). Of these, 50 patients (64.9%) had previously
been exposed to asbestos at work. Histological examination showed
that 59 MPMs (77%) were epithelioid, 10 (13%) biphasic, and 8
(10.4%) sarcomatoid. In total, 41 patients had stage II tumours, 30
stage III tumours, and 6 stage IV tumours. Eastern Cooperative
Oncology Group PS was 0–2 in 68 patients, and 42 in nine
patients. In all, 41 patients were treated with platinum plus
pemetrexed and 22 with platinum alone; 14 received no treatment
because their PS was 42 or because they refused.
Follow-up data were collected from 74 patients (three were lost
to follow-up). In all, 15 patients were still alive at June 2012 with a
median follow-up of 24.5 months (range 14–39 months). The
median DSS of the cohort as a whole was 12.5 months (range 1–39
months).
Mutation analysis. Mutations in the EGFR downstream pathway
were identified in nine patients (12%): five in the KRAS gene, three
in the BRAF gene, and one in the PIK3CA gene. No mutations were
detected in the EGFR gene by direct sequencing or the Scorpions-
ARMS assay, even though the latter has a sensitivity of 1% (vs the
10–20% of direct sequencing).
Table 1. Sequences of primers and PCR reaction protocol
Gene Primer
name Sequence Cycle Length
KRAS codon 12 (outer) 3F 50-ACTGAATATAAACTTGTGGTAGTTGGACCT–3095 1C10 min; (95 1C30 s, 501C1 min,
72 1C1 min) 20 cycles; 72 1C3min
143
10B 50-ACTCATGAAAATGGTCAGAGAAACCTTTAT-30
KRAS codon 13 (outer) 9F 50-ACTGAATATAAACTTGTGGTAGTTGGCCCTGGT-3095 1C10 min; (95 1C30 s, 54 1C1 min,
72 1C1 min) 20 cycles; 72 1C3min
113
10B 50-ACTCATGAAAATGGTCAGAGAAACCTTTAT-30
KRAS codon 12 (inner) 3F 50-ACTGAATATAAACTTGTGGTAGTTGGACCT-30
95 1C10 min; (95 1C30 s, 54 1C1 min,
72 1C1 min) 45 cycles; 72 1C3min
143
14B 50-TCAAAGAATGGTCCTGGACC-30
KRAS codon 13 (inner) 9F 50-ACTGAATATAAACTTGTGGTAGTTGGCCCTGGT-30113
4B 50-TCAAAGAATGGTCCTGCACC-30
EGFR exon 18 EGFR18F 50-TCCAGCATGGTGAGGGCTGAG-30
50 1C2 min; 95 1C10 min; (95 1C40 s,
58 1C40 s, 72 1C35 s) 40 cycles;
72 1C3min
242
EGFR18R 50-GGCTCCCCACCAGACCATG-30
EGFR exon 19 EGFR19F 50-TGGGCAGCATGTGGCACCATC-30217
EGFR19R 50-AGGTGGGCCTGAGGTTCAG-30
EGFR exon 20 EGFR20F 50-CCTCCTTCTGGCCACCATGCG-30296
EGFR20R 50-CATGTGAGGATCCTGGCTCC-30
EGFR exon 21 EGFR21F 50-CCTCACAGCAGGGTCTTCTC-30229
EGFR21R 50-CCTGGTGTCAGGAAAATGCT-30
BRAF exon 15 BRAF15F 50-TCATAATGCTTGCTCTGATAGGA-30951C10 min; (95 1C15 s, 52 1C30 s,
72 1C30 s) 45 cycles; 72 1C3min
224
BRAF15R 50-GGCCAAAAATTTAATCAGTGGA-30
PIK3CA exon 9 PIK3CA9F 50-GGGAAAAATATGACAAAGAAAGC-30
95 1C10 min; (95 1C35 s,
251
PIK3CA9R 50-CTGAGATCAGCCAAATTCAGTT-30
56 1C30 s, 72 1C30 s) 40 cycles;
72 1C10 min
PIK3CA exon 20 PIK3CA20F
PIK3CA20R
50-CTCAATGATGCTTGGCTCTG-30
50-TGGAATCCAGAGTGAGCTTTC-30
241
Analysis of EGFR gene and pathway in malignant mesothelioma BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2013.130 1745

KRAS and BRAF gene mutational profiling. The KRAS gene was
successfully amplified in all of the samples, five of which showed
mutations: two patients had the GGT-GtT point mutation in
codon 12 leading to a glycine-to-valine amino-acid substitution
(G12V); two had the GGC-GaC point mutation in codon 13
leading to a glycine-to-aspartic acid substitution (G13D); and one
had the rare GGC-aGC mutation in codon 13 leading to a
glycine-to-serine substitution (G13S). As shown in Table 2, three of
the five mutations occurred in patients with epithelioid MPMs
(G12V, G13D, and G13S), one in a patient with a biphasic MPM
(G13D), and one in a patient with a sarcomatoid subtype (G12V).
All five patients with KRAS mutations reported previous occupa-
tional asbestos exposure.
The BRAF gene mutational analysis showed the classical valine-
to-glutamic amino-acid substitution in codon 600 (V600E) in three
patients: two with epithelioid MPMs and one with a biphasic
tumour (Table 2). None of them reported previous occupational
asbestos exposure.
PI3KCA gene mutational profiling. The DNA of exons 9 and 20
of the PIK3CA gene was successfully amplified from 75 of the 77
specimens. A point mutation was detected in only one case: it
occurred in exon 20, and led to a methionine-to-isoleucine
substitution in position 1040 (M1040I). The patient had a biphasic
mesothelioma and no previous occupational asbestos exposure
(Table 2).
Statistical analysis. The correlations between the presence/
absence of gene mutations and demographic, clinical, and
pathologic features (gender, age, occupational asbestos exposure,
history of previous cancer, histological type, ECOG PS, treatment)
were investigated, without finding any significant differences
(Table 3).
The Kaplan–Meier analysis of the influence of some variables
on long-term outcomes revealed no difference in DSS between
the patients with and without gene mutations (P¼0.552).
Moreover, separate evaluation of the patients with KRAS and
BRAF mutations did not indicate any advantage in terms of
DSS (P¼0.363 and 0.752) and, within the mutated group, no
mutation significantly correlated with DSS (KRAS,P¼0.363;
BRAF,P¼0.187).
Interestingly, the patients with KRAS gene mutations reported
occupational asbestos exposure, whereas those with BRAF and
PI3KCA gene mutations did not. When the DSS of the patients
with reported asbestos exposure was considered, the five KRAS
gene-mutated patients had a worse prognosis than those with wild-
type KRAS (n¼42), although the difference was not statistically
significant (mean survival, 9.20±6.91 vs 15.6±10.39 months;
P¼0.188) (Figure 1A). On the contrary, the DSS of the patients
without reported occupational asbestos exposure was better in the
BRAF gene-mutated patients (n¼3) than in those without BRAF
mutations (n¼22), although, once again, the difference was not
statistically significant (mean survival, 20.33±12.06 vs 12.1±8.37
months; P¼0.140) (Figure 1B).
DISCUSSION
As EGFR is involved in the carcinogenesis of MPM, it is possible
that EGFR-targeted therapies may be efficacious in MPM patients
(Barbieri et al, 2011). Epidermal growth factor receptor TKI
inhibitors, such as gefitinib and erlotinib, inhibit MPM cell
migration and proliferation, enhance the response to radiation of
human MPM cell lines, and reduce motility and invasion in MPM
Table 2. Characteristics of patients with gene mutations
Patient Gene Amino-acid
substitution Gender Age Histotype Asbestos
exposure DSS
1KRAS G12V Male 81 Sarcomatoid Yes 4
2 G12V Male 55 Epithelioid Yes 14
3 G13D Male 82 Epithelioid Yes 4
4 G13S Male 60 Epithelioid Yes 5
5 G13D Male 77 Biphasic Yes 19
6BRAF V600E Female 51 Epithelioid None 9
7 V600E Male 57 Biphasic None 19
8 V600E Male 73 Epithelioid None 33
9PIK3CA M1040I Male 68 Biphasic None 7
Abbreviation: DSS ¼disease-specific survival.
Table 3. Statistical correlation between demographic, clinical,
pathological data, and gene mutations
Wild type
(n¼68)
Mutations
(n¼9) P-value
Age, mean±s.d. (years) 66±21 67±12 0.89
Gender (male proportion) 49/68 8/9 0.491
Previous cancer 5/68 2/9 0.385
Asbestos exposure 44/68 6/9 0.799
Histological subtype
Epithelial 54/68 5/9 0.241
Biphasic 7/68 3/9 0.135
Sarcomatoid 7/68 1/9 0.623
ECOG score
0–2 60/68 8/9 0.892
42 8/68 1/9 0.617
Clinical stage
II 37/68 4/9 0.689
III–IV 31/68 5/9 0.576
Treatment type
None 13/68 1/9 0.623
Platinum 19/68 3/9 0.876
Platinum þpemetrexed 36/68 5/9 0.776
Abbreviations: ECOG ¼Eastern Cooperative Oncology Group; s.d. ¼standard deviation.
BRITISH JOURNAL OF CANCER Analysis of EGFR gene and pathway in malignant mesothelioma
1746 www.bjcancer.com | DOI:10.1038/bjc.2013.130

cell lines (Kurai et al, 2012). However, the promising results
obtained in in vitro studies were not reproduced in two phase II
trials involving patients with pleural and peritoneal mesotheliomas,
although it should be noted that neither study evaluated the
mutation status of the EGFR gene and its downstream signalling
transduction pathway (Govindan et al, 2005; Garland et al, 2007).
As in the case of colorectal cancer and lung adenocarcinoma,
this lack of molecular selection could explain the therapeutic
failure.
The few studies that have sought mutations in the tyrosine
kinase domain of the EGFR gene in patients with malignant
mesotheliomas involved small populations and used a relatively
insensitive method (the direct sequencing of exons 18–21) (Cortese
et al, 2006; Velcheti et al, 2009; Enomoto et al, 2012). The primary
objective of our study was to look for EGFR gene mutations in a
larger series of patients (n¼77) using two molecular methods: all
of the cases were first screened using Scorpion-ARMS technology,
which is capable of detecting 1% of mutated cells against a 99%
background of wild-type cells, followed by direct sequencing to
find rarer mutations or mutations that cannot be detected using
the first method. However, despite this, we did not find any
mutations in the TK domain of EGFR: in addition to confirming
previous findings (Cortese et al, 2006; Velcheti et al, 2009), this
also indicates that, unlike in the case of lung adenocarcinomas,
mutations cannot be detected even when real-time PCR is used to
increase sensitivity (Allegrini et al, 2012).
On the contrary, Enomoto et al (2012) have recently studied 38
patients and found EGFR missense mutations in exons 18 (n¼1),
20 (n¼3), and 21 (n¼1) in six (16%) patients with pleural (n¼3)
or peritoneal mesotheliomas (n¼3). Epidermal growth factor
receptor gene mutations have been previously found in peritoneal
mesotheliomas (Foster et al, 2009, 2010), but this is the only
published report of EGFR gene mutations in MPM. However, the
study involved Japanese patients, who are characterised by more
frequent EGFR gene mutations in lung adenocarcinoma than
Western patients (Endo et al, 2005). Furthermore, some of the
detected mutations had never been reported before, and their
biological and clinical significance is still unknown.
An alternative method of blocking EGFR is to use mABs, which
may be extremely useful as it has been demonstrated that MPM
patients show EGFR gene amplification (Dazzi et al, 1990; Destro
et al, 2006; Okuda et al, 2008). No published studies have assessed
the in vivo effects of anti-EGFR mAbs on MPMs, although one
recent study has found that cetuximab is highly efficacious in
cultured MPM cell lines (Kurai et al, 2012). It has been
demonstrated that mutations in EGFR downstream pathways can
affect the efficacy of EGFR mABs in other tumours such as
colorectal adenocarcinoma (Jonker et al, 2007), and we found nine
patients (11.7%) with missense mutations involving the KRAS
(n¼5), BRAF (n¼3), and PIK3CA genes (n¼1). Few other
studies have separately investigated the presence of mutations in
KRAS,BRAF, and PIK3CA genes in MPM samples and
mesothelioma cell lines without success (see review by Agarwal
et al, 2011), but, to the best of our knowledge, ours is the first to
investigate these alterations systematically in a large series of MPM
patients.
Various reasons may explain these discrepant results. We
screened a large number of samples (n¼77), whereas the other
studies were based on smaller series and may have underestimated
the real frequency of such mutations. Furthermore, we analysed
KRAS gene mutations using an ME-PCR technology whose
sensitivity is 0.1% (Molinari et al, 2011), and so it is possible that
the percentage of KRAS gene-mutated cells is very low in MPM
and that more widely used sequencing methods are unable to
detect small clones.
Our findings show that, although infrequent, mutations in
EGFR downstream pathways can be found in MPMs, thus
supporting the hypothesis that EGFR mABs may be clinically
effective in the majority of patients. On the other hand, patients
with a molecular profile indicating putative resistance to EGFR
mABs (because of the presence of KRAS or BRAF or PIK3CA
mutations) may be directed towards new targeted therapies. One
recent study has shown that vemurafenib is promising not only in
patients with metastatic melanoma but also in patients with non-
small-cell lung cancer carrying a BRAF mutation (Gautschi et al,
2012), and selumetinib and BYL-719, which target KRAS and
PIK3CA mutations, are currently being evaluated in several clinical
trials (http://ctmagnifier.org/). Our data therefore underline the
importance of the molecular characterisation of patients with
MPM.
The clinical implications of the gene mutations detected in our
study are not clear. DSS was no different in the patients with or
without gene mutations (whether analysed together or separately).
Interestingly, all of the patients with KRAS gene mutations
reported occupational asbestos exposure, but none of those with
BRAF or PIK3CA gene mutations. Comparison of mean DSS in the
KRAS and BRAF gene-mutated patients vs wild-type patients
previously exposed to asbestos or not, showed that the KRAS gene-
mutated patients (n¼5) tended to have a worse prognosis than the
wild-type patients (9.20±6.91 vs 15.6±10.39 months), and the
BRAF gene-mutated patients (n¼3) tended to have a better
1.0
KRAS mutation
Complete
Censored
Wild-type KRAS
Mutated KRAS
Complete
Censored
Wild-type BRAF
Mutated BRAF
Log-rank test, P = 0.188
BRAF mutation
A
B
Log-rank test, P = 0.140
0.9
0.8
0.7
0.6
Disease-specific survival (proportion)
0.5
0.4
0.3
0.2
0.1
0.0
1.0
0.9
0.8
0.7
0.6
Disease-specific survival (proportion)
0.5
0.4
0.3
0.2
0.1
0.0
06121824
Months
30 36 42
06 121824
Months
30 36 42
Figure 1. Kaplan–Meier DSS curves for MPM patients with KRAS
mutation vs wild-type (A) and with BRAF mutation vs wild-type (B).
Analysis of EGFR gene and pathway in malignant mesothelioma BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2013.130 1747

prognosis (20.33±12.06 vs 12.1±8.37 months). However, the
differences were not statistically significant and our findings need
to be confirmed in larger series of MPM patients.
In conclusion, our extensive molecular characterisation of EGFR
pathways may explain the failure of TKI administration and may
open up the possibility of developing new targeted therapies.
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Analysis of EGFR gene and pathway in malignant mesothelioma BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2013.130 1749