Oncogene (2004) 23, 554558

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Mutations of BRAF and KRAS2 in the development of Barretts

adenocarcinoma
Florian Sommerer1,6, Michael Vieth2,6, Annett Markwarth1, Knut Rohrich1, Susanne Vomschlo!1,
Andrea May3, Christian Ell3, Manfred Stolte4, Ulrich R Hengge5, Christian Wittekind1 and
Andrea Tannapfel*,1
1

Institute of Pathology, University of Leipzig, Liebigstra!e 26, Leipzig 04103, Germany; 2Institute of Pathology, Otto-von-Guericke
University, Magdeburg 39120, Germany; 3Clinic of Internal Medicine II, HSK-Clinics Wiesbaden, Wiesbaden 65199, Germany;
4
Institute of Pathology, Klinikum Bayreuth, Bayreuth 95445, Germany; 5Department of Dermatology, Heinrich-Heine-University,
Dusseldorf, Germany

Activation of the Raf/MEK/ERK (MAPK) signal transduction cascade by RAS mutations has been found in a variety of
human cancers. Mutations of BRAF provide an alternative
route for activation of this signalling pathway. To determine
the role of mutations in BRAF and KRAS2 in the neoplastic
progression of Barretts adenocarcinoma, we analysed both
genes for common mutations. After microdissection, DNA of
19 Barretts adenocarcinomas, 56 Barretts intraepithelial
neoplasias (n 29 low-grade intraepithelial neoplasia (LGIN)
and n 27 high-grade intraepithelial neoplasia (HGIN)), 30
Barretts mucosa without neoplasia and normal squamous, as
well as gastric epithelium, were analysed for BRAF and
KRAS2 mutation. Activating BRAF mutations were identified in 2/19 Barretts adenocarcinomas (11%) and in 1/27
HGIN (4%). KRAS2 mutations were found in four out of 19
(21%) Barretts adenocarcinomas examined and in three
cases of HGIN (11%). In LGIN as well as in normal gastric
or oesophageal mucosa, neither BRAF nor KRAS2 mutations
were detected. All lesions with KRAS2 mutations had an
intact BRAF gene. The status of mismatch-repair proteins
was neither related to BRAF nor KRAS2 mutations. These
data indicate that RAS or BRAF mutations are detected in
about 32% of all Barretts adenocarcinomas. We conclude
that the disruption of the Raf/MEK/ERK (MAPK) kinase
pathway is a frequent but also early event in the development
of Barretts adenocarcinoma.
Oncogene (2004) 23, 554558; doi:10.1038/sj.onc.1207189
Keywords: BRAF; KRAS2; barrett; neoplasia

Introduction
Barretts adenocarcinoma arises from Barretts oesophagus, in which an intestinal-type epithelium (specia*Correspondence: A Tannapfel; E-mail: tana@medizin.uni-leipzig.de
6
Florian Sommerer (FS) and Michael Vieth (MV) contributed equally
to the study
Received 31 July 2003; revised 4 September 2003; accepted 8 September
2003

lized intestinal metaplasia) replaces oesophageal

squamous epithelium damaged by gastro-oesophageal
reflux disease. The development of cancer in Barretts
oesophagus follows a multistep pathway. Histologically,
there is a progression from intestinal metaplasia
(Barretts mucosa) through low- (LGIN) and high-grade
intraepithelial neoplasia (HGIN) to adenocarcinoma
(Barrett et al., 1996; Spechler, 2002; Tselepis et al.,
2002).
To date, the exact cellular and molecular mechanisms
leading to neoplastic progression in Barretts epithelium
are still not fully understood (Barrett et al., 1999; Dunn
et al., 2003). Increasing evidence exists that carcinogenesis must be understood in terms of an accumulation of
mutations in regulatory genes, including activation of
oncogenes and inactivation or loss of the tumoursuppressor genes (Morales et al., 2002). Since the
discovery of the role of RAS oncogenes in tumorigenesis, an increasing focus has been set in research of the
signal transduction area (Liu et al., 2003). A key RAS
effector pathway involves the kinase cascade RAF/
MEK/ERK (MEK: MAP/ERK kinase; ERK: extracellular signal-related kinase) (Mercer and Pritchard,
2003). In the quest to understand how RAS transmits
extracellular growth signals, the MAP kinase (MAPK)
pathway has emerged as an important route between
membrane-bound RAS and the nucleus (Weber et al.,
2001; Leder et al., 2002).
Signalling through the MAPK cascade is transduced
by GTP loading of RAS leading to the activation of
RAF kinase. In mammalian cells, there are three
isoforms of RAF: A-RAF, B-RAF, and C-RAF (Liao
et al., 2001). Although all three of the raf isoforms share
a common function with respect to MEK phosphorylation, studies have shown that these proteins might be
differentially activated by oncogenic ras (Peyssonnaux
and Eychene, 2001; Smalley, 2003). Recently, mutations
of BRAF have been described in about 15% of all
human cancers, especially in malignant melanomas,
papillary thyroid cancer, as well as ovarian cancer
(Davies et al., 2002; Cohen et al., 2003; Dong et al.,
2003; Kimura et al., 2003; Singer et al., 2003; Soares
et al., 2003). Previous studies of ras mutations in

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F Sommerer et al

555

Barretts adenocarcinoma have reported varying results;

mutation rates up to 88% have been described (Arber
et al., 2000).
In the present study, we analysed the status of the
BRAF gene together with KRAS to elucidate a possible
role of these genes in the carcinogenesis of Barretts
adenocarcinoma.
Materials and methods
Patients and tissue samples
Between February 2000 and September 2001, 19 patients with
well-differentiated Barretts adenocarcinoma, 56 patients with
Barretts epithelium and intraepithelial neoplasia (n 29 with
LGIN and n 27 with HGIN), and 30 patients with Barretts
mucosa without neoplasia were included in this retrospective
study. The normal squamous cell epithelium as well as gastric
mucosa were obtained from five patients.
All patients with Barretts neoplasia received endoscopic
mucosal resection. Barretts mucosa without neoplasia was
obtained from biopsies of patients without neoplasia.
The present study was in accordance with the ethical
standards of the Committee on Human Experimentation of
the University of Leipzig. All samples were taken during
treatment procedures under therapeutical intent.
Each tumour was re-evaluated with regard to typing (WHO,
2000). In all cases, H&E-stained slides were re-examined
independently by three experienced GI pathologists (MV, MS,
AT) without any knowledge of clinical data. In case of
conflicting results of grading intraepithelial neoplasia, microscopical re-evaluation was obtained until concordance of
opinion was obtained.
DNA samples
For each sample, the histopathological lesions of interest were
first identified on routinely stained sections. Microdissection
was performed as described previously (Figure 1) (Weber et al.,
2003). The approximate number of cells was estimated to be at
least 1000 per sample for PCR analysis. For DNA extraction,
standard methods were used: after incubation with proteinase
K at 371C overnight, the tissue was extracted twice in phenol
and twice in chloroform, followed by ethanol precipitation.

Mutation analysis
All pre-PCR tissues were handled in an environment free of
PCR products. All samples were coded and the investigator
was blinded to all patient clinical details. Deparaffinized tissue
was recovered by a 15 min incubation with xylene, followed by
centrifugation for 5 min at 14 000 r.p.m. two times. The tissue
pellet was then washed twice in absolute ethanol, followed by
two washes in phosphate-buffered saline. The pellet was
incubated with 10 pellet volumes (approximately 500 ml) of
lysis buffer (0.32 M sucrose, 10 mM Tris-HCl, 1% (v/v) Triton
X-100) and 0.2 volumes of proteinase K (final concentration
400 mg/ml) for 23 days at 371C. DNA was phenolchloroform
extracted and precipitated in ethanol using conventional
techniques. The resulting DNA pellet was resuspended in
50 ml TE buffer, pH 7.4, (10 mM Tris-HCl pH 7.4, 1 mM EDTA
pH 8.0). DNA samples were stored at "201C.
Using these matched samples (tumour and normal squamous epithelium from the same patient), screening for exon 2
18 BRAF mutations was performed using an ABI PRISM 3100
Genetic Analyser. PCR primers were designed to amplify the
exon plus at least 50 bp of the flanking intronic sequence,
according to previously published protocols. The primers were
adopted from those published in the literature to omit
analysing the BRAF pseudogene (Davies et al., 2002; Naoki
et al., 2002; Yuen et al., 2002).
The resulting traces were analysed to identify samples that
produced a shift in peak migration relative to the matched
normal control from the same individual or a standard normal
control, indicating the presence of a putative sequence
variation.
The first exon of KRAS2 was amplified by PCR using
primers designed to avoid amplification of the KRAS2
pseudogene. The primers used were 50 -ATTATAAGGCCTGCTGAAAATG-ACTGA-30 (upstream primer) and 50 ATATGCATATTAAAACAAGATTTACCT-CTA-30 (downstream primer), giving a 155 bp product. Amplification was
performed using a touchdown PCR technique (Tannapfel et al.,
2003; Weber et al., 2003) from 63 to 531C over 10 cycles,
followed by 30 cycles at 94, 53, and 721C.
PCR products were purified using the Qiaquick PCR
purification kit (Qiagen, Hilden, Germany) and sequenced
using dye primer cycle sequencing and AmpliTaq polymerase
FS on an Applied Biosystems DNA sequencer (ABI PRISM
3100; Applied Biosystems-Perkin-Elmer/Cetus, Norwalk).

Figure 1 Microdissection of the Barretts mucosa and adenocarcinoma compartments. Microdissection of the Barretts mucosa with
HGIN (ac) and Barretts adenocarcinoma (pT1m) (df). The outlined areas (b, e (green arrows)) were microdissected (c, f) by the laser
system (PalmsMicrobeamSystem) and catapulted as described in Materials and methods
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As a positive control for RAS mutation analysis, DNA from
colon carcinoma cell lines SW480 (Clontech, Palo Alto, CA,
USA) and HCT116 (American Type Culture Collection,
ATCC, Rockville, MD, USA) with known KRAS2 mutations
at codon 12 (GTT) and codon 13 (GAC), respectively, were
used. Negative controls, without DNA, were run as controls
for contamination.
If a mutation was detected, it was confirmed by amplification and sequencing of a fresh DNA sample using the
upstream primer. Any sequences, which proved difficult to
read, were reamplified and resequenced.
Immunohistochemistry of active MAPK and MLH1/MSH2
The immunohistochemical analysis was performed as described recently (Tannapfel et al., 2003; Weber et al., 2003). In
all cases, the tumour and non-neoplastic epithelium were
examined.
For the immunohistochemical visualization of ERK and
active ERK, we used the antibodies anti-p44/42 MAPK and
antiphospho-p44/42 MAPK (Anti-ACTIVER MAPK Ab;
PromegaR, Madison, WI, USA). Antiphospho-p44/42 MAPK
specifically recognizes the dually phosphorylated, active form
of MAPK (also known as p44/ERK1 and p42/ERK2)
enzymes. The working dilution was 1 : 200.
To analyse the MSI status of our tumours, immunohistochemistry of the mismatch repair proteins hMLH1 and hMSH2
was performed according to previously published standardized
protocols (Ruschoff et al., 1998; Stahl, 2000) on formalin-fixed,
paraffin-embedded specimens, using the standard avidinbiotin
method (Tannapfel et al., 2003; Weber et al., 2003) with the
following antibodies: MLH1 (Clone G 168-15, dilution 1 : 100,
BD PharmingenR,Germany) and MSH2 (Clone GB12, dilution
1 : 10, Oncogene-CalbiochemR, USA).
The positive controls included were sections known to
express the investigated antibodies. The negative controls were
obtained by omitting the primary antibodies. For the
estimation of nuclear hMSH2 and hMSH2 protein expression,
the sections were subjected to microwave epitope retrieval.
DAKOR TechMate 500 slide-processing equipment was used
for all immunohistochemical procedures.

tions. Somatic BRAF mutations were found in 2/19

Barretts adenocarcinomas (11%) and in 1/27 (4%) of
HGIN. All mutations were within exons 11 and 15
(Table 1), with a predominant nucleotide change
(Figure 2). The mutation of HGIN was found in exon
11 (G1403T; resulting in the substitution of glycine to
valine at 468). The two mutations of Barretts adenocarcinomas were T1796A (resulting in the substitution
of valine 599 by glutamate), a previously documented
hotspot. In all three cases, the normal epithelium of the
same patient exhibits wild-type BRAF. Samples with
LGIN, Barretts mucosa, and normal gastric and
squamous cell epithelium were wildtype as well.
We failed to detect a significant correlation between
the mutation status of BRAF, grade or other histopathological factors (vascular density, multiplicity,
desmoplastic reaction, grade of peritumourous inflammation) in Barretts neoplasia.
KRAS2 status
PCR amplification and DNA sequencing enabled the
detection of heterozygous mutations in 4/19 Barretts
adenocarcinomas (21%). Two patients had a mutation
of codon 12 and two of codon 13. In HGIN, three

Results
BRAF gene alterations
Genomic DNA from Barretts adenocarcinoma and
precursor lesions was analysed for BRAF gene muta-

Figure 2 Mutation analysis of the BRAF gene. Electropherograms

of the DNA sequences of normal mucosa and Barretts adenocarcinoma of patient no. 8

Table 1 BRAF and KRAS2 alterations in Barretts mucosa, intraepithelial neoplasia and adenocarcinoma
KRAS2
Nucleotide change
No of points

BRAF mutation

Codon 12

Codon 13

Barrett mucosa

30

0/30

0/30

0/30

Intraepithelial neoplasia (IN)

Low grade
High grade

29
27

0/29
1/27

0/29
2/27
GGT-GAT
GGT-GCT

0/29
1/27
GGC-TGC

Adenocarcinoma (pT1m)

19

2/19

2/19
GGT-GTT
GGT-TGT

2/19
GGC-TGC
GGC-GAC

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KRAS2 mutations were found (11%): two in codon 12,

and one in codon 13. No patient had multiple mutations
(Table 1). In normal Barretts mucosa without intraepithelial neoplasia and in the corresponding normal
squamous and gastric epithelium, KRAS2 was wildtype
in all cases. We failed to observe an association between
KRAS2 mutation pattern and histopathological variables.
We failed to detect a simultaneous BRAF and RAS
mutation within the same tumour specimen. However,
the number of mutations identified in either gene is too
small to evaluate a genetic interaction.
Immunohistochemistry
Active, that is, phosphorylated ERK was observed in
14/19 (74%) Barretts adenocarcinomas with a nearly
homogeneous intratumoural expression (Figure 3b, d, f),
while unphosphorylated ERK showed baseline levels
(Figure 3a, c, e). The active protein was also observed in
the 12/27 HGIN (44%) and in 5/29 (17%) samples of
LGIN and 5/30 (17%) of Barretts mucosa without
neoplasia. Especially, globlet cells and regenerative
immature cells were positive for phosphorylated ERK.
In the normal squamous cell epithelium as well as gastric
mucosa, unphosphorylated ERK was the predominant
detected protein. In general, active ERK immunostaining was more intense in areas with inflammation and
also in lesions with intraepithelial neoplasia, as compared to unphosphorylated ERK (Figure 3).
To analyse the status of MSI, immunohistological
assessment of MLH1 and MSH2 was performed in all

Figure 3 Immunostaining of the anti-p44/42 MAPK (ERK)

(a, c, e) and antiphospho-p44/42 MAPK MAPK (active ERK)
(b, d, f). (a, c, e) Weak perinuclear and slight nuclear ERK staining
(red/brown reaction product) in Barretts mucosa (a), HGIN and
Barretts adenocarcinoma (original magnifications: # 20). (b, d, f)
Weak to absent antiphospho-ERK staining in Barretts mucosa.
Strong staining of anti-ERK in HGIN and Barretts adenocarcinomas (original magnification: # 20; insets: # 60)

cases. The normal staining pattern for MLH1 and

MSH2 is nuclear, and nuclei in stromal cells were used
as internal positive controls. Some cases showed weak
cytoplasmic staining for MLH1, but only the nuclear
staining was recorded. We could detect both proteins in
all cases of Barrett mucosa as well as LGIN. In HGIN,
immunostaining for both proteins occurred in 26 cases.
In one patient with HGIN and two patients with
Barretts adenocarcinoma, we failed to detect a specific
staining for MLH1 and MSH2. These patients had
neither BRAF nor KRAS2 mutations.
Discussion
In the study presented, we examined the frequency of
mutations of BRAF and KRAS2 in Barretts adenocarcinoma and precursor lesions. Both, BRAF and KRAS2
are members of the RAS-RAF-MEK-ERK-MAP kinase pathway, which mediates cellular response to
growth signals (Hakimi et al., 2002). Whereas KRAS2
was examined in a variety of human malignancies (for a
review, see Mercer and Pritchard, 2003), this is the first
study of the mutational status of BRAF in Barretts
neoplasia. In contrast to melanoma (Davies et al., 2002;
Pollock et al., 2003), colon (Rajagopalan et al., 2002),
(low-grade serous) ovarian (Singer et al., 2003), and
thyroid cancer (Kimura et al., 2003), we detected BRAF
mutation only in a small number of Barretts adenocarcinomas and precursor lesions. The predominant
BRAF mutations occurred at nucleotide 1796, leading to
a T to A change at exon 15 of the BRAF gene. It has
been shown, previously, that this mutation in the
activation segment, leading to a conversion of valine
599 to glutamic acid, is a hotspot for BRAF mutation in
human cancer (Davies et al., 2002; Yuen et al., 2002).
This mutation results in the insertion of a negatively
charged residue adjacent to a site of regulatory
phosphorylation at T598, which may mimic regulatory
phosphorylation, thus leading to constitutive activation
of BRAF independent of RAS (Satyamoorthy et al.,
2003).
According to our results, KRAS2 and BRAF mutations never occur simultaneously in Barretts adenocarcinoma or HGIN. Since oncogenic KRAS2 activates
wild-type BRAF, but mutated BRAF does not require
KRAS2 for growth induction (Davies et al., 2002;
Hakimi et al., 2002), a simultaneous BRAF and KRAS2
mutation in the same tumour may be redundant. Both
KRAS2 and BRAF influence the same effector pathway
(promoting cell survival by activating the MAPK
pathway); a simultaneous double mutation may not
confer a selection advantage for a single tumour cell. To
prove this hypothesis, further studies are necessary,
especially to look for RAS and BRAF alterations in
preneoplastic lesions or early tumour stages.
In the present study, we show that most Barretts
adenocarcinomas signal through a constitutively activated Erk pathway. One may speculate that, for
Barretts carcinoma, neither RAS nor BRAF mutations
may be a prerequisite for the activation of Ras-RAFOncogene

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558

MEK-Erk pathway. Alternative ways of activation of

ERK have recently been detected. The inhibition of
Rac1 or Cdc42 signalling led to MAPK Erk activation
via a pathway involving PI(3)K, Akt, Raf, and MEK
(Aguirre-Ghiso et al., 2003). Interestingly, Cdc42 was
implicated in the activation of p38, which, in relation to
the activation status of Erk, determined whether a
tumour cell was actively proliferating (high Erk/p38
ratio) or was dormant (low Erk/p38 ratio) (AguirreGhiso et al., 2003). Although the prevalence of BRAF
mutations in Barretts adenocarcinomas is relatively low
compared to colon, thyroid, low-grade ovarian carcinomas or melanomas, the inhibition of the RAS-RAFMEK-ERK-MAP kinase pathway may be a new
diagnostic or even therapeutic strategy in Barretts
adenocarcinomas (Fitzgerald, 2003). The successful

application of inhibitors of activated kinases was

reported for STI571, an inhibitor of the BCR-ABL
kinase, which is activated in chronic myeloid disorders
or gastrointestinal stroma tumours, and may facilitate a
new therapeutic approach for Barretts adenocarcinomas and their precursor lesions. If BRAF inhibitors are
found to be effective in these tumours, they should also
be considered for (adjuvant) treatment of BRAF-mutant
Barretts adenocarcinomas and precursor lesions, especially HGIN.
Acknowledgements
This paper was supported by the Bundesministerium fur
Bildung und Forschung (BMB F), Interdisciplinary Centre
for Clinical Research (IZKF) at the University of Leipzig
(01KS9504/1, project D01).

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