Mutagenesis vol. 25 no. 4 pp.

351358, 2010
Advance Access Publication 12 March 2010

doi:10.1093/mutage/geq012

Lifestyle factors and p53 mutation patterns in colorectal cancer patients in the
EPIC-Norfolk study

Jin Young Park, Panagiota N. Mitrou1,, Jennifer Keen2,,

Christina C. Dahm1, Laura J. Gay2, Robert N. Luben,
Alison McTaggart1, Kay-Tee Khaw, Richard Y. Ball3,
Mark J. Arends4,* and Sheila A. Rodwell1,

*To whom correspondence should be addressed. Department of Pathology,

University of Cambridge, Addenbrookes Hospital, Cambridge CB2 2QQ, UK.
Tel: 44 1223 217813; Fax: 44 1223 216980; Email: mja40@cam.ac.uk
Received on December 2, 2009; revised on February 16, 2010;
accepted on February 17, 2010

The tumour suppressor p53 is one of the most commonly

altered genes in colorectal cancer (CRC) development.
Genetic alterations in p53 may therefore be associated with
postulated lifestyle risk factors for CRC, such as red meat
consumption. In the European Prospective Investigation into
Cancer and Nutrition-Norfolk study, we examined whether
detailed estimates of dietary and lifestyle factors measured at
baseline related to later development of p53 mutations in
CRCs. After 10-year follow-up, there were 185 incident CRCs
of which 34% had somatic p53 mutations (p531). We observed
significantly higher mean intakes of alcohol, total meat and red
meat, in the group with p53 mutations and advanced Dukes
stage disease (daily alcohol intake was 7 and 12 g for p532 and
p531 cases, respectively, P 5 0.04; daily total meat intake was
69 and 100 g for p532 and p531 cases, respectively, P 5 0.03
and daily red meat intake was 39 and 75 g for p532 and p531
cases, respectively, P 5 0.01). Each 50 g/day increment in
total meat intake was associated with having p53 mutations in
cases with advanced Dukes stages [odds ratio (OR): 3.43, 95%
confidence interval (CI): 1.477.96]. Similarly, each 50 g/day
increment in red meat intake was also significantly associated
with having consistent p53 mutations in cases with advanced
Dukes stages (OR: 2.42, 95% CI: 1.184.96). These effects of
total meat or red meat intake and advanced Dukes stages
were independent of age, sex, body mass index, smoking and
alcohol intake. Furthermore, P values for interaction between
daily total meat or red meat intake and Dukes stages were
statistically significant in multivariable models (Pinteraction
< 0.001). Our results suggest that p53 mutations accelerate
progression of CRC to advanced Dukes stage in association
with higher meat especially red meat intakes.

Colorectal cancer (CRC) was the second most common cancer

in Europe in 2006 (1), and
100 new cases of CRC were
diagnosed each day in the UK with almost equal distribution
between men and women (2).
It has been suggested that both genetic and environmental
factors play a major role in CRC development. Many CRC
cases develop from normal colonic epithelium through intermediate adenomatous neoplasms often with sequentially
worsening degrees of dysplasia, during which somatic
mutations, deletions or other changes are acquired (3,4). One
of the most commonly altered genes identified in human
tumours to date is the p53 tumour suppressor gene, with
GC.AT transitions at CpG dinucleotides being the most
frequent mutation type (5,6). According to the proposed
genetic model for colorectal tumourigenesis, in which alterations to different oncogenes and tumour suppressor genes play
a sequential role based upon the concept of progression from
adenoma to carcinoma, mutations in the p53 tumour suppressor
gene are proposed to occur either just before or concurrently
with the transition to malignancy (7,8). However, in some
cases, the mutation might have also been present from the onset
of the lesion, in particular if the mutation is due to a direct or
indirect effect of exposure to exogenous risk factors. Mutations
in the p53 gene could provide a growth advantage to a potential
cancer cell by allowing survival despite DNA damage that
would otherwise be lethal and thus allow tumourigenic
proliferation to proceed (9).
Red and processed meat intake has been associated with
increased risk of sporadic CRC (10,11). GC.AT transitions at
the CpG dinucleotides in CRC may be characteristic of the
effects of alkylating agents and can be induced by N-nitroso
compounds (NOC) formed endogenously in the presence of
haem, while CG.TA transitions have been linked with
endogenous deamination mechanisms (1215). DNA alkylating
agents, such as some NOCs, also generate O-6-methylguanine
adducts, which, if not repaired, may play a significant role in
mutagenesis, and these have been detected in human colonic
tissue (15). It has also been suggested that the levels of nitrogen
species were elevated in inflammation, which may link to
carcinogenesis (16).
Alcohol drinking is associated with several malignant
diseases (11). Although not itself carcinogenic, a metabolite
of ethanol, acetaldehyde, is able to form stable DNA adducts,
interfering with DNA repair and may therefore trigger mutation
(1719). Alcohol is also known to alter levels of cytokines,
which may be pro-inflammatory, in certain situations (20).
Cigarette smoke contains a wide variety of highly mutagenic
compounds, including polycyclic aromatic hydrocarbons,
heterocyclic amines and N-nitrosamines, which are absorbed
from the lungs into the blood (21,22). The various carcinogens
in cigarette smoke may be metabolized by the liver to active

The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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351

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Department of Public Health and Primary Care, University of Cambridge,

Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 8RN,
UK, 1Medical Research Council Centre for Nutritional Epidemiology in Cancer
Prevention and Survival, University of Cambridge, Strangeways Research
Laboratory, Worts Causeway, Cambridge CB1 8RN, UK, 2Medical Research
Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building,
Cambridge CB2 0XY, UK, 3Norfolk and Waveney Cellular Pathology
Network, Norfolk and Norwich University Hospital NHS Foundation Trust,
Colney Lane, Norwich NR4 7UY, UK and 4Department of Pathology,
Addenbrookes Hospital, University of Cambridge, Cambridge CB2 2QQ, UK,
y
Both the authors contributed equally to this work.
yy
Professor Rodwell (professionally known as Bingham) read an initial draft of
this manuscript, but sadly passed away in June 2009.

Introduction

J. Y. Park et al.

metabolites that are transported to colorectal tissues where they

may mutate cancer-related genes (23).
These lifestyle factors that may generate mutations in the
p53 gene may contribute to CRC growth and progression (24).
However, very few prospective studies have explored the
association of these lifestyle factors with p53 mutations in CRC
and less is known about possible joint actions of genetic
mutations and such environmental factors on the progression of
colorectal carcinogenesis.
This study aimed to explore the p53 mutation patterns in
CRC and to investigate the associations of dietary and other
lifestyle factors with p53 mutations and how these relate to
different clinicopathological features including tumour site,
tumour differentiation and tumour progression using Dukes
staging information in patients from the Norfolk arm of the
European Prospective Investigation into Cancer and Nutrition
(EPIC)-Norfolk Study. Emphasis was placed on using highquality methods for the detection of mutations from independent polymerase chain reaction (PCR) products as well as
for the rigorous collection and analysis of lifestyle factors.

Study population
The participants in this study were part of a prospective population study of
25 639 individuals (11 607 men and 14 032 women) aged between 40 and
79 years who were residing in Norfolk, UK. The design and study methods
have been described previously (25). Briefly, the cohort was recruited between
1993 and 1997 from agesex registers of general practices (which, because of
the UK National Health Service, serve as a population register) as part of
a 10-country collaborative study, the EPIC (26). Baseline examination
comprised a clinic visit for a health examination that included anthropometric
measurements and blood sampling as well as completion of a detailed
questionnaire to assess health and lifestyle factors. Ethical approval was
received from the Norwich Local Research Ethics Committee.
Case ascertainment and tissue samples
Incident CRC cases (International Statistical Classification of Diseases and
Related Health Problems 9th revision, 153.0153.9, 154.0 and 154.1) were
ascertained by matching all participants to the East Anglian Cancer Registry
and Information Centre (ECRiC) and the UK Office for National Statistics,
which provided notification of all cancer registrations and deaths for the cohort.
Where available, archival tumour material, in the form of formalin-fixed
paraffin-embedded (FFPE) tissue blocks, and diagnostic histopathological
reports were obtained from the Norfolk and Norwich University Hospital. The
clinicopathological information was obtained from a combination of the cancer
reports and information provided by the ECRiC. As of June 2004, 291
participants were reported as having been diagnosed with CRC. All cases that
were diagnosed within the first year after baseline recruitment were excluded
from the study. Tumour material was available from 190 cases at the Norfolk
and Norwich University Hospital. Five cases were excluded from the study:
three had only adenomas, one had a rare carcinoid tumour with a neuroendocrine pattern of differentiation and the fifth case because very few cancer cells
were present in the biopsy sample. The remaining 185 cases were analysed for
this study.
DNA extraction
Consecutive 5-lm sections were cut from each FFPE block for 185 CRC cases
and mounted onto glass slides. Sections were deparaffinized in xylene and then
rehydrated in a series of graded ethanol solutions and finally ultra pure water.
One section from each block was stained with haematoxylin and eosin using
standard protocols. The stained sections were examined by a histopathologist
and the areas of tumour tissue were marked to act as a guide for the manual
microdissection of tumour tissue away from any normal epithelial mucosa,
muscle, necrosis or other non-cancerous tissue. Corresponding areas of tumour
from each of the remaining tissue sections were scraped off into a sterile tube.
The sections were digested with Proteinase K (Sigma) in Promega PCR Gold
buffer with MgCl2 (2.5-mM final volume) at 55C with shaking for up to
5 days. The genomic DNA concentration and purity were measured using
a nanodrop spectrophotometer and diluted 1:10 for use as a DNA template
directly in PCR.

352

Lifestyle and other exposure assessment

At the baseline health examination from 1993 to 1997, height (metre) and
weight (kilogram) were measured by trained nurses and body mass index (BMI,
kilogram per square metre) was then calculated. Information on smoking status
and family history of CRC was obtained from the baseline health and lifestyle
questionnaire. Smoking status was derived from yes/no responses to the
questions Have you ever smoked as much as one cigarette a day for as long as
a year? and Do you smoke cigarettes now? and was categorized as never-,
former- and current-smokers. Pack-years were derived from questions how
many cigarettes were consumed each day at the age of 20, 30, 40, 50 and now?
and calculated as pack-years with one pack-year being equal to smoking
20 cigarettes/day for 1 year (27). Habitual physical activity was assessed using
questions referring to present activities (28). For the purposes of the current
study, we dichotomized the participants into low physical activity (a sedentary
job with no recreational activity, a sedentary job with ,0.5 h of recreational
activity per day or a standing job with no recreational activity) and high
physical activity (any activity levels above the former). Educational status was
based on the highest qualification attained and was regrouped into low
educational level (O-level or equivalent, less than O-level or no qualifications)
and high educational level (degree or equivalent, A-level or equivalent) (29).
Dietary assessment
Dietary data were obtained using 7-day food diaries that were completed at the
end of the baseline health check. This nutritional method has been validated
and described elsewhere (30). Briefly, 7-day food diaries comprised a 45 page
A5 colour booklet containing food portion photographs and detailed
instructions in which the description, preparation and amounts of foods eaten
at main meals, snacks and between meals over a week could be recorded
(30,31). Alcohol and nutrient intakes were calculated using a custom-designed
dietary assessment software programme, DINER (Data Into Nutrients for
Epidemiological Research) programme (31,32). Meat variables were grouped
into categories of total meat, processed meat and red meat. The total meat
variable included red and processed meat, poultry, game and meat dishes, while
the red meat variable included beef, lamb, pork and veal dishes. The processed
meat category was defined as meat products and processed meat, such as ham,
corned beef, sausages, bacon and beefburgers.
Statistical analysis
For the present casecase analysis, all 185 CRC cases were categorized into
two groups: p53 mutation present (p53) or absent (p53).
The p53 cases and p53 cases were compared according to selected
characteristics, including lifestyle and tumour characteristics. Selected
characteristics included age (years, continuous), family history of CRC (no,

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Materials and methods

p53 Mutation analysis

The extracted genomic DNA was used as a substrate to amplify by PCR exons
58 from p53. Each primer was designed in the adjacent intronic region to cover
the intronexon boundary and ensure full coverage of the exons. The primers
used with PCR annealing temperatures and product sizes were as follows: exon
5 sense 5#-CGTCTTCCAGTTGCTTTATC-3# and antisense 5#-CACTCGGATAAGATGCTGAG-3#, 58C, 367 bp; exon 6 sense 5#-AGCTGGGGCTGGAGAGAC-3# and antisense 5#-GGGAGGTCAAATAAGCAGCA-3#, 58C,
295 bp; exon 7 sense 5#-CCCTGCTTGCCACAGGTGT-3# and antisense 5#TGATGAGAGGTGGATGGGTAGTAG-3#, 61C, 295 bp and exon 8 sense
5#-ATGGAGCCTGGTTTTTTAAATG-3# and antisense 5#-AAAGAGGCAAGGAAAGGTGATAAA-3#, 55C, 305 bp. Because of the fragmented nature
of genomic DNA extracted from the FFPE blocks, each exon was amplified
separately as the DNA was of such quality that it was not possible to PCR
amplify DNA lengths .400 bp. Forward and reverse strands were both
sequenced. A positive control from a cancer cell line, HT29, was routinely used
in each PCR and the products were identified by electrophoresis on a 1.5%
agarose gel containing 1 lg/ml ethidium bromide and visualized under
ultraviolet light. Any products of the correct size were purified using a Millipore
multiscreen-PCRl96 filter plate. The purified products were then sent for
sequencing using ABI technology and Big Dye Terminator Chemistry based on
the Sanger dideoxy chain termination method using the same primers as above.
The subsequent chromatogram traces were analysed for DNA mutations using
the Genome Assembly Programme (GAP4) and wild-type reference sequences
for each p53 exon from Ensemble were used as a sequence comparison. Any
DNA mutations found were entered into a database recording the sample
number, p53 exon and the precise location and type of mutation (base
substitution or frameshift). All DNA mutations were confirmed by resequencing from an independent PCR product from the same exon in the same sample.
We defined p53 cases as having an identical DNA base change at the same
codon position in a given case, confirmed by sequencing at least two and often
more than three independent PCR products.

Lifestyle and p53 mutations in CRC

Results
Among the 185 analysed cases of CRC, a total of 62 cases were
identified with confirmed p53 mutations (supplementary Table I
is available at Mutagenesis Online). The most frequently
observed types of mutations were C to T and G to A transitions
at GC base pairs (GC to AT, 67%). Patterns of p53 mutation hot
spots are shown in Figures 1 and 2.
Table I shows the baseline lifestyle and tumour characteristics of the 185 CRC cases by sex and p53 mutational status.
Both genders were approximately equally represented. Men

more frequently reported being former-smokers and longer

term smokers, as well as having a higher educational level
compared with women, and these differences were statistically
significant. When comparisons were made between patients
tumours with (p53) and without p53 mutations (p53),
patients with p53 tumours less frequently reported a family
history of CRC, but more frequently presented tumours located
at a distal site, and the tumours more frequently showed poor
differentiation. None of these differences, however, were
statistically significant (P , 0.05).
The distributions of selected lifestyle factors including
smoking, alcohol and meat variables among CRC cases with
and without p53 mutations are presented in Table II according
to sex, Dukes stage and tumour site. Patients with p53
mutations showed longer pack-years and higher intakes of
alcohol, total meat, red meat and processed meat. A
significantly higher mean intake of alcohol, total meat and
especially red meat intake was observed in the advanced
Dukes stage group (Dukes C&D) with p53 compared to
p53 cases (daily alcohol intake of 7 and 12 g for p53 and
p53 cases, respectively, P 5 0.04; daily total meat intake of
69and 100 g for p53 and p53 cases, respectively, P 5 0.03
and daily red meat intake of 39 and 75 g for p53 and p53
cases, respectively, P 5 0.01), while similar or lower means
were observed among those with p53 mutations and early-stage
CRC, i.e. Dukes A&B (daily alcohol intake of 8 g for both
p53 and p53 cases, P 5 0.67; daily total meat intake of
90 and 67 g for p53 and p53 cases, respectively, P 5 0.07
and daily red meat intake of 61 and 37 g for p53 and p53
cases, respectively, P 5 0.06). p53 mutational status did not
vary significantly by sex or tumour site (proximal or distal) in
any of the lifestyle factors analysed (Table II).
We repeated this analysis for other lifestyle variables,
including BMI and physical activity, and other dietary
variables, such as folate, fat and fibre that have been associated
with risk of CRC; however, none of the differences were
statistically significant between p53 and p53 cases (data not
shown). To address the possibility that preclinical CRC
symptoms might have influenced observed results, we
conducted further sensitivity analyses by excluding 12 CRC
cases incident within 2 years of baseline (total N 5 175), and
the findings were not substantially altered (data not shown).
We further explored the odds of having p53 mutations
among these 185 CRC patients with increased total meat and
red meat consumption (per 50 g/day increase,
1 SD) stratified
by Dukes A&B and Dukes C&D by fitting the age- and sexadjusted model and a multivariable model (Table III). In the
multivariable model, each 50 g/day increment in total meat

Fig. 1. p53 mutation hot spots in colorectal tumours in the EPIC-Norfolk study.

353

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yes), BMI (kilogram per square metre), physical activity (low, high), cigarette
smoking status (never, former, current), pack-years of smoking (years,
continuous) and educational level (low, high). Regarding tumour characteristics, tumour site groups were determined by splitting cases into proximal and
distal groups. Proximal tumours included caecal, ascending colonic and
transverse colonic cancers. The distal group included all descending colonic,
sigmoid colonic and rectal cancers. Appendix cancer cases were excluded from
the analysis. Tumour differentiation was determined by histopathological
examination and was graded as well, moderate, moderate poor and poor, both at
the time of diagnosis and at the review. Dukes staging was used to describe the
degree of spread of a tumour (33). We dichotomized Dukes staging
information into Dukes A&B and Dukes C&D groups.
Selected lifestyle factors including smoking, alcohol and meat variables
were compared between p53 cases and p53 cases according to sex, Dukes
stage and tumour site. In addition, the above-listed lifestyle and tumour
characteristics were compared for cases with GC.AT transition, which has
been shown to be the most frequently occurring type of mutation in p53, versus
other base changes. We conducted further analyses by stratifying GC.AT
transitions into those occurring at CpG dinucleotides versus those at non-CpG
dinucleotides and results were compared according to the lifestyle and tumour
characteristics listed above.
Differences in mean values of the continuous variables between patients
with p53 and p53 cases were tested with the Students t-test. Because data
on pack-years of smoking, alcohol, total meat, red meat and processed meat
intakes tended to be skewed, those variables were compared by Wilcoxon ranksum test (MannWhitney U-test). The distributions in categorical variables
between patients with p53 and p53 cases were tested with the Pearsons
v2 test, using the Fishers exact test when the smallest expected value was ,5,
respectively.
The independent relationship of having consistent p53 mutations among
these 185 CRC patients with increased daily meat or red meat (per 50 g/day
increase,
1 SD) consumption was further examined by fitting logistic
regression models. Odds ratios (ORs) and 95% confidence intervals (95% CIs)
were estimated from the age- and sex-adjusted model and a multivariable model
that additionally adjusted for BMI, smoking and alcohol intake. These analyses
were stratified by Dukes A&B and Dukes C&D groups. Tests for interaction
were performed with the likelihood ratio test of models with and without
interaction terms.
To address the possibility that preclinical CRC symptoms might influence
both lifestyle and tumour characteristics, we excluded cases incident within
2 years of baseline and all analyses were repeated. All statistical tests were
two sided, and all statistical analyses were performed with the statistical
software package STATA (version 10, Stata Corporation, College Station,
TX, USA).

J. Y. Park et al.

intake was significantly associated with having p53 mutations

(OR: 3.43, 95% CI: 1.477.96) among cases with advanced
Dukes stages (Dukes C&D). Similarly, each 50 g/day
increment in red meat intake was significantly associated
with having consistent p53 mutations (OR: 2.42, 95% CI:
1.184.96) among cases with advanced Dukes stages (Dukes
C&D stage). These increased ORs by high total or red meat
intake were independent of age, sex, BMI, smoking and
alcohol intake and were not seen among p53 cases with early
Dukes stages (Dukes A&B stage). When we tested for
interaction, Pinteraction for daily total meat intake and Dukes
stage was ,0.0001 and Pinteraction for daily red meat intake
and Dukes stage was 0.0002 in multivariable models
(Table III).
Table IV presents selected lifestyle and tumour characteristics of CRC cases by different types of p53 mutations
comparing GC.AT transition with other base changes and by
GC.AT transitions occurring at CpG dinucleotides with those
not. Of 185 CRC cases, 38 cases had GC.AT transition, 25 of
which occurring at CpG dinucleotides. Analysis of observed
mutations showed that cases with GC.AT transitions more
frequently reported being female compared with those with
other base changes (P 5 0.05). Cases with GC.AT transitions
found at CpG dinucleotides showed higher alcohol intake and
lower total meat, red meat and processed meat intake compared
with those occurring at non-CpG dinucleotides. However, none
of these differences were statistically significant.
Discussion
To our knowledge, this is the first UK population-based study that
has explored the role of lifestyle factors on p53 mutational patterns
in colorectal tumours. In our study, higher daily total meat and red
meat intake were significantly associated with having p53
mutations in CRC patients with advanced Dukes stages after
multivariable adjustment. We found a significant interaction
between total meat or red meat intake and Dukes late stage.
According to the genetic basis of CRC development
hypothesized in an early concept of the adenoma to carcinoma
354

progression, p53 mutations appear to function at the stage of

transition to malignancy (7). It may be that lesions with p53
mutations progress much more rapidly, leading to their clinical
detection when they are at an advanced stage as compared with
those with no mutation. However, the mutation might have also
been present from the onset of the lesion, in particular if the
mutation is due to a direct or indirect effect of exposure to
exogenous risk factors. It is well known that meat, especially red
meat intake, increases CRC risk (11). However, there has been
no attempt made to investigate how p53 mutations could
influence CRC development or progression in relation to certain
lifestyle risk factors. Our results suggest that the carcinogenic
effect of meat, including red meat intake, was associated with
having p53 mutations that contribute to formation of more
aggressive tumours, which are detected as late Dukes stage
CRC, confirming a significant role of meat intake in CRC
progression. In contrast, no such association was detected in
early Dukes stage cases, suggesting that accumulation of p53
mutations in early stage may be the result of other environmental factors rather than an effect of meat-related carcinogens.
The general distribution of p53 mutational hot spots in exons
58 in our study showed the most common hot spots at codons
175, 245, 248 and 273 (Figure 1). These sites were comparable
to the distribution pattern of p53 mutation hot spots in CRC in
the International Agency for Research on Cancer TP53
mutation database (version R14, November 2009), which
comprises 24 810 somatic mutations worldwide (34) (Figure 2).
Previous studies have suggested that the p53 mutation
spectrum in CRC shows a high proportion of GC.AT transitions
(23,3538) and that GC.AT transitions predominantly occurred
at CpG dinucleotides that contain 5-methylcytosine (6). Our
study extends such work by providing data on the link between
lifestyle factors and p53 mutational status as well as mutational
patterns within CRC.
The GC.AT transitions have been significantly associated
with increased levels of inducible nitric oxide synthase
[(iNOS)/NOS2] activity when compared with tumours with
other types of mutations (39). The level of iNOS has also been
positively correlated with the degree of inflammation, possibly

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Fig. 2. p53 mutation hot spots in colorectal tumours in the International Agency for Research on Cancer database (34).

Lifestyle and p53 mutations in CRC

Table I. Baseline characteristics of study participantsa

Cases by p53 status

Cases by sex
Total

p53 cases

Pc

76
66.0  6.6

62
64.9  8.5

0.38

64 (84.2)
12 (15.8)
26.7  4.3

57 (91.9)
5 (8.1)
27.6  4.5

0.17

0.41

54 (71.1)
22 (28.9)

41 (66.1)
21 (33.9)

0.53

0.001

31
33
9
10

23
30
8
13

(37.7)
(49.2)
(13.1)
 20

0.85

40 (52.6)
36 (47.4)

36 (58.1)
26 (41.9)

0.52

0.20

29 (42.7)
39 (57.3)

19 (32.2)
40 (67.8)

0.23

69 (84.2)
13 (15.8)

0.64

59 (88.1)
8 (11.9)

46 (80.7)
11 (19.3)

0.26

43 (51.8)
40 (48.2)

0.40

36 (54.6)
30 (45.4)

33 (61.1)
21 (38.9)

0.47

Men

Women

92
65.9  7.4

93
65.3  7.3

84 (91.3)
8 (8.7)
26.9  3.7

82 (88.2)
11 (11.8)
27.5  4.8

0.48

59 (64.1)
33 (35.9)

65 (69.9)
28 (30.1)

27
53
9
15

(30.3)
(59.6)
(10.1)
 19

51 (55.4)
31 (33.7)
10 (10.9)
8  14

44 (47.8)
48 (52.2)

63 (67.7)
30 (32.3)

0.006

27 (31.4)
59 (68.6)

35 (40.7)
51 (59.3)

72 (86.8)
11 (13.2)
45 (58.4)
32 (41.6)

0.61

0.39

,0.001

(42.5)
(45.2)
(12.3)
 15

0.26

0.53

Mean (SD) or number (%).

P values relate to two-sided t-tests of equality of the means or v2 tests of association between men and women, as appropriate.
c
P values relate to two-sided t-tests of equality of the means or v2 tests of association between p53 cases and p53 cases, as appropriate.
d
One pack-year is equal to smoking 20 cigarettes/day for 1 year, and P values are from Wilcoxon rank- sum test.
e
Total number does not add up to 185 due to missing data.
f
One patient had two different tumours in proximal and distal colon and was therefore excluded from this analysis.
b

increasing the risk of developing cancer (40). Furthermore,

these iNOS-expressing cancer cells with mutated p53 have an
accelerated tumour growth (41).
iNOS may produce increased amounts of nitric oxide from
the substantial increase in dietary protein with either red or
white meat consumption (39) and this increased level of
nitrogen species by the inflammatory process may in turn react
with NOC precursors present in the colon to produce increased
levels of NOC (42). Haem in red meat can also facilitate
endogenous NOC formation, which has been thought to induce
the GC.AT transition (43).
However, in this limited sample we observed no significant
difference in any lifestyle factors including daily red meat and
processed meat intake comparing cases with GC.AT transitions
occurring at CpG dinucleotides and those not. Nor was there
a significant difference in our study in alcohol and smokingrelated variables. Alcohol is known to play a role in the immune
system possibly through the cytokine cascade, modifying the
inflammatory response in a number of tissues (20,44). Although
less is known about the effect of a modified inflammatory
response due to alcohol or meat intake on CRC risk, there
is evidence that use of non-steroidal anti-inflammatory drugs
protects against CRC (45) and patients with inflammatory bowel
disease have an increased risk of CRC (46,47).
Two previous studies using a casecontrol design have
examined associations between lifestyle factors and p53
mutations in colon carcinomas (24,48). In one, p53 cases

compared with controls were slightly more likely to consume

a Western diet than p53 cases compared with controls (48).
In the other, colon cancer risks in cases with and without p53
mutations compared with population-based controls were not
significantly different for most dietary factors, except for fat
intakes (24). A casecontrol study design is, however,
susceptible to misclassification of either genetic or environmental exposures, which may be differential by disease
status.
The lack of association or the discrepancies observed in
these studies might indicate a complex interplay of disease
pathways but may also be attributed to the different populations
studied and various methodologies employed for both
genotypic and phenotypic data collection. In our prospective
study, by using a case-only analysis that can provide better
control over the impact of potential misclassification errors, we
were able to investigate the effect of certain dietary factors on
p53 abnormalities among cases. Case-only study design can
also suffer from uncontrolled confounding, exposure misclassification or non-response bias when cases are collected
retrospectively. However, EPIC-Norfolk is a population-based
prospective study of a relatively homogenous population. It
may, therefore, be less prone to residual confounding factors.
Nonetheless, caution is warranted in interpreting results as the
significant associations were from a relatively small sample
size. The investigated associations in our study, however, were
mainly hypothesis driven. Due to the limited sample size, we
355

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Selected characteristics
185
Age (years)
65.6  7.3
Family history of colorectal cancer
No
166 (89.7)
Yes
19 (10.3)
27.2  4.3
BMI (kg/m2)
Physical activity
Low
124 (67.0)
High
61 (33.0)
Cigarette smoking status
Never
78 (43.1)
Former
84 (46.4)
Current
19 (10.5)
11  17
Total pack-yearsd
Educational level
Low
107 (57.8)
High
78 (42.2)
Tumour characteristics
Tumour sitee,f
Proximal
62 (36.1)
Distal
110 (63.9)
Tumour differentiatione,f
Moderate/well
141 (85.5)
Moderate poor/poor
24 (14.5)
e,f
Dukes stage
A&B
89 (55.3)
C&D
72 (44.7)

p53 cases

J. Y. Park et al.

Table II. Selected lifestyle factors stratified by sex, Dukes stage and tumour
site according to p53 mutation statusa
p53 cases

Pb

Dietary factor
10  15

13  20

0.53

12  14
8  15

17  22
9  15

0.80
0.62

11  16
8  15

11  17
14  20

0.94
0.45

11  16
8  13

17  23
13  18

0.65
0.35

8  13

12  17

0.04

13  17
47

18  20
69

0.23
0.16

8  14
7  11

8  12
12  13

0.67
0.04

7  12
9  15

12  18
12  17

0.16
0.19

82  55

83  55

0.90

93  61
72  47

84 50
82  61

0.82
0.77

90  61
69  46

67  57
100  41

0.07
0.03

77  54
89  56

87  53
80  58

0.71
0.37

55  52

56  52

0.83

65  56
46  47

55  49
57  57

0.48
0.42

61  56
39  39

37  44
75  51

0.06
0.01

50  53
60  52

61  56
53  53

0.38
0.54

23  18

27  23

0.41

25  21
22  16

35  24
19  18

0.06
0.21

23  17
22  19

27  26
26  17

0.87
0.41

26  19
22  18

28  20
28  24

0.95
0.24

Mean (SD).
P values relate to two-sided Wilcoxon rank-sum test of medians between wild
p53 cases and p53 cases, as appropriate.
c
One pack-year is equal to smoking 20 cigarettes/day for 1 year.
b

were unable to analyse colonic and rectal cancer cases

separately, although it has been suggested that the aetiology
of the two cancers may be different (49). Further investigation
in larger studies is therefore warranted.
356

OR for p53
versus p53
cases (95% CI)

Age- and sex-adjusted model

Total daily meat intake (/50g)
Dukes stage
A&B
0.66
C&D
2.55
Daily red meat intake (/50g)
Dukes stage
A&B
0.59
C&D
2.38
a
Multivariable model 1
Total daily meat intake (/50g)
Dukes stage
A&B
0.63
C&D
3.43
Daily red meat intake (/50g)
Dukes stage
A&B
0.53
C&D
2.42

P value for
interaction with
Dukes stage

(0.431.02)
(1.255.23)

0.0002

(0.351.00)
(1.214.71)

0.0006

(0.410.98)
(1.477.96)

,0.0001

(0.310.93)
(1.184.96)

0.0002

Age, sex, BMI, smoking and alcohol intake adjusted.

The methodological strengths of this study include high

standard methods, applied for both mutational analysis and
lifestyle assessment. Mutational analysis was conducted
rigorously and DNA was sequenced at least twice, more than
three times in most cases, to ensure reliability in the reported
mutations. We were also able to reduce the degree of
diagnostic error or inconsistency by having a uniform
pathological review and classification of all lesions.
Furthermore, we used nutritional information from 7-day
food diaries. A sensitivity analysis conducted using dietary
data from food frequency questionnaire (FFQ) completed by
the same participants concomitantly with the 7-day food diaries
did not observe any significant differences between risk factors
on p53 mutational patterns, whereas 7-day food diaries did
(data not shown). Epidemiological studies often employ an
FFQ as their measurement method for the dietary factors;
however, the degree of error associated with FFQ measurements is considerably larger than previously suspected (50). In
a recent validation study, strong associations between biomarkers and intakes were found as assessed by food diaries,
and coefficients were markedly attenuated for data obtained
from the FFQ (32). Therefore, it is possible that our study may
provide more valid results than those using only an FFQ. It is
also unlikely that dietary information obtained for our study
was affected by knowledge of cancer diagnosis as our
185 tumour samples were collected from cases incident at
least a year after diary completion and further sensitivity
analysis that excluded cases incident within 2 years of baseline
did not materially change our results.
In summary, higher total meat and red meat consumption
were significantly associated with p53 abnormalities in latestage CRC cases suggesting that p53 mutations accelerate
progression of CRC to the advanced stage in association with
higher meat, especially red meat, intakes. The relationship
between lifestyle factors and p53 mutations in colorectal
tumours clearly merits further exploration, especially with
regard to possible underlying mechanisms.

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Pack-yearsc
Total pack-years of
smoking (n 5 76/62)
Sex
Male (n 5 34/33)
Female (n 5 42/29)
Dukes stage
A&B (n 5 36/33)
C&D (n 5 30/21)
Tumour site
Proximal (n 5 29/19)
Distal (n 5 39/40)
Alcohol (g/day)
Total intake
(n 5 76/62)
Sex
Male (n 5 34/33)
Female (n 5 42/29)
Dukes stage
A&B (n 5 36/33)
C&D (n 5 30/21)
Tumour site
Proximal (n 5 29/19)
Distal (n 5 39/40)
Total meat (g/day)
Total intake
(n 5 75/62)
Sex
Male (n 5 34/33)
Female (n 5 41/29)
Dukes stage
A&B (n 5 36/33)
C&D (n 5 29/21)
Tumour site
Proximal (n 5 28/19)
Distal (n 5 39/40)
Red meat (g/day)
Total intake
(n 5 75/62)
Sex
Male (n 5 34/33)
Female (n 5 41/29)
Dukes stage
A&B (n 5 36/33)
C&D (n 5 29/21)
Tumour site
Proximal (n 5 28/19)
Distal (n 5 39/40)
Processed meat (g/day)
Total intake
(n 5 75/62)
Sex
Male (n 5 34/33)
Female (n 5 41/29)
Dukes stage
A&B (n 5 36/33)
C&D (n 5 29/21)
Tumour site
Proximal (n 5 28/19)
Distal (n 5 39/40)

p53 cases

Table III. Odds of having consistent p53 mutations among 185 CRC patients
with a 50 g/day increment in total meat and red meat consumption stratified
by Dukes stage and P values for interaction

Lifestyle and p53 mutations in CRC

Table IV. Selected characteristics including lifestyle factors and tumour characteristics by various types of p53 mutationsa
p53 cases
P

GC.AT

Other base
changeb

38 (66.7)
65.3  8.3

19 (33.3)
62.8  9.3

17 (44.7)
21 (55.3)
28.0  5.1

14 (73.7)
5 (26.3)
27.0  3.2

0.05

27 (71.1)
11 (28.9)

10 (52.6)
9 (47.4)

0.17

18 (72.0)
7 (28.0)

18 (48.7)
16 (43.2)
3 (8.1)
10.5  16.8
13  19
86  62
65  60
24  18

4 (21.0)
11 (57.9)
4 (21.1)
21.0  24.7
13  13
85  44
44  35
31  19

0.10

11 (45.8)
10 (41.7)
3 (12.5)
10.6  15.2
15  22
80  66
59  63
24  16

12 (32.4)
25 (67.6)

7 (41.2)
10 (58.8)

0.53

6 (24.0)
19 (76.0)

6 (50.0)
6 (50.0)

0.11

29 (82.9)
6 (17.1)

14 (77.8)
4 (22.2)

0.72

20 (87.0)
3 (13.0)

9 (75.0)
3 (25.0)

0.39

21 (63.6)
12 (36.4)

8 (47.1)
9 (52.9)

0.26

14 (70.0)
6 (30.0)

7 (53.9)
6 (46.1)

0.35

0.30

0.43

0.07
0.21
0.75
0.29
0.15

GC.AT transitions
at CpG
dinucleotides

GC.AT transitions
at non-CpG
dinucleotides

25 (65.8)
65.0  8.5

13 (34.2)
66.0  8.4

11 (44.0)
14 (56.0)
27.5  4.3

6 (46.2)
7 (54.8)
29.0  6.3

0.99

9 (69.2)
4 (30.8)

0.99

7 (53.8)
6 (46.2)
0 (0.0)
10.1  20.4
8  10
98  54
77  55
25  23

0.73

0.37

0.68
0.47
0.89
0.30
0.16
0.76

a
Mean (SD) or number (%), P values relate to two-sided t-tests of equality of the means or v2 tests of association between GC.AT and other base change or
GC.AT at CpG dinucleotides and GC.AT at non-CpG dinucleotides using the Fishers exact test when the smallest expected value was ,5, as appropriate.
b
Information on base changes was not available for five cases and they were excluded in this analysis.
c
One pack-year is equal to smoking 20 cigarettes/day for 1 year.
d
P values relate to two-sided Wilcoxon rank-sum test of medians between GC.AT and other base change or GC.AT at CpG dinucleotides and GC.AT at
non-CpG dinucleotides, as appropriate.
e
One patient had two different tumours in the proximal and distal colon and was therefore excluded from this analysis.

Supplementary data
Supplementary Table 1 is available at Mutagenesis Online.
Funding
Cancer Research UK and Medical Research Council to EPICNorfolk cohort study group; Cancer Research UK to MJA.
Acknowledgements
We thank all the participants and the entire EPIC-Norfolk team.
Conflict of interest statement: None declared.

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