Clinicopathological and Molecular Profiles of
Colorectal Tumours with BRAF mutation
Weiqi Li BSc
THE UNIVERSITY OF WESTERN AUSTRALIA
2006
This thesis is presented for the degree of Master of Medical
Sciences at the University of Western Australia
Supervisor: Associate Professor Barry Iacopetta
School of Surgery and Pathology, University of Western Australia
2
Abstract
Introduction
BRAF is a member of the RAF family that encodes serine/threonine kinases of
the RAS/RAF/MAP kinase pathway. Recently, the BRAF V600E hotspot
mutation has been implicated in about 10% of colorectal cancers (CRC). It
occurs frequently in CRC with microsatellite instability (MSI+) caused by
promoter hypermethylation of the mismatch repair gene hMLH1, but has never
been observed in MSI+ tumours from patients with the familial CRC syndrome
referred to as hereditary nonpolyposis colorectal cancer (HNPCC). This opens
the possibility of using BRAF mutation screening to assist in the detection of
HNPCC individuals at the population level. BRAF mutations are inversely
associated with KRAS mutations and could define a subgroup of CRC with
distinctive phenotypic features.
Aims
The primary aim of this study was to identify the clinicopathological and
molecular features of CRC with BRAF mutation. The secondary aim was to
determine the frequency of BRAF mutation in CRC from younger patients who
were being screened as part of a population-based study into the prevalence of
HNPCC in the state of Western Australia.
Methods
A consecutive and well characterized series of 275 stage I-IV colorectal
tumours was evaluated for BRAF, KRAS and TP53 mutations, as well as MSI. A
large (n=780) series of CRCs from young (<60 years) patients was also
analyzed for BRAF mutation and MSI. All mutations and MSI status were
3 determined using fluorescent-single stranded conformation polymorphism (F-
SSCP) analysis.
Results and Conclusions
BRAF mutations were identified in 8.4% of a consecutive series of CRC. These
were mutually exclusive with KRAS mutations but no clear association with the
presence of TP53 mutation was observed. Mutations in BRAF were 5-10-fold
more frequent in tumours located in the proximal colon and having poor
histological grade, mucinous appearance and the presence of infiltrating
lymphocytes. BRAF mutant tumours were also 10-fold more likely to be MSI+
and frequently methylated. Such morphological features remained after
stratification for MSI and methylator phenotypes, suggesting that BRAF
mutation identifies a CRC subgroup with distinctive phenotypic properties
independently of MSI status.
Amongst 55 MSI+ cases identified in younger (<60 yrs) patients from the
HNPCC screening study, only 5 (9%) harboured a BRAF mutation. These could
therefore be excluded from further follow-up as possible HNPCC individuals.
Similar strong associations between BRAF mutation and proximal tumour site,
poor histological grade and mucinous appearance were found for younger and
older patients. In contrast, BRAF mutations were far more common in MSI+
tumours from older patients (50% vs 9%, P<0.0001). This important observation
suggests that the molecular phenotype of MSI+ tumours varies according to
patient age.
4 Our study has clarified the clinicopathological and molecular features of CRC
with BRAF mutations. It also provides evidence that associations between
BRAF mutation and MSI+ are age-related. Incorporation of BRAF mutation
analysis for young (<60 years) CRC patients could aid in further refinement of
population-based screening programs for HNPCC.
5 PUBLICATIONS ARISING FROM THIS THESIS
1. Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B
(2006) BRAF mutations are associated with distinctive clinical,
pathological and molecular features of colorectal cancer independently of
microsatellite instability status. Molecular Cancer 5:2
2. Iacopetta B, Li WQ, Grieu F, Ruszkiewicz A, Kawakami K (2006) BRAF
mutation and gene methylation frequencies of colorectal tumours with
microsatellite instability increase markedly with patient age. Gut (in
press)
6 ACKNOWLEDGEMENTS
It has been a great pleasure conducting my Master of Medical Science in the
Department of Surgery and Pathology. The past year and a half has been a
wonderful journey filled with joy and warmth brought about by many people.
First of all, I would like to extend my gratitude to my supervisor Barry Iacopetta
for his excellent supervision that is not only filled with scientific knowledge but
also with an abundance of patience, kindness and motivation. His amicable
spirit has created an excellent studying environment for my master degree.
I would also like to thank Fabienne Grieu for her inexhaustible assistance
around the laboratory. Her ever-smiling face made lab work felt so much less
tedious and there is always plenty of coffee and cookies to nourish my tired
brain! Thank you for your wonderful friendship!
Natasha Watson for her assistance in gathering the tissue samples.
Not forgetting Sophia Ang for her friendship, thought-provoking discussions and
lunch companionship!
Everyone else in the department including Shaoying Li, Norman Rong and
Maggie Weedon for assisting me in one way or another.
The team at the Oncology Research Institute, National University of Singapore,
for their advice and friendship, including Dr. Richie Soong, Nur Diyanah Anuar,
Peiyi Chong, Swee Siang Ng, Michelle Goh and Tiling Chang.
7 SPECIAL DEDICATION
In Loving Memory of My late Mother,
Even though you knew you were losing the battle to cancer, your lovely smile
never ceases. That, together with your love, has always been an inspiration to
me. I miss you Mum!
To My Dad and Little Brother,
The past two years have been extremely difficult with mum’s passing. However,
your undying love, support and belief in me have carried me through the tough
times. I am blessed to have the both of you.. Thank you for staying strong for
me! I love you both!
To my Fiancé Ben,
You are the reason for me to smile again! Thank you for all the laughter and
optimism, and above all, your love for me!
8 ABBREVIATIONS
APC Adenomatous polyposis coli
CGP Cancer Genome Project
CIMP CpG island methylator phenotype
CRC Colorectal cancer
DNA Deoxyribonucleic acid
DNTP Dioxynucleotide triphosphate
FAP Familial adenomatous polyposis
F-SSCP Fluorescent-Single strand conformation polymorphism
GSWA Genetic Services of Western Australia
HNPCC Hereditary nonpolyposis colorectal cancer
HP Hyperplastic polyp
ICG-HNPCC International Collaborative Group on HNPCC
IHC Immunohistochemistry
MAPK Mitogen-activated protein kinase
MMR Mismatch repair
MSI Microsatellite instability
MSI-H Microsatellite instability-high
NCI National Cancer Institute
PCR Polymerase chain reaction
TILs Tumour infiltrating lymphocytes
TP53 Tumour suppressor protein 53
9 TABLE OF CONTENTS CHAPTER 1: Introduction 1.1 Mitogen-activated protein kinase (MAPK) cascade 14
1.2 BRAF oncogene in human cancers 15
1.3 Genetics alterations in colorectal cancer 17
1.4 Hereditary nonpolyposis colorectal cancer
1.4.1 Clinical features 19
1.4.2 Genetics of HNPCC 19
1.4.3 Guidelines for detection of HNPCC 20
1.4.4 BRAF in HNPCC 24
1.5 Aims 25
CHAPTER 2: Materials and Methods 2.1 Case selection 27
2.2 Ethics approval 27
2.3 DNA extraction from paraffin embedded tissue sections 28
2.4 PCR for MSI screening 28
2.5 PCR for KRAS mutation screening 29
2.6 PCR for TP53 mutation screening 29
2.7 PCR for BRAF mutation screening 30
2.8 Screening for CpG island methylation 30
2.9 Fluorescent-single strand conformation polymorphism
(F-SSCP) analysis
31
2.10 Statistical analysis 33
CHAPTER 3: Results BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status 343.1 Introduction 35
3.2 Results 36
3.3 Discussion 43
3.4 Conclusion 45
10
CHAPTER 4: Results BRAF mutation in tumours from patients aged <60 years 464.1 Introduction 46
4.2 BRAF mutations and clinicopathological features of
tumours in patients aged <60 years 48
4.3 Clinicopathological characteristics of tumours with BRAF
mutations: comparison between young and old
colorectal cancer patients 50
4.4 Discussion 52
CHAPTER 5: General Discussion 5.1 BRAF mutations and phenotypic properties of CRC 55
5.2 BRAF mutations and screening for HNPCC 57
5.3 Limitations of this study 59
5.4 Conclusions 60
5.5 Future work 61
References 63
11 LIST OF TABLES
Table 1.1 Amsterdam I and II criteria for the identification of
HNPCC cases (source: Vasen et al., 1991; Vasen et
al., 1999)
22
Table 1.2 Bethesda guidelines for testing colorectal tumours
for MSI (source: Rodriguez-Bigas et al., 1997; Umar
et al., 2004)
23
Table 2.1 Primer sequences, annealing temperatures and
PCR product sizes
31
Table 2.2 SSCP gel conditions for the mutation analyses of
BAT-26, BRAF, KRAS and TP53
32
Table 3.1 Associations between BRAF mutation and
clinicopathological features of colorectal cancer
39
Table 3.2 Associations between BRAF mutation and
molecular features of colorectal cancer
40
Table 3.3 Clinicopathological and molecular features of BRAF
mutant colorectal cancers stratified according to
microsatellite instability status
41
Table 3.4 Clinicoptahological and molecular features of BRAF
mutant colorectal cancers stratified according to
methylator phenotype status
42
Table 4.1 Associations between BRAF mutation and
clinicopathological features of colorectal cancer in
patients aged <60 years
49
12 Table 4.2 Clinicopathological characteristics and MSI status of
tumours with BRAF mutations in young (<60 yrs)
and old (≥60 yrs) colorectal cancer patients
51
13 LIST OF FIGURES
Figure 1.1 The general structure of (a) MAPK pathway and
(b) ERK pathway (source: Kolch, 2000)
15
Figure 1.2 The BRAF protein and signal transduction
(Source: Pollock & Meltzer, 2002)
16
Figure 2.1 SSCP analysis of BRAF, KRAS, BAT-26 and
TP53 genes
33
Figure 3.1 (A) Representative F-SSCP gel used to detect
BRAF mutations in colorectal cancer. WT, wild
type; M, mutation. (B) DNA sequencing gel result
confirms the presence of a 1799T to A mutation
giving rise to the V600E mutation.
38
14 CHAPTER 1 INTRODUCTION
1.1 Mitogen-activated protein kinase (MAPK) cascade
Cancer is a disease of the genome, triggered by the accumulation of genetic
errors that eventually transform a normal cell into a tumour cell (Pollock &
Meltzer, 2002a). Multiple physiological processes are governed by the mitogen-
activated protein kinase (MAPK) cascade (Figure 1.1a) - a conserved signaling
system that transduces extracellular signals into the nucleus via a cascade of
kinases (Kolch, 2000; Peyssonnaux & Eychene, 2001). Tumorigenesis takes
place when genes encoding key components of this pathway are mutated,
resulting in inactivation of tumour-suppressor genes or the activation of
oncogenes (Pollock & Meltzer, 2002b).
Among the different MAPK cascades, the RAS/RAF/MEK/ERK module (Figure
1.1b) is a key signal transduction cascade through which cell proliferation,
differentiation, survival and apoptosis are regulated by changes in gene
expression in response to extracellular signals (Kolch, 2000). Extracellular
signals such as growth factors first activate the small G protein Ras which then
recruits Raf to the plasma membrane for activation. Activated Raf proteins then
activate MEK, which in turn activates a third protein kinase called ERK (Ikenoue
et al., 2004).
As part of the Cancer Genome Project (CGP), Davies and colleagues began to
analyze the entire genome in DNA samples from a wide array of human
cancers in order to study every gene for oncogenic mutations. Given the
15 importance of signal transduction in regulating cellular growth, particular
attention was paid to genes that encode components of the MAPK pathway.
Figure 1.1 The general structure of (a) MAPK pathway and (b) ERK pathway.
(Source: Kolch, 2000)
1.2 BRAF oncogene in human cancers
BRAF gene is a member of the RAF family that encodes cytoplasmic
serine/threonine kinases which are components of the MAPK cascade (Mercer
& Pritchard, 2003; Rajagopalan et al., 2002). In a large-scale screen for genes
mutated in human cancers, BRAF was found to be mutated in a wide variety of
tumours, suggesting it is a proto-oncogene (Davies et al., 2002). Recent studies
found that somatic BRAF mutations occur in approximately 66% of malignant
melanomas, 15% of colorectal cancer (CRC), 30% of ovarian cancer, 50% of
papillary thyroid carcinomas but a lower frequency (1-3%) in other cancer types
(Davies et al., 2002; Rajagopalan et al., 2002; Singer et al., 2003; Brose et al.,
2002; Cohen et al., 2003; Garnett & Marais, 2004).
16 The predominant mutation that accounts for 80% of all BRAF mutations is a
single-base substitution in exon 15 (T1796A) that leads to a substitution of
valine by glutamic acid at codon 600 (V600E; Davies et al., 2002). This
mutation occurs in the activation segment, which together with the glycine-rich
loop forms the BRAF kinase domain (Ikenoue et al., 2003). BRAF activity in
normal cells is controlled by mitogens and RAS proteins (Figure 1.2), however
the V600E mutation is thought to mimic phosphorylation and lead to constitutive
activation independently of RAS. As a result, ERK signaling is constitutively
active and this leads to cellular growth in favor of tumour development (Davies
et al., 2002; Ikenoue et al., 2003; Dibb et al., 2004). Wan and colleagues
reported V600E as the most active BRAF mutant with an in vitro kinase activity
~500-fold greater than wild-type (Wan et al., 2004).
Figure 1.2 The BRAF protein and signal transduction. (Source: Pollock &
Meltzer, 2002a)
17 1.3 Genetic alterations in colorectal cancer
The two main forms of genetically predisposed syndromes that account for
approximately 2-5% of all colorectal cancers are familial adenomatous
polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC; Smith
et al., 2002; Kinzler & Vogelstein, 1996). In FAP, the primary genetic alteration
is germline mutation of the tumour-suppressor gene adenomatous polyposis
coli (APC) (Kinzler & Vogelstein, 1996; Lindblom, 2001). An inherited defective
DNA mismatch repair (MMR) system is the underlying cause of HNPCC (Marra
& Boland, 1995; Jass et al., 2002a). When this MMR system fails, the length of
repetitive nucleotide sequences, known as microsatellites, present throughout
the genome is altered (Jacob & Praz, 2002). This phenotype is known as
microsatellite instability (MSI+) and is a hallmark of HNPCC (Boland et al.,
1998).
The remaining 95-98% of colorectal cancers arises sporadically and
approximately 12% of these are also characterized by MSI+ (Smith et al., 2002;
Kinzler & Vogelstein, 1996). Unlike the HNPCC MSI+ cases, sporadic MSI+
tumours are caused by somatic inactivation of a MMR gene (Kinzler &
Vogelstein, 1996). Traditionally, the adenoma-carcinoma sequence proposed
by Vogelstein and Fearon has been the accepted model for non-MSI+
colorectal carcinogenesis. The transition from normal epithelium to malignant
tissue requires the inactivation of a variety of tumour suppressor genes, such as
APC and TP53, and subsequent cumulative activation of oncogenes such as
KRAS. On the other hand, the initiation and pathogenic progression of sporadic
MSI+ remains unclear (Jass et al., 2002a; Hawkins & Ward, 2001; Park et al.,
2003).
18 It was recently proposed that a subset of hyperplastic polyps (HP) predisposes
to sporadic MSI+ cancers and these may represent the premalignant lesion in
the controversial “serrated neoplasia pathway” (Hawkins et al., 2002b). The
supporting evidence comes from studies that have associated sporadic MSI+
colorectal cancers with the CpG island methylator phenotype (CIMP)
characterized by methylation of multiple CpG islands (Toyota et al., 1999;
Hawkins et al., 2002a; Whitehall et al., 2002). CpG islands are rich in cytosine-
guanosine dinucleotides and are found in the promoter region of about half of all
human genes (Toyota et al., 1999). In normal tissues, these CpG islands are
virtually unmethylated. During carcinogenesis, however, simultaneous promoter
methylation of multiple CpG islands takes place, resulting in transcriptional
silencing of genes (Toyota et al., 2000; Esteller et al., 2001). Hypermethylation
of the hMLH1 promoter region results in the lack of expression of this MMR
protein and accounts for the large majority of sporadic MSI+ CRC (Herman et
al., 1998; Peltomaki, 2003).
Support for the serrated neoplasia pathway as a distinct pathway to CRC was
further reinforced when several studies found that BRAF mutation was
frequently observed in hyperplastic polyps, serrated polyps and sporadic MSI+
tumours, all of which are associated with aberrant CpG island methylation
(Yang et al., 2004; Kambara et al., 2004).
19 1.4 Hereditary nonpolyposis colorectal cancer
1.4.1 Clinical features
HNPCC, also known as Lynch syndrome, is the most common form of
hereditary colorectal cancer (Lynch & Chapelle, 2003). It is an autosomal,
dominantly inherited cancer syndrome and accounts for approximately 2% of all
CRCs. HNPCC predisposes affected individuals to an approximate 80% lifetime
risk of CRC, principally in the proximal colon and with a higher risk in males
(Burt & Neklason, 2005; Lynch & Chapelle, 2003; Lynch et al., 2003; Kambara
et al., 2004). Synchronous and metachronous colorectal tumours are also
common (Lynch & Chapelle, 1999; Lynch et al., 2003). In addition, carcinomas
in extracolonic tissues such as the endometrium, ovaries, upper urothelial tract,
small bowel, pancreas, brain, hepatobiliary tract and stomach have been
reported to occur with an increased frequency (Lynch & Chapelle, 1999).
1.4.2 Genetics of HNPCC
HNPCC is caused by an inherited mutation in one of at least five mismatch
repair (MMR) genes: (a) MLH1, (b) MSH2, (c) PMS1, (d) PMS2, and (e) MSH6
(Loukola et al., 2001; Peltomaki, 2003). Germline mutations in MLH1 and MSH2
constitute about 90% of all MMR gene mutations (Lynch & Chapelle, 1999;
Domingo et al., 2004b). These genes are normally responsible for correcting
errors in the length of microsatellites (nucleotide repeat regions) produced
during the replication of DNA (Lynch & Chapelle, 2003). The presence of
somatic alterations in the length of microsatellites (referred to as microsatellite
instability, or MSI+) and the absence of MMR protein expression detected by
immunohistochemistry (IHC) are both hallmarks of HNPCC (Wang et al., 2003;
Baudhuin et al., 2005).
20 Colonoscopy screening performed at 3-year intervals is able to reduce CRC-
related mortality by about 65% in HNPCC families (Jarvinen et al., 2000). At
present, the International Collaborative Group on HNPCC (ICG-HNPCC)
recommends that ‘at-risk’ individuals in HNPCC families undergo colonoscopic
surveillance every 1-2 years, beginning at the age of 25 years or 5 years
younger than the youngest affected family member, whichever is earliest
(Vasen et al., 1993). Faecal occult blood testing is offered in alternate years or
to subjects unwillingly to undergo colonoscopy. Screening for endometrial
carcinoma is recommended from 30-35 years of age (Jarvinen et al., 1995).
Extended surgery has been recommended for patients with proven HNPCC
because of the increased risk of metachronous CRC.
Testing for germline mutation of MMR genes is important as it allows exclusion
of healthy family members carrying the wild-type allele from unnecessary
surveillance programs (Wolf et al., 2005). The following guidelines have been
proposed to help identify patients with a high probability of a MMR germline
mutation.
1.4.3 Guidelines for detection of HNPCC
The detection of suspected HNPCC cases is often difficult as the syndrome
lacks well-defined pre-symptomatic characteristics (Lynch et al., 2003; Aaltonen
et al., 1998). HNPCC is usually recognized by the occurrence of cancers over
multiple generations and at an early age of onset (average age of onset <45
years). A strong family history has therefore become the primary diagnostic tool
(Lynch & de la Chapelle, 2003). In order to standardize diagnostic criteria, the
21 ICG-HNPCC developed the original Amsterdam criteria (I) as shown in Table
1.1 below (Umar et al., 2004; Aaltonen et al., 1998). Since then, revision has
been made to include small families (Amsterdam criteria II). These criteria were
pivotal in identifying kindreds that eventually led to association of the HNPCC
syndrome with germline MMR gene mutations (Rodriguez-Bigas et al., 1997).
22 Table 1.1 Amsterdam I and II criteria for the identification of HNPCC cases
(source: Vasen et al., 1991; Vasen et al., 1999)
Amsterdam Criteria I Amsterdam Criteria II
1. Three or more family members
with CRC and all of the following
features:
2. One is a first-degree relative of the
other two
1. At least three relatives must have
a cancer associated with HNPCC
(CRC, endometrial, stomach,
ovary, ureter or renal-pelvis,
brain, small bowel, heptobiliary
tract, or skin 3. At least two successive
generations must be affected 2. One must be a first-degree
relative of the other two 4. At least one of the relatives with
CRC must have received the
diagnosis before the age of 50
years
3. At least two successive
generations must be affected
4. At least one of the relatives with
cancer associated with HNPCC
should have received the
diagnosis before the age of 50
5. FAP should have been excluded
in any relatives with CRC
5. FAP must have been excluded.
6. Tumours should be pathologically
verified whenever possible
In clinical practice, MSI testing is used as a marker for underlying MMR
dysfunction. To identify which patients are appropriate for MSI testing, the
National Cancer Institute (NCI) developed a set of criteria known as the
Bethesda Guidelines (Table 1.2) during the International Workshop on HNPCC
in 1996 and later revised in 2002 (Rodriguez-Bigas et al., 1997).
23 Table 1.2 Bethesda guidelines for testing colorectal tumours for MSI
(source: Rodriguez-Bigas et al., 1997; Umar et al., 2004)
Bethesda Guidelines Revised Bethesda Guidelines
1. Amsterdam I criteria met
2. Individuals with more than one
HNPCC cancer
3. CRC and first-degree relative with
CRC/HNPCC cancer, one cancer
younger than 45 years or one
adenoma younger than 40
4. CRC/endometrial cancer younger
than age 45
5. Right-sided CRC, undifferentiated,
younger than 45
6. Signet ring CRC younger than 45
7. Adenomas younger than 40 years
1. CRC diagnosed in a patient under
the age of 50
2. Presence of synchronous,
metachronous colorectal, or other
HNPCC-associated tumours,
regardless of age
3. CRC with the MSI-H histology
diagnosed in a patient who is less
than 60 years of age
4. CRC diagnosed in one or more
first-degree relatives with an
HNPCC-related tumour*, with one
of the cancers being diagnosed
under age 50 years
5. CRC diagnosed in two or more
first- or second-degree relatives
with HNPCC-related tumours,
regardless of age.
*HNPCC-related tumours include colorectal, endometrial, stomach, ovarian,
pancreas, ureter and renal pelvis, biliary tract, and brain (usually glioblastoma
as seen in Turcot syndrome) tumours, sebaceous gland adenomas and
keratoacanthomas in Muir-Torre syndrome, and carcinoma of the small bowel
(Lin et al., 1998).
24 Despite the availability of improved diagnostic criteria and guidelines for
identification and molecular testing, the detection of HNPCC patients at the
population level remains difficult. The sensitivity of the Amsterdam criteria is
compromised by the amount of time and resources needed to obtain a
comprehensive family history required to assess the possible genetic risks.
These lead to inaccuracies when reporting people at risk of CRC (Mitchell et al.,
2004). It has been estimated that only 10-20% of individuals at high risk for
HNPCC are being referred for further evaluation (Terdiman et al., 2002). The
specificity of MSI testing is limited by the occurrence of MSI+ in 15% of sporadic
CRC cases. These arise due to somatic inactivation of the MMR genes,
particularly methylation-induced transcriptional silencing of MLH1 (Thibodeau et
al., 1993; Umar et al., 2004).
Current recommendations for the detection of HNPCC includes an initial testing
of tumours for the presence of MSI+ combined with IHC for the absence of
MMR protein expression. If loss of gene expression is found, this allows
germline mutation testing to be targeted to the relevant gene (Salovaara et al.,
2000; Domingo et al., 2004a). This complementary MSI/IHC approach may
increase the sensitivity and specificity when diagnosing HNPCC. However, this
molecular-based approach may not be sufficiently efficient, cost effective or
available in routine clinical practice to allow HNPCC screening in the entire
colorectal cancer population (Halvarsson et al., 2004; Domingo et al., 2004b).
1.4.4 BRAF in HNPCC
Recently, investigators have reported a strong association between V600E
BRAF mutation and MMR deficiency (Rajagopalan et al., 2002; Davies et al.,
25 2002; Yuen et al., 2002). This association was seen exclusively in sporadic
MSI+ tumours, but not in MSI+ tumours from HNPCC (Deng et al., 2004; Wang
et al., 2003; Koinuma et al., 2004; Kambara et al., 2004; Nagasaka et al., 2004;
Domingo et al., 2004a; Miyaki et al., 2004; McGivern et al., 2004). This
exclusivity of BRAF mutation for sporadic but not familial MSI+ CRC could
therefore be used as a strategy to help identify HNPCC families.
1.5 AIMS
Hereditary nonpolyposis colorectal cancer is the most common form of familial
bowel cancer (Lynch et al., 2003). It is often left undiagnosed due to the lack of
distinguishing morphological features, thus leaving other family members with
germline mutations to the risk of cancer at an early age (Lynch et al., 2003;
Aaltonen et al., 1998). There is firm evidence that routine colonoscopic
screening, improves the survival rate of individuals with HNPCC syndrome
(Jarvinen et al., 2000), thus justifying the need to detect this genetic condition in
the population.
Currently, suspected HNPCC cases are identified primarily based on family
history as defined by the Amsterdam criteria (Vasen et al., 1991; Vasen et al.,
1999). However, the feasibility and accuracy of following these guidelines have
been proven less than ideal (Mitchell et al., 2004). An alternative approach
which involves complementary IHC-based screening for loss of MMR protein
expression and molecular screening for MSI+ has been proposed (Halvarsson
et al., 2004). Although this approach increases the sensitivity and specificity in
diagnosing HNPCC suspects, its implementation at the population level needs
26 to be evaluated for cost-effectiveness (Halvarsson et al., 2004; Domingo et al.,
2004b).
The recent finding that V600E BRAF mutation is frequently present in sporadic
MSI+ but not HNPCC MSI+ tumours suggests a possible strategy that may
simplify the detection of HNPCC families. The aims of this work are therefore:
1. To evaluate the clinical, pathological and molecular phenotype of
colorectal tumours with BRAF mutations
2. To compare the clinical, pathological and molecular features of colorectal
tumours with BRAF mutation in younger and older patients
3. To determine the frequency of BRAF mutation in the younger patient
population that is likely to be the target of screening for HNPCC
27 Chapter 2 MATERIALS & METHODS
2.1 Case selection
A consecutive series of 275 stage I - IV colorectal tumours investigated in this
study were obtained from the Colorectal Unit of the Royal Adelaide Hospital.
The tumour samples were snap frozen in liquid nitrogen within 20-40 min after
resection and stored at -70ºC prior to DNA extraction.
Another series of 780 stage I - IV paraffin-embedded colorectal tumour samples
were also studied. These were obtained from three major public teaching (Sir
Charles Gairdner, Royal Perth, Fremantle) and two private hospitals (St John of
God at Murdoch and Subiaco) in Western Australia. Cases selected from these
five institutes were diagnosed between 2000 and 2004 and a patient age at
diagnosis of <60 years was the basis for selection.
Clinical data available for both colorectal tumour series included patient age and
sex, while pathology data included nodal involvement, tumour site, histological
grade, mucinous histology and the presence of infiltrating lymphocytes.
2.2 Ethics approval
Ethics approval was been obtained from each of the West Australian public
teaching (Sir Charles Gairdner, Royal Perth, Fremantle) and major private
hospitals (St John of God) for access to archival paraffin-embedded tumour
blocks for the purposes of phenotypic analysis.
28 2.3 DNA extraction from paraffin-embedded tissue sections
Two 25 µm sections of paraffin embedded tissues from each case were placed
in a 1.5 ml Eppendorf tube containing 300 µl of digestion buffer (50 mH Tris
HCL, 1 mM EDTA, 0.5 % Tween 20 at pH 8.5). The tubes were heated at 94°C
for 10 min in a water bath to melt the paraffin before centrifuging for 10 min at
12,000 rpm to separate the tissues from the paraffin. The tubes were allowed to
cool at 4°C for approximately 2 hours until a firm crust of paraffin was formed.
This was removed and the tissue transferred to a new 1.5 ml Eppendorf tube
containing 200 µl of fresh digestion buffer. Twenty µl of Proteinase K from a 20
mg/ml stock dissolved in digestion buffer were then added and the mixture
incubated in a rotating oven at 55°C for 48 hours. The Proteinase K reaction
was then inactivated by heating the tubes at 94°C in a water bath for 10 min.
The samples were centrifuged and the resulting clear solution containing DNA
was transferred into a new 1.5 ml Eppendorf tube and stored at 4°C for use
within several weeks.
2.4 PCR for MSI screening
The MSI status of each tumour was evaluated by fluorescent-single stranded
conformation polymorphism (F-SSCP) analysis of the BAT-26 mononucleotide
repeat (Iacopetta et al., 1998). The PCR reaction was carried out in a 16 µl
reaction mix containing 1x polymerization buffer, 1x Q-Solution, 200 µM of each
dioxynucleotide triphosphate (dNTP), 3 mM MgCl2 (Qiagen, Melbourne), 0.5 µM
of each HEX-labeled BAT-26 primer (Geneworks, Adelaide; primer sequences
listed in Table 2.1) and 0.5U Taq DNA Polymerase (Qiagen, Melbourne).
Reactions were ‘hot started’ by the addition of 1 µl genomic DNA at 94°C prior
to commencement of cycling. PCR amplification was carried out using the
29 following conditions: 35 cycles of 94°C for 30 sec, 46°C for 30 sec, and 70°C for
30 sec; followed by a final extension at 70°C for 10 min.
2.5 PCR for KRAS mutation screening
Mutations in KRAS codons 12 and 13 were detected by F-SSCP analysis
(Wang et al., 2003a). The KRAS gene was amplified in a 14 µl mix containing
1x polymerization buffer, 1x Q-Solution, 200 µM of each dNTP, 3 mM MgCl2
(Qiagen, Melbourne), 0.5 µM of each HEX-labeled KRAS primer (Geneworks,
Adelaide; primer sequences listed in Table 2.1), 0.5U Taq DNA Polymerase
(Qiagen, Melbourne) and 1 µl of DNA. Amplification was performed using the
same cycling conditions as described in Section 2.4 except that the annealing
temperature was 54°C.
2.6 PCR for TP53 mutation screening
Exons 5, 7 and 8 of TP53 tumour suppressor gene were screened for mutations
using F-SSCP as described previously (Soong & Iacopetta., 1997). The reaction
mix for each exon was 14 µl containing 1x polymerization buffer, 1x Q-Solution,
200 µM of each dNTP, 2.5 mM MgCl2 (Qiagen, Melbourne), 0.5 µM of each
HEX-labeled primer (Geneworks, Adelaide; respective primer sequences listed
in Table 2.1), 0.5U Taq DNA Polymerase (Qiagen, Melbourne) and 1 µl of DNA.
Amplification was carried out using the same cycling conditions as described in
Section 2.4 except the extension time for TP53 exon 5 was 45 seconds and the
annealing temperatures were 60°C, 60°C and 56°C, respectively, for TP53
exons 5, 7 and 8.
30 2.7 PCR for BRAF mutation screening
A hotspot V600E mutation site in exon 15 of the human BRAF gene was
identified in previous studies (Davies et al., 2002). This mutation was identified
here by F-SSCP analysis. The BRAF gene was amplified in a 14 µl mix
containing 1x polymerization buffer, 1x Q-Solution, 200 µM of each dNTP, 3 mM
MgCl2 (Qiagen, Melbourne), 0.5 µM of each HEX-labeled BRAF primer
(Geneworks, Adelaide; Davies et al., 2002; primer sequences listed in Table
2.1), 0.5U Taq DNA Polymerase (Qiagen, Melbourne) and 0.8 µl of DNA.
Amplification was carried out following the same cycling conditions as described
in Section 2.4 except that the annealing temperature was 60°C.
2.8 Screening for CpG island methylation
CIMP phenotype of tumours included in this work was determined in a previous
study headed by A/Prof Iacopetta (Kawakami et al., 2003).
31 Table 2.1 Primer sequences, annealing temperatures and PCR product
sizes.
Primer Sequence
Annealing
Temperature (ºC)
PCR product size (bp)
BAT26 F 5’-TTGGATATTGCAGCAGTCAG-3’ 46 136 BAT26 R 5’-GCTCCTTTATAAGCTTCTTCA-3’ BRAF F 5’-TCATAATGCTTGCTCTGATAGGA-3’ 60 224 BRAF R 5’-GGCCAAAAATTTAATCAGTGGA-3’ KRAS F 5’-GACTGAATATAAACTTGTGG-3' 54 107 KRAS R 5’-CTATTGTTGGATCATATTCG-3'
TP53
Exon 5 F 5'-TCTTCCTGCAGTACTCCCCT-3' 60 205 Exon 5 R 5'-AGCTGCTCACCATCGCTATC-3' Exon 7 F 5'-TTGTCTCCTAGGTTGGCTCT-3' 60 136 Exon 7 R 5'-GCTCCTGACCTGGAGTCTTC-3' Exon 8 F 5'-TCCTGAGTAGTGGTAATCTA-3' 56 157 Exon 8 R 5'-GCTTGCTTACCTCGCTTAGT-3'
2.9 Fluorescent-single strand conformation polymorphism (F-SSCP)
analysis
The Single Strand Conformation Polymorphism (SSCP) technique is based on
the differential electrophoretic migration in non-denaturing acrylamide gels of
single stranded DNA molecules having different primary sequences and
therefore different secondary structures (Grieu et al., 2004). In the current
study, a fluorescent Gel-Scan 2000 system (Corbett Research, Sydney) was
used to detect HEX-labeled fluorescent primers used in the amplification of
BAT-26 and the KRAS, BRAF and TP53 genes.
32 In summary, 3 µl of amplified fluorescent-labeled PCR product was mixed with
9 µl of deionized formamide loading buffer containing 0.05 % w/v Bromophenol
blue and 0.5M EDTA, and denatured by heating at 94°C for 5 min. One µl of
this mix was then loaded onto a non-denaturing polyacrylamide gel (8%
polyacrylamide/2% glycerol) and run on the Gel-Scan 2000 real-time DNA
fragment analyzer according to manufacturer’s instruction (Corbett Research,
Sydney)s. The optimum SSCP gel condition for individual gene products was
determined empirically and is listed in Table 2.2. Once loaded into the wells,
samples were pulse loaded for 20 sec at 1400V, the wells were then rinsed
thoroughly and the gel was run for 120 min at 1400V in 0.8x TBE buffer at a
constant temperature of 25°C. ONE-D scan software (Scanalytics, Billerica,
USA) was used to enhance contrast of the electrophoretogram and thus
facilitate the reading of aberrant bands (Figure 2.1).
Table 2.2 SSCP gel conditions for the mutation analyses of BAT-26, BRAF,
KRAS and TP53.
Gene SSCP gel a
BAT-26 8/2 BRAF 8/2 KRAS 12/2 TP53 exons 5, 7 and 8
8/2
a % polyacrylamide/% glycerol content
33
BAT-26
* * *
BRAF
* *
KRAS
* * * * * *
TP53 Exon 5
* * * *
* * *
Figure 2.1 SSCP analyses of BRAF, KRAS, BAT-26 and TP53 genes.
* Samples with aberrant bands indicative of mutation.
2.10 Statistical analysis
All statistical analyses were performed using SPSS Version 12.0 (Chicago,
Illinois, USA). Differences in frequencies were evaluated using the Fisher’s
exact or Pearson’s chi-squared tests as appropriate. Multivariate analysis was
performed using binary logistic regression with BRAF mutation as the
dependent variable.
34 CHAPTER 3 RESULTS
BRAF mutations are associated with distinctive clinical, pathological and
molecular features of colorectal cancer independently of microsatellite
instability status
Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B
Molecular Cancer (2006) 5:2
35 3.1 Introduction
BRAF is a member of the RAF family of kinases that acts upstream of the
MEK1/2 kinases in response to RAS signals. Activating mutations in BRAF
have been reported in 5-15% of colorectal carcinomas (CRC), with by far the
most common mutation being a T to A transversion at nucleotide 1796 leading
to a V600E substitution (Davies et al., 2002; Rajagopalan et al., 2002; Yuen et
al., 2002). The BRAF V600E hotspot mutation is strongly associated with the
microsatellite instability (MSI+) phenotype but is mutually exclusive with KRAS
mutations (Oliveira et al., 2003; Deng et al., 2004; Nagasaka et al., 2004; Yang
et al., 2004). Interestingly, BRAF mutations are found only in MSI+ sporadic
tumours that result from aberrant MLH1 promoter methylation and do not occur
in MSI+ tumours from hereditary non-polyposis colorectal cancer (HNPCC)
patients (Deng et al., 2004; McGivern et al., 2004; Miyaki et al., 2004; Domingo
et al., 2004a), thus providing a convenient discriminator between sporadic and
familial cases. The majority of MSI+ sporadic tumours belong to a larger CRC
group referred to as the CpG island methylator phenotype (CIMP+) that is
characterised by widespread hypermethylation of CpG islands located with
gene promoter regions (Toyota et al., 1999). Both MSI+ and CIMP+ tumours
are thought to arise from large hyperplastic polyps and serrated adenomas
(Hawkins et al., 2001; Jass et al., 2002b) and recent work has demonstrated a
high frequency of BRAF mutations in these lesions (Yang et al., 2004; Chan et
al., 2003; Kambara et al., 2004).
Although the positive association with MSI+ and inverse association with KRAS
mutation have been well documented, little is known about other phenotypic
properties of tumours with BRAF mutation. In the present study we analysed for
36 BRAF V600E mutations in a consecutive series of 275 CRCs that were well
characterised for the major pathological and molecular features of this disease.
Our results demonstrate that oncogenic BRAF mutation occurs preferentially
within a subgroup of CRCs that have distinctive features. It could therefore be
used as a convenient marker for the further characterisation of these tumours,
particularly in relation to their prognosis and response to adjuvant
chemotherapy.
3.2 Results
Figure 3.1A shows representative Fluorescent-SSCP results for the screening
of BRAF mutations in this CRC series, while Figure 3.1B shows DNA
sequencing confirmation of the 1799 T to A transversion resulting in the V600E
mutation. The overall frequency of BRAF mutation was 8.4% (23/275),
comparing favorably with frequencies of 9-11% reported for other large studies
of this tumour type (Nagasaka et al., 2004; Koinuma et al., 2004; Samowitz et
al., 2005). The mean age of patients with and without BRAF mutation was
identical (Table 3.1). Strong associations were observed between BRAF
mutation and tumour origin in the proximal side of the large bowel, poor
histological grade, mucinous appearance and the presence of infiltrating
lymphocytes. Higher frequencies of BRAF mutation were also observed in
females and in node negative tumours but these did not reach significance.
BRAF mutations showed no association with TP53 mutations and were mutually
exclusive with the presence of KRAS mutations (Table 3.2). In contrast, BRAF
mutations were approximately 10–fold more frequent in MSI+ and CIMP+
tumours compared to tumours without these phenotypes. A strong association
37 was also seen with methylation of the MLH1 gene promoter and in particular
with methylation of its proximal region. The methylation status of 7 different
CpG islands in this CRC series has been reported previously (Kawakami et al.,
2003). The mean number of these methylated sites was 3–fold higher in
tumours with BRAF mutation compared to those without (2.6 + 1.7 vs 0.8 + 1.0;
P<0.001). Multivariate analysis revealed that MSI+ was the only significant
independent predictor of BRAF mutation (RR=6.3, 95%CI [1.2-32.3]; P=0.028)
in a model that included CIMP+, tumour site, histological grade, presence of
infiltrating lymphocytes and mucinous appearance.
We next examined whether the characteristic features of tumours with BRAF
mutation were still apparent following stratification into MSI and CIMP
phenotypes. Although the statistical power of this subgroup analysis was
limited, the morphological features of infiltrating lymphocytes, poor histological
grade and mucinous appearance were clearly associated with BRAF mutation
regardless of tumour MSI status (Table 3.3). Similarly, these features were each
more common in tumours with BRAF mutation in both the CIMP- and CIMP+
subgroups (Table 3.4). Similar to previous observations in a separate CRC
cohort (van Rijnsoever et al., 2002), the frequency of KRAS mutation was lower
in MSI+ compared to MSI- tumours (P=0.034; Table 3.3), while the frequency of
TP53 mutation was also considerably lower in MSI+ tumours with wildtype
BRAF than in MSI- tumours with wildtype BRAF (P=0.014).
38
Figure 3.1 (A) Representative F-SSCP gel used to detect BRAF mutations in
colorectal cancer. WT, wild-type; M, mutation. (B) DNA sequencing gel result
confirms the presence of a 1799 T to A mutation giving rise to the V600E
mutation.
(A) BRAF V600E mutation screening using F-SSCP
(B) DNA sequence showing BRAF 1799 T to A mutation
WT M M M WT WT WT M WT
39 Table 3.1 Associations between BRAF mutation and clinicopathological
features of colorectal cancer.
Feature (n) a BRAF wild-type (%)
BRAF mutation (%) P
Total (275) 252 (92) 23 (8)
Age (yrs) 68.4 + 13.0 68.4 + 20.7 NS
Gender Men (132) 124 (94) 8 (6) Women (100) 87 (87) 13 (13) 0.068
Infiltrating lymphocytes
Negative (199) 190 (95) 9 (5) Positive (21) 11 (52) 10 (48) <0.0001
Nodal involvement
Negative (128) 115 (90) 13 (10) Positive (70) 66 (94) 4 (6) NS
Tumour site
Proximal (93) 79 (85) 14 (15) Distal (126) 122 (97) 4 (3) 0.0015
Histological grade
Well/moderate (140) 133 (95) 7 (5) Poor (29) 22 (76) 7 (24) 0.0006
Mucinous
Negative (159) 150 (94) 9 (6) Positive (27) 20 (74) 7 (26)
a Data was unavailable for gender in 43 cases, infiltrating lymphocytes in 55
cases, nodal involvement in 77 cases, tumour site in 56 cases, grade in 106
cases and mucinous appearance in 89 cases.
0.0005
40 Table 3.2 Associations between BRAF mutation and molecular features of
colorectal cancer.
Feature (n) aBRAF
wild-type (%) BRAF
mutation (%) P
Total (275) 252 (92) 23 (8) MSI
Negative (204) 195 (96) 9 (4) Positive (31) 19 (61) 12 (39) <0.0001
Methylation status
CIMP- (150) 145 (97) 5 (3) CIMP+ (42) 31 (74) 11 (26) <0.0001
MLH1 distal region Negative (168) 159 (95) 9 (5) Positive (24) 17 (71) 7 (29) <0.0001
MLH1 proximal region Negative (179) 169 (94) 10 (6) Positive (13) 7 (54) 6 (46) <0.0001
KRAS
Wild-type (156) 134 (86) 22 (14) Mutant (93) 93 (100) 0 (0) <0.0001
TP53
Wild-type (183) 166 (91) 17 (9) Mutant (66) 62 (94) 4 (6)
a Data was unavailable for MSI status in 40 cases, methylation status in 83
cases, KRAS mutation in 26 cases and TP53 mutation in 26 cases
NS
41 Table 3.3 Clinicopathological and molecular features of BRAF mutant
colorectal cancers stratified according to microsatellite instability status.
MSI- MSI+
Feature BRAF WT BRAF M P BRAF WT BRAF M P
(n=192) (%) (n=9) (%) (n=19) (%) (n=12) (%)
Age (years) 68.5 ±12.6 58.2±26.5 NS 67.5±16.8 76.1±10.9 NS
Females 39 44 NS 63 75 NS
TILS positive a 3 44 0.0004 28 60 0.08
Node negative 62 62 NS 81 89 NS
Proximal site 36 67 0.05 72 89 NS
Poor grade 15 40 0.12 25 56 0.11
Mucinous 12 53 0.05 6 44 0.04
CIMP+ 15 50 0.03 40 88 0.03
MLH1 meth. distal 7 12 NS 40 75 0.10
MLH1 meth. prox. 1 0 NS 33 75 0.06
KRAS mutant 43 0 0.008 21 0 0.12
TP53 mutant 29 11 NS 5 18 NS
a Tumour-infiltrating lymphocytes
42 Table 3.4 Clinicopathological and molecular features of BRAF mutant
colorectal cancers stratified according to methylator phenotype status.
CIMP- CIMP+
Feature BRAF WT BRAF M P BRAF WT BRAF M P
(n=145) (%) (n=5) (%) (n=31) (%) (n=11) (%)
Age (years) 68.3±13.5 71.0±11.0 NS 71.7±11.8 65.4±26.3 NS
Females 37 60 NS 42 45 NS
TILS positive 2 40 0.008 17 45 0.06
Node negative 63 60 NS 65 82 NS
Proximal site 35 60 NS 74 80 NS
Poor grade 13 66 0.05 20 40 NS
Mucinous 9 25 NS 25 45 NS
MSI+ 6 20 NS 19 64 0.01
MLH1 meth dist. 3 0 NS 42 64 NS
MLH1 meth. prox 0 0 NS 23 55 0.05
KRAS mutant 43 0 0.06 55 0 0.001
TP53 mutant 26 0 NS 29 20 NS
43 3.3 Discussion
The BRAF V600E mutation has already been proposed as a convenient marker
to discriminate between MSI+ tumours that are sporadic or HNPCC in origin
(Deng et al., 2004; McGivern et al., 2004; Miyaki et al., 2004; Domingo et al.,
2004a). This is a very important issue for population-based screening programs
that aim to identify CRC associated with the HNPCC syndrome. Compared to
the analysis of MLH1 promoter methylation, mutation at the BRAF V600E
hotspot is relatively simple to detect using DNA sequencing, RFLP or the SSCP
method used in the present work (Figure 3.1).
Similar to other studies (Oliveira et al., 2003; Deng et al., 2004; Domingo et al.,
2004a; Koinuma et al., 2004; Samowitz et al., 2005) we observed BRAF
mutation frequencies of 4% in MSI- tumours and 39% in MSI+ tumours (Table
3.1). The highest frequencies were seen in tumours showing methylation of the
MLH1 promoter proximal region (46%) and in tumours with infiltrating
lymphocytes (48%). BRAF mutation frequencies of up to 70-80% have been
reported in sporadic MSI+, CIMP+ and MLH1-methylated CRC and polyps
(Yang et al., 2004; McGivern et al., 2004; Kambara et al., 2004; Koinuma et al.,
2004). For reasons that are still unclear, BRAF mutations are approximately 5-
10–fold more frequent in tumours that have characteristic features of sporadic
MSI+ (ie. MLH1 methylated) and CIMP+ phenotypes. These include proximal
colon location, poor differentiation, mucinous histology and infiltrating
lymphocytes (Jass et al., 2002a; Hawkins et al., 2002a; van Rijnsoever et al.,
2002). Interestingly however, in the present study BRAF mutations never
occurred in association with KRAS mutation, were present in only 3% of CIMP-
tumours and showed no association with TP53 mutation (Table 3.2). The
44 observation that BRAF mutations occur only very rarely in HNPCC-related MSI+
CRC demonstrates that defective DNA mismatch repair is not involved in
causing this genetic alteration.
In order to determine whether the characteristic clinicopathological features of
tumours with BRAF mutation were due to their close association with MSI+ and
CIMP+, we stratified tumours according to these phenotypes. Despite having
only 9 MSI-/BRAF mutant and 5 CIMP-/BRAF mutant tumours, the results
showed that associations between BRAF mutation and the morphological
properties of tumour-infiltrating infiltrating lymphocytes, poor histological grade
and mucinous phenotype were retained (Tables 3.3 and 3.4).
The frequencies of BRAF mutation observed in MSI- (4%) and MSI+ (39%)
tumours in the present study compare favorably (5% and 52%, respectively) to
those reported recently in another large, population-based study (Samowitz et
al., 2005). Although BRAF mutations are much more frequent in MSI+ tumours,
the comparative rarity of this phenotype means that a considerable proportion
occur in MSI- tumours. In the present study, 43% of all BRAF mutations
occurred in MSI- tumours compared to 48% in the study by Samowitz et al
(Samowitz et al., 2005). BRAF mutations were reported to show prognostic
significance in MSI- but not in MSI+ CRC (Samowitz et al., 2005). The lack of
follow-up information on CRC patients in the current study and the small
number of BRAF mutations (n=21) meant that we were unable to evaluate the
prognostic significance of BRAF mutation according to MSI status.
45 3.4 Conclusion
Findings from the present study and from previous work indicate that BRAF
mutation is likely to be a convenient marker for the identification of a subset of
CRCs with distinctive clinical, pathological and molecular features and which
may originate in hyperplastic polyps and serrated adenomas (Yang et al., 2004;
Chan et al., 2003; Kambara et al., 2004). In view of the strong associations
between BRAF mutation and specific pathological (site, grade, mucinous,
infiltrating lymphocytes) and molecular (methylated MLH1, MSI+, CIMP+,
wildtype KRAS) features, it will be interesting in future studies to determine the
predictive significance of this marker for response to adjuvant therapies in CRC.
46 CHAPTER 4 RESULTS
BRAF mutation in colorectal tumours from patients aged <60 years
4.1 Introduction
Molecular screening for MSI+ and complementary IHC-based screening for loss
of MMR protein expression have been recommended as a first-line testing
strategy for suspected HNPCC cases (Muller et al., 2004; Liljegren et al., 2004;
Shia et al., 2005). MSI testing provides evidence of defective MMR and IHC
confirms this and pinpoints the responsible gene, hence, directing the specific
mutational analysis to be performed (Baudhuin et al., 2005). Several studies
have shown this approach to be highly sensitive (~90%) and specific (100% in
most cases) for the detection of HNPCC (Lindor et al., 2002; Ruszkiewicz et al.,
2002; Debniak et al., 2000). Since a low prevalence of MMR gene mutations is
expected even in younger CRC patients, preselection by means of IHC and/or
MSI analysis is justified before carrying out expensive germline mutation
analysis (Niessen et al., 2006). However one of the complications to arise from
routine MSI and IHC screening is the detection of sporadic cases with defective
MMR due to methylation of the hMLH1 gene. This results in a reduced
specificity from the use of these tests.
Previously, technically challenging methylation analysis was required to
determine if cases with loss of hMLH1 expression were sporadic or due to a
germline mutation in this gene. The discovery that BRAF mutations were often
found in sporadic MSI+ tumours, but rarely in HNPCC MSI+ tumours (Deng et
al., 2004; Wang et al., 2003; Koinuma et al., 2004; Kambara et al., 2004;
Nagasaka et al., 2004; Domingo et al., 2004a; Miyaki et al., 2004; McGivern et
47 al., 2004), allowed simplification of population-based screening for HNPCC. As
indicated in Chapters 2 and 3, BRAF mutation screening is relatively
straightforward using PCR-based techniques such as SSCP.
Following an earlier tissue microarray study of MLH1 and MSH2 expression in
more than 1000 consecutive colorectal tumour specimens (Chai et al, 2004), a
patient cut-off age of 60 years was arbitrarily chosen for a population-based
HNPCC screening program in the state of Western Australia. This age was
chosen to maximize the capture of HNPCC cases while at the same time
limiting the detection of sporadic MSI+ cases due to hMLH1 methylation.
Approximately 22% of all colorectal cancer patients are aged less than the 60
yrs cut-off at diagnosis, meaning that less than one quarter of patients undergo
routine MSI and IHC screening.
All colorectal cancer patients in Western Australia aged <60 yrs at the time of
diagnosis in the years 2000-2004 inclusive were identified from pathology
records and their archival surgical or biopsy tumour blocks retrieved. They were
analyzed for MSI using the BAT-26 mononucleotide marker and for BRAF
mutations using fluorescent SSCP. The aim of the project is to determine the
prevalence of HNPCC in a population-based setting and in the absence of any
information on family history of cancer. By simultaneously screening all tumours
for BRAF mutations, this has allowed sporadic MSI+ cases to be identified and
excluded from further follow-up as possible HNPCC. Additionally, this has
allowed the phenotypic properties of BRAF-mutant tumours from younger
patients (<60 years) to be compared to those of older patients (≥60 years)
derived from the consecutive tumour series described in Chapter 3.
48 4.2 BRAF mutations and clinicopathological features of tumours in
patients aged <60 years
Of the 780 cases assessed as part of the population-based screening program
for HNPCC in younger patients, 54 (6.9%) cases harbored a BRAF mutation
(Table 4.1). The mean age of patients with and without BRAF mutation did not
differ (50.5 yrs and 51.0 yrs, respectively), nor was there a significant difference
in the frequency of BRAF mutation between males and females (8.2% and
6.1%, respectively).
Tumours originating in the proximal side of the large bowel showed a much
higher frequency of BRAF mutation compared to distal tumours (P<0.0001).
BRAF mutation was also strongly associated with poor histological grade,
advanced tumour stage and mucinous phenotype. Higher frequencies of BRAF
mutation were also observed in tumours with infiltrating lymphocytes and the
MSI+ phenotype, however these associations did not reach significance.
Of the 780 tumour samples, 55 tumour samples were found to be MSI+ and
thus potentially from HNPCC-affected individuals. BRAF mutation was found in
5 of these cases (9.1%), allowing them to be excluded from further patient
follow-up as possible HNPCC.
49 Table 4.1 Associations between BRAF mutation and clinicopathological
features of colorectal cancer in patients aged <60 years
Feature (n)
BRAF wild-type (%)
BRAF mutation (%)
P
Total (780) 726 (93.1) 54 (6.9)
Age (years) 50.97 50.46 0.68
Gender Males (462) 434 (93.9) 28 (6.1) Females (318) 292 (91.8) 26 (8.2) 0.25
Tumour site Proximal colon (222) 189 (85.1) 33 (14.9) Distal colon (544) 524 (96.3) 20 (3.7) <0.0001
Histological grade Well/moderate (597) 570 (95.5) 27 (4.5) Poor (99) 78 (78.8) 21 (21.2) <0.0001
Tumour stage Stage I (127) 124 (97.6) 3 (2.4) Stage II (175) 166 (94.9) 9 (5.1) Stage III (221) 205 (92.8) 16 (7.2) Stage IV (78) 63 (80.8) 15 (19.2) <0.001
Infiltrating lymphocytes Negative (748) 697 (93.2) 51 (6.8) Positive (32) 29 (90.6) 3 (9.4) 0.21
Mucinous Negative (628) 594 (94.6) 34 (5.4) Positive (152) 132 (86.8) 20 (13.2) 0.0007
MSI Negative (722) 674 (93.4) 48 (6.6) Positive (55) 50 (90.9) 5 (9.1) 0.15
50 4.3 Clinicopathological characteristics of tumours with BRAF
mutations: comparison between young and old colorectal cancer
patients.
The clinicopathological features of tumours with BRAF mutation from young
patients (<60 yrs) in the HNPCC screening program were compared to those
from older patients (age ≥60 yrs) derived from the consecutive tumour series
described in Chapter 3 (Table 4.2). The frequency of BRAF mutation was higher
in older patients, although this did not reach significance (10% vs. 6.9%;
P=0.16).
In both age groups, BRAF mutations were approximately 3-6-fold more frequent
in proximal, poorly differentiated and mucinous tumours. The associations with
infiltrating lymphocytes and MSI+ were however much stronger in older
patients. Interestingly, BRAF mutations were more frequent in node-positive
tumours in younger patients but in node negative tumours in older patients,
although neither association reached statistical significance.
The difference in BRAF mutation frequency between MSI+ tumours from young
and old patients (9.1% vs 50%, respectively) was highly significant (P<0.0001).
These results are shown in bold in Table 4.2. Similar differences in the
frequency of gene promoter methylation have also been reported recently by
our laboratory (Iacopetta et al, Gut, in press 2006).
51 Table 4.2 Clinicopathological characteristics and MSI status of tumours with
BRAF mutations in young (<60 yrs) and old (≥60 yrs) colorectal cancer patients
BRAF mutation (%)
Feature (N1,N2) <60 yrs old ≥ 60 yrs old
Total (780, 180) 54 (6.9) 18 (10) Gender
Males (462, 104) 28 (6.1) 8 (7.7) Females (318, 76) 26 (8.2) 10 (13.2)
P=0.25 P=0.23 Tumour Site
Proximal colon (222, 81) 33 (14.9) 12 (14.8) Distal colon (544, 87) 20 (3.7) 3 (3.4)
P<0.0001 P=0.008
Histological grade Well/moderate (597, 111) 27 (4.5) 6 (5.4) Poor (99, 21) 21 (21.2) 7 (33.3)
P<0.0001 P<0.0001
Nodal involvement Negative (302, 107) 12 (4) 12 (11.2) Positive (299, 48) 31 (10.4) 3 (6.3)
P=0.002 P=0.16
Infiltrating lymphocytes Negative (748, 153) 51 (6.8) 9 (5.9) Positive (32, 16) 3 (9.4) 7(43.8)
P=0.21 P<0.0001
Mucinous Negative (628, 126) 34 (5.4) 9 (7.1) Positive (152, 21) 20 (13.2) 6 (28.6)
P=0.16 P=0.003
MSI Negative (722, 156) 48 (6.6) 6 (3.8) Positive (55, 24) 5 (9.1) 12 (50)
P=0.15 P<0.0001
N1: patients aged <60 yrs
N2: patients aged ≥60 yrs
52 4.4 Discussion
In the previous chapter, study of a consecutive colorectal tumour series found
that BRAF mutations identified a subgroup with distinctive phenotypic properties
independently of MSI or CIMP status (Li et al., 2006; Chapter 3). In this chapter,
BRAF mutations were investigated in a relatively young patient cohort in the
context of population-based HNPCC screening. This study also allowed us to
determine if patient age affected the associations between BRAF mutation and
the clinicopathological and molecular features observed in Chapter 3, in
particular with MSI+ status.
The frequency of BRAF mutation in tumours from <60 yr old patients was
slightly lower than that of older patients (6.9 vs 10%), but this did not reach
significance (Table 4.1). The strong associations with proximal site, poor
histological grade and mucinous appearance (Table 4.1) reflect those observed
for the consecutive series (Table 3.1). Although a progressive increase in the
frequency of BRAF mutation with advancing stage was observed in younger
patients (Table 4.1), a trend for inverse association with nodal involvement was
seen in older patients (Table 4.2). The reasons for this are unclear, but it
suggests that BRAF mutations are likely to have prognostic significance in
younger patients.
BRAF mutations were strongly associated with the presence of infiltrating
lymphocytes (TILS) in older patients but not in younger patients (Table 4.2).
This observation suggests that age can influence on the molecular phenotype of
tumours with TILS. In addition to this morphological feature, the frequency of
BRAF mutation was also much higher in MSI+ tumours from older patients
53 (Table 4.2). It is unclear why BRAF mutations are quite frequent in MSI+ and
TILS positive tumours from older patients (50 and 44%, respectively), but
relatively infrequent in the same tumours from younger patients (9% each).
Recent work also shows that gene methylation frequencies are markedly higher
in MSI+ tumours from older compared to younger patients (Iacopetta et al,
2006, in press). This work suggests that BRAF mutation is linked to aberrant
gene promoter methylation rather than to the MSI+ phenotype.
The V600E BRAF mutation has already been proposed as a convenient
discriminator between MSI+ tumours from HNPCC or sporadic origin (Deng et
al., 2004; Domingo et al., 2004b). Based on these earlier findings, the relatively
small proportion (9%) of MSI+ cases with BRAF mutation found in young CRC
patients (<60 years) could therefore be confidently excluded from further follow-
up as possible HNPCC individuals. Given the prior finding that use of a 60 yr
age limit for HNPCC screening already greatly reduces the number of sporadic
MSI+ cases (Chai et al., 2004), it is reasonable to propose routine screening for
BRAF mutation in younger patients in order to further refine the target group for
HNPCC genetic testing. Although BRAF screening excludes only about 10% of
suspicious cases from the need for further follow-up, the relative ease of this
assay is likely to justify its routine use.
In summary, the current study found that:
1. Patient age influences the associations between BRAF mutation and
some clinicopathological features of colorectal tumours.
2. Patient age is a major determinant of the frequency of BRAF mutation
observed in MSI+ CRC tumours.
54
3. Routine screening for BRAF mutation in MSI+ tumours from CRC
patients aged <60 yrs allows further refinement of population-based
screening for HNPCC.
55 Chapter 5 General Discussion
5.1 BRAF mutations and phenotypic properties of CRC
Somatic mutations in the BRAF oncogene occur in approximately 7-12% of
CRC (Davies et al., 2002; Rajagopalan et al., 2002; Singer et al., 2003; Brose et
al., 2002; Cohen et al., 2003; Garnett & Marais, 2004). The large majority of
BRAF mutations comprise a T1796A substitution in codon 600 (V600E; Davies
et al., 2002). The V600E mutation greatly increases the activity of the
RAF/MEK/ERK pathway resulting in stimulation of cell growth, suppression of
apoptosis and therefore contribution towards colorectal tumourigenesis (Ikehara
et al., 2005).
During the development of CRC, the gene that is often mutated in the
RAS/RAF/MEK/ERK signaling pathway is KRAS (Ikehara et al., 2005). Several
studies have investigated for simultaneous occurrence of BRAF and KRAS
mutations in colorectal tumours (Davies et al., 2002; Rajagopalan et al., 2002;
Yuen et al., 2002; Miyaki et al., 2004). As found in the present study (Table 3.2),
no tumours with concomitant mutations of the two genes were found. The
current work therefore supports the notion that BRAF and KRAS mutation are
functionally equivalent in their tumourigenic effects for CRC (Rajagopalan et al.,
2002).
In agreement with other studies, CRC with BRAF mutations exhibit many of the
characteristic features of sporadic tumours with the MSI-H phenotype such as
location in the proximal colon, poor differentiation, mucinous histology and
infiltrating lymphocytes (Jass et al., 2002a; Hawkins et al., 2002a; van
Rijnsoever et al., 2002). Further stratification according to MSI and CIMP
56 phenotypes has revealed for the first time that some of these distinctive
clinicopathological features occur independently of both MSI and CIMP status
(Tables 3.3 and 3.4). Interestingly, the associations between BRAF mutation
and the features of nodal involvement, TILS and MSI were strongly influenced
by patient age (Table 4.2). It is not clear what the driving force is for these
phenotypic associations, although one strong possibility is the age-related
increase in aberrant DNA methylation. In agreement with previous work
(Rajagopalan et al., 2002), BRAF mutations were strongly linked to MSI
tumours that have aberrant hMLH1 gene promoter methylation.
A recent large, population-based study reported prognostic significance for
BRAF mutations in MSI-, but not MSI+, CRC (Samowitz et al., 2005). The
frequencies of BRAF mutations observed in MSI- (4%) and MSI+ (39%)
tumours in the current study compare favourably with those of the Samowitz
study (5% and 52%, respectively). The prognostic value of BRAF mutations
could not be determined in the present study however due to a lack of
information on patient follow-up. Prospective studies that also take into account
MSI and adjuvant treatment status are required to evaluate the prognostic value
of BRAF mutation in CRC patients.
In summary, the present study has elucidated the clinical, pathological and
molecular features of CRC with BRAF mutations. These features appear to be
independent of both the MSI and CIMP phenotypes. Further studies are
required to determine the prognostic significance of BRAF mutations and their
predictive value for response to adjuvant chemotherapy.
57 5.2 BRAF mutations and screening for HNPCC
Although there is increasing evidence that routine colonoscopic screening can
improve the survival rate of HNPCC patients, the mortality rate from this
disease remains high (Jarvinen et al., 2000; Lynch et al., 2003; Aaltonen et al.,
1998). This is due to the fact that tumours from HNPCC patients lack
characteristic clinical and pathological features that would help to distinguish
them from sporadic cases. The identification of all individuals in the population
with pathogenic germline mutations in mismatch repair gene would enable them
to undergo regular surveillance and hence improve their survival.
It is known that MMR deficiency is the molecular basis for tumour development
in HNPCC individuals (Luokola et al., 2001; Peltomaki, 2003). The presence of
MSI and the absence of MMR protein expression are the hallmarks of defective
MMR. Hence, molecular screening for MSI+ and complementary IHC-based
screening for loss of MMR protein expression have been recommended as a
first-line testing strategy for suspected HNPCC cases (Muller et al., 2004;
Liljegren et al., 2004; Shia et al., 2005). However, the specificity of these
approaches is greatly comprised by the fact that defective MMR also occurs in
sporadic CRC (Baudhuin et al., 2005), particularly in older patients. Although
the finding of DNA methylation in the hMLH1 MMR gene can be used to
exclude sporadic cases, the assay is technically challenging and not widely
available for routine clinical use.
A recent observation made with BRAF mutations in CRC has been of enormous
significance in helping to distinguish sporadic from HNPCC MSI+ tumours.
BRAF mutations are often found in sporadic MSI+ tumours, but rarely in
58 HNPCC MSI+ tumours (Deng et al., 2004; Wang et al., 2003; Koinuma et al.,
2004; Kambara et al., 2004; Nagasaka et al., 2004; Domingo et al., 2004a;
Miyaki et al., 2004; McGivern et al., 2004). This finding has been employed in
the current study to investigate the feasibility of routine BRAF mutation
screening in relatively young (<60 yr old) CRC patients with a view to refining
the target group for HNPCC genetic testing. The major advantage of studying
this age group is that it enriches for possible HNPCC cases while at the same
time limiting the detection of MSI+ cases that are due to hMLH1 methylation.
Only 9% (5/55) of the MSI+ cases detected in this population-based study of
<60 yr old patients were found to have a BRAF mutation. These could then be
confidently excluded from further follow-up as possible HNPCC individuals,
since tumours from this familial cancer syndrome have never been shown to
contain a BRAF mutation.
When the patient data was reviewed, these five MSI+ patients with BRAF
mutation were aged between 54 and 59 at the time of diagnosis. This suggests
that BRAF mutation is very unlikely to occur in young (<50 yr old) MSI+ patients
and hence it will probably not be worthwhile to test such young patients. The
current work does indicate however that routine BRAF mutation testing can be
proposed for MSI+ patients aged >50 years where it can avoid unnecessary
follow-up of sporadic cases.
Although the overall percentage of MSI+ cases with BRAF mutation is relatively
small in the younger (<60 yr old) patient cohort studied here, the analysis is
reliable and easy to perform at the same time as MSI testing using PCR-based
techniques. Previous work has also shown that BRAF hotspot mutation analysis
59 is a low-cost, effective strategy for simplifying HNPCC genetic testing, further
justifying its incorporation for routine screening (Domingo et al., 2004b).
5.3 Limitations of this study
The major limitations of the current study into the investigation of BRAF
mutations in CRC can be summarized as follows:
1. The statistical power of the stratification analyses for BRAF mutation
presented in Chapter 3 was limited by the small number of MSI+ and
CIMP+ cases. Study of a larger series (>500 cases) will be required to
obtain more accurate information on the features of tumours with BRAF
mutations in MSI-/MSI+ and CIMP-/CIMP+ subgroups.
2. The prognostic and predictive values of BRAF mutations could not be
evaluated in this study because of the lack of survival and adjuvant
treatment information on CRC patients.
3. DNA sequencing was not performed for all BRAF mutation cases and
thus it could not be determined if all mutations were T1796A substitution.
However the identical SSCP banding patterns that were observed
strongly indicates they were all the same hotspot mutation. Other
oncogenic BRAF somatic mutations found in exon 11 were not screened.
However, these BRAF mutants were reliant on RAS for activation,
suggesting they may have functionally different properties from CRC
tumorigenesis.
60
4. The Results in Chapter 4 on BRAF mutations and population-based
screening for HNPCC were obtained on a series of retrospective patients
in which there was no information available on family history of cancer.
All MSI+ cases, with the exception of the 5 cases with concurrent BRAF
mutation, are now being actively investigated for evidence of family
history and germline MMR mutations.
5. IHC data for expression of the MMR proteins (hMLH1, MSH2 and
hMSH6) in the MSI+/BRAF wildtype cases presented in Chapter 4 was
not yet available. This information will confirm the MSI result and may
also indicate which of the MMR genes contains a germline mutation.
5.4 Conclusions
The major conclusions that can be drawn from this study are:
1. BRAF mutations occur most frequently in CRC with proximal location in
the colon, poor histological grade, mucinous appearance and with large
numbers of infiltrating lymphocytes. Most of these associations appear to
be independent of MSI and CIMP status, although this should be
confirmed in a larger tumour series.
2. Confirming other studies, KRAS mutations were mutually exclusive with
the presence of BRAF mutations, suggesting functional equivalence for
these alterations in colorectal tumourigenesis.
61
3. Patient age is a major determinant of the frequency of BRAF mutation
observed in MSI+ CRC tumours, with older patients showing much
higher frequencies of mutation.
4. The frequency of BRAF mutation in MSI+ tumours from young patients (<
60 yrs) is very low at only 9%. No BRAF mutations were found in MSI+
patients aged <50 yrs, suggesting this test may not be required for young
CRC cases.
5. Routine screening for BRAF mutation in MSI+ tumours from CRC
patients aged > 50 years is technically feasible and should assist with the
population-based screening for HNPCC.
5.5 Future work
1. The prognostic and predictive significance of BRAF mutations needs to
be evaluated in prospective studies to determine whether this molecular
marker can be used to helping to guide the use of adjuvant
chemotherapy.
2. High-throughput assays such as denaturing high performance liquid
chromatography (DHPLC) that require minimal post-PCR manipulation
(Ellis et al., 2000) need to be developed and validated for possible use in
the routine clinical setting.
62
3. Young MSI+ patients (<60 yrs) with wildtype BRAF identified in this study
have been referred to Genetic Services Western Australia for further
follow up, including ascertainment of detailed family history and germline
testing for consenting patients.
4. Mutant BRAF could be exploited as a possible target for small molecule
inhibitory drugs.
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