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.

63 REFERENCES

Aaltonen LA, Salovaara R, Kristo P, et al. (1998) Incidence of hereditary

nonpolyposis colorectal cancer and the feasibility of molecular screening for the

disease. N Engl J Med 338(21): 1481-1487

Aarnio M, Sankila R, Pukkala E, et al. (1999) Cancer risk in mutation carriers of

DNA-mismatch-repair genes. Int. J. Cancer 81: 214-218

Baudhuin LM, Lawrence JB, Leontovich O and Thibodeau SN (2005) Use of

microsatellite instability and immunohistochemistry testing for the identification

of individuals at risk for Lynch syndrome. Familiar Cancer 4: 255-265

Boland CR, Thibodeau SN, Hamilton SR, et al. (1998) A National Cancer

Institute Workshop on Microsatellite Instability for cancer detection and familial

predisposition: development of international criteria for the determination of

microsatellite instability in colorectal cancer. Cancer Res 58(22): 5248-5257

Brose MS, Volpe P, Feldman M, et al. (2002) BRAF and RAS mutations in

human lung cancer and melanoma. Cancer Res 62:6997-7000

Burt R and Neklason D (2005) Genetic testing for inherited colon cancer.

Gastroenterology 128:1696-1716

Chai SM, Zeps N, Shearwood AM, Grieu F, Charles A, Harvey J, Goldblatt J,

Joseph D, Iacopetta B (2004) Screening for defective DNA mismatch repair in

64 stage II and III colorectal cancer patients. Clin Gastroenterol Hepatol

2(11):1017-1025

Chan TL, Zhao W, Leung SY and Yuen ST (2003) BRAF and KRAS mutations

in colorectal hyperplastic polyps and serrated adenomas. Cancer Research 63:

4878-4881

Cohen Y, Xing M, Mambo E, et al. (2003) BRAF mutation in papillary thyroid

carcinoma. J Natl Cancer Inst 95: 625-627

Davies H, Bignell GR, Cox C, et al. (2002) Mutations of the BRAF gene in

human cancer. Nature 417(6892): 949-954

Debniak T, Kurzawski G, Gorski B, et al. (2000) Value of pedigree/clinical data,

immunohistochemistry and microsatellite instability analyses in reducing the

cost of determining hMLH1 and hMSH2 gene mutations in patients with

colorectal cancer. Eur J Cancer 36: 49-54

Deng G, Bell I, Crawley S, et al. (2004) BRAF mutation is frequently present in

sporadic colorectal cancer with methylated hMLH1, but not in hereditary non-

polyposis colorectal cancer. Clinical Cancer Research 10: 191-195

Dibb NJ, Dilworth SM and Mol CD (2004) Switching on kinases: oncogenic

activation of BRAF and the PDGFR family. Nat Rev Cancer 4(9): 718-727

65 Domingo E, Espin E, Armengol M, et al. (2004a) Activated BRAF targets

proximal colon tumours with mismatch repair deficiency and MLH1 inactivation.

Genes, Chromosomes & Cancer 39: 138-142

Domingo E, Laiho P, Ollikainen M, et al. (2004b) BRAF screening as a low-cost

effective strategy for simplifying HNPCC genetic testing. J Med Genet 41: 664-

668

Domingo E, Niessen RC, Oliveira C, et al. (2005) BRAF-V600E is not involved

in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and

MSH2 genes. Oncogene 24(24): 3995-3998

Dunlop MG, Farrington SM, Carothers AD, et al. (1997) Cancer risk associated

with germline DNA mismatch repair gene mutations. Human Molecular Genetics

6(1): 105-110

Ellis LA, Taylor CF and Taylor GR (2000) A comparison of fluorescent SSCP

and denaturing HPLC for high throughput mutation scanning. Human Mutation

15:556-564

Esteller M, Corn PG, Baylin SB and Herman JG (2001) A gene

hypermethylation profile of human cancer. Cancer Res 61(8): 3225-3229

Garnett MJ and Marais R (2004) Guilty as charged: B-RAF is a human

oncogene. Cancer cell 6: 313-319

66 Grieu F, Joseph D, Norman P and Iacopetta B (2004) Development of a rapid

genotyping method for single nucleotide polymorphisms and its application in

cancer studies. Oncology Reports 11(2): 501-504

Halvarsson B, Lindblom A, Rambech E, et al. (2004) Microsatellite instability

analysis and/or immunostaining for the diagnosis of hereditary nonpolyposis

colorectal cancer? Virchows Arch 444:135-141

Hawkins NJ and Ward RL (2001) Sporadic colorectal cancers with microsatellite

instability and their possible origin in hyperplastic polyps and serrated

adenomas. J Natl Cancer Inst 93:1307-1313

Hawkins N et al. (2002a) CpG island methylation in sporadic colorectal cancers

and its relationship to microsatellite instability. Gastroenterology 122:1376-1387

Hawkins NJ, Bariol C and Ward RL (2002b) The serrated neoplasia pathway.

Pathology 34:548-555

Herman JG, Umar A, Polyak K, et al., (1998) Incidence and functional

consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.

Proc Natl Acad Sci USA. 95:6870–6875.

Iacopetta B and Hamelin R (1998) Rapid and non-isotopic SSCP-based

analysis of the BAT-26 mononucleotide repeat for identification of the replication

error phenotype in human cancers. Hum Mutat 12(5): 355-360

67 Ikehara N, Semba S, Sakashita M, et al. (2005) BRAF mutation associated with

dysregulation of apoptosis in human colorectal neoplasms. Int. J. Cancer 115:

943-950

Ikenoue T, Hikiba Y, Kanai F, et al. (2003) Functional analysis of mutations

within the kinase activation segment of B-Raf in human colorectal tumors.

Cancer Res. 63(23): 8132-8137

Jacob S and Praz F. (2002) DNA mismatch repair defects: role in colorectal

carcinogenesis. Biochimie 84: 27-47

Jarvinen J et al (2000) Controlled 15-year trial on screening for colorectal

cancer in families with hereditary nonpolyposis colorectal cancer.

Gastroenterology 118: 829-834

Jarvinen HJ, Mecklin JP and Sistonen P (1995) Screening reduces colorectal

cancer rate in families with hereditary nonpolyposis colorectal cancer.

Gastroenterology 108:1405-1411

Jass JR (2004) HNPCC and sporadic MSI-H colorectal cancer: a review of the

morphological similarities and differences. Fam Cancer 3(2):93-100

Jass JR, Walsh MD, Barker M, et al. (2002a) Distinction between familial and

sporadic forms of colorectal cancer showing DNA microsatellite instability. Eur J

Cancer 38: 858-866

68 Jass JR, Whitehall VL, Young J, Leggett BA (2002b) Emerging concepts in

colorectal neoplasia. Gastroenterology 123:862-876

Kakar S, Burgart LJ, Thibodeau SN, et al. (2003) Frequency of loss of hMLH1

expression in colorectal carcinoma increases with advancing age. Cancer 97(6):

1421-1427

Kambara T, Simms LA, Whitehall VLJ et al. (2004) BRAF mutation is

associated with DNA methylation in serrated polyps and cancers of the

colorectum. Gut 53: 1137-1144

Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Watanabe G, Iacopetta B

(2003) The folate pool in colorectal cancers is associated with DNA

hypermethylation and with a polymorphism in methylenetetrahydrofolate

reductase. Clin Cancer Res 9:5860-5865.

Kinzler KW and Vogelstein B. (1996) Lessons from hereditary colorectal cancer.

Cell 87:159-170

Koinuma K, Shitoh K, Miyakura Y, et al. (2004) Mutations of BRAF are

associated with extensive hMLH1 promoter methylation in sporadic colorectal

carcinomas. Int J Cancer 108: 237-242

Kolch W. (2000) Meaningful relationships: the regulation of the

Ras/Raf/MEK/ERK pathway by protein interactions. Biochem. J. 351:289-305

69 Li WQ, Kawakami K, Ruszkiewicz A, et al. (2006) BRAF mutations are

associated with distinctive clinical, pathological and molecular features of

colorectal cancer independently of microsatellite instability status. Mol Cancer

(in press)

Liljegren A, Olsson L and Lindblom A. (2004) MSI test to distinguish between

HNPCC and other predisposing syndromes – of value in tailored surveillance.

Disease Markers 20: 259-267

Lin KM, Shashidharan M, Thorson AG, et al. (1998) Cumulative incidence of

colorectal and extracolonic cancers in MLH1 and MSH2 mutation carriers of

hereditary nonpolyposis colorectal cancer. J Gastrointest Surg 2(1): 67-71

Lindor NM, Gurgart LJ, Leontovich O, et al. (2002) Immunhistochemistry versus

microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol

20: 1043-1048

Lindblom A. (2001) Different mechanisms in the tumorigenesis of proximal and

distal colon cancers. Curr Opin Oncol. 13(1): 63-69

Loukola A et al. (2001) Microsatellite marker analysis in screening for hereditary

non-polyposis colorectal cancer (HNPCC). Cancer Research 61: 4545-4549

Lynch HT & Chapelle A (1999) Genetic susceptibility to non-polyposis colorectal

cancer. J Med Genet 36: 801-818

70 Lynch HT & Chapelle A (2003) Hereditary colorectal cancer. N Engl J Med 348:

919-932

Lynch HT, Riley BD, Weismann S, et al. (2003) Hereditary non-polyposis

colorectal carcinoma (HNPCC) and HNPCC-like families: Problems in

diagnosis, surveillance, and management. Cancer 100(1): 53-64

Marra G and Boland CR. (1995) Hereditary nonpolyposis colorectal cancer: the

syndrome, the genes, and historical perspective. J Natl Cancer Inst 87(15):

1114-1125

McGivern A, Wynter CVA, Whitehall VLJ, et al. (2004) Promoter

hypermethylation frequency and BRAF mutations distinguish hereditary

nonpolyposis colon cancer from sporadic MSI-H colon cancer. Familial Cancer

3: 101-107

Mercer KE and Pritchard CA. (2003) Raf proteins and cancer: B-Raf is identified

as a mutational target. Biochim Biophys Acta. 1653(1): 25-40

Mitchell RJ, Brewster D, Campbell H, et al. (2004) Accuracy of reporting of

family history of colorectal cancer. Gut 53: 291-295

Miyaki M, Iijima T, Yamaguchi T, et al. (2004) Both BRAF and KRAS mutations

are rare in colorectal carcinomas from patients with hereditary nonpolyposis

colorectal cancer. Cancer Letters 211: 105-109

71 Muller A, Edmonston TN, Dietmaier W, et al. (2004) MSI-testing in hereditary

non-polyposis colorectal carcinoma (HNPCC). Disease Markers 20: 225-236

Nagasaka T, Sasamoto H, Notohara K, et al. (2004) Colorectal cancer with

mutations in BRAF, KRAS and wild type with respect to both oncogenes

showing different patterns of DNA methylation. Journal of Clinical Oncology

22(22): 4584-4594

Nakagawa H, Nuovo GJ, Zervos EE, et al. (2001) Age-related hypermethylation

of the 5’ region of MLH1 in normal colonic mucosa is associated with

microsatellite-unstable colorectal cancer development. Cancer Research 61:

6991-1995

Niessen RC, Berends MJW, Wu Y, et al. (2006) Identification of mismatch

repair gene mutations in young colorectal cancer patients and patients with

multiple HNPCC-associated tumours. Gut (in press)

Oliveira C, Pinto M, Duval A, et al. (2003) BRAF mutations characterize colon

but not gastric cancer with mismatch repair deficiency. Oncogene 22: 9192-

9196

Park SJ et al. (2003) Frequent CpG island methylation in serrated adenomas of

the colorectum. Am J Pathol 162: 815-822

Peltomaki P (2003) Role of DNA mismatch repair defects in the pathogenesis of

human cancer. J Clin Oncol 21(6): 1174-1179

72 Peyssonnaux C. and Eychene A. (2001) The Raf/MEK/ERK pathway: new

concepts of activation. Biology of the Cell 93(1-2): 53-62

Pollock PM and Meltzer PS (2002b) A genome-based strategy uncovers

frequent BRAF mutations in melanoma. Cancer Cell 2(1): 5-7

Pollock PM and Meltzer PS (2002a) Lucky draw in the gene raffle. Nature 417:

906-907

Rajagopalan H, Bardelli A, Lengauer C, et al. (2002) Raf/Ras oncogenes and

mismatch-repair status. Nature 418: 934

Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. (1997) A National Cancer

Institute workshop on hereditary nonpolyposis colorectal cancer syndrome:

meeting highlights and Bethesda guidelines. Journal of the National cancer

Institute 89(23): 1758-1762

Ruszkiewicz A, Bennett G, Moore J, et al. (2002) Correlation of mismatch repair

genes Immunohistochemistry and microsatellite instability status in HNPCC-

associated tumours. Pathology 34: 541-547

Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA,

Wolff RK, Slattery ML (2005) Poor survival associated with the BRAF V600E

mutation in microsatellite-stable colon cancers. Cancer Res 65:6063-6069.

73 Salovaara R, Loukola, Kristo P, et al. (2000) Population-based molecular

detection of hereditary nonpolyposis colorectal cancer. Journal of Clinical

Oncology 18(11): 2193-2200

Shia Y, Klimstra DS, Nafa K, et al. (2005) Value of immunohistochemical

detection of DNA mismatch repair proteins in predicting germline mutation in

hereditary colorectal neoplasms. Am J Surg Pathol 29(1): 96-104

Singer G, Oldt R 3rd, Cohen Y, et al. (2003) Mutations in BRAF and KRAS

characterize the development of low-grade ovarian seous carcinoma. J Natl

Cancer Inst 95: 484-486

Smith G, Carey FA, Beattie J, et al. (2002) Mutations in APC, Kirsten-ras, and

p53-alternative genetic pathways to colorectal cancer. Proc Natl Acad Sci USA

99:9433-9438

Soong R and Iacopetta B (1997) A rapid and nonisotpoic method for the

screening and sequencing of p53 gene mutations in formalin-fixed, paraffin-

embedded tumours. Mod Pathol 10(3): 252-258

Terdiman J et al. (2002) Hereditary nonpolyposis colorectal cancer in young

colorectal cancer patients: high-risk clinic versus population-based registry.

Gastroenterology 122: 940-947

Thibodeau SN, Bren G and Schaid D (1993) Microsatellite instability in cancer

of the proximal colon. Science 260:816-819

74 Toyota M, Ahuja N, Ohe-Toyota M, et al. (1999) CpG island methylator

phenotype in colorectal cancer. Proc Natl Acad Sci 96:8681-8686

Toyota M, Ohe-Toyota M, Ahuja N and Issa JJ (2000) Distinct genetic profiles in

colorectal tumors with or without the CpG island methylator phenotype. PNAS

97(2): 710-715

Umar A et al. (2004) Revised Bethesda guidelines for hereditary non-polyposis

colorectal cancer (Lynch Syndrome) and microsatellite instability. Journal of the

National Cancer Institute 96(4): 261-268

Vasen HF, Mecklin JP, Khan PM, et al. (1991) The International Collabortive

Group on hereditary nonpolyposis colorectal cancer (ICG-HNPCC). Dis Colon

Rectum 34: 424-425

Vasen HF, Mecklin JP, Watson P, et al (1993) Surveillance in hereditary

nonpolyposis colorectal cancer: an international cooperative study of 165

families. The International Collaborative Group on HNPCC. Dis Colon Rectum.

36(1):1-4.

Vasen HF, Watson P, Mecklin JP, et al. (1999) New clinical criteria for

hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed

by the International Collaborative group on HNPCC. Gastroenterology 116:

1453-1456

75 van Rijnsoever M, Grieu F, Elsaleh H, Joseph D, Iacopetta B (2002)

Characterisation of colorectal cancers showing hypermethylation at multiple

CpG islands. Gut 51: 97-802.

Vogelstein B, Fearon ER, Hamilton SR, et al. (1998) Genetic alterations during

colorectal-tumour development. N Engl J Med 319(9): 525-532

Wan PT, Garnett MJ, Roe SM, et al. (2004) Mechanism of activation of the

RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116(6):

855-867

Wang C, van Rijnsoever M, Grieu F, Bydder S, Elsaleh H, Joseph D, Harvey J,

Iacopetta B. (2003a) Prognostic significance of microsatellite instability and Ki-

ras mutation type in stage II colorectal cancer. Oncology 64(3):259-265.

Wang L, Cunningham JM, Winters JL, et al. (2003b) BRAF mutations in colon

cancer are not likely attributable to defective DNA mismatch repair. Cancer

Research 63: 5209-5212

Watson P and Lynch HT (2001) Cancer risk in mismatch repair gene mutation

carriers. Fam Cancer 1: 57-60

Whitehall VL, Wynter CV, Walsh MD, et al. (2002) Morphological and molecular

heterogeneity within nonmicrosatellite instability-high colorectal cancer. Cancer

Res 62: 6011-6014

76 Wolf B et al. (2005) Efficiency of the revised Bethesda guidelines (2003) for the

detection of mutations in mismatch repair genes in Austrian HNPCC patients.

International Journal of Cancer 118(6): 1465-1470

Yang S, Farraye FA, Mack C, et al. (2004) BRAF and KRAS mutations in

hyperplastic polyps and serrated adenomas of the colorectum. Am J Surg

Pathol 28(11): 1452-1459

Yuen ST, Davies H, Chan TL, et al. (2002) Similarity of the phenotypic patterns

associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer

Research 62: 6451-6455