Colorectal Cancer Microsatellite lnstability
by
Robert Gryfe
A thesis submitted in confomity with the requirements
for the degree of Doctor of Philosophy.
fnstitute of Medical Science,
University of Toronto
@Copyright by Robert Gryfe 2001.
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Colorectal Cancer Microsatellite lnstability
by
Robert Gryfe
A thesis submitted in conformity with the requirements for the degree of Doctor of
Philosophy, hstitute of Medical Science, University of Toronto, 200 1.
Abstract
Background: Colorectal cancer is the third most common cancer in both sexes and the
second leading cause of cancer-related deaths in Canada. Microsatellite DNA sequences.
common throughout the human genome, are subject to high rates of mutation in
approximately 15% of colorectal cancers due to deficiency in DNA mismatch repair.
Mutations of the Adenornatous Putyposis Coli (APC) gene are thought to initiate colorectal
neoplasia The APC 11307K pol ymorphism, common in the Ashkenazi Jewish population,
contains an (A)8 microsatellite repeat. The dinical relevance of mismatch repak deficiency
and microsatellite instability in colorectai cancer is incompletely unde r s t a as is the cancer
risk of the APC 11307K polymorphism. Methods: Tumors From a population-based series of
607 young patients with colorectai cancer fiom were screened for microsateLite instability
and 476 Ashkenazi Jewish patients with colorectai neoplasms were screened for the APC
113MK polymorphism. Results: High-fkquency microsatellite instability @HI-El) was
observed in 17% of the cancm €rom the population-based series. Patients with MSI-H
colorectal cancers were found to have significantly better survîvai in multivariate andysis
and MSI-H cancers were l e s likely to metastasue after controlling for the extent of -or
invasion. The APC Il3MK poIymorphism was present in more than 10% of Ashkenazï
Jewish patients with colorectal neoplasia The (Ah microsatellite sequence of the APC
II307K polymorphism was subject to a very high rate of somatic mutation by a mechanism
apparendy not related to mismatch repair deficiency. An additional polymorphism, APC
EHI 7Q, was identified in this series, but did not appear to be associated with a significant
risk for colorectal neoplasia Conclusions: MSI-H is relatively cornmon in colorectal
cancers and defines a distinct disease subtype with an improved survivai and a demased risk
of metastasis. The APC 11307K polymorphism is a risk factor for colorectal neoplasia due to
somatic mutation of the (Ah polymorphic microsateIlite sequence.
Acknowledgements
1 would iike to acknowledge and thank several people for their guidance, support and
contribution to the work presented in this thesis. Many thanks to the members of the
Gallinger and Redston laboratories, including Kazy Hay, Geeta Lal, CoUeen Ash, Mol1 y
Pind Laura Mirabelü-Rimdahl, Eugene Hsieh and most especiaily Heyja Kim and Nando
DiNicola for al1 their help, advice and toierance. Thanks to the numerous technicians,
graduate students, post doctoral fellows and investigators of the Samuel Lunenfeld Research
Institute who have helped me both directly and indirectly throughout my research training.
I am grateful for the efforts of Nelson Chong. Darlene Dale and Eric Holowaty of the
Ontario Cancer Registry, Susan Bondy and Marc Thenault of the Institute of Clinical
Evaluative Science, and the numerous physicians and hospital staff throughout the province
of Ontario who helped me obtain the clinical information and materiais needed for the
research contained within this thesis. I am especially indebted to the more than one thousand
individuals with colorectal cancer and adenornatous poiyps who served as the subjects for
these studies.
I wouid iike to thank Steven Nami for his excellent insights, suggestions and
participation on my thesis cornmittee and examination. Additionai th& to Joyce
Slingerland, Zane Cohen, Serge Jothy and Stan Hamilton for reviewing my thetis manuscript
and participating in my examination.
1 wish to thank Mark Redston, an individual of incredible knowledge, insight and
expertise. Mark has been a fantastic mentor, advisor. critic and Friend for which 1 am
eterndiy gratefitl.
There is insufficient space here for me to properly thank my supmisor Steve
Gailinger. During the course of my training, Steve has not ody served as an outstanding
mentor, motivator, advisor, role mode1 and human being, he has become one of my closest
fkiends. I am confident that my graduate shdies and surgical nsidency mark only the
beginning of our long and rewarding professional and personal friendship. Steve, th& you.
Finaily, I would Iike to thank my family who has supported me throughout my Iife
and in particular my lengthy pst-secondary school education. My wife and best Fnend
Elana, and children Manhew and Marley, are the glue that holcls me together. Their patience,
support, encouragement and endless love have made aIl of this possible.
1 acknowledge and th& the Surgical Scientist Program, Department of Surgery and
Division of General Surgery, University of Toronto for allowing me the time to pursue my
research shidies and thank the National Cancer Institute of Canada and the American Society
of Colon and Rectal Surgeons for their generous financial support.
Table of Contents
Abstract ii
Ac knowledgements iv
Table of Contents vi
List of Tables xi
List of Figures xii
List of Abbreviations xiii
Oissemination of thesis content xvii
Chapter One: Introduction 1
Colorectd cancer microsatellite instability 2
Background 2
ne adenorna to carcinoma sequence 2
Clinical aspects of colorectal cancer 7
Colorectal cancer presentation, treatment and prognosis 7
Clinicai screening for colorectai cancer 8
Cancer genes, gatekeepers and caretaicers 10
Gatekeeper genes 10
Caretaker gens 11
Molecular genetic pathways of coiorectal carcinogenesis: chromosomal and
microsateIlite instability 11
The chmosomai instability gatekeeper pathway 14
The M C gene and familial adenornatous polyposis 15
Other genetic targets of the gatekeeper pathway 19
The mismatch repair &€icient, microsatellite instability caretaker pathway 21
hstability of micfosateliite DNA 21
Dynamic trinucleotide expansion diseases 23
Hereditary nonpol yposis colorectal cancer 23
Microsatellite instability in colorectal cancer 25
MSI-H and mismatch repair deficiency in hereditary nonpolyposis colorectai
cancer 30
MSI-H and mismatch repair deficiency in sporadic colorectal cancer 34
Non-mismatch repair deficient causes of MSI-H colorectal cancer 36
DNA mismatch repair 36
MSI-H and rnismatch repair deficiency in extracolonic cancers 40
Somatic genetic targets of MSI-H and mismatch repair deficiency 41
Transforming growth factor B receptor II 41
APC, PCatenin and TCF-4 43
M C II307K: microsatellite instability in a caretaker gene 44
Other genetic targets of the MSI-H pathway 45
MSI-H, mismatch repair deficiency and colorectal cancer phenotype 47
The Bethesda criteria for hereditary nonpolyposis coIorectai cancer
screening 48
MSI-H colorectal cancer and patient survival 50
Thesis overview 51
Chapter Two: Tumor microsatellite instability and clinicel outcome in young
patients with colorectal cancer 53
s-ary 54
Introduction 55
Methods 57
Study population 57
Clinical database 57
DNA preparation, microsatellite testing, and analysis 59
Statistical anal ysis 60
Results 61
Clinical characteristics associated with MSI-H 61
MSI-H and standard cünical propostic factors for survival 66
Discussion 70
Chapter Three: Somatic instabiltty of the APC 11307Kallele in colorectal
ne0 plasia 73
s-=Y 74
Introduction 75
Methods 75
Tumor samples 75
Somatic mutation analysis 76
Microsatellite andysis 77
Statistical rnethods 77
Discussion 82
Chapter Four: lnherited colocectal polyposls and cancer risk of the APC
Il3Otl( polyrnorphism 88
s m a r ~ 89
Introduction 90
Methods 92
Cohort and phenotypic data 92
M C I1307K germ-line andysis 93
Statisticai methods 93
Resuits 94
Discussion 101
Chapter Fhre: The APC €13110 polymorphism does not preâispose carriers to
colorectal adenornatous or hyperplastic polyps 107
introduction 109
Cohort and phenotypic data 1 10
Genetic testing 111
Statistical methods 111
Discussion 116
Chapter Six: Cotorectal cancer microsatellite instabillty, conclusions and
future directions 119
Summary 120
The clinical phenotype of MSI-H colorectal cancer 121
APC I1307K and the risk of colorectal neoplasia 123
APC E1317Q does not predispose to colorectai neoplasia 125
List of Tables
Table 1-1:
Table 1-2:
Tabie 1-3:
Table 1 4
Table 2-1 :
Table 2-2:
Table 2-3:
Table 2-4:
Table 3-1:
Table 4 4 :
Table 4-2:
TabIe 5-1:
Classification of colorectal adenomatous polyps 4
Classification of colorectal adenocarcinorna 5
Colorectal cancer molecular pathway s 13
Microsatellite loci for evaluation of microsatellite instability in colorectal
cancer 29
Characteristics of 587 patients with colorectal cancer evduated for
microsatellite instability 64
Multivaxiate analysis of predictive factors for metastases to regional1 ymph
nodes or distant organs in 587 patients with colorectal cancer 65
Univariate analysis of predictive factors for srtnnval in 587 patients with
colorectal cancer 68
Significant predictive factors for survivai in a Cox proportional-hazards
anaiysis of 587 patients with colorectal cancer 69
Somatic mutations in APC II3O7K c d e r colorectal cancers and adenomatous
p0lYPs 79
M C 11307K carrier rates 97
APC 11307K carrier and tumor phenotype 98
M C El31 7Q carrier phenotype 114
List of Figures
Figure 1-1:
Figure 1-2:
Figure 2-1:
Figure 2-2:
Figure 3-3:
Fig~re 4-1:
Figure 4-2:
Figure 4-3:
Figure 5-1:
Figure 5-2:
The polymerase-template slippage mechanism of insertion and
deletion mutations in microsatellite DNA 22
Schematic representation of human pst-replication DNA mismatch
repaïr 39
Coiorectal cancers with MSI-H and MSS 63
Kaplan-Meier survival curves for patients with colonctal cancer,
stratified according to microsatellite status 67
Reverse primer sequence of APC 11307K h m carrier colorectal
cancers 80
BAT-26 microsatellite anal ysis of APC 11307K carriers colorectal
-
Gatekeeper inactivation in colorectal carcinogenesis 87
SSCP and reverse primer sequence analysis of M C codons 1303-
Cumulative distribution of time until colorectai nunor diagnosis for
APC Il307K carriers and non-carriers 99
APC II307K carrier rate and number of coIorectal neoplasm 100
SSCP and reverse primer sequence anaiysis of APC codons 1303-
1317 f 13
Reverseprimer sequence of APC El31 7Q from a camer colorectal
cancer 115
AGA
BAX
BUB 1
DCC
List of Ab breviations
polyadenine nucleotide repeat
attenuated aâenomatous polyposis COL
American Gastroenterology Association
Adenornatous Polyposis Coli
Apolipoprotein B
American Society of Clinical Oncology
polyadenine-thymidine nucleotide repeat
PZ-Microglobulin
Bcl2-Associated X pmtein
Breast Cancer 1
Breast Cancer 2
Budding Uninhibited by Benzimidales
polycytosine repeat
polycytosine-a&nine nucleotide repeat
poiycytosine-adenine-guanine nucleotide repeat
carcinoembryonic antigen
congenitai hypertrophy of the retind epithelium
confidence interval
Avian Myelocytomatosis Viral ûncogene Homologue
Cyclooxygenase 2
Catenin, B 1
Deleted in Colon Cancer
DUG
E1317Q
FAP
FEN1
(G).
GSK3B
GTBP
HNPCC
11307K
IGF2
insA
K-ras
LEF
LOH
MCR
MBD4
MLHl
MMP
ml
MSH2
MSI
MSI-H
MSI-L
Divergently transcribed Upstream Gene
glutaniic acid to glutamine at codon 1317
familial adenornatous polyposis
Flap Endonuclease 1
polyguanine nucleotide repeat
glycogen synthase kinase 38
G R Base Pau
hereditary nonpolyposis colorectal cancer
isoleucine to lysine polymorphism at codon 1307
Insuiin-Like Growth Factor
single adtnine nucleotide insertion
Kirsten Rat Sarcoma 2 Viral Oncogene Homologue
Lymphoidenhancer Factor
loss of heterozygosity
mutation cluster region
methyl-CpG binding protein 4
MutL homologue 2
microsatellite rnutator phenotype
Mismatch Repair Protein I
MutS homologue 2
microsatelIite instability
high-ftequency mimsateiIite instabiüty
low-fresuency microsatellite instability
xiv
MSS
Mutsût
MutSb
NOS
P53
PCNA
PCR
pmsl
po13
PPmG
PTEN
Rgs2
RB
RER+
RIZ
RPA
SE
SMAD2
S M .
SSCP
STRS
mimsateilite stable
MIHI-PMS2 heterodimer
MSm-MSH6 heterodimer
MSH2-MSH3 heterodimer
not otherwise specified
Tumor Pmtein 53
Pro tiferating Ce11 Nuclear Antigen
polyrnerase chah reaction
post-meiotic segregation
polymerase 3
Peroxisome Proliferator-Activated Receptor 6
Phosphatase and tensin homologue deleted on chromosome ten
Rostaglandin G H synthase 2
Retinoblastoma
replication emrs
Retinoblastoma Protein-Binding Zinc Finger
Repücation pmtein A
standard error
a merger of the Cumorhbditis elegans srna (srnail) gene and the Drosuphila
melmiogn~rer mothers against decapentaplegic gene
see S M . 2
single-strand confornation po1ymorphism
short tandem repeats
TCF T Ceil Factor
TGF-8 RII TGF-$ type II receptor
TIS twnor in situ
TNM xumor invasion, 1 ymph Node metastases, distant organ Met astases
USM ubiquitous somatic mutations
VWL von Hippel Lindau
Dissemination of thesis content
The work arising h m this thesis has been pubüshed as follows:
Chapter two appears with the permission of the Massachusetts Medical Society and was
published as detailed below:
Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty El, Bull SB, Redston M.
Gailinger S. Tumor microsatellite instability and clinical outcome in young patients
with colorectal cancer. NEngIJMed. 2000; 342:69-77.
Chapter three appears with permission of the Amencan Association of Cancer Research and
was published as detailed below:
Gryfe R, Di Nicola N, Gallinger S, Redston M. Somatic instability of the APC
11307K allele in colorectal neoplasia. Cancer Res. 1998; 58:4040-3.
Chapter four appears with the permission of the University of Chicago Press and was
pubüshed as dctailed below:
Gryfe Et, Di Nicola N, Lal G, Gallinger S, Redston M. Inherited colorectal polyposis
and cancer risk of the APC U307K polymorphism. Am JHum-Genet. 1999; 64378-
84.
Chapter One:
t ntroduction
Colorectal cancer microsatellite instability
Background
With 16,600 new cases and 3,400 deaths estimated for 1999, colorectal cancer is the
third most common malignancy and the second leading cause of cancer-related deaths in
Canada (1). Several predisposing risk factors have been identified for this common
malignancy including a personal or family history of colorectal cancer (2-5), adenornatous
polyp ( 6 3 or inflammatory bowel disease [ulcerative colitis, Crohn's disease; (QI.
Individuals with a family history of colorectal cancer, but no currently identifiable inherited
genetic syndrome account for 15-20% of cases (5,6). The inherited genetic syndrome of
hereditary nonpolyposis colorectal cancer (HNPCC) accounts for approximately 2% of
colorectal cancer (9,lO). Approximately 1% of colorectal cancer is attributable to the familial
adenomatous polyposis (FM) syndrome, other polyposis syndromes (Peutz-Jeghers
syndrome, JuveniIe Polyposis) and sequelae of infi ammatory bowel disease (10-13). Thus,
approximately 75% of colorectal cancers occur in individuais without obvious predisposing
risk factors and are currently classified as "sporadic" cancers (12).
The adenoma to carcinoma sequence
CIinicai and histopathologicd observations suggest that most colorectal cancers arise
h m benign adenomatous po1yps (10,13). m e n referred to as the adenoma to carcinoma
sequence, the morphologie stages of colorectal neoplastic progression that take years,
possibly decades to occur, include:
1. Abarant crwt foci (ACF), which are very early colonic lesions, visible by methylene
blue staining and microscopie examination without sectioning or histologic preparation
(14.15). Dysplastic ACF, aiso termed microadenornas, are putative adenoma and cancer
precursors. Hyperplastic ACF, akin to hyperplastic polyps, are thought to have minimal
malignant potential.
2. Adenornatous polps which are categorized on the basis of size, gross and histologic
morphology, and degree of dysplasia (Table 1-1). The malignant potential of a polyp is
directiy related to polyp size, villous component and degree of glandular atypia
[dysplasia (16,17)]. The gross morphology of an adenoma detemiines the ability to fully
mat the lesion - Bat and sessile adenornas king more difficult to fully remove
endoscopicall y.
3. Adenocarcinornas which are categorized by histologic grade (differentiation), ceIl type,
and TNM Chimor invasion, lyrnph gode metastases, distant organ metastases) pathologie
stage Fable 1-2; (1 8.19) 1. Greater TNM stage, pwrer grade, and ceH types other than
adenocarcinorna not otherwise specified (Le. signet-ring cell, undifferentiated,
mucinous), have been associated with reduced sumival (18,2020).
Table 1 -1 : Classification of coIo rectal adenornatous polyps.
A. Size
clcm
1-2 cm
2-3
>3 cm
B. Gross Morphology
Pedunculated with a stalk
Sessile without a staik
Rat not raised above the smunding epithelium
C. Histologie Morphology
Tubulas straight or branched tubules of dysplastic tissue
Villous fingerlike projections of dysplastic epithelium
Tubulovillous both epithelial tubules and villi present
D. Dysplasia
Miid nuclei are crowded, but small and limited to the cell base
Moderate nuclear enlargement and more marked pseudostratification
Severe prominent, ovoid nuclei, many located near the cell apices
Table 1-2: Classification of colorectal adenocarcinorna (1 8).
A Stage Classification
Primary Tumor Invasion
TX
TO
Tis
TI
T2
T3
T4
primary tumor invasion cannot be assessed
no evidence of primary tumor
carcinoma in situ: intraepithelial or invasion of the lamina propria
tumor invades the submucosa
turnor invades the muscularis propria
-or invades through the muscularis propria
tumor dinctly invades other organs, structures or perforates the viscerai peritoneum
Regional Lymph Nodes
NX regional Iymph nodes cannot be assessed
NO no regional lymph node metastasis
NI metastasisinf-3regionallymphnodes
N2 metastnsisin~4ngionallymphnodes
N3 metastasis in any lymph node dong the course of a named vascula. tnmk or in the
apicai lymph node
Distant Metastasfs
MX presence of distant metastasis cannot be assessed
MO no distant metastasis
M 1 distant metastasis present
Stage Grouping
Stage O Tis NO MO
Stage 1 Tl, T2 NO MO
Stage II T3, T4 NO MO
Stage III A ~ Y T Nl,N2,N3 MO
Stage N A ~ Y T A ~ Y N MI
A
B
C
'D'
B. Histopatholog ic Classification
Adenocarcinoma in situ
Adenocarcinorna
Mucinous adenocarcinorna (colloid type >50% mucinous carcinoma)
Signet-ring ce11 carcinoma (>50% signet-ring cell)
Squamous ce11 (epidermoid) carcinoma
Adenosquamous carcinoma
Small ce11 (oat ceiI) carcinoma
Undifferentïated carcinoma
Carcinoma, NOS (not otherwise specified)
C. Histopathologic Grade
GX grade cannot be assessed
G 1 well differentiated
0 2 moderately differentiated
G3 poorly differentiated
G4 undifferentiated
Clhical aspects of colorectal cancer
Colorectal cancer presentation, treatment and prognosis
The signs and symptoms of colorectal cancer rnay be varied and nonspecific.
Colorectal cancers arising proximal to the splenic flexure often present subacutely with
stigmata of iron deficiency anemia due to occult blwd loss. Lower abdominal or back pain,
change in bowel habits, constipation and gross rectal bleeding rnay be associated with cancer
of the left colon and rectum. Less commonl y, colorectal cancer may present with weight loss,
fever or septicemia. Colorectal cancer may present acutely with obstruction, perforation or
gastrointestinal hemorrhage. In some individuais, symptoms of metastatic disease such as
ascites or jaundice may be the fint signs of colorectal rnalignancy.
Preoperatively, individuals with colorectal cancer should undergo visualization of
their entire colon for assessrnent of synchronous adenornatous polyps and cancers. Routine
screening for metastases using abdominal computed tomography, ultrasound or senun liver
function tests and c hest x-ray and s e m carcinoembryonic antigen (CEA) level
determination is currently recommended (21).
The mainstay of colorectal cancer treatment is surgicd resection. Curative intent
surgery involves segmental resection of the involved colon dong with its venous and nodal
drainage. In addition to curative procedures, surgical resection is commonly employed to
prevent or deviate obstruction or bteeding in incurable cases. Adjuvant 5-fluorourad based
chemotherapy has been shown to be Me pmlonging in colorectai cancers that have
metastasized to regionai lymph nodes CïNM stage m, (22,23)3. Adjuvant (24) and
neoadjwant (25) radiotherapy has been shown to àecrease local recunence rates in TNM
stage II and III cancers. Selecied case series have demonstrated modest five-year survival
rates for surgical resection of hepatic and pulmonary metastases (26-29).
Rognosis of colorectal cancer is highly associated with TNM stage at diagnosis (18).
Early stage cancers may be resected with a high iikelihood of long-term survival, while
colorectal cancers that have metastasized to distant organs are rarely curable. In addition to
TNM stage, a number of other factors rnay offer some degne of prognostication inciuding
tumor site (303 l), histologie differentiation (20,32), ce11 type (2033-35), extramural venous
invasion (20,36-39), ploidy status (20,40), and patient age (3 8,4 1).
Clinicar screening for colorectal cancer
The rationde for asymptomatic clinical screening for colorectal cancer is based on two
observations. First, as described, most colorectd cancers are believed to arise h m benign
neoplastic precursor Iesions. Second, survivd from colorectal cancer is closely related to
stage of disease at diagnosis. Current clinical screening techniques include fecd occult blood
testing, air contrast barium enema, sigmoidoscopy and colonoscopy. In 1994, the Canadian
Task Force on the Periodic Health Examination examined the issue of colorectai cancer
screening and concluded that insufiCient evidence existed to support the inclusion or
exclusion of any specific ciinical screening program for individuals not affected by rare
dominantiy inherited colorectal cancer syndromes (42). More recently the American
Gastroenterology Association has adopted more aggressive screening guidelines (13):
A. For average risk hdividuais, one of the following scnxning options should be
commenced at 50 years of age:
1. Annud fecd occuIt blood testing. Two samples should be submitted h m three
consecutive stools,
2. Flexible sigmoidoscopy every 5 years.
3. Flexible sigmoidoscopy every 5 years cornbined with annual fecal occult blood
testing.
4. Air conhast barium enema every 10 years.
5. Colonoscopy every 10 years.
A positive result h m any of the above screening techniques mandates investigation of the
entire colorectum.
B. Guideline for individuals at increased risk for colorectal cancer:
1. Individuais with a farnily history of colorectal cancer should be offered the same
screening as average risk individuals, but screening should commence at 40 years
of age.
2. hdividuals who have had an adenomatous polyp removed should have repeat
coIonoscopy after 3 years. If q a t exam is nomal or reveds only a small
adenorna, colonoscopy should be repeated 5 years later.
3. Individuals who have undergone curative intent colorectal cancer resection should
undergo colonoscopy within one year of surgery. If this exam is nomal.
colonoscopy should be repeated every 5 years.
4. Individuais with a family history of familial adenomatous polyposis should
receive genetic comseling and testing to determine if they are carriers. At risk
individuals should have flexibIe sigmoidoscopy every 12 months begînnîng at
puberty. If poIyposis is detected, colectomy shouid be planned
S. Individuais with a family history of hereditary nonpolyposis colorectal cancer
based on the Amsterdam criteria should have a complete examination of their
colorectum every I to 2 years beginning at 30 years of age and annual
examination after 40 years of age.
6. Individuals with inflammatory bowel disease should have colonoscopy with
random biopsies every 1 to 2 years after 8 years of pancolitis or after 15 years of
left-sided oniy disease. The presence of dysplasia or carcinoma is an indication
for colectomy.
Cancer genes, gatekeepers and caretakers
We have recently reviewed the molecular genetic events that give rise to colorectal
cancer (43). The following nview will update and expand upon issues relevant to my thesis
researc h.
Gatekeeper genes
It is now commonly believed that al1 cancers arise due to the accumulation of a
number of consistent genetic alterations that impart a growth advantage for neoplastic ceils
(44). Certain genetic events are observed in benign cancer precursor lesions and in the
ediest cancers studied and are thus Iikely instrumental in neoplastic initiation. This group of
genes has k e n referred to as "gatekeeper" genes and includes the M C (Adenornatous
Polyposis Coli) gene in colorectal cancer, the VHL (von Hippel Lindau) gene in rend ceil
carcinoma, and the RB (Retinoblastoma) gene in retinoblastoma (10).
Caretaker genes
The spontaneous mutation rate of DNA in normal human cells, estimated at
approximately 1.4 x mutation~ucIeotide/cell gemeration is likely inadequate to account
for the number of genetic aiterations observed in most human cancers (45). It has therefore
been suggested that destabilization of the genorne rnay be a prerequisite to drive tumor
m s s i o n (10,45,46). For example, disruption of mutagen metabolism. replication
proficiency, post-replication DNA repair, or repair of DNA hydrolysis, oxidation or
nonenzymatic methylation may d l be expected to increase the rate of acquisition of genetic
mutations. When disrupted during carcinogenesis, this group of genes has been referred to as
''caretalce? genes and includes the DNA mismatch repair genes MSH2 (MutS homologue 2)
and MLHl (MU& homologue 2) in colorectal cancer and the double stand break repair genes
BRCAl and BRCA2 (Breast Cancer L and 2) in breast cancer (IO). Mismatch repair gene
deficiency, present in approximately 15% of colorectal cancers, Ieads to more than a one
hundred-fold increase in the mutation rate of short repetitive DNA repeats known as
microsatellites (47). While many gatekeeper and caretaker gens were initially identified
through rare, inherited cancer syndromes such as familial adenomatous polyposis and
heredîtary nonpolyposis colonctal cancer, many of these genes are also kquently involved
in the more common sporadic €omis of cancer.
Molecular genetic pathways of colorectal carcinogenesis:
chromosomal and microsatellite instability
Due to the relaàvely high incidence of colorectal cancer and adenomatous polyps in
Western society and the relative ease of access to these lesions offered by endoscopic and
secd methods, the molecuiar genetic aiterations that accompany the adenorna to
carcinoma sequence have been studied extenàvely. It is now apparent that colorectal cancers
arise nom (at Ieast) two separate genetic pathways. The first pathway involves genomic
instability at the level of the chromosome, inactivation of the gatekeeper M C gene and is
chmctenzed by the inhented familial adenomatous polyposis syndrome. The second
pathway involves microsatellite instability, loss of DNA mismatch repair function and is
characterized by the inherited hereditary nonpolyposis colorectai cancer syndrome.
Differences between these colorectal cancer mutational pathways are surnmarized in Table 1-
3 and discussed below.
Table 1-3: Colorectal cancer molecular pathways.
Instabüity Pathway Chromosomal Microsatellite
Chromosomd Alterations
DNA Nucleutide Alterations
Cause of Instability
Genes
Prevaience
Genetic Targets
Tumor Initiation
Tumor Progression
Ploidy
Right Colon Cancer
Pwr Differentiation
Common
Rare
Spindle Checkpoint Deficienc y?
BUBI, BUBRl
Sporadic 88%
FAP 4%
APC
K-ras. p53,18q, COX-2
Ofken Aneuploid
-30%
Rare
Rare
Common
Mismatch Repair Deficiency
MLHI, MSH2,
MSH6, MSH3. PM=, PMSI, MLH3
Sporadic 10%
HNPCC 2%
APC, B-catenin
TGF-$RI& BAX, fGF IIR
Near Diploid
-70%
Comrnon
See text for details for further details.
The chromosomal instability gatekeeper pathway
Macro-genetic instability characterized by chromosomal alterations tapifies the fmt
elucidated and more common genetic mutationai pathway in colorectal cancer. The
chromosomal instability carcinogenic pathway that characteristicalIy targets chromosome 5q
and the APC gatekeeper gene for mutation, is estimated to account for approximately 85% of
aU colorectal cancer (10,48). More than 65% of these colorectal cancers have been observed
to be aneuploid by flow cytometry (49) and undergo allelic loss (LOH) at an average of 25%
of chromosomd a m (50). Furthemore, more than 75% of these tumors display
chromosome l8qZ loss of heterozygosity (5 152). In addition to Ioss of heterozygosity, gene
amplification detected by comparative in situ hybridization is frequently observed in
colorectal cancers with chromosomal instability (53). Finally, the number of ailelic and
karyotypic abnormalities observed in these tumors increases with progression from adenoma
to carcinoma (5455).
While the underlying cause of chromosomal instability is not well understood, this
instability appears to be associated with mitotic checkpoint dysfunction (56). Dimption of
mitotic checkpoint function abrogates arrest that would norrnally occur with the acquisition
of chromosomal alterations Dominant negative functional mutations of two human
homologues of Saccharomyces cerevisiae mitotic checkpoint gene BUBl (Budding
Uninhibited by BenPrnidales) have been observed in aneuploid co1orecta.I cancer ce11 lines
(56). Similady, a cornplementation experiment fusing chromosomal stable and unstable
colorectal cancer ce11 lines has demonstrated the dominant effects of chromosomd instability
(57). However, specific mueons of mitotic checkpoint gens other than the p53 utunor
Protein 53) gene have only m l y been demonstrated in coiorectal cancers. It has dso been
hypothesized that global hypomethylation, often evident in colorectal cancer, may contriiute
to chromosomd instability by inhibiting chromosome condensation and potentiaily leading to
rnitotic non-disjunction (58-61).
Interestingly, while some authors have argued that chromosornai instability may be
the cause of aneuploidy (62.63). direct experimentai evidence does not support that abnomal
chromosome numbers Iead to chmmosomal instability (57). Fwthermore, while mutations of
the p53 cell-cycle gene are common in aneuploid colorectal cancers (64), they are unlikely to
be responsible for chromosornai instability as similarp53 mutations arr observed in
colorectal cancers with stable chromosome number (65). Further arguing against the
causative role ofp53 mutations in chromosomal instabiiity. p53 mutations generally occur in
carcinomas, but not adenomas (54,66), whereas alletic Ioss has k e n observed in early
adenomas (54).
The APC gene and familial adenomatous polyposis
While a generaiized inmase in point mutations is not apparent in colorectal cancers
with chromosomd instability (67). mutations involving specific genes located at regions of
most fiequent alleiic Ioss. such as chromosomes 5q and L7p, have been observed. Colorectai
cancer aüelotyping in combination with Iinkage anaiysis of familial adenomatous polyposis
families strongiy implicated a m o r suppressor Iocus on chromosome 5q21-22 as an eady
alteration in colorectal neoplasia (68-71). Further studies identified the APC gene as this
chromosome 5q target in both familial adenornatous polyposis and sporadic co~orectai
cancers and adenomatous polyps (72-75). While gem-he APC mutations causing familid
adenomatous polyposis are rare occlming in approximately IffûOû individuals and
accounting for 0.3% of colorectai cancer, somatic APC mutations are observed in more than
80% of di colorectd cancers and a similin percentage of adenornas (10). Further implicating
the role of APC in colorectal cancer initiation, mutations of this tumor suppressor gene have
also been identified in dysplastic ACF (76).(77) Consistent with Knudson's two-hit
hypothesis (78), both alleles of the APC gene are usually lost either through somatic biallelic
inactivation in sporadic tumors or germ-line mutation followed by somatic Ioss in familial
adenomatous polyposis adenornas and cancers. Virtually ail intragenic mutations of APC are
predicted to generate a tmncated protein pmduct and usually ûuncate the APC gene in the 5'
half of its open reading hune (79).
The APC gene is a large housekeeping gene and is expressed in most tissues. The
gene is composed of 19 exons, six of which are aitematively expressed. APC encodes a
protein of 2843 amino acids. The APC protein interacts with diverse protein partners
including a p-catenin (CTNNBI) regulating complex that includes B-catenin (80,81), GSK38
[giycogen synthase kinase 38 ; (82)J and axin (83). Additionally, the amino terminai of APC
binds to itself [homooligomerization; (84)], while 3' amino acid motifs interact with the
microtubule component of the cytoskeleton (85), EB 1 (86,87) and the human disc large
protein (88). Mutant forms of APC observed in colorectal cancer most commonly retain
homooligomerization and some batenin binding domains, while losing other B-catenin, and
EBI. microtubule and human disc large binding domains (79).
The roIe of APC in the regdation of B-catenin appears to be of primary importance in
colorectal carcinogenesis. Wild-type APC binds batenin in a multimeric complex and
phosphory1ation by GSK@ leads to p-catenin degradation and dom-regdation of
Wxngiess/WNT-1 signalling (80-82,89-91). Tnmcated mutant forms of APC observed in both
familial adenomatous polyposis and sporadic colorectal cancers usually retain their ability to
bind katenin, but Iose their ability to effect degradation of this substrate (79). The
accumulation of B-catenin in the cytoplasm that results fiom most truncating APC mutations,
leads to increased nuclear 6-catenin concentrations and subsequently to transcriptional
upregulation through the TCFLEF enhancers Cell Factor l Lymphoidenhancer Factor,
(89-91)J. Recent work suggests that important transcriptional targets of this pathway include
c-Myc [Avian Myelocytomatosis Viral Oncogene Homologue; (92)], cyclin D 1 (93) and
PPARb Iperoxisome Roliferator-Activated Receptor, (9411. Cytoplasmic overexpression of
b-catenin has also been implicated in impaired ce11 to ce11 adhesion, cytoskeletal anchoring
and signalling through p-catenin interactions with E-cadherin (80,81,95). Further supporting
the importance of batenin's role in APC gatekeeper huiction is the observation that
stabilizing batenin mutations have been observed in cancers that lack APC mutations
[(89,9 1); discussed m e r , below].
While alteration of batenin regulation has been offered as a key mie for APC
mutation in colorectal carcinogenesis, other hinctional consequences of APC dimption may
also be important. One such consequence is the effect of APC on EB 1. EBI interaction with
APC is pledicted to be lost in most tnmcated foms of mutant APC observed in colorectai
cancer. Mutant EBl has recently ken observed to aboiish a delay that occurs when yeast
mitotic spindes are rnisaligned (96). It is therefore intriguing to postdate that in addition to
its colonic epitheliai gatekeeper d e , the APC gene may be implicated as a caretaker of
chromosornai stabüity. Another potentialiy interesting role for APC can be appreàated h m
studies that have show that expression of full-length M C leads to apoptosis in colorectd
cancer ceU iines normaily dencient in this protein (97).
In addition to fiinctional analyses of the APC protein, genotypic-phenotypic studies of
individuais with fami1ia.l adenomatous polyposis have offend a different mechanism to
explore the consequences of alteratioas of APC hctional domains. Attenuated adenornatous
polyposis coii (AAPC) is typically associated with Iater age of onset or a reduced frequency
of colorectal adenomas cornparnt to familial adenomatous polyposis (98,99). In attenuated
adenornatous polyposis coli families, gem-line APC mutations tend to cluster at the ends of
the APC gene, either 5' to codon 157 (98.99) or 3' to codon 1597 (100). IntRguihgly, it has
recently ken suggested that certain attenuated adenomatous polyposis coli mutations may
restrict possible mutational spectra of the wiId-type APC allele, further implicating APC as a
potential caretaker gene (101,102).
In contmt to attenuated adenomatous polyposis coli, some familial adenomatous
polyposis families manifest an aggressive, profuse polyposis phenotype [>5000 colorectd
adenomatous polyps; (103,104)1. Familial adenomatous polyposis kindreds with an
aggnssive polyposis phenotype tend to have gem-line APC mutations in the mutation
cluster region @KR, codons 1286-15 13), the region of APC most commonly af5ected by
somatic mutations. Funher supporthg the role of APC as a potential gatekeeper gene,
mutation cluster region alterations of APC tend to be accompanied by loss of the second APC
allele, while APC mutations outside this region are o€ten observed in colorectal tumors with
biallelic intmgenic mutations (105).
Among extracolonic manifestations associated with familial adenomatous polyposis,
congenital hyperûmphy of the ~t inai epitheiium (CMRlPE) serves as one of the eariiest
clinical diagnostic indications of disease penetrance and is asscsciated with mutations
between codons 463 and 1387 ( I o . Approximately two-thuds of familid adenomatous
polyposis famiües manifest CHRPE (107). In a distinct phenotypic variant of familid
adenornatous polyposis previously known as Gardner's syndrome (108), coiorectal polyposis
is associated with osteornas, epidermoid cysts and skin fibromas. Tnmcating mutations
between codons 1403 and 1578 are associated with Gardner's syndrome, and these patients
do not exhibit CHRPE. Recent evidence has dso dernonstrated that the majority of Turcot's
syndrome families, characterized by colorectal cancer and medulloblastoma, are indeed a
variant of familial adenornatous polyposis and c q germ-line APC mutations (109).
Other genetic targets of the gatekeeper pathway
While APC is thought to be the colorectal cancer gatekeeper gene and is therefore
likely one of the earliest targets and possibly the rate-lirniting step of the chromosomal
instability mutation pathway, co1orecta.I cancers axising h m this pathway have also been
associated with a number of other consistent genetic alterations. Activating K-ras (Kirsten
Rat Sarcoma 2 Viral Oncogene Homologue; chromosome 12q) mutations have been reported
commonly in both colorectal adenomas and carcinomas (54,110.1 11). Interestingly, while K-
ras mutations have been observed in coionic ACF lacking dysplasia, they are rarely observed
in ACF with dysplasia (77). It has thmfore been hypothesked that K-ms mutation, while
important in progression of the cancer phenotype, is not sufficient to initiate dysplasia [i.e, K-
r u is not a gatekeeper; (IO)].
Chromosome 18q21 has been reported to be one of the most common sites of delic
deletion in both co1orecta.i adenomas and cancers (54,112). While the genetic target of
chromosome 18q l o s of heterozygosity has not been definitively demonstrateci, the DCC
Peleted in Colon Cancer, (1 1311 SMAD4 (114) and SMAD2 genes (1 15) [a rnerger of the
Caenothabditis elegmrs srna (smdl) gene and the Drosophila melanogarter mothers agaiiist
decapentaplegic gene; (1 1611, ail located on chromosome 18q, have been postdated as
potential colorectai cancer tumor suppressors. Additionally, as previously mentioned, the pS3
gene appears to be the target of chromosome L7p allelic loss observed fresuently in invasive
colorectal carcinomas (64). In addition to the above-mentioned alterations, regions of
chromosomes lq, 4p. 6p, 8p, 9q and 22q have been observed to be lost in 2530% of
coIorectal cancers (68).
Another intriguing finding associated with chromosornai instability is the frequent
overexpression of COX-2 (Cyclwxygenase 2) in colorectal adenomas and carcinomas (1 17).
While neither genetic mutations, nor any other cause for COX-2 dysngulation has yet k e n
found. significant experimental evidence exists linking COX-2 with colorectal carcinogenesis
meviewed in (1 18)]. When Apc knockout mice were crossed to produce offspring with either
homozygousl y or heterozygousl y inac tivated Ptgs2 mstaglandin G/H s ynthase 2, the
mouse CUX-2 homologue), polyp numbers were significantly decreased in a dose dependent
fashion compared to Apc knockout mice with wild-type Prgs2 (1 19). Furthemore, in addition
to polyp number, the size of the polyps was significantly nduced in the Ptgs2 knockout
mice. Prospective trials of non-selective pharmacologicai inhibition of COX-2 have shown a
decnased risk for colorectal cancer in the g e n d population (120) and the ability to suppress
polyposis in familial adenornatous polyposis (121). Selective COX-2 inhibitors have recently
been introduced in Canada and the United States (CeIebrex, GD Searle and Company. Vioxx,
Merck Laboratones hcorporated) and may hold even fimher promise for the
chemoprevention of coloreaal adenomas and cancers. These compounds have the advantage
of not causing the gastrointestinal side-effects cornmon with non-seiective cyciooxygenase
inhibition by nonstemidal anti-infiammatory agents.
The mismatch repair deficient microsatellite instability caretaker
pathway
lnstability of microsatel lite DNA
ALI DNA has limited stability and may be modifed through various mechanisms.
DNA alterations may be produced by exogenous forces such as chemical mutagens and
gamma radiation, or endogenous events such as hydrolysis, oxidation and nonenzymatic
methylation (122). Additionally, mismatched nucleotides may arise due to DNA polymerase
misincorporation errors (123) or genetic recornbination producing mispaired heteroduplexes
(124). Repetitive DNA elements such as microsatellite (1-5 base pair) repeats (also known as
short tandem repeats. STRs) display a greater degree of instability than non-repetitive
sequences (125). The (A). mononucleotide and (CAX, dinucleotide repeats are predicted to be
the most abundant microsatellites in the human genome (126). Insertions and deletions in
microsatellite DNA are thought to arise due to uncomcted polymerasc-template slippage
dttring replication of repetitive DNA elements mgtue 1-1; (ln)]. A high d e p e of
polymorphic informativeness and their relative abundance have made microsatellite markers
very useful in genetic tinkage, forensic science, phylogenetic mapping and somatic tirnior
analyses. Because microsatellite DNA is intrinsically unstabIe. it is iikeiy that genetic
pressures have kept these sequences under-represented in translated regions of the genome
(128)-
Figure 1-1 : The polymerase-template slippage mechanism of insertion and deletion
mutations in microsatellite D NA.
Temp late - CACACACACACA - DNA (CA), - GTGTGTGTGTGT -
DN A Template Poly merase Slippage Stippage
Leadtng - CACACACACACA - - CACACACACACA - Leadfng Strand (CA), - GTGTGTGTGTGT - - GTGTGTGTGTGT - Strand (CA),
W g i W - CACACACACA - - CACACACACACACA - Lagging Stmnd (CA), - GTGTGTGTGT - - GTGTGTGTGTGTGT - Strand (CA),
Deletion Mutation
Insertion Mutation
Dynamic trinucleotide expansion diseases
Though often insignificant, mutation of mimsatellite DNA is not dways without
consequence. Genn-line and somatic trinucleotide repeat expansions have ken implicated in
a number of inherited neurodegenerative diseases including Fragile X (129), spinobulbar
muscuiar atrophy (1301, myotonic dystrophy (13 1). Huntington's disease (132).
spinocerebellar ataxia type 1 (133), hereditary dentatombrd-pallidolusian atrophy (134) and
Machado-Joseph disease (135). Each of these diseases appears to be due to inhentance of a
large, unstable expansion of a specific tnnucleotide microsatellite repeat. Mechanistically, it
appears that these trinucleotide microsatellite loci undergo uncontrolled and frequent
expansion mutation when their sequence size evolves to a critical length that physically or
chemically prevents FENl (Flap Endonuclease 1) recognition and repair of repeat slippage in
Okazaki hagrnenu (136).
Hereditary nonpolyposis colorectal cancer
in 1913. Warthh reported a family with frequent occurrence of endometrial, gastnc
and colon cancers (L37). More than half a century later, Lynch re-discovered this family
(Family G) in addition to two other families and observed that they had an autosomal
dominant inheritance pattern of colon cancer in the absence of polyposis (138,139). Further
investigations of sirniIar kindreds reveaied that some were affected with only colorectal
cancer, while other f d e s displayed both coIorectal and extracolonic cancers. initiaily
named cancer family syndrome, the terms Lynch syndrome 1 and II were proposed to
distinguish between f d e s thaî either Iacked or displayed extracoIonic malipancies in
addition to nonpolyposis coIorectaI cancer (140). More recently, the term hereditary
nonpoIyposis colorectal cancer or HNPCC syndrome has ken used to describe both Lynch 1
and II families. Phenotypically, hereditary nonpolyposis colorectal cancers were observed to
arise at an early age (rnean age approximately 44 years), were Frequently observed proximal
to the splenic flexure (approximately 70%). were ofien p r l y differentiated or of mucinous
cell type and cytogenetically were usually near diploid (141). WhiIe the occurrence of
cancers at many different ext.racolonic sites has ken reported in hereditary nonpolyposis
colorectal cancer families, significant excesses of endometrial, stomach, small intestine.
biliary, upper urinary tract and ovarian cancers have been verified in epidemiologic studies
(142-145).
In order to increase both clinicd and research specificity, the Amsterdam aiteria, a
formal clinical definition of hereditary nonpolyposis colorectal cancer, were intmduced in
199 1 by the International Collaborative Group on Hereditary Nonpolyposis Colonctal
Cancer (146).
These critena are:
1. At least three relatives with histologically verified colorectal cancer. one of them is
the first degree relative of the other two. Familial adenomatous polyposis is excluded.
2. At least two successive generations are fiected
3. Colorectd cancer is diagnosed under 50 years of age in at least one of the relatives.
The Amsterdam criteria were recentiy expanded to include extracolonic cancers of the
endometnum, mal1 bowel, rend pelvis or meter in addition to colorectal cancer [Amsterdam
criteria & ((147)l. However, various shortcoming of these clinical criteria still exist, including
lack of recognition of coiorectd adenomatous polyps, as well as multigeaerationai, multi-
individual reqdments that bias against diagnosis in small or poorly documented families.
Microsatellite instability in colorectal cancer
Enonnous advances in the understanding of hereditary nonpolyposis colorectal cancer
genetics began in 1993 when thme groups simultaneously deSCI"ibed an apparent second
molecular pathway in colorectal cancer that involved pneraiïzed somatic microsatellite
instability (505 1,148). In cornparison to trinucleotide repeat expansion neurodegenerative
syndromes that affect a single locus per disease or individual, microsatellite instability in
colorectai cancer was obsewed to be ubiquitous and predicted to be indicative of more than
100,000 such mutations per cancer (148). Definitions of this molecular pathway and the
timing of its discovery have been of some debate (149,150) and will be presented in order of
publication submission &te. Perucho and colleagues initially observed that 1296 of colorectal
cancers displayed somatic instability of (A). (polyadenine) sequences and that these tumors
arose more commonly in young individuais, were more commonly nght sided in origin,
usuall y presented wi thout metastases, were often poorl y di fferentiated and infrequentl y
displayed K-ras or p53 mutations (148). Interestingiy, this group had alluded earlier to the
discovery of this instability in a publication describing a subset of coIorectaI cancers
displaying somatic deletions of a few nucleotides length when amplifed by arbitraniy
primed PCR [polymeme chah reaction; (151)]. However, no m e r details of the nature or
frequency of these findings were provided in their earlier report. Thibodeau's laboratory,
similarly obseinred that 13% of sporadic colorectd cancers displayed somatic instability at
multiple (CA). dinucleotide DNA elements and that these cancers were often Iocated in the
nght colon, were associated with improved patient sufvivd and infiequentiy displayed l o s
of hetcrozygosity at the common tumor suppressor loci on chromosomes Sq, 17p or 18q (SI).
At the same time the labonitories of de ta Chappelle and Vogelstein geneticdy ünked two
hereditary nonpolyposis colorectal cancer kindreds to the D2S123 (CA), locus on
chromosome 2p (152). While searching for correspondhg somatic loss of heterozygosity at
this microsatellite locus, they unexpectedly observed that 1 1114 (79%) of tumors (12
colorectal cancers, 1 adenoma, 1 ovarian cancer) fmm these hereditary nonpolyposis
colorectal cancer patients displayed shifted alleles (microsatellite instability) rather than
alleiic loss (50). In addition to these hereditary nonpolyposis colorectal cancer patient
tumors, they fond that 13% of sporadic colorectal cancers displayed sirnilar somatic
instability at multiple (CA), and (CAG), microsatellite loci and that these tumors with
microsatellite instability were commonly diploid (50) and displayed very smail fractions of
alleüc loss (4% of chromosomes).
Known variously as ubiquitous somatic mutations (USM), mi*crosatellite instability
(MSI), microsatellite mutator phenotype (MMP) and replication emrs (RER+), consensus
testing and nomenclature definitions of this molecuiar phenomenon have rezently been
adopted by the National Cancer Institute (153). High-fkquency microsatellite instability
(MSI-H) was fonnall y defined as instabili ty at least 2/5 or 4/10 (240%) speci ficall y
designated microsatellite loci (Table 1.7.3-1). InstabiLity at 1-3/10 of these loci was defined
as low-kquency microsatellite instabiiity (MSI-L) and instability at 06 of these loci was
defined as mimsateUite stable (MSS). Interestingîy, the phenomenon of MSI-H had been
previously describeci, but not appreciated by the Vogelstein group when in 1990 they
observed that 10/94 (1 1%) of colorectal cancer ceil Iines, xenografts and primary tumors
displayed somatic expansions in a pdymorphic intronic (AT). domain of the DCC gene
(1 13).
As mentioned, in addition to hereditary nonpolyposis colorectal cancers, MSI-H is
observed in sporadic colorectal cancer cases. To date the only large, prospective, multicentre
estimate of MSI-H in colorectal cancer cornes from Finland where 63/509 (12%) cancers
were MSI-H (9). Seven of these patients (1 1% of MSI-H and 1% of total) had family
histories fulfilling the Amsterdam criteria of hereditary nonpolyposis colorectal cancer.
Small studies of colitis associated neoplasia have documented MSI-H prevalence sirnilar to
those reported for sporadic colorectal cancer with MSI-H king observed in 847% of colitis-
associated colorectal cancer or dysplasia specimens (154,155). In contrast, the prevalence of
MSI-H in colorectal tumors from individuals with familial adenomatous polyposis appcars to
be less than 1% (156).
In addition to its prevalence in colorectal cancer, the MSI-H molecular phenotype has
been studied in benign colorectal cancer precursor lesions. MSI-H has been observed in ACF
h m 2/10 [20%; (1 57)] and 2/20 [IO%; (1 58) J colorectal cancer patients. Unfortunatel y,
neither of these studies reported the microsatellite status of the colorectal cancers of the
patients h m which these ACF were obtained, or whether the ACF were dysplastic or
hyperplastic. MSI-H has k e n observed in 57-100% of adenomatous polyps From patients
with hereditary nonpolyposis colorectai cancer (156,159-161). In contrast, an initial study
reporteci MSI-H in 0146 sporadic colorectal adenomatous potyps (162). Subsequent studies
have confinneci a low rate (0.7%) of MSI-H in sporadic adenornas (IS6,lS!J,l63-166).
Differences in testing techniques (how many and which microsateIlite loci were used) or
poor tumor celIuiarity may explain the Iow observed frequency of MSI-H in sporadic
aâenomas cornpared to invasive colorectal cancers. However, since the overd prevalence of
sporadic colorectal adenornas to carcinomas is approximately 10: 1 (10). the observed
Table 14: Microsatellite loci for evaluation of microsatelîiie instability in colorectal
cancer (1 53).
High-frequency microsatellite instability (MSI-H) is defined as insertion or deletion
mutations in at Ieast 40% of at least five of these loci. Low-Frequency microsatellite
instability (MSI-L) is defined as insertion or deletion mutations in Iess than 40% of at Ieast
ten these loci. Microsatellite stability (MSS) is defined as no insertion or deietion mutations
in at least five of these loci.
Reference Panel Alternative Loci
BAT25 BATLU) D 18S58 DlOS197 D M 6 9
BAT26 BAT34C4 Dl8S61 D13S175 D13S153
DSS346 TGF-B RI1 D 18S64 D 17S588 D 17S787
D2S 123 ACTC D 18S64 D5S 107 D7S5 19
D 17S250 D18S55 D3S 1029 D8S87 D20S 100
MSCH and mismatch repair deficiency in hereditary nonpolyposis
colorectal cancer
The description of MSI-H in colorectal cancers was simila. to that previously
observed for Escherichia coli mutant in either mutL or mut3 pst-replication DNA mismatch
repair (168) and this prompted microsatellite mutation analyses of the Saccharontyces
cermkiae DNA mismatch repair gene [Pm1 (pst-meioric segregation I ) , mlhl and msM]
mutants (169). These yeast mutants wen found to display several hunclred-fold inmases in
(CA). deletion and insertion mutation rates and the authors hypothesized that similar
deficiencies might be respon sible for the MSI-H identified in most hereditary nonpolyposis
colorectal cancers and a subset of sporadic colorectal cancers. Interestingly, prior to any
description of MSI-H in colorectal cancer, Loeb, the originator of the cancer mutator
phenotype hypothesis (46), suggested that deficiencies in rnismatch repair might be centrai to
carcinogenic mutagenesis (45). Within months of MSI-H being observed in hereditary
nonpolyposis and sporadic colorectal cancers, the chromosome 2p mismatch repair gene
MSH2, a human homologue of bacterial mutS, was cloned independently by two groups
(170,171). Inherited mutations of MSHZ w m observed in affected individuais with
hereditary nonpolyposis colorectal cancer (170,171) and biailelic inactivation was observed
with somatic mutation of the second MSH2 dele in a colorectal cancer From a germ-line
carrier (171). At the same time, Iinkage anaiysis of two hmditary nonpolyposis colorectal
cancer families identified a second locus, D3S 1029 on chromosome 3p as another hereditary
nonpolyposis colorectai cancer locus and two colorectal cancers h m these families
displayed the MSI-H moIecular phenotype (172). FoiIowing this, the same groups involved
in the discovery of MSH2 cloned the chromosome 3p mismatch repair gene MLHI, a human
homologue of bacterial mutL (173.174). M M gexm-he mutations were observed in
affected individuais with hereditary nonpoiyposis colorectal cancer (173). Furthemore, a
colorectd cancer ce11 line was found to express no wild-type MLHl product indirectly
implicating the necessity for bialleiic mismatch repair gene inactivation (174). hterestingiy,
while wiàespread loss of heterozygosity is rare in MSI-H colorectd cancers, specific somatic
l o s of the MLHI allele appemd to be nlatively comrnon in colorectal cancers h m MLHI
gem-line carriers and sporadic MSI-H colorectai cancers (175).
Since the discovery of MSH2 and M W , a number of additional human homologues
of prokaryote mutS and mutL have been cloned and implicated in cminogenesis. The PMSI
(chromosome 2q) and PMSZ (chromosome 7p) genes are two human mutL homologues
simila. to Saccharomyces cerm*siae pml(176). A nonsense germ-line mutation of P M I
was identified in one hereditary nonpolyposis colorectal cancer patient with wild-type MSH2
and MLHI, while genn-line and tumor mutations of PMSZ were observed in another
hereditary nonpolyposis colorectal cancer patient (176).
A fifth human DNA mismatch =pair gene. GTBP (GR Base Pair, now known as
MSHo, chmmosome 2p) was cloned and found to be mutated in a colorectai cancer ce11 line
and an alky Iation-resistant 1 ymphoblastoid ce11 Iine (177,178). Interestingly, MSK6 mutant
ce11 Iines displayed & widespread microsatellite instability than that observed with MSH2
or MLHI mutation. This was primarily evident by (Ah, and oniy infiequent (CA). instabiüty.
Subsequently. two Japanese groups demonstrated mincating MSH6 genn-Iine mutations in
co1orecta.i cancer patients from two cancer families not fulfiliing the Amsterdam aiteria for
h d t a r y aonpoIyposis colorectai cancer (179,180). Both groups observed presumed
bialleüc mutation of MSH6. Recent studies have found gem-line MSH6 mutations in 6/91
(7%) familial (non-Amsterdam critena) colorecfal cancer patients (181) and 4118 (22%)
familial colorectal cancer patients with MSI-L tumors (182). In conhast, 0th- auîhors have
failed to demonstrate that germline M W 6 mutations are associated with MSI-L cancers
(1 83).
Two additional human mismatch repair genes, MSH3 [originally DUG, Divergently
transcribed Upstream Gene, dso known as MRPI, Mismatch Repair Rotein; (184,185)l and
M M 3 (186) have thus far been cloned However, no germ-line mutations of either of these
genes have yet been reported. Interestingl y, both MSHo and MSH3 contain translated
mononucleotide repeats that appear to be frequent targets for sornatic mutation in MSI-H
colorectal cancers (187). In a recent review of 120 human germ-line mismatch repair gene
mutations, 52 (43%) were reported in the MSHZ gene, 65 (54%) in M L H I , 2 (2%) in PMS2
and 1 (1%) in PMS2 (188). Seventy-seven percent of mutations were predicted to alter
protein length and 23% were missense mutations.
Most studies of affected individuals from hmditaiy nonpolyposis colorectal cancer
kindreds fulfilling the Amsterdam criteria have identified gem-line DNA mismatch repair
gene mutations in approximately 2550% of subjects (189-197). VirtuaHy al1 of these
investigations have concentrated on MSH2 and MLHI mutation analysis only. Utilization of
recently reported novel mutational techniques may allow for inmased detection rates in the
future (198).
Using standard mutation detection methods, a s m d number of groups have reponed
high cietection rates of germ-line mismatch gene mutation. For example, Finnish
Uivestigators have observed germ-he mutations in 30/35 (86%) of hereditary nonpolyposis
colorectal cancer families (157)- However, 22 (73% of mutations, 63% of hereditary
nonpolyposis colorectal cancers) of these famiües had one of two rccumnt, founder MLHI
mutations (199,200). Similady, a muiti-cenüe, prospective study of 509 F i ~ i s h coiorectd
cancer patients found gem-line mutations in 100% of seven patients whose histories fulfilled
the Amsterdam criteria (9). in Finland 2% of prospectively studied, unselected colorectal
cancer patients were found to have gem-line mutations of either MLHI or MSH2, and 60%
of these mutations (1% of d l colorectal cancer) were one of the two MLHl founder
mutations (9). Founder mutations of MLHI have also been described in Korean hereditary
nonpolyposis colorectai cancer families (192).
Similar to the high rate of mutation detection in the Finnish population, a three
continent study involving New Zealand, Europe and North Amenca reported gem-line DNA
mismatch repair gene mutations in 34/48 (70%) of kindreds fulfilling the Amsterdam criteria.
Success in finding gem-line mutations in these families was in part be due to testing P W ,
PMS2 and MSH6 in addition to analyses of MSH2 and MLHI. Inclusion of these genes
incnased mutation detection by 6%. Furthemore, (and of p a t e r significance) only
hereditary nonpolyposis colorectal cancer families with MSI-H cancers were included in
gem-üne analyses. in order to appreciate this prescreening bias, consider a fifteen year
population-based senes of 1.83 1 Itaiian colorectal cancer patients (20 1). In this study,
eighteem (1%) families hilnlled the Amsterdam criteria, Uiree of which (17%) had gem-iine
MSH2 or M M mutations. These gem-line mutations were identified in 3/ 11 (27%) patients
wîth MSI-H cancers compared to Of7 patients with MSS cancers. Thus, a 10% mutation
detection Merence would have occurred if the authors had @ormeci germ-Iine analyses
ody on patients who both fulnlled the Amsterdam criteria and had MSI-H colorectal cancer.
More subtle biases of a case series approach may be appreciated when one considers that the
authors of the pooulation-based studv which reported a 17% gem-line mutation rate of
MSRZ and MLHl(201) found mutations in 41% of 17 Itaüan hereditary nonpolyposis
colorectd cancer kinhds in a case series (195), nearly a 25% difference in mutation
detection.
MSI-H and mismatch repair deficiency in sporadic colorectal
cancer
Although, approximately 12- L 5% of sporadic colorectal cancers display the MSI-H
phenotype, the hallmark of mismatch repair de ficienc y, searc hes for gem-line and somatic
mismatch repair gene mutations in sporadic colorectal cancers have ken largely
unsuccessful. Germ-line mismatch repair gene mutations account for less than 10% of
MSI-H colorectal cancer fiom individuals without a farnily history of colorectal cancer
(9,19 1,202-204). Not surprisingi y, a higher kquenc y of gem-Iine mutations have been
observed in studies of colorectal cancer patients with family histories of colorectd cancer
which fdl short of completely fulfilling the Amsterdam critena (142,191,205-207). However,
while not satisfying the strict definition of the Amsterdam criteria, these MSI-H colorectai
cancers canot be considered "sporadic".
In smaU case senes' of sporadic MSI-H colorectal cancers, somatic MSH2 or MLHI
mutations have been observed in 442% (202,203,208) of turnors andyzed Interestingly,
some of these sporadic MSI-H cancers were noted to lack either MLHl or MSH2 expression
despite the lack of gm-iine or somatic mutations (202,îW). Subsequently it was observed
that lack of MLHI protein expression in these tumors conelated with MLHl promoter
hypermethylation (210). In a later stady of 42 MSI-H colorectd cancers not defined in terms
offamily history (Le. likely sporadic colorectal cancers), 38 (90%) were fond to lack MLKl
expression while only 2 (5%) did not express MSH2 and 2 (5%) had intact expression of both
gene products (21 1). While not explored directly, these results suggest that the majority of
MSI-H sporadic colorectal cancer may be due to hypemethylation of the MLHl prornoter
resulting in gene silencing. Results of direct examination of MLHl protein expression and
pmmoter hypemethylation in smaller studies of MSI-H colorectal cancers have supported
this hypothesis (212-214). Of importance in establishing causality, inhibition of de novo
methylation by 5-aza-2'-deoxycytidine has been show to lead to demethylation of the
MLHI promoter and restoration of rnismatch repair activity (212). Interestingly. promoter
hypermethylation and gene silencing appear to be a generalized feature in MSI-H colorectal
cancers (61,215). implying that a hypermethylator phenotype may precede (Le. cause) the
global mutator phenotype in sporadic MSI-H cancer. From an epidemiologic perspective, this
hypermethylator phenomenon does not appear to be inherited since sporadic MSI-H
colorecrd cancers do not occur at significantly younger age (21 1) or in the context of a
family history of colorectai cancer (2 16).
Loss of imprinting, another epigenetic phenomenon, appears to be associated with
sporadic MSI-H colorectal cancers (217). Loss of imprinting of the IGFZ (Insulin-Like
Growui Factor) gene was fond in 101 1 1 (91%) MSI-H cancers, but oniy 2/16 (12%) MSS
colorecral cancers. Of greater ciinical relevance, Ioss of imprinting was evident in normal
colonic mucosa of 10110 patients whose tumors dispfayed this epigenetic phenomenon. In
cornparison, 105s of imprinting in normal tissue was only found in 1112 (8%) colorectal
cancer patients without this featlrre in their tumor and m 2/15 (13%) controls without
colorectal cancer. These results indicate that loss of imprinting in normal tissue may be
usefil as a d n g rnethod to detect individuah who will develop "'spocadic" MSI-H
colorectal cancer and shodd therefore undergo more rigomus endoscopie screening.
Non-mlsmatch repair deficient causes of MSbH colorectal cancer
Loeb's original mutatoi phenotype in cancer hypothesis predicted that emr-prone
polymerases, rather than deficient DNA repair machinery, may be the source of an increased
mutation rate in cancer (46). In Succharomyces cerevisiae studies, po13 (polymerase 3, a
homologue of Pol ymerase 6) mutants displayed an intermediate level of microsatellite
instabiiity compared to genetic mismatch repair mutants (169). Pol-6 variants have ken
observed in the DLD-1, HCT-116 and SW48 human colorectal cancer ce11 lines which ail
display MSEH (218,219). However, al1 these ce11 lines also carry DNA mismatch repair gene
mutations suggesting that deficiency in mismatch repair may underlie mutation of the ~ o l - 6
gene (similar to somatic mutation of MSH3 and MSHo in MSI-H colorectai cancers, see
below). However, missense aiterations of Pot-b have also been reported in the gerrn-lines of
three colorectd cancer patients without significant cancer family histones, indicating that
polymemse aiterations may predispose to colorectai cancer (21 8 2 19).
DNA mismetch repair
The complex field of DNA mismatch repair has been extensively reviewed elsewhere
(198,220-224) and wili be summarized ùriefly here (Figure 1.7.7-1). Initial characterization
and conservation of DNA mismatch repait in prokaryotes and Iower eukaryotes and the
avdabüity of specific mutants, has assisted tremendously in detineating human mismatch
repair. The MSH2 and MSH6 proteins are initiaiiy tightly associated as a heterodimer,
MUSOC, and recognize DNA mismatches introduced into the newly synthesized strand by the
replication complex (l70,U l,I77,178,ZS>26). While strand dismimination is made by
transient undermethyIation of the newly synthesized strand in Eschenchia d i (227228), the
mechanism of this recognition in human ceEs remains unresolved It is hypothesized that an
association between mismatch repair proteins and the replication complex protein PCNA
(Proliferating Ceii Nuclear Antigen), may play a role in the process (229,230). Furthemore,
in vitro, a single nick in DNA is recognized by misrnatch repair machinery and rnay explain
how this process works in Okazaki fragments, but not in the Ieading strand (23 1). After
binding of the hetetodimer to heteroduplex DNA, MutSu undergoes an ATP-dependent
conformational change and moves dong the DNA double helix (232-235).
MSH2 mutations lead to deficiencies in repair of base-base mismatches, srnaIl
insertioddeletion loops and instability in mono-, di- tri- and tetranucleotide repeats (236). In
contrast, mutant MSHo cells are deficient in base-base mismatch and mononucleotide
insertioddeletion bop repair, but proficient in repairing insertioddeletion Ioops of
dinucleotides or pa te r (178). This suggests that another oligomer of MSH2, possibl y the
MSH2-MSH3 heterodimer (known as Mut@) may act ~dundantly to recognize di-, tri- and
tetranucleotide insertioddeletion loops (237). However, in human cells, Ioss of MSH3
expression does not appear to lead to microsatellite instability (238). While more work is
requkd to M y elucidate this pmess, it does appear that MutSu and MutSB are somewhat
functionaIIy redundant with M u s a operating as the prllnary protein complex in mismatch
repair (239,240).
A k r heteradimer recognition of DNA mismatches, it is beiïeved that the
MutLû~ hetemdimer, consisting of the MLHl and PMS2 proteins, oIigomerizes with MutSû~
(241). While no human data yet exists, MLH3 may under certain cUcumstances oligomezïze
with MLHl creating a redundancy a b to that noted for MSH2/MSH6 and MSHUMSH3
(198). Bi-directional threading of DNA through the MutW heteroduner continues until the
mismatch repair complex contacts the replication complex PCNA protein (229). This is
hypothesized to lead to replication arrest, dissociation of POI-6 and 3' exonucleolytic
degradation of the error-containing primer (242). The resultant single stranded DNA is
believed to be protected by RPA [Replication protein A; (243)]. Following exonucleolytic
degradation of the DNA mismatch, the mismatch repair complex (Mutsa and M u U )
dissociates and PCNA bound at the end of the primer once again remits the replication
complex proteins to repair the excised sequence. The mismatch repair biochernistry described
has Iargely been demonstrated in lower organisms. Direct evidence for much of this process
in human cells still needs to be done (242). The biochemical function of the Mut .
homologue PMS 1 remains unknown (176.1 86).
In addition to repairing mismaiches and insertioddeletion Iwps of microsatelIite
DNA, mismatch repair proteins have been found to be involved in meiosis (244),
homologous recombination (245). transcription (246) and G2 cell-cycle arrest (247).
Figure 1-2 Schematic representation of human post-replication DNA mismatch
A. Double stranded DNA with (i) a base-base mismatch, and (ii and iii) a two nucleotide
insertion loop. B. The MutSa heterodimer of MSH2 and MSH6 recognizes both base-base
mismatches and insertiodde Ietion loops w hile MutS B (MSH2 and MSH3) recognizes
insertioddeletion loops ody. C. and D. The MutLa heterodimer of MLHl and PMS2
wociates with MutSa or MutSp and leads to the excision of the post-replication mismatch.
E. DNA is resynthesized by replication cornplex (not show).
MSI-H and mismatch repair deficiency in extracolonic cancers
Afier the initial description of mismatch repair deficiency in colorectal cancer, a
number of investigators sought to determine whether MSI-H is also observed in tumors of
non-colonic origin. Unfortunately. MSI-H and its synonymous t e m have often ken used
inappropriately to describe tumors (colonic and extracolonic) that display infrequent, rather
than generalized microsatellite instability or instability at longer microsatelIite loci (Le.
tetranucleotides). Some studies have erruneously included al1eIic imbaiance as evidence of
"instability". Furthemore, many authors have not included any definition of the number of
shifted loci that constituted microsatellite instability in their studies. Interpretation of studies
of exûacolonic tumors is made additionally difficult by the fact that the cumnt National
Cancer Institute definition of MSI-H was developed specifically for colorectal cancers and
may not be appropriate for evaluating extracolonic neoplasia (153). The specific
microsatellite loci recommended and the 40% instability rate among loci tested currently
endorsed for MSI-H in colorectal cancer, may Iack sensitivity or speQficity for evaluating
extnicolonic cancers. Although the prevaience of MSI-H in extracolonic cancers has ken the
subject of two recmt literature reviews (248249). these analyses have provided Iittle to
clarify the subject It appears that a significant, but a variable number of the sporadic
extmcoIonic neoplasms associateci with hereditary nonpolyposis colorectal cancer display the
MSI-H molecular phenotype. For example, MSI-H has been observed in 9-3246 of sporadic
gastric cancers (250-254) and 16-2396 of sporadic endometrial cancers (250,255-257).
Converseiy. MSI-H has been noted both fkequentiy [33-50%; (251,258)l and infrrquently
[4%; (25911 in sporadic panmatic cancer and is rately seen in sporadic ovarian (251,260)
and transitional ce11 carcinomas (261). Tumor MSI-H and germ-line mismatch vair
deficiencies have been observed in both Turcot's syndrome [glial cancers and co1orecta.l
cancers; (109,262)] and Muir-Torre syndrome [sebacmus gland and viscerd organ tumors;
(263,264)], establishing these syndromes as hereditary nonpolyposis colorectal cancer
(Lynch II) variants. Furthennore, homozygous MLHI mutations have been associated with
nelimfibromatosis type I and hematological malignancies Opphorna and leukemia) in two
families (265,266). Additionally. it appears that a variable number of extracolonic cancers
such as lung, bteast. prostate, esophagus and head and neck cancers may display the MSI-L
molecular phenotype (248). However, similar to the case in colorectal cancer, the molecular
genetic and clinical significance of MSI-L in these tumors remains unknown.
Somatic genetic targets of MSI-H and mismatch repair deficiency
Transforming growth factor receptor II
While the majority of repetitive rnicrosateilite DNA is situated in non-coding regions
of the human genome, some microsatellite repeats do exist in exons. The first recognized
coding microsatellite region subject to somatic mutation in MSI-H colorectal cancers is an
(A),* repeat in the TGF-B type II receptor ( T G F ~ Rn) gene (267). Colorectal cancers with
mutations in the (A)io region do not express the TGF-p RII protein on their ceU surface.
Ftïrthermore, restoration of wiId-type TGF-p RI1 to a MSI-H colorectd cancer ce11 iine
diminishes tumorigenkity (268). One or two base-pair insertions and deletion mutations of
TGF-#? RL?, predicted to yield a tnmcated protein, were observed in 1011 1 1 (90%) MSI-H
colonctal cancers h m individuais with herectitary nonpolyposis colorectal cancers, as well
as sporadic and xenografted cancers (269). Smaller studies have confirmed the prwence of
muent frameshift mutations in TGF-fi RU in both sporadic and hereditary nonpolyposis
colorectal cancers with MSI-H (270-272). TGF-fi MI (A)io mutations have been observed
significantiy more often than mutation of other (A)io repeats (269). Using single-strand
conformation polymorphism (SSCP) analysis, second somatic mutations, presumably causing
biallelic inactivation. werr obsmed in 14/20 (70%) MSI-H hereditary nonpolyposis
colorectal cancers and adenornas with TGF# RII (A) instabilitr, 101 14 (7 1%) involved
homozygous mutation of the (A)io repeat, 3 (21%) carrîed missense mutations in the kinase
subdomain XI and one (7%) contained a rnissense mutation of the kinase subdomain VIII
(273). No Ioss of heterozygosity was evident in these tumors. Similar TGF# HI (A)
mutations appear to be common in MSI-H gastric (274) and glial cancers (275). but are rare
in MSI-H endomerrial (274,276) and pancreatic cancers (277-280).
Subsequent to the identification of TGF# MI (A)io mutations, it has become
apparent that the rpgions of the TGF-8 RII gene outside of this repetitive element are
targeted by cancers without microsatellite instability. T h e MSS colorectal cancer ce11 lines
were reported to be resistant to TGF-B-mediated growth inhibition (28 1). Two of these cell
iines were observed to have biallelic mutations of the kinase subdornain XI. One of these
cancers was aIso found to have biallelic mutations of the Smud4 gene, an intracellular
component of the TFG-8 signaling pathway. The third colorectal cancer cell iine displayed
separate mutations of the kinase snbdomaui iX and the extracytoplasrnic region of the
receptor. Studies of genomic DNA h m twnors confirmed that these mutations were not
tissue culture artif'&, and germ-line analysis of two of the patients h m which the ce11 lines
were derived, estabiïshed that the mutations w m somatic, Furùiermore, transfection of TGF-
f l Rn (and Srnad4 when appropriate) restored TGF-$ signaling and tumor suppressor
activity.
Recentiy, a TGF$ RII gemi-line mutation in the kinase subdomain N was
identified in a familial colorectal cancer kindred that did not Mly satisQ the Amsterdam
criteria and lacked MSH2 or MLHI mutation (282). Bialletic inactivation of T G F d RII was
evident in a colorectal cancer h m this family and this tumor was MSS and demonstrated
loss of heterozygosity of chromosomes Sq, 17p and 18q. In addition to studies of colorectal
cancers. inactivating somatic mutations of TGF-fl RI1 have been reported in head and neck
squamous ceIl cancers (283), small cell lung cancers (284). and cutaneous T-cell Iymphoma
cells (285).
APC, p-catenin and TCF-4
Somatic mutational analyses of the APC gene in MSI-H colorectai cancers have
yielded seemingly paradoxical results. WIe the mutation prevalence of APC is greater than
80% in colorectai cancer, 045% of MSI-H colorectal cancers have been reported to harbor
M C mutations (156,286-289). However, despite a Iower Frequency of APC mutation, the
somatic mutation sr~ctrum of MSI-H colorectal cancers appears to be unique with small,
fnuneshift mutations of repeat sequences more common in these cancers (287,290).
Functionat consequemes of this &que mutational spectrurn may potentiaily explain why
hereditary nonpolyposis colorectal cancer is not a polyposis syndrome despite mismatch
repair deficiency specifically targeting the APC gene. More recently, it has ken appreciated
why 50% or more of MSI-H colorectai cancers lack APC gene mutation altogether - the
majority of these cancers have stabilizing mutations of B-catenîn (291-293). FinaIly, ment
work has show that a dowustream target of clba bat en in signalling, the TCF-4 gene was
mutated at an (A)9 q a t in 40% of 57 MSI-H colorectal cancers and ce11 lines (294).
However, the (Ab repeat of TCF-4 is located downstnam of its functional domains, in a
region that normally encodes altematively spliced transcripts. Thus, the functional
consequence of frameshift (A)9 TCF-4 mutations remains unclear.
APC 11307Ki microsatellite instability in a caretaker gene
In 1997, the Vogelstein laboratory cloned an APC gene polymorphism, comrnon in
Ashkenazi Jews. in which lysine had been substituted for isoleucine at codon 1307 (295).
This polymorphism, present in 6-746 of dl Ashkenazim (295,296), was observed in 10-2898
of Jewish individuds with colorectal cancers or adenomas and a family history of these
tumors. While the polymorphisrn itself encoded a semi-conserved amino acid substitution.
more intriguing was the actual genomic DNA nucleotide change in which an (A)s
mononucleotide repeat was introduced into the gatekeeper APC gene. Somatic andysis of the
APC gene in 23 APC 11307K c h e r colo~ctal cancers and adenomas reveded 11 (48%)
mutations in codons 1296-1322 including 6 (26%) of the (A)s repeat, Therefore, unlike
familial adenornatous polyposis where carriers inherit a rnutated copy of the M C gene, APC
11307K caniers inherit a susce~tible allele (a bbpremutation"). A subsequent study in a series
of breast cancer patients showed that the APC II3MK polymorphism was present in 10-2096
of B R W or BRCAL carriers (297). However, somatic instability of APC II3WK has not
been observed in breast cancers. While the poiymorphism encodes a polyadenine repeat, the
association of APC II307K instability and MSI-H in colorectal cancex has not been
investigated
Interestin@y7 an instability mechanism simila. to APC 11307K has been observed to
transcriptionally restore the reading frame of an ApoB (Apolipoprotein B) mutation
(128,298). In this case, despite a germ-line fhneshift mutation that created an (AI8 in the
ApoB gene, full-length ApoB protein was synthesized, and a reduced hyperlipidemia
phenotype was observed.
Other genetic targets of the MSGH pathway
Similar to the (A)io of T G F 4 H Z , a number of other coding microsatellite repeats
have bem found to be sornaticaily mutated in MSI-H colorectal cancers. However, it has yet
to be clearly established whether these mutations are merely bystander effects of generaiized
instability of short tandem repeat DNA in these tumors. Sixteen of41 (39%) and 12/40
(30%) MSI-H colorectal cancers were found to have fiameshift mutations of (A)s and (C)8
repeats located in coding regions of the DNA mismatch repair genes MSH3 and MSHo
respectively (187). These mutations appeared to be heterozygous and fkequentiy occumd in
cancers in which other mismatch repair genes (MSH2 or MLHI) had already been
demonsaited to be inactivated Somatic frameshift mutations in 21/41 (5 1%) and 4/43 (9%)
MSI-H colorectal cancers have ken observed in coding (Ci)* npeats of the BAX Pc12-
Associated X protein; (299)l and ZGFIlR msulin-Like h w t h Factor II Receptor, (300)J
genes, respectively. While ZGmR mutations were aiways heterozygous (300), four cancers
(19%) with BAX mutations showed bidelic inactivation associated with absent protein
expression (299). Both homozygous and hetemzygous, i n - h e somatic shifts of a (CAG)o
hinucleotide repeat fouad in the coding region of the E2F-4 transcription factor have been
reporteci in 2/2 and 13/31 (42%) MSI-H colorectal cancers (301,302). Relinsnary data also
suggests that both short repetitive and non-qetitive elements in the D M (82-
Microgiobulin) gene may be fkquently mutated in MSI-H colorectal cancers and less
frequently in MSS tumors (303-305).
More recently, severai additional genetic targets of the MSI-H pathway have ken
propose& These include fnuneshift mutations of an (A)** in the MBD4 (methyl-CpG binding
protein) gene in 10123 (43%) MSI-H coiorectd cancers and celi ünes (306). (& and (A)8
mutations of the PïïW (Phosphatase and tensin homologue deleted on chromosome ten)
gene in 6/32 (19%) MSI-H colorectal cancers (307) and (A)* and (A)9 mutations of the RIZ
(Retinoblastoma Rotein-Binding Zinc Finger) gene in 15135 (43%) of MSI-H colorectal
cancers and ce11 lines (308). The majority of these mutations appeared to be heterozygous.
However, in the case of RIZ, reduced transaipt expression was seen in some mutated
cancers. Furthemore, restoration of full-length Rn transcript in one mutant ce11 line resulted
in G 2 M cell cycle arrest and apoptotis.
Establishing the relevance of exonic microsatellite repeat mutation is problematic.
The presence of mutations in any of these genes does not establish their role in colorectal
carcinogenesis. However. compared to the high rate of intronic microsatellite mutation.
coding microsatellites were infrequently mutated in MSI-H colorectal cancers suggesting that
exonic mutations observed may be important (187,269,299,300,302,308-3 1 1). The National
Cancer Institute workshop on microsatellite instability made the foLiowing recommendations
for establishing the role of exonic microsateIlite mutations (153):
1) a hi& fresuency of inactivation in MSI-H cancer;
2) biallelic inactivation by simultaneous aiteration of the other allele's q a t tract,
point mutation, or 10s;
3) involvement of the target gene in a bona fide growth suppressor pathway;
4) inactivation of the same growth suppression pathway in MSS tumors through
inactivation of the same gene, or another gene within the same pathway; and
5) functional suppressor studies in in vitro or in vivo models.
To date, the oniy candidate gene to fulfil ail or most of these requirements is the TGF4 RIZ
gene.
When the MSI-H molecular phenotype was f i t described in colorectal cancer it
appeared that allelic l o s of chromosome 18q and mutations of the K-ras and p53 genes
occumd rare1 y in these cancers in cornparison to MSS colorectai cancers (5 1,148). While
investigators have since reported relatively fiequent activating mutations of the K-ras
oncogene in MSI-H colorectal tumors (16 1 .270T3 12.3 13). rnost studies have confimed that
mutations of the p53 gene occur in only 043% of MSI-H colorectal cancers (156.3 12.3 14).
Similady. chromosome 18q allelîc loss and mutation of the DCC gene are rare in MSI-H
cancers (156,2703 15). Additiondly COX-2. often overexpressed in colorectal cancers with
chromosomal instability, does not appear to be up-repuiated in MSI-H colorectal cancers
(3 16).
MSCH, mismatch repair deficiency and colorectal cancer
phenotype
Given that deficiencies in mismatch npair lead to microsateIlite instabiiity and target
specific gnies by unique mutational spectra to cause colorectai cancer, it seems iikely that
these cancers should be cIinicaHy distinct from those that arise due to the chromosomal
mstabrlity M C gatekeeper pathway. The occurrence of right-si&d colon cancers (proximal
to the splenic flexrne) predominates in individuals with a family history of hereditary
nonpolyposis colorectd cancer (141), gem-line mismatch repair gene mutation camers
(9,270) and sporadic MSI-H colorectal cancers (9,51,148,3 17). Approximately, 70-80% of
MSI-H coloreztal cancers are right-sideci, and fewer than 5% of sporadic le&-sided and rectal
cancers display MSI-H (9).
In addition to gmss pathological distinctions, a number of histopathologic ciifferences
have been noted in MSI-H and mismatch repair colorectal cancers. Approximately 3040%
of MSI-H colorectal cancers are poorly differentiated (1483 173 18), 35-5096 are mucinous
(3 173 18) and 3040% display marked intratumoral or pexitumoral lymphocytic infiltration
(3 173 18). Additionally, MSI-H colorecial cancers frequently display an expanding (as
opposed to an infiltrating) invasion pattern (3 172 18).
The Bethesda criteria for hereditary nonpolyposis colorectal
cancer screening
The Amsterdam criteria were established in an era when the genetic causes of
hereditary nonpolyposis colorectal cancer were unknown and the criteria were developed to
ensure specifkity in identifying individuds with hereditary nonpolyposis colorectal cancer.
Now that a number of genes that cause hereditary nonpolyposis colorectal cancer have been
identifie& these criteria may not be adequately sensitive to be utilized in screening for
hereditary nonpolyposis colorectal cancer. For this reason the National Cancer Institute has
recentty offered the foiiowing Bethesda guidelines for ~creening purposes (3 19):
1, Individuals with cancer in families that meet the Amsterdam criteria
2. hdividuds with two hereditary nonpolyposis colorectai cancer-related cancers
(colorectal, endometrial, ovarian, gastric, hepatobiiiary, small bowel, rend pelvis
or ureter transitional cell)
3. Individuds with colorectal cancer and a h t degree relative with a colorectal
cancer, colorectal adenoma or hereditary nonpolyposis colorectal cancer-related
cancer, one of the cancers diagnosed at age <45 years, and the adenoma
diagnosed at age <40 years.
4. Individuals with colorectal or endometrial cancer diagnosed at age <45 years.
5. Individuais with right-sided colorectal cancer with an undifferentiated pattern on
histopathology diagnosed at age c45 years.
6. Individuals with a signet-ringceII-type colorectal cancer diagnosed at age <45
years.
7. Individuais with adenornas diagnosed at age c4û years.
hdividuals Mfilling any of these criteria should have microsatellite testing of their turnor
and if the MSI-H phenotype is identifieci, germ-line mismatch npair gene testing should be
offered. Jass and colleagues have proposed that tumor histopathology alone may be
diagnosticaiiy distinguishing as a screening method for MSI-H colorectal cancer (3 17).
Similarly, a recent study by Fodde found that the following clinical features:
1. Age of colorectai cancer diagnosis,
2, FulfiUment of the Amsterdam criteria, and
3. A f d y history of endometrial cancer,
were independent predictors of germ-üne mismatch repair gene mutation and thus should be
used m a decision-making Screening algorithm (142).
MSI-H colorectal cancer and patient survival
It was first suggested in 1978 that hereditary nonpolyposis colorectal cancer might be
associated with improved survival(320) and latex studies have often (32 1322,322-324), but
not always (325-328) supported this finding. m i l e these studies were based on famiIy
history of hereditary nonpoIyposis colorectal cancer, a recent population-based series has
shown improved sunrival of MLHl carriers with colorectai cancer (329). None of these
studies however, have addressed whether or not a survival advantage exists for al1 MSI-H
colorectai cancer, of which hereditary nonpolyposis colorectal cancers are in the rninority.
To date, at lecrst 14 snidies have sought to detennine whether MSI-H is prognostic of
improved survivaI(49.5 1,203,272-3 13,330339). Eight (57%) of these studies included fewer
than 100 subjects (513 13,330-332,337-339), and 5 (36%) pooled MSI-L and MSI-H data in
their sumival anal ysis (49,203 ,33033 133 8). In these survival analyses, 61 14 (43%) found no
signifcant univariate association between MSI-H and survivd (2723 13 3 3 1,333,335339).
and 3/14 (21%) did not observe a signifiant muitivariate association (49,334,337).
AdditionaIIy, 3/8 (38%) studies that reported a univariate sumival advantage of MSI-H did
not mclude multivariate analyses (330,332338). Thus, 12/14 (86%) studies to date have not
found MSI-H to be a multivariate predictor of improved survival. Of the two studies that dÏd
find MSI-H to be an independent prognosticator, one pooled MSI-L and MSI-H cancers h m
a single institution case series (203) and one utilized an achowIedged biased subset of cases
from multiple diffcrrnt chemotherapy triais (336).
As previously detailed, the mutationai pathway (chromosomal versus microsatellite
instability) of a partïcular colorectd cancer may determine a number of moIecuiar and
clinical features of the tumor. Interestingly, a number of these feanires have previously been
postulated as pmgnosticators in colorectai cancer includmg chromosome 18q 10s (52),
widespread alletic loss (MO), aneuploidy (40)- decreased DCC expression (341) and p53
mutation (342,343). If MSI-H is indeed an independent predictor of improved survival, these
markers rnay have previously sewed as crude determinants of tumor microsatellite statu in
the previously noted studies.
Thesis overview
nie research contained within this thesis was undertaken to explore various aspects of
microsatellite instability in human colorectal cancer. Specifically, 1 have sought to:
I. Clan@ the role of MSI-H in colorectal cancer prognosis, and
2. Establish the risk of the APC 11307K polymorphism in human colorectal
carcinogenesis and the relationship of this polymorphism to the MSI-H molecular
phenotype.
Chapter two of this thesis, "Turnor microsatellite instabiiity and clinical outcome in
young patients with colorectal cancer", describes a population-based analysis of over 600
young colorectal cancer patients h m Cennal-East Ontario and explores the prognostic role
of MSI-H in conjunction with cuffentty accepted clinical prognostic faftors. Chapter three
and four, "Somatic instability of the APC II307K aIIele in colorectai neoplasia" and
"Inhented colorectal polyposis and cancer risk of the M C II3OZK polymorphism", outline
analyses of neariy 500 Ashkenazi Jewish colorectal cancer and adenornatous polyp patients
treated at Mount Sinai Hospital, Toronto, and assesses the relative risk of the APC I I 3 O X
polymorphism for colorectai neoplasia using genetic and phenotypic methods, Using similar
methods, chapter five, "The M C EI317Q polymorphism does not predispose cimiers to
colorectd adenornatous or hyperplastic polyps", assesses the relative nsks of a second APC
polymorphism, EIJI 7Q in the Mount Sinai colorectal cancer patient series. The final chapter
of this thesis (six), ''Colorectal microsatellite instability? conclusions and future directions",
s rna r izes my thesis research, places these results in the context of the most recently
pubiished literature and discusses the future implications of this work.
C hapter Two
Tumor microsatellite instability and clinicat outcome
in young patients with colorectal cancer
Summary
Background: Colorectal cancer cm arise through two distinct mutational pathways:
microsatellite instabüity or chromosomal instability. We tested the hypothesis that colorectd
cancers arising from the mimsateiIite-instabiüty pathway have distinctive ciinical attributes
that affect clinical outcome. Methods: We tested specimens of colorectal cancer h m a
population-based series of 607 patients (50 years of age or younger at diagnosis) for
microsatetlite instability. We compared the clinical features and survivai of patients who had
colorectai cancer characterized by MSI-H with these characteristics in patients who had
colorectal cancers with MSS. Results: We found MSI-H in 17% of the coiorectal cancers in
607 patients, and in a rnultivariate analysis, microsatellite instability was associated with a
significant survivd advantage independently of a11 standard prognostic factors, including
tumor stage (hazard ratio, 0.42; 95% confidence interval. 0.27 to 0.67; Pc0.00 1).
Furthmore, regardless of the depth of tumor invasion, colorectai cancers with MSI-H had a
decreased 1ikeIihood of metastasizing to regional Iymph nodes (odds ratio, 0.33; 95%
confidence interval, 0.2 1 to 053; P< 0.00 1) or distant organs (odds ratio, 0.49; 95%
confidence intenral. 0.27 to 0.89; P=0.02). Conclusions: MSI-H in colorectai cancer is
indepemdentiy predictive of a relatively favorable outcome and, in addition. reduces the
likelihood of metastases.
Introduction
Colorectai cancer is the third rnost common cancer in Western society (1,344).
Despite advances in screening, diagnosis, and treatment, it is still the second leading cause of
cancer-dated death in North America (1,344). Much has k e n learned over the past decade
about the moleculai genetic alterations that give rise to colorectal cancer. However, this
knowledge has yet to affcct its clinical management substantially, and pathological staging
remahs the basis for prognostication and decisions about therapy (18).
It is now commonly believed that al1 cancers arise as a nsult of the accumulation of
genetic alterations that allow the growth of neoplastic cells (10.44). However, the rate of
random mutational events alone cannot account for the number of genetic alterations found
in most cancers in humans (45). For this reason, it has been suggested that destabilization of
the genome May be a prerequisite early in carcinogenesis (45,46). This "mutator phenotype"
is best understood in colorectal cancer, in which there are two separate destabilizing
pathways (4857). The more common of these mutational pathways involves chromosomal
instability (48,54) characterized by allelic losses (LOH), chromosomal amplifications, and
translocations in colorectai-cancer cells. In the second mutationai pathway, colorectat cancers
display inmased rates of intragenic mutation, characterized by generaiized instability of
short, tandemiy repeated DNA sequences known as microsatellites (50,s 1.148). MSI-H
(instability at 40% or more of microsatellite loci) has k n found in most cases of hereditary
nonpolyposis colorectal cancer (930) as defined by the Amsterdam criteria [which require
that at Ieast three persons from at lest two successive generations have colorectai cancer and
that the disease be diagnosed in at least one of these persons by the age of 50; (14611. In
addition, MSI-H occws in approximately 15% of sporadic cases of colorectai cancer
(951,148).
Normally, mismatches of nucleotides that occur when DNA polymerase inserts the
wrong bases in newly synihesized DNA are repaired by mismatch-repair enzymes. Defects in
mismatch lepair lead to MSI-H in colorectal cancer (169-17 l,l73,L74). Inherited gem-line
mutations of mismatch-repair genes have k e n found in approximately 50% of persons with a
family history that fulfills the Amsterdam critena (144,197). Alterations of the MSH2 and
MLHI mismatch-repair genes account for more than 90% of these cases (10,144). In
addition, acquired, non-inherited alterations of the MLHI gene occur in most sporadic cases
of colorectd cancer with MSI-H (2L09212).
Although colorectal cancer continues to be regardcd as a single distase, it is possible
that colorectai cancers with MSI-H constitute a clinically distinct subtype. A number of
studies have shown that MSI-H occurs relatively Frequendy in colorectal cancers that arise
proximal to the splenic flexure (5 1, Mg), in poorly diffmntiated cancers or those of the
mucinous-ceil type, and in cancers with peritumoral lyrnphocytic infiltration (3 18).
Furthemore, it has been suggested that the survival of patients with colorectal cancers that
have arisen h m the high-fkequency microsatellite-instability pathway is longer than the
srirvival of patients with cancers that have MSS [Tht latter cases constitute the majority of
colorectai cancers; (495 1 203,332336)]. However, these resuits were O btained from small,
uncontrolled, or potentidy biased analyses. We therefore conducted a population-based
study to determine whether MSI-H is an independent predictor of improved survivai in
patients with colorectai cancer.
Methods
Study population
Through the Ontario Cancer Registry, we identified a population-based series of al1
newly diagnosed cases of histopathoIogically confimed colorectai adenocarcinorna in
patients 50 years of age or younger who were residing in CentraEast Ontario (an area with a
population of approximately 4.7 million) between January 1,1989, and December 3 1,1993.
The age of diagnosis of fifty years or less was chosen in order to increase the number of
hereditary nonpol yposis colorectal cancers. Identification through the Ontario Cancer
Registry has been estimated to identify 96% of al1 Ontario residents with a diagnosis of
colonctal cancer (345). After obtaining permission to contact subjects from the physicians
who treated the patients, we collected information on family history and clinical screening
h m the patients or their next of kin, or by reviewing medical charts.
We excluded patients from the study if they did not undergo resection of the primary
colorectal adenocarcinoma or if pathological review did not confirm invasion of the tumor to
at least the level of the submucosa (stage Tl or higher). In total. 640 patients treated at 41
hospitais were eligible for inclusion in the study. Specimens of colorectal cancer h m 607 of
the patients (95%) were available for retrievai and testing. The snidy was approved by the
Human Ethics Committee of the University of Toronto.
Clinicat database
A clinical data base was pnpared by persons with no howledge of the results of
rnolecular genetic testing of each patient's cancer. The date of the patient's fim biopsy or
resection uiat provided a histologie diagnosis of adenocarcinoma of the colon or rectum was
rexorded as the &te of diagnosis of cancer. We classified cancers according to seved gross
and histologie features. With the exception of the preoperative level of serum
cafcinoembryonic antigen, we included aIl College of Amencan Pathologists category 1
factors (pathological stage, tumor ce11 type, tumor grade, and presence or absence of
extramural venous invasion), which are well supported by the literature and are generally
used in patient care (20). Ail specimens underwent histopathological review by a single
pathologist, who was unaware of the results of molecular genetic testing. In accordance with
the classification of tumors by the World Health Organization (19), we defined tumors as
signetdng ce11 or mucinous if 50% or more of the tumor displayed the specified ceU type
and as undifferentiated if featmes of tumor-ce11 differentiation were absent. Other turnors
were classified as "adenocarcinorna, not otherwise specified" or, in rare cases,
adenosquamous carcinoma, if malignant squamous and glanddar components were present.
Distant metastases were judged to be present if they appeared in a histopathological
specimen or if they were identified by the Ontario Cancer Registry within 120 days after
diagnosis. In total, 103 of the 138 cases of distant-organ metastases (75%) were confmed by
histopathological examination.
Radiation treatment in Ontario is provided exclusively at nine speciaiized oncologic-
treamient centers that report to the Ontario Cancer Registry. Data on radiation treatment
initiated within 120 days afk r diagnosis were extracted h m Ontario Cancer Registry
records and were available for ai l study patients. chemotherapy for cancef may be
administered either in oncologic-treannent centers or in other hospitals and clinics in the
province. Information on chemotherapy initiated within 120 days after diagnosis was
acquired h m the data bases of both the Ontario Cancer Registry and the Ontario Institute for
Clinicat Evaluative Sciences and was available for 392 of the 607 sîudy patients (65%).
DNA preparation, microsatellite testing, and analysis
BIocks of surgicdy resected cancerous tissue that had ken fixed in fonnalin and
embedded in pdn were requested h m the relevant pathology departments for ai l
patients. For each specimen, regions of invasive cancer with the highest proportion of
neoplastic cells (median, 80%; range, 40 to 100%) and normal tissue were rnicrodissected,
and DNA was extracted b y proteinase K digestion (293). Samples of genomic DNA were
used to ampli@ sequences @y the polymerase chah reaction) from 5 to 10 of the following
mononucleotide and dinucleotide microsatellite loci: BAT-25, BAT-26, D5S346, D2S 123,
D 17S250, BAT-40, TGF-p RII, D 18SS8, D l8S69, and D US787 (Human MapPairs,
Research Genetics, Huntsville, Na.). These specific microsatellite loci were denved from the
National Cancer Institute reference and alternative laci panel in order to ensure standardized
findings (153). Rimer sequences and conditions of the PCR assay and gel electrophoresis
have k e n published previously (293,346).
The pnsence of additiona1 bands in the PCR product h m tumor DNA, not observed
in DNA from normal tissue from the same patient, was scond as instability at that particula.
locus. In accordance with the National Cancer Institute consensus on microsatellite instability
(153). any pair of samples of normal DNA and tumor DNA that displayed instability at two
or more of five loci was scored as having MSI-H, whereas a sample pair with no instability at
five Ioci was scored as having MSS. Any sampte pair observeci to have instability at one of
five mtmsatelIite Ioci mderwent a second test at that locus. If instability was confirme&
additional loci, up to a maximum of 10, were tested to determine whether the phenotype of
the sample was MSI-L - instability at 1 to 3 of 10 loci assayed - or MSI-H - instability at 4 or
more loci.
Statistical analysis
The primary outcome of this study was overail survivai, measured from the date of
histologie diagnosis of colorectal cancer. The study was designed to determine the prognostic
importance of MSI-H in addition to known prognostic factors. Because the genetic basis of
MSI-L remains poorly understood, 34 and because the incidence of MSI-L was too low in
our series to allow for meaningfd statistical testing, we excluded h m the study 20 patients
(3%) with colorectal cancers characterized by MSI-L before we performed the statistical
anal ysis.
The univariate associations between the presence or absence of MSI-H and base-line
prognostic factors were anaiyzed with a chi-square test for categoricai variables and an
unpaired Student's t-test for continuous factors. The associations of microsatellite status and
the depth of tumor invasion with metastases to regional 1 ymph nodes and distant organs were
evaiuated with multivariate logistic regression. Survival c w e s were prepared according to
the method of Kaplan and Meier (347), and univariate survival distributions were cornpared
with use of the log-rank test. All patients were followed fmm diagnosis until death or until
data were censored (and the patient considered to be alive) as of September 30,1998. A
multivariate sunival analysis was evaîuated according to the Cox proportionai-hazards
modei (348). A mode1 obtained with step-down variable selection, in which alI prognostic
factors were initially entered into the mode1 and m which nonsignificant factors (PAU) were
successively rejected, was compared with the primary model, which included al1 pmgnostic
factors regardess of their measured significance. Ail factors were treated as simple
categorical variables with the exception of age at diagnosis, which was analyzed as a
continuous variable. AU reported P values are two-sideci, and P values of less than 0.05 were
considered to indicate statistical significance.
Results
Clinical characteristics associated with MSI-H
Of the 607 specimens of colorectal cancer that we tested, 102 (17%) were
characterized b y high fmluenc y microsatellite instability, 20 (3 %) had MSI-L, and 485
(80%) had MSS (Figure 2-1). Colorectal cancers with MSI-H were more likely to be poorly
differentiated and located proximal to the splenic flexure than were cancers with MSS (Table
2-1). The patients with coiorectal cancer with MSI-H were more likeiy io have muItipIe
synchronous or metachronous colorectal cancers and received a diagnosis at a younger age
than the patients with colorectal cancers with MSS.
Aithough colorectal cancers with MSI-H were diagnosed at a significantly p a t e r
depth of tumor invasion, these turnors had a significantly Iowa overall pathologicaf stage
than cancers with MSS fiable 2-1). Multivariate logjstic regression demonstrated that both
MSI-H and a Iesser depth of -or invasion were independentiy associated with a decreased
ükelihood of metastases to either regional lymph nodes or distant organs (Table 2-2).
To ensure that treatment did not Mer in an era when the benefits of adjuvant thmpy
were stin king estabiished, we compared the use of chemotherapy and radiation therapy in
patients with colorectai cancer with MSI-H with their use in patients whose cancers had
MSS. Although a trend toward more frequent use of chemotherapy and radiation treatment
was evident in the care of patients whose cancm had MSS (Table 2-11, we found no
signifiant ciifferences in treatment patterns after contmlling for pathologicd stage (P=0.60
for chemotherapy and W. 14 for radiation therapy, according to logistic-regression
anaiysis).
Figure 2-1: Colorectal cancers with MSI-H (MSI) and MSS.
The MSI colorectal cancer displays shifted bands in tumor DNA (T) as cornparrd with
normal (N) DNA at the BAT-25, BAT-26, D2S 123, DSS346, and D17S250 microsatellite
loci. The MSS colorectai cancer has identical bands in tumor and normd DNA at the BAT-
25, BAT-26, D2S 123, and D5S346 microsatel tite loci. In addition, the MSS colorectal cancer
displays los of heterozygosity at the D17S250 locus - that is, a loss of the top (larger) allele
in turnor DNA as compared with norrnai DNA.
N T N T N T N T N T N T N T N T N T N T
Table 2-1 : Characteristics of 587 patients with colorectal cancer evaluated for
ncatmcllt - no. (%)
Table 2-21 Muitivariate analysis of predictive factors for metastases to regional
fymph nodes or distant organs in 587 patients with colorectal cancer.
Mimsatciiïtc scatus M W MSI
Tumor invasion1 Tl§ T2 T3 T4
*CI denotes confidence intcnnl, MSS c o l o d canccr with mieromtel- lice stability, MSI c d o d cancer wich hi&-Gcqucncy mkmatciiitc in- stability, and NA not 3s~~sscd.
$The P ducs d t e d h m th~hypotbcsis &t the o<Ms +O as deta- minai by muithiate I w c icpesion c q d d l l
m o t hasion was chdicd accoirluig a, the8Amaiaii Joint Corn- rnittccan.C;in~tfjasdcfai'btdh;r~tabtcto'l[ablcl~ -
MSCH and standard ciinicar prognostic factors for survival
In total, 272 of the 587 patients (46%) died during a mean follow-up period of
7M.1 years after diagnosis. The sUnnval of patients with colorectal cancers with MSI-H
[mean (6E) five-year s w i v d , 7W%] was significantly better than that of patients with
cancers with MSS [five-year survival, S M % ; P<O.ûûl; (Figure 2-2)]. Colorectal cancers
with mucinous, signet-ring, and undifferentiated ce11 types, poorer grade, higher pathological
stage, or extramural venous invasion were associated with significantly lower s u ~ v a i (Table
2-3).
Information on family history was available for 84 (82%) of the 102 patients who had
colorectai cancer with MSI-H, including 21 of the 29 patients (72%) who died during follow-
up. In total, 13 of these 84 patients (15%) had farniiy histories that fulfilled the Amsterdam
criteria for hereditary nonpolyposis colorectai cancer. Among the patients who had cancer
with MSI-H, no significant difference in survivai was found between those who fulfilled the
Amsterdam criteria (five-year surYival, 77*12%) and those who did not (five year survival,
786%; M.41). Of the 84 patients, only 1 (whose family history did not fuifill the
Amsterdam aiteria) was asymptomatic when a diagnosis was made by clinicai screening.
In a stepdown m~ltiva~ate andysis, the microsatellite status, pathologicai stage,
Nmor grade, and histologic type of the cancer were found to be significantly and
independentiy associated with survivd (Table 2-4). The &val advantage of MSI-H over
MSS was similar in the mode1 that included aiI 12 prognostic variables Iisted in Table 2-1,
regardles of their measured significance (hazard ratio, 0.42; 95% confidence interval, 0.27
to 0.67; Pd.001). The proportionality of the survivd advantage associated with MSI-H cm
be seem in KaplamMeier s w i v d curves stratified according to disease stage (Figure 2-2).
Figure 2-2: Kaplan-Meier sunBml curves for patients with colorectal cancer,
stratified according to microsatellite status.
Tabk 2-3: Univariate analysis of predictive factors for sunrival in 587 patients with
colorectal cancer.
Table 2-4: Significant predictive factors for suMval in a Cox proportional-hazards
anabsis of 587 patients with colorectal cancer.
- . , - 7
, TABLE 4. SI^^ P R E D I ~ T ~ ~ E ~ FACTORS FOR SURWAL IN A Cox P R O P ~ K ~ O N A L - ~ ANALms OF 5- PATEMS
W ~ I COLO~C~TAL Cm-* t
Discussion
Because most cancers are thought to arise from an accumulation of genetic
alterations. it is not surprising that cancers that emerge h m different mutational pathways
should differ ciinicaily. We have found this to be the case for the subgroup of colorectal
cancers that are characterized by MSI-H. In our population-based series. MSI-H was
associated with prolonged survival independently of classic ciinicai prognostic factors.
inciuding the disease stage. Eighty-five percent of the patients who had cancer with MSI-H
did not have a family history suggestive of hereditary nonpolyposis colorectal cancer. For
this reason, the considerable survival advantage confened by MSI-H appears to be applicable
to both heritable and sporadic types of colorectal cancer. Furthermore, in only one of the
patients whose cancer had MSI-H was the cancer diagnosed by clinical screening when he
was asymptomatic; this k t eliminates lead-tirne bias as a Iikely cause of the survivd
advantage. The association of MSEH with improved clinicai outcome has ken suggested
previously (5 1). In other studies. however, no survival advantage was detected
(3 13,334335,333, and a recent National Cancer Institute workshop concluded that
microsatellite instability had not yet ken shown conclusive1 y to be an independent predictor
of prognosis (153). Furthermore, since the first descriptions of MSI-H (50), the literature has
been complicated by inconsistent and confusing definitions of this molecular phenotype
(153). The tem "high-kquency microsatellite instability" is meant to descni a generahed
(not occasional) instability of microsatellite DNA in cancers that aimost dways lack the
ab- to cepair mismatched bases in DNA. For this reason. the National Cancer Institute has
defined MSI-H, MSI-L, and MSS in colorectd cancer in terrns of how many microsatellite
loci and which spocific loci need to be tested and shown to be altered (153). In our study we
used these consensus definitions.
We found a 17% incidence of MSI-H, but in a ment large series reported by
Aaitonen et al. (9), a 12% incidence was found There was a similar difference in incidence
among patients whose family histories fulfilled the Amsterdam critena for henditary
nonpolyposis colorectal cancer (15% in our series and 11% in the study by Aaltonen et al.
(9)). Thus, the differences noted are likely to reflect the fact that our population was
relatively young (dl received a diagnosis at 50 years of age or younger) and thus may have
included a greater proportion of patients with hereditary nonpolyposis colorectal cancer.
Despite their relatively young age, Iess than 1046 of the patients in our cohort had colorectd
cancer associated with hereditary nonpolyposis colorectal cancer, familial adenornatous
polyposis, or infiammatory bowel disease.
Revious case-control studies reported that 58% (349) and 4795 (332) of colorectal
cancers in patients 35 years of age or younger and 40 years of age or younger, nspectively,
had MSI-H. These results highlight the need for unbiased methods of case ascertainment to
use as a basis for calculating accurate frrquencies of motecular markers such as MSI-H.
In addition to MSI-H, we found that the pathological stage of colorectal cancer was
an independent and powemil ptedictor of cünical outcome. This is not surprising, because
the pathologicd stage is the main determinant of outcome for most cancers (18). The fact that
MSI-H was stmgly wociated with a lower stage of cancer, even after we controlled for the
depth of -or invasion, is intnguing. These resdts indicate that MSI-H contributes to
irnproved slwivai in two separate ways. Fit, MSI-H is proguostic of impruved survival
independentiy of other pmgnostic factors, including pathoIogica1 stage. Second, MSI-H is
independently predictive of lower pathologicd stage, thus firrther contributing to the
improved sirrvival through tumor dom-staging.
The mechanism by which MSI-H influences clinical outcome is unknown, but it may
be related to the kinds of mutations or the genetic targets involved in colorectal cancers that
are deficient in DNA-mismatch repair. For example, colorectd cancers with MSI-H have
fewer mutations of the APC (156) andp53 (148,156) genes and more Frequent mutations of
the B-catenin [CTNNBI; (89292)l and transfortning growth factor B receptor type 11 (267)
genes than colorectal cancers with MSS. Distinct clinical and pathological features, such as
the intense lymphocytic infiltrates observed in tumors with MSI-H (3 18). may result h m
these unique genetic alterations and contribute to the less aggressive nature of these cancers.
In addition, the therapeutic effects of DNA-damaging chemotherapeutic agents, such
as fluorouracil, are likely to be influenced by the underlying mutational mechanism. In vitro,
cell lines with MSI-H are less responsive than cell lines with MSS to various
chemotherapeutic agents (350). Furthemore, the targeting of DNA cells that are deficient in
mismatch repair may offer a spedic intervention that does not affect nomal tissues that
ntain mismatch-repair func tion (47).
In conclusion. we detected MSI-H in 17% of colorectal-cancer specimens from a
population-based series of relatively young patients. In most of these patients, there was no
family history suggestive of hereditary nonpolyposis colorectd cancer. MSI-H was found to
be an independent predictor of improved suwival, and tumors with this genetic phenotype
were less like1y to metastasize than those characterized by MSS.
Chapter Three
Somatic instability of the APC 11307Kallele in colorectal neoplasia
Summary
Background: The APC gene is proposed to function as a gatekeeper of colorectal
neoplasia. A germ-Line variant of this gene, the APC II307K allele, is present in
appmximately 6% of the Ashkenazi Jewish population. Methods: To mess the role in
tumorigenesis of the variant (Al8 tract produced by this allele. we undertook a somatic
mutation analysis of the region surroundhg codon 1307 in colorectal tumors h m APC
11307K carriers. Results: Somatic mutations involving the variant (A)8 tract were identified
in 53 of 127 (42%) tumors h m APC II307Kcaniers compared with 5 of 127 (4%)
mutations involving the wild-type dlele of these tumors (P4.0001). Loss of heterozygosity
of the wild-type allele was significantiy more common in tumors with APC 11307K allele
mutations (25 of 41,6146) compared with APC II307K carrier tumors without mutation of
the variant (A)8 tract (12 of 53,2356; Pc0.0005). This somatic biallelic APC inactivation
m e r confirrns the biological importance of the 11307K gem-line variant. The vast
majority of APC 11307K somatic mutations consisted of a single adenine insertion (insA)
involving the variant (A)8 tract This insA mutation was mutualIy exclusive of the presence
of microsatellite instability with O of 49 tumors with insA displaying BAT-26 instability
compared with 9 of 78 himors without insA (P=0.01). Conclusions: These findings
support a mode1 where somatic instability of the (A)8 tract produced by the APC 11307K
ailele leads to increased APC gene inactivation and directiy accounts for 42% of the
coloreçtal neoplasms o c c d n g in APC II307K carriers.
Introduction
The M C II307K polymorphism (codon 1307 isoleucine to lysine), carried by 6.1%
of the Ashkenazi Jewish population, has been observed at an increased frequency in
colorectal cancer patients with a family history of colorectal cancer (295). Analysis of a
srnail number of colorectal tumors from carriers has suggested that the polymorphism may be
a target of incrnwd somatic mutation (295). The wild-type sequence that gives rise to APC
II307K is a T to A transversion altering (AhT(A)4 to an (A)8 repeat. Mononucleotide repeat
sequences have been show previously to undergo somatic mutation in colorectal tumors
with MSI-H (269). MSI-H has been observed in the majority of tumors from individuals with
hereditary nonpolyposis colorectal cancer (50) caused by rnismatch repair deficiency and in
10-208 of sporadic colorectal cancers (51,148). To clarify the mechanistic role of the APC
11307K polymorphism in colorectal carcinogenesis and investigate its association with
MSI-H, we have analyzed a large number of colorectal cancers and adenornas from M C
I I 3 W K carriers. Somatic mutation of the (A), mononucleotide repeat of the APC 11307K
polymorphism was identified at an exceptionally high Çnquency and consisted almost exclu-
sively of a single adenine frameshift insertion. This instability was observed to be mutually
exclusive of the presence of tumor MSI-H. Thus, sequence-specific hypermutability and
bialleüc APC gene inactivation likely lead to colorectal tumor initiation in APC 11307K
carriers.
Methods
Tumor samples
A consecutive series of colorectai cancer patients was screened at our institution to
i&nm APC II3O7K carriers. In accordance with genetic testing guidelines (351) and
institutional approval, tumor samples were obtained h m all subjects, codeci, and stripped of
iâentifiers prior to genetic analysis. In total, we characterized 47 colorectai cancers ruid 80
adenornatous polyps h m germ-line APC 11307K carriers. Residual adenoma adjacent to
CRC was available h m 15 of the colorectal cancers. In addition. nine hyperplastic poiyps, a
colorectal lesion not associated with si gni ficant neoplas tic progression, were identified for
analysis.
Genomic DNA was isolated from rnicrodissected paraffin-embedded samples by
standard pmteinase K digestion (52). Only tumor samples with at least 50% neoplastic
cellularity were used for somatic mutation anaiysis. We have demonstrated previousiy lhat
this threshold allows for reliable mutation detection by direct sequencing (352).
Somatic mutation analysis
Codons 1303-13 17 of the APC gene were PCR amplified from midssected
genornic -or DNA template using fornard primer (5'-AGATTCTGCTAATACCCTGC-
3') and reverse primer (5'-GAACTTCGCTCACAGGATC-3'), and the 83-bp product was
dhctly sequenced (ThennoSequenase; Amenham) using the =verse primer. Tumor DNA
was aIso sequenced using the fornard primer to confm the allele affkcted by somatic
mutation as nee&d. Loss of heterozygosity was cietennineci only in -or samples of greater
than 70% neoplastic cellularity and was judged by eye or cornputerized scanning
densitometry with ImageQuant software where ambiguous. Loss of heterozygosity was
considered to be present by densitometry if the ratio (Al/A2):(T1lT2) was p a t e r than 2 (Al,
the polymorphic adenine of U3O7K; A.2, the immediately adjacent adenine in the sequencing
reaction; Tl, the wild-typ thymidine at codon 1307; R, the nearest thymidine in the
sequencing reaction). Tumor samples of less than 70% neoplastic ceilulanty were considered
non-informative for Loss of heterozygosity analysis.
Microsatellite analysis
Microdissected tumor DNA was analyzed for microsatellite instability at the
polyadenine BAT-26 mononucleotide locus by PCR amplification and denaturing PAGE
using primm and conditions publis hed previous 1 y (269). This locus has been demonstrated
to be 96% sensitive, 100% specific, and highly reproducible for MSI-H (346,353). the
hailmark of misrnatch repair deficiency. Furthemore, because BAT-26 is quasimonomorphic
and alterations consist of large deletions. tumor DNA need not be paired with normal DNA
for analysis (353).
Statistical met hods
Somatic mutation data proportions were compared by 2 test or Fisher's exact test.
Results
In total, 127 separate colorectat tumors (47 invasive cancers and 80 adenomatous
potyps) h m APC U3MK carriers were anaiyzed. Somatic aiterations of M C were
identified in 76 (60%) of 127 tumors. Inactivation of both APC deles was observed in 29
(3 1%) of 94 informative tumors. Mutations predicted to yield a tmncated protein product
were observed in 53 (42%) of the M C 11307K deles, compared with 5 (4%) of the wild-
type deles ~0.0001; Table 3-1). Identical dteraîions in the M C II307K allele were
detected in 15 of 15 residual adenornas adjacent to co1orectaI cancers, contirming that these
mutations occurred earfy during neoplastic progression, prior to the deveIopment of
carcinoma No clifferences were observed in the fiequency or s p e c t - of mutations in
colorectal cancers compared with adenornas (data not shown). APC 11307K did not appear to
contribute to the development of hyperplastic polyps because no somatic mutations were
detected in any of the nine hyperplastic pol yps.
Forty-nine (92%) of 53 predicted truncating APC 11307K mutations consisted of a
single adenine insert (insA) frameshift in the (A)s repeat (Figure 3-1). Inactivation of the
wiid-type allele was observed in 25 of 38 (66%) informative APC I2307K tumors with insA
somatic mutation. Wild-type allelic loss was observed in 25 of 41 (61%) informative tumors
with intragenic II307K alleie mutation compared with 12 of 53 (23%) infonnative tumors
without 11307K mutation (P4.0005). Furthemore, loss of heterozygosity of wild-type
(n=12) and 11307K (n = 10) alleles was similar in 53 infonnative tumors that did not undergo
intragenic mutation of the 11307K allele.
BAT-26 microsatellite instability was present in 9 of 127 (7.1%) tumors (Figure 3-2).
Surprisingly, tumor microsatellite instability and somatic insA mutation of the APC II307K
were rnutually exclusive with microsatellite instability present in 9 of 78 (12%) tumors
without somatic insA compared with O of 49 tumors with insA (W.01). Interestingly, the
oniy tumor with a single adenine deletion in the (A)s repeat tract dernonstrated microsatellite
instability. APC loss of heterozygosity was observed in O of 5 informative tumors wÎth
microsatellite instability compared with 47 of 89 (53%) informative nimors without
mimsateiiite mstability ( P 4 . O .
Table 34: Somatic mutations in A f C 11307K carrier colotectal cancers and
adenornatous polyps.
Table I &ma& nzumhll~ ùi APC 11307K c u d r colorecrcJ c(uu:em Md , arknumotow porrps
1
A M to TAA 1308 GAA CO T M 1306
Total inüagenic mutationsa WH'
>76% tumor cellularity in which no frameshift, subUcitution. or LOH was observe& <70% nimot celIulMty in which ao fnuneshift or substitution was observed.
Figure 3-1 : Reverse primer sequence of APC 11307Kfrom camer colorectal
cancers.
The arrowhead points to the gem-line M C W O 7 K T to A polymorphism. The double
arrowhead points to the start of the somatic fiameshifi single adenine insertion (insA) in the
(A)* mononucleotide q a t of the APC I1307K pol ymorphism. A. Colorectal cancer with
insA of the 11307K allele and no wiid-type loss of heterozygosity. B. Colorectal cancer with
insA of the 11 3O7K allele and wild-type loss of heterozygosity. loss of heterozygosity of the
wild-type ailele is evident, with loss of the "A" corresponding to a thymine nucleotide in the
forward seqwnce.
A. B.
ACGT A C G T
81
Figure 3-2: BAT-26 microsatellite analysis of APC ll3O7K carriers colorectal tumors,
The mwhead points to the nomid BAT-26 product size. Deletions can be seen in lanes 8
and 12 (double arrowhead), both products of tumors without insA somatic mutation. Nomial
BAT-26 product in lane 8 cornes fiom nsidual normal tissue in the tumor sample.
Discussion
We have observed an exceptionally high rate of somatic mutation specifically
targeting the APC 11307K sequence. Although instability of some repeated sequences rnay
occur without functional significance, particdarly in tumors with MSI-H, there are five
separate lines of evidence strongly supporting the biological functional significance of the
APC 11307K insA mutation:
(a) the insA mutation predicts a protein tnincation in the mutation cluster region,
using the same termination codon as other common APC frameshift mutations (354);
(b) the insA mutation is represented as a monoclonal alteration in tumors and is
nadily detected by sequencing bulk DNA without the necessity of subcloning;
(c) IOSS of heterozygosity of the wild-type allele occurs at a very high rate in
association with the insA mutation, confrming biallelic inactivation. This biallelic
inactivation also suggests that the APC 11307K variant has no functional effect per se,
which is further supported by the similar rates of loss of heterozygosity of I M V K
and wild-type alleles in the absence of somatic 11307K mutation;
(d) identical alterations were identified in adenornas adjacent to carcinomas,
consistent with APC inactivation during the noninvasive stage of colorectal neoplasia;
and
(e) no mutations were identified in hyperplastic polyps, a Iesion shown previously to
harbor cIond Eras gene mutations but not APC mutations (7'7).
The mutation rate of the APC II3MK variant is remarkable given that it may be
attributed to a single base pair substitution in an 8.5-kb gene. The relative increase in APC
II307K mutability can be estimated by cornparison with other mutation rates quantifid in
our study. The most Frequent wild-type allele framestiift mutation obsenred in APC 11307K
carrier tumors was codon 1309delAAAAG. This somatic alteration has been reported
previously as one of the most common somatic frameshift APC mutations in colorectd
cancer (288,354). and ou. 1.2% mutation rate of alleles tested is similar to those reported
previously. Our data suggest that the mononucleotide repeat (A)8 of M C Z2307K is
approximately 32 times more mutable than one of the most mutable wild-type APC
sequences (95% confidence interval, 10.4-102.8).
The mutual exclusivity of the APC I1307K insA mutation and the presence of
microsatellite instability is a stnking finding, suggesting that the tumorigenic effects of the
M C 11307K variant are entirely separate From the MSI-H carcinogenic pathway. This
finding is consistent with observations in both yeast and human experiments that
mononucleotide tract deletions. rather than insertions, pndominate in mismatch repair
deficiency (169,288346,355). The relatively Iow rate of frameshift deletion of the M C
11307K (A)8 tract in tumors with microsatellite instability is also interesting. In mismatch
repair deficiency, (A)8 repeats are approximateIy 75 times more mutable than (A)4 tracts
(356). Furthemore, somatic mutation rates of greater than 90% have been desaibed for the
translated (A),* repeat of the transforming growth factor receptor type 11 gene in tumors
with MSI-H (269,288). Our Iow APC mutation rate and the absence of APC Ioss of
hetemzygosity in tumors with microsateMite instability provide further evidence that M C
gene inactivation is a l e s common event in colorectal cancers chanrterized by MSI-H,
compared with those with chromosomal instability (288). These results are also consistent
with ment findings that mutations in the APC bindmg protein B-catenin, rather than APC
itself, are presemt in approximately 50% of tumors with MSI-H (292).
In addition to the APC 11307' insA mutation, we observed two tumors with nonsense
mutations at codon 1308 of the II307K allele, a mutation that has not been reported
previously. In contrast, the two nonsense mutations on the wiId-type ailele were at codon
1306, a mutation that is reported to account for approximately 1% of somatic APC mutations
(354). These findings provide some evidence that the variant (AlB tract couid alter the rate or
profile of specific nucleotide substitutions in addition to the profound effects on frameshift
mutagenesis.
The APC 11307K polymorphism may be targeted for somatic mutation in specific at-
risk individuais or may npresent a sequence variant that is the target of mutation to a sirnila.
degree in most carriers. The predominance of a single (insA) mutation, as opposed to other
aiterations, raises the possibility that there could be an accompanying specific DNA repair
deficiency. For instance, yeast deficient in plymerase delta proofreading are prone to base
pair insertions (356). Although we have not found evidence for involvement of mismatch
repair deficiency in APC 11307K instability, subtle alterations in other DNA repair pathways
cannot be excluded
At Ieast two other possibilities may account for the predominance of a single base
pair insertion in the M C 11307K allele:
(a) conformational structures of DNA andlor repair proteins couid favor (stabilize)
misalignment intemediates tbat lead to insertions. This possibility is supported by
the observation that insertions predominate in mononucleotide repeats in DNA
repair-pmficient yeast (355). Accordingly, the hypermutability codd mereiy be a
reff ection of the instability associated with this mononucIeotide repeat, rather than
a specific DNA repair deficiency;
@) it is possible that a functional ciifference exists between the insertion and deletion
frameshift APC product However, both the insertion and deletion mutations are
predicted to produce a tnincated protein of similar size. Interestingly, the common
APC 1309delAAAAG mutation uses the same termination codon as the more
prevaient APC 11307K insA mutant.
Hereditary factors contribute significanti y to coIorectal tumorigenesis (6). and APC
11307K represents a novel mechanism of cancer predisposition compared with other comrnon
syndromes (Figure 3-3). In familial adenornatous polyposis, because one fmctionally
inactivated copy of APC is inherited, any single inactivating mutation of the second allele
leads to complete abrogation of APC. Inactivation of the APC gatekeeper gene is believed to
initiate colorectd tumorigenesis, and thus familial adenornatous polyposis patients are
characterized by the development of hundreds to thousands of adenornas (10). Mismatch
vair gene inactivation, either due to genn-line inactivation of one allele followed by
somatic loss of the second copy in hereditary nonpolyposis colorectai cancer. or somatic
bidlelic inactivation in sporadic colorectd cancer, leads to a profoundly increased genomic
mutation rate (47). Although adenoma formation may be slightly increased, there is an
accelerated acquisition of mutations requïred for progression to carcinoma (10).
We propose a mode1 in which the APC U3O7K ailele contributes a proportional
increase in the potential mutability of APC via (A)s repeat instabiüty. Fotzy-two percent of
tumors h m APC II307K carriers may be duecdy attnbuted to specific mutation of the
polymorphic Sequence. Compareci with hereditary nonpolyposis coiorectd cancer, this
increased mutation rate is modest and affects only the APC locus. Furthemore, in conarst to
familial adenornatous polyposis, the APC II307K variant m u t still undergo somatic bialleüc
APC inactivation. Thus, the APC II3WK variant may be thought of as a susceptible
gatekeeper allele. With a much reduced iikelihood of turnor initiation compared with familial
adenornatous polyposis and a much slower rate of acquisition of other genetic alterations
compared with hereditary nonpolyposis colorectal cancer, it is not stuprising that the
penetrance of the APC 11307K aiiele should be modest compared with these other inherited
colorectal cancer syndromes. The identification of the APC 11307K allele and other common
low penetrance cancer predisposition alleles will allow intermediate risk target populations to
be recognized both for cost-effective endoscopie screening prognuns and for risk
modification by dietary, chemopreventive. or other measures.
Figure M Gatekeeper inactivation in colorectal carcinogenesis.
Germ-iine FAP APC mutation (R) is followed by somatic inactivation of the second APC
allele and the formation of hundreds to thousands of adenornatous polyps. Gem-Line APC
I1307K carriers undergo somatic mutation of both the hypermutable I1307K allele (N) and
the remaining wild-type allele (0) and have an increased rate of adenoma formation
compared to sporadic tumorigenesis in w hich bidlelic APC inactivation occurs.
Germline Somatic Events Adenoma Formation
Chapter Four
lnherlted coloreetal polyposis and cancer risk
of the APC Il3OïK polymorphism.
Summary
Background: Gemi-line and somatic tnmcating mutations of the APC gene are thought to
initiate colorectal tumor formation in familial adenomatous polyposis syndrome and sporadic
colorectal carcinogenesis, respectively. Recently, an isoleucine to lysine polymorphism at
codon 1307 (11307K) of the APC gene has been identified in 6-7% of the Ashkenazi Jewish
population. Methods: To assess the risk of this common APC ailelic variant in colorectai
carcinogenesis, we have anaiyzed a large cohort of unselected Ashkenazi Jewish subjects
with adenomatous polyps andlor colorectai cancer, for the APC 11307K polymorphism.
Results: The APC 11 3O7K allele was identi fied in 48 (10.1 %) of 476 patients. Compared
with the frequency in two separate population control groups, the APC Il307K allele is
associated with an estimated relative risk of 1.5-1.7 for colorectai neoplasia (both Pcû.0 1).
Furthemore, cornparrd with non-carriers, APC 11307K carriers had increased numbers of
adenornas and colorectal cancers per patient (P=0.03), as well as a younger age at diagnosis.
Conciuslons: We conclude that the M C 11307K variant leads to increased adenoma
formation and directly contributes to 396-496 of ail Ashkenazi Jewish colorectal cancer. The
estimated relative risk for carriers may justify specific dinical screening for the 360,000
Americans expected to harbor this allele, and genetic testing in the setting of long-tem
outcome studies rnay impact significantly on colorectal cancer prevention in this popuIation.
Introduction
Approximately L5-20% of colorectal cancer, the second leading cause of cancer death
in North America, occurs in familial aggregations (6) (344). Familial adenomatous polyposis
caused by an inherited functional mutation of one copy of the APC gene is thought to account
for Iess than 1% of al1 colorectal cancer (10). Gem-line mutation of a DNA mismatch-repair
gene, causing hereditary nonpolyposis colorectal cancer, is beiieved to be responsible for
approximately 2% of colorectal cancer (9). The majority of the remaining hereditary
colorectd cancer is unexplained. It is plausible that relatively comrnon but less penetrmt
alleles may account for a significant proportion of inhented or even seemingly sporadic
colorectal cancer. The isoleucine to lysine polymorphisrn at codon 1307 of the APC gene
(APC II307K) recently has been reported to be carried by 6.1% of New York Ashkenazi
Jewish individuals (295) and 7.0% of Washington, DC, Ashkenazim (296). In contrast with
these control populations, the carrier fiequency of this ailele was significantly elevated, to
2846, in 28 Ashkenazi persons with a persona1 and farnily history of colorectal cancer (295).
Bayesian analysis of genetic iinkage in these families confvmed thîs increased risk of
colorectai cancer (357).
MechanisticaiIy, the sequence encoded by the APC 11307K polymorphism is
hypemutable compared with wild-type M C sequence (295,358). This mutationai
susceptibility leads to somatic bialleiic inactivation of the APC gene and to colorectal
tumorigenesis (358). Despite APC II307K encoding a polyadenine nucleotide repeat,
hypermutability of this sequence was not accounted for by tumor rnimsateIIite instability
(358). Aithough previous findings clearly support the somatic hypermutability of the APC
11307K genornic sequence, subtie functional impairment of the APC II3O7K gene pmduct
cannot be excluded.
The actuai risk of APC 11307K for colorectd neoplasia remains controversid. When
&ta h m patients with a family history of colorectal cancer were combined with data from
additional individuals not ascertained by family history, a 10.4% APC II307K c h e r
fiequency among 21 1 patients with colorectal cancer was observed (295). These findings
support a modest odds ratio of 1.8 [95% confidence interval (CI) 1 .OSZ.8; W .O31 for
subjects with colorectal neoplasia, compared with controls. A similar but statistically
insipnificant risk estimate for colorectal cancer (Odds Ratio 1.9; 95% CI 0.84-4.2) was
observed among 55 individuals with colorectal cancer and 5,026 unaffected controls (296).
The APC I1307K polymorphism was originally identified in an individual with eight
colorectal adenornatous poIyps (295). More recently, the polymorphism was found to be
carried by three (38%) of eight British Ashkenazi Jews with multiple adenornas (359).
However, to date then has ken no large-scde systematic study of the phenotypic efiects of
the APC II307K allele. Thus, the relative nsk of APC II307K for colorectal polyposis and
cancer rernains incompletely understaxi, but it is of great importance to more than 360,000
American Ashkenazi Jews estimated to carry this allele.
To elucidate the attributable risk (attributable risk = exposed population incidence -
unexposed population incidence) and phenotypic effkcts of the M C ll3O7K polymorphism,
we have evduated a large cohort of unselected Ashkenazi Jewish patients with either
colonctai cancer or ahornatous polyps. The APC 113MK variant was present in a
significantiy increased proportion of this colorectal himor population, compared with that in
controls, and caniers w a e observed to have significantly elevated numbers of maiignant and
prernatignant co1orectd neoplasms, compared to nonaniers. These findings support a
significant biological role for this allele in colorectai cancer predisposition.
Methods
Cohort and phenotypic data
Subjects were identified by searching Mount Sinai Hospital records for Jewish
patients who had been admitted for surgery during 1977-97 and who had a diagnosis of
colorectd adenocarcinorna anaor adenomatous polyps. Additionally, a case series of Jewish
patients with either panmatic and ampullary adenocarcinorna who previously had been
analyzed for the BRCA2 mutation (360) were m e r investigated for the cunent study. It is
estimated that greater than 90% of Jews h m the greater Toronto area are of Ashkenazi
ongin [J. Brodbar (Jewish Federation of Greater Toronto, United Jewish Appeai Canada),
personal communication]. Al1 pathology specimens for each subject were reviewed, and a
database was prepared W h patient- and pathology- phenotypic features. Individuals with
familial adenomatous polyposis were excluded h m analysis, and patients with un&rIying
inflammatory bowel disease were analyzed separately. Because accurate and consistent
assesment of family cancer histories codd not be ensured by retmspective chart review of
this cohort, no attempt was made to obtain this information.
Representative normal and tumor tissues from each individual were identified from
pafaffin-embedded surgical pathology samples, and unstained and hematoxylin and eosin-
stained sections were prepared for DNA analysis. In accordance with genetic-testing research
guidelines (351) and institutional approvai, these sarnples were stripped of identifie= and
weze coded to anonymously link them to the phenotypic database ppared @or to blinded
genetic testing. Patients with the same smame were given consecutive DNA-sample
numbers, to indicate that they might be related. Normal genomic DNA was isoiated h m
microdissected paraffin-ernbedded tissues, by standard proteinase K digestion (52).
APC 11307K germ-line analysis
Codons 1303-1317 of the APC gene were amplified by PCR, h m normal genornic
DNA template, with the following primers: forward, S'-AGA~cI:GCTAATACC~GC-3';
and reverse, 5'-GAACrrrCGCTCACAGGATC-3'. Single-strand conformation
polymorphism analysis of the denatund 83-bp PCR product was perforrned by means of 9-
L O watt electmphontic separation on an 8% polyacrylarnide gel witb 5% glycerol, at 47 O C
for 16 hours. Ail positive samples were confirmed by dideoxy chah-termination reaction
ThennoSequenase (Arnersharn) sequencing of an independent PCR amplification product
h m a newiy prepared second genornic DNA sample from the sarne case. Given the unlinked
nature of the samples, neither camers nor non-carriers were notified of the test results.
Statistical methods
APC IljlWK carrier rates in patients with colorectd tumor and in control populations
were compared by either $ or Fisher's exact test, as were other categorical factors (Le.,
gender and matornical site of colorectd cancer) in M C I H O X carrier and non-carrier
patients with colorecial -or. M C II307K carrier and non-carrier continuous variables (Le.
colorectal and extracolonic ttxnor number pet patient and age at diagnosis) were compared
by Student's t-test, wîth Welch's correction for unequal variances when appropriate. Canier
and noncarrier cumulative colorectai-tumor distributions by age at diagnosis were estimated
by the Kaplan-Meier method and were compared by the log-rank test.
Results
We identified 3,535 patients who had undergone surgical resection for either
colorectal cancer or adenorna, and, in their admitting record, 49 1 (13.9%) of these
individuais indicated that they were Iewish. Blocks were retrieved, and amplifiable DNA was
obtained in 476 (96.9%) of of ese 49 1 cases (Figure 4- 1). The APC 11307K polymorphism
was detected in the germ line of 48 (10.1%) of these 476 patients (Table 4-1). The frequency
of this APC alleüc variant was simila. in both the patients with cancer and the patients with
only ademoma Although no colorectal cancer family history data was available, none of the
48 APC II3O7K carriers had redundant surnames.
M C Il307Kcarrier demographic features and tumor phenotype were compared with
those of noncarriers (Table 4-2). No significant difference was found in either patient gender
or anatomic location of colorectal cancer. Mean age at colorectal cancer diagnosis was
approximately 2 years younger in APC 11307K carriers than in non-carriers (W.13).
Cornparison of the cumulative distribution curves @gure 4-2) revealed a significantly
younger age at tumor diagnosis in APC II307K carriers compared with non-carriers (hazard
ratio 1.45; 95% CI 1.09-221; P-O.01)-
Anaiysis of tumor numbers revealed striking ciifferences between APC I1307K
cimiers and non-cmîers (Table 4-2)- The 48 APC If3O7K carriers were fond to have 139
colorectaî cancers and adenornatous polyps, cornparrd with 835 colorectal neoplasms
identified m 428 non-carriers. In temis of odds ratio, this corresponds to a relative-risk
estimate of 1.48 (95% CI 1 .OS-2- 10; W -03) for colorectal neoplasia in M C 11307K carriers.
Furthermore, the APC ZI307K carrier rate steadily inmased with colorectai neoplasm
number (F5gure 4-3; for the trend P=0.001). Of the four patients with more than ten colonic
neoplasms, two were found to be APC 11307K carriers. In conaast to familial adenornatous
polyposis, no microscopie adenornas were identified in routine histologic sections of flat
mucosa from any of the APC II307K carriers. APC 11307K carriers were no more likely to
have extracolonic cancers than were non-carriers (Table 4-2). The only extracolonic cancers
present in carriers were one breast cancer and one bladder cancer, and these were aiso
present, at a similar frequency, in noncarriers (seven breast cancers and eight bladder
cancers). Al1 other extracolonic cancers in the non-carriers were present at Frequencies less
than 1%.
To assess the contribution of APC 11307K to colitis-associated neopiasia and
pancreatic and ampuilary carcinomas. we separately anaiyzed Ashkenazi Iewish individuais
affected by these conditions. Of seven patients with underlying inflammatory bowel disease
and either colorectd cancer or adenoma/dysplasia, none were found to be APC 11307K
carriers. Two (5.7%) of 35 patients with pancreatic cancer and O of 6 patients with ampullary
cancer were identified as king APC 11307K canïers.
Figure 4-1 : SSCP and reverse primer sequence analysis of APC codons 1303-
1317.
a SSCP anaiysis of APC codons 1303-13 17. Lanes N, PCR product h m patients without
APC aiteration. Lane C, PCR product h m a carrier of the APC 11307K alteration. The
arrowhead points to the APC II307K band with aitered electrophoretic mobility. b. Reverse-
primer sequence of APC of genn-line tissue h m an APC II3O7K carrier. The m w h e a d
points to the polymorphic T to A substitution.
Table 4-1 : APC 11307Kcarrier rates.
Tabfe 1
A PC 11307K Carrier Rates .
No. (%)
11307K Carriers Total
Patients with CRC 41 (10.1) 404 Patients wi th adenoma 7 (9.7)
Total 48 (10.1) 476
Table 62: APC ll3OïK carrier and tumor phenotype.
Table 2
APC 11307K Carrier and Tumor Phenotype
Gendes: Male Female
CRC site: Right Le ft Rectum
Mean age of patients f SE (years) Tumot types (per patient + SE):
CRCs Adenornasa
TotaP Extracolonic cancers
Thexe was a signifiant diffetence bmeen APC Il3O7K a i e r s and noncarriers (P < .OS).
Figure 4-2: Cumulative distribution of tinte untii colorectal tumor diagnosis for APC
11307Kcarriers (a) and non-carriers (A).
The time until coIorectaI himor diagnosis in APC I13MK carriers is significantly shifted to a
younger age, compared with that in noncarriers e . 0 1 ) .
30 40 50 60 70 80 90 100
Age of Diagnosis (years)
Figure 4-3: APC 11307Kcamer rate and number of colorectal neoplasms.
A significant correlation between APC IHWK carrier fiequency and an inmasing number of
colorec ta1 neoplasms is observable (W.00 1).
1 2 3 4-5 6-8 r9
Colorectal Neoplasms
Discussion
Our study provides several lines of evidence that APC ZI307K is associated with a
modest but clinicaily agnificant increase in the risk of colorectal cancer. The 10.1% APC
LI307K carrier frequency observed in our unselected patients with either colorectd cancer or
adenoma is significantly elevated compared with the previousl y published 6.1 % (47n66)
carrier rate in non-colorectal cancer Ashkenazi Jewish controls ascertained through a New
York Tay-Sachs screening program [P=0.01; (29511. On the basis of the odds ratio. the
estimated relative risk for colonctd neoplasia in APC 11307K carriers is 1.72 (95%CI 1.13-
2.61). Recentiy, a Washington, DC, senes, which included both unaffected individuals and
those with a variety of cancers, demonstrated an APC I1307K cadet rate of 7.0% (32614,635
(296)). Compared with this controi estimate, our observed rate of APC II307K in patients
with colorectal tumor is significantiy elevated, to a similar degne (odds ratio 1.48; 95% CI
1.08-2.04; M . 0 1). Furthermore, because the APC 11307K pol ymorphism predisposes to
adenoma formation that is often asymptomatic, the carrier rate in control populations may
overestimate the tme unaffected-carrier rate - and thus lead to an underestimate of the risk of
APC 11307K for colorectal neoplasia.
Testing of a large Toronto control group to establish the local carrier rate of M C
11307K was not possible for the current study. However. results of previous studies of
founder mutations in North AmeBcan Ashkenazi populations make feasible the cornparison
of our Toronto APC II3WK &ta with data on previousl y pubüshed controls. Fit, no
significant ciifference was observed in APC ZI3OZK control carrier fresuencies cierived h m
the large New York and Washington, DC, stuclies (295,296). Second, previous stuclies have
reveaied that the rates of three founder mutations of the hexosamùiidase gene were similar in
five urban Amencan and Canadian Ashkenazi populations, including that of Toronto (361).
Third, the rates of three BRCAl and BRCA2 founder mutations have been obsewed to be
similar in different Amerïcan Ashkenazi control populations (295362f 63). Resumably,
carrier rates of aü these allelic variants are similar in various urban centers because they
arose in Ashkenazi ancestors long ago and were not significantly influenced by later
migration to North America
Our odds ratio estimate of 1.72 is slightiy lower than previous risk estimates of the
M C II307K allele but is supported by both a tighter confidence interval and more-powemil
statistical association. The number of patients with colonctal tumon who were included in
the present study is mort than double that in previous studies, and the inclusion of only
unselected cases avoids the potential biases of family history selection (295) and volunteers
(296) that are present in previous APC 11307K colorectal cancer analyses. Our 10.1% carrier
frequency is significantiy lower than either the 28% rate observed in individuals with a
personal and family history of coIorectal cancer w . 0 2 ; (295)] or the 38% rate observed in
persons with multiple adenornas w.04; (359)l. However, both these selected populations
w m very small and may have been influenced by other environmental or genetic modifiem.
Two additional independent findings support a predisposition to colorectal neoplasia in
APC II307K carriers. Fit, cumulative colorectal-tumor distribution by age at diagnosis was
significantiy shifted to a younger age in carriers, compared with that in non-carriers.
Although this effect was small in terms of absolute mean age at diagnosis, it supports a
defimte biologicd effect and is in agreement with previous findings (295). Second, carriers
were found to have an inmase in the number of colorectal neoplasms, with an estïmated
relative risk of 1.48. This value closely p d e l s the relative risk predicted h m camer-rate
data and introduces a novel and potentially powemil method to independently quantify risk
of colorectai-tumor initiation h m low-penetrant exposures (either genetic or
environmentai). If it is assumed that colorectd cancer risk in the Ashkenazi Jewish
population is the same as that in the general Amencan population, then the estimated relative
risks suggest that the iifetime risk that APC 11307K carriers will develop colorectal cancer is
approximately 9-10% (344). Although these relative-risk estimates are consistent with a low-
penetrant predisposition allele, there is a signifcant associated clinical effect due to the
comrnon occurrence of this allele in the population. In fact, both our carrier frequency and
our previously pubüshed allele-specific sornatic mutation rate (358) suggest that 3-4% of d l
Ashkenazi Jewish colorectal neoplasia may be directly attributable to APC II307K. Thus,
althou@ Lifetime risks to the individual carrier are on1 y 9-IO%, this likel y represents a more
significant contribution to the overall burden of colorectal neoplasia in this population than is
imparted by familial adenornatous polyposis and herecütary nonpolyposis colorectal cancer
combined However, one consequence of the relatively Iow penetrance of the APC 11307K
ailele predicted h m the present study is that analyses of allele frequency in probands not
ascertained for colorectal cancer - such as those analyses that ncently have been published
(296,364,365) - are unlikely to &tect a significant association with a positive family history
of colorectal cancer, unless very large numbers of individuais are tested.
The overrepresentation of carriers in the subgroup of individuals with multiple
coiorectal neoplasms is pmticularly interesting h m a chical viewpoint. Overall, there were
59 patients with four or more colorectal neoplasms, who represented 12% of the total cohort,
and 13 (22%) of these 59 were APC II3O7K carriers. Of the four patients with more thaa 10
colorectal neoplasms (range 1&19), two were found to be M C 1.3O7K carriers. Attenuated
adenomatous polyposis coli has previously been charactenzed by both increased adenoma
formation (range 1CL99) and nonsense gem-line mutations at the extreme 5' and 3' ends of
the APC gene (98.366). Our findings suggest that, in addition to these previously observed
AAPC mutations, other mechanisms may contribute to the appearance of multiple adenornas.
We predict that a significant fraction of patients with multiple polyps may have an important
inherited predisposïtion due to either Iess-penetrant APC alterations or other modifier genes.
Results of our study indicate that carefbl examination of the pathological phenotype may be a
powefiul method to accurately identify these patients and characterize the contribution From
their in hented predisposition.
Using several independent lines of evidence, we have demonstrated relative-risk
estimates of approximately 1.5-1.7 for colorectal neoplasia in APC 11307K carriers. These
results raise important public-health issues ngarding clinical screening and genetic-testing
recommendations. Our risk estimates are similar to those that have been calculated for
persons with a family history of either colorectal cancer or adenoma in a first-depe relative
(5,7), for whom the Arnerican Gasûoenterology Association (AGA) has advocated more-
stxïngent chical screening (13). The colorectal cancer age-at-diagnosis data h m the present
study (Figure 4-2) suggest that standard AGA clinical screening beginning at age 50 (13) is
likely to be effective for APC 11307K carriers who do not have a significant family history
but who do have a modestly increased risk for development of colorectal cancer. However, in
view of previous findings in indivïduals with a family history of either colorectd cancer or
adenomatous polyps (295). more rigorous clinical screening beginning at a younger age
should be recommended to APC IUWK carriers who have a significant family history.
Geaetic testing for the APC 11307K polymorphism in the Ashkenazi Jewish population
is a more contentious issue and of great importance to the 360.000 predicted carriers in the
United States. h u e s surmunding positive genetic tests - such as potential negative
psychological effects and difficulties in obtaining insurance - have led the American Society
of Clinical Oncology (ASCO) to support genetic testing for cancer predisposition only in the
setting of a strong family history (367). Such histories are likely to be absent, with the lower
penetrance of APC 11307K. Furthemore, neither a positive nor a negative genetic result for
this allele is Likely to have an impact on clinical screening recommendations in individuals
with a stmng family history. However, several additional factors must be considered before
recommendations regarding testing for the APC 11307K allele are made. Flrst, colorectal
cancer is a common and often fatal disease that is potentially preventable by endoscopic
screening. Second, pa t e r than 80% of colorectal cancer occurs in the absence of a family
history for this disease (6). Third, in accordance with ASCO guidelines (367). APC 11307K
testing is easiiy interpreted, and positive test results are likely to influence medical
management in the majority of individuals. Despite both the common occurrence of
colorectai cancer and the effectivenes of colonoscopy, screening compfiance rernains poor
(368). However, individuals without a family history of colorectal cancer who are aware of
their APC 11307Kcanier status and who are at modestly increased risk for development of
colorectaI cancer may be more motivated to undergo colonoscopie screening. Our data
indicate that genetic testing of Ashicenazi Jews with or without a family history of colorectal
cancer, foiIowed by appropriate clinical screening, might sipnificandy benefit the 940% of
carriers expected to &veIop colorectd cancer. Similarly, screening of these individuak could
potentially lead to either the prevention or early diagnosis of appmximately IO% of ail
Ashkenazi lewish colorectai cancer. Because the full impact of genetic testing on this
population is not currently known, it should only be conducted in conjunction with pre- and
pst-test genetic counseling and in the setting of long-tem outcome research studies. Further
studies will be required to accurately determine how family history and other genetic and
enWonmental factors could influence neoplasia rkks in APC 11307K carriers.
Chapter Five
The APC El3170 polymorphism does not predispose
carriers to colorectal adenornatous or hyperplastic polyps
Summary
Background: Truncating germ-line mutations of the APC gene cause the inherited fstailiai
adenomatous polyposis syndrome characterized by the development of at least one hundred
colorectal pol yps and if left untreated., colorectal adenocarcinorna. Some missense
polymorphisms of the APC gene, such as the codon 1307 isoleucine to lysine. also predispose
carriers to colorectal neoplasia, albeit, at a much reduced penetrance compared to classic
familial adenomatous polyposis mutations. The M C codon 13 17 glutamic acid to glutamine
(E1317Q) has been reported to be carried by small numbers of individuals with colorectal
cancer. adenornas and hyperplastic polyps suggesting a mie for this APC variant in genetic
predisposition to neoplasia Methods: We screened 476 patients with coIorectal cancer or
adenomatous polyps for the APC El 31 7Q gem-line polymorphisrn. Clinical characteristics
of carriers were compared with non-carriers. R B S U ~ : We have observed the APC E1317Q
gem-line variant in 2.3% of476 patients with colorectal cancers a d o r adenomatous polyps.
Carriers of the APC EI 3 1 7Q variant were diagnosed with colorectal neoplasms at an older
age than non-carriers. Furthemore, M C EI317Q carriers had significantly fewer colorectal
neoplasms than non-carriers. No somatic mutations of the variant APC EI317Q sequence
were observed in eleven carrier colorectal tumors studied, Concf usions: These results
suggest that the M C EI3I7Q germ-Iine po1ymorphism does not significantly predispose
carriers to colorectal neoplasia
Introduction
The APC gene is thought to act as the gatekeeper to colorectal epithelial neoplasia
with inactivation of APC initiating adenomatous polyp formation (10). Truncating gem-line
mutations of APC give rise to the highly penetrant familial adenomatous polyposis
syndrome, classicdly characterized by the formation of hundreds to thousands of polyps and
ultimately, if untreated, colorectal cancer. Similar truncating somatic mutations of APC have
ken observed in the majority of sporadic colorectal cancers and adenornas. Recently, it has
ken postulated that some gem-line missense polymorphisms of APC may predispose to
colorectal adenoma and cancer formation, albeit with much reduced penetrance compared to
classic familial adenomatous p l yposis (295,369). One such APC variant, codon 1307
isoleucine to lysine (11307K), has been well characterized and predisposes carriers to an
approximate 50-70% inmase in colorectal cancer nsk due to the introduction of a somatic
mutational hot spot by the 11307K pofymorphic nucleotide sequence (295,358,370).
A second APC germ-line variant, codon 13 17 glutamic acid to glutamine (E1317Q),
has been postulated to predispose to coforectal neoplasia (359,371). Interestingty, one study
has also suggested that the EI317Q polymorphism may contribute to hyperplastic polyp
formation (359), a colorectal lesion that unlike the adenornatous polyp, is not believed to be a
pracursor to cancer formation. While the APC 11307K has been obsewed almost exclusively
in the Ashkenazi Jewish population (295). APC El31 7Q has ken noted in both Jewish and
non-Jewish individuais (359,364).
In coneast to the claims that M C EIJI 7Q predisposes carriers to coloreaal tumor
formation, a recent study found no statisticd différence m the prevdence of this allele in
populations with or without colorectal cancer (372). However, this study lacked statisticai
power to reject even a substantially increased cancer risk for this APC allele. In fact. given
the case prevalence of 0.5-2.446 observed for M C E13I7Q (372). (359)at least 2000 cases
and 2000 controls would be reqWred for sufficient (80%) power to detect a doubling of
colorectal cancer nsk. Thus, it remains incompletely understood if indeed this polymorphism
does in fact predispose carriers to colorectal tumor formation.
In order to assess the risk of colorectal neoplasia conferred by the APC EI317Q
allele, we have screened a large, unselected cohort of Ashkenazi Jewish patients with
colorectal cancers and adenomatous polyps for this polymorphism. The phenotype of carriers
was compared with that of non-caniers and colorectai tumors h m carriers were analyzed for
somatic mutations of the APC EI3I 7Q poIymorphism.
Methods
Cohort and phenotypic date
As previously detailed (370). an unselected case series of 476 Jewish patients with
colorectai cancers (n-404) and adenomatous polyps (n=72) treated at Mount Sinai Hospital.
Toronto, were studied. All pathology specimens for each subject were reviewed and a
database prepared with patient and pathology phenotypic features. Representative normal and
tumor tissues for each individual w a identified h m paraffin-ernbedded surgicd pathology
samples and unstained and hematoxylin and eosin stained sections were prepared for DNA
andysis. In accordance with genetic testing research guidelines (35 1) and institutional
approval, these sampIes were stripped of identifiers and coded to anonymously ünk them to
the phenotypic database prepared pior to bünded genetic testing.
Genetic testing
DNA was isolated h m microdissected pdn-embedded tissues by standard
proteinase K digestion (293). Codons 1303-13 17 of the APC gene were amplified by
polymerase chah reaction h m normal genomic DNA template with the primers: forward
5' GATI'CTGCTAA
TACCCTGC-3' and reverse 5'-GAACTTCGCTCACAGGATC-3. Single strand
conformation polymorphism anaiysis of the denatured 83 base pair PCR product was
perfomed using 9-10 Waîî electrophoretic separation on 8% polyacrylamide gel with 5%
glycerol at 4OC x 16 hom. All samples with altered SSCP bands were directly sequenced by
ThennoSequenase (Arnenham) dideoxy chah-termination reaction of an independent PCR
amplification product h m a newly prepared second genomic DNA sample from the same
case.
Colorectal cancers and adenomatous polyps h m APC EI3I 7Q carriers were
microdissected. PCR amplified and directly sequenced for somatic mutation using the above
primen. In addition a second downstream reverse primer, 5'-CAGTCTGCTGGATLTGG
TTC-3', was used for PCR based sequencing in order to detect mutations spanning codons
1303-1330 of the M C gent. Tumor samples of pa t e r than 70% were considered
informative and loss of heterozygosity was judged present if either polymorphic cytosine
nucleotide or the wild-type guanine were Iess than half the intensity of the other.
Statistical methods
APC E13I7Q carrier and noncarrier tumor number and age at diagnosis were
compared using Mann Whitney non-parametric testing.
Results
Anaiysis of germ-üne DNA from 476 unselected Jewish colorectal tumor patients
reveaIed eleven (2.3%) heterozygous carriers of the M C El31 7Q polymorphism (Figure 5-
1). A p t from these and 48 (10.1%) APC 11307K carriers previousl y reported (370), no other
polymorphîsms were identified in codons 1303 to 13 17 of the APC gene in these individuds.
The number of adenornatous polyps and colorectal cancers observed in El31 7Q camers was
significantly smaller than those observed in either wild-type APC or 11307K carriers (Table
5-1). Furthemore, in a trend similar to that noted in the tumor number data, the mean age of
colorectal tumor diagnosis was more advanced in these APC El31 7Q carriers compared to
non-cmiers Fable 5.4-2). In total. we observed nine colorectal cancers and three
adenomatous polyps in eleven El31 7Q carriea. No trend was observed in anatornic site of
these tumors (right colon: 4, left colon: 5, and rectum: 4) or in colorectal cancer stage (TiS:
1, AJCC Stage 1: O, II: 3, and IIk 4). No hyperplastic polyps were noted in APC EI317Q
carnier colonic pathology specimens and no extracolonic cancers were diagnosed in any of
these eleven carriers.
In order to examine a potential role for the El31 7Q nucleotide sequence in somatic
mutation, we dllectly sequenced codons 1303 to 1330 from microdissected tumor DNA h m
ail nine colorectal cancers and two of thne adenomatous polyps iâentified in the eleven APC
EI3I7Q carriers. None of these eleven tumors revealed somatic mutation of the APC
E13I7Q sequence. Interestingly, of six informative tumors, three had allelie loss of the wild-
type aIlele while none were observed to have lost the El3170 alIeIe (Figure 5-2).
Ftgure 5-1 : SSCP and reverse primer sequence analysis of APC codons 1303-
1317.
(A) SSCP andysis of APC codons 1303-1317. Lanes WT, PCR product h m patients
without M C alterations. Lanes E13I7Q and II307K, PCR products f'm M C El3I7Q and
11307K carrim. respectively. @) Reverse-primer sequence of germ-line M C from an M C
E13I7Q carrier. The arrowhead indicates the polyrnorphic G to C substitution.
B
ACGT
Table 5-1: APC El31 70 carrier phenotype.
N 11 (2.3%) 417 (87.6%) 48 (10.1%)
Patient age (yr. i SE) 76.3 k 3.3 72.1 t 0.5 70.2 t 1.2
Coiorectai tuniors I SE^ 1.09 f 0.09 1.97k0.09 2.90f0.42
wild-type sequence for APC codons 1303-13 17
' fiom reknce (370)
E1317Q versus Wild-type, @.03; El31 7Q versus II307K. @.O07
Figure 5-2: Reversaprimer sequence of APC €13170 from a carrier colorectal
Cancer.
Wild-type M C ailelic loss is evident, with loss of the "C" corresponding to a guanine
nucleotide in the forward sequence (arrow head).
ACGT
Discussion
The contniution of a relatively common, Iow penetrant cancer dele may
dnuaatically outweigh the disease burden of a rare, highly penetrant genetic predisposition.
However, proving the disease association of a low penetrant variant is chaüenging. Linkage
andysis for this type of predisposition is likely to be problematic as low penemce rnakes
identification by significant family history unlikely. Casecontrol studies may overcome this
problem. Our observed 2.3% prevalence of APC EI3l7Q was not significantly elevated
compared to previously reported rates of 0-0.796 in unaffected individuals (359,37 1,372). or
the 0.7% and 2.5% prevalences reported in patients with breast (364) and ovarian cancers
(373). respectively. However, our study, like those previously published (372,373) lacked
sufficient power to definitively conclude that APC E1317Q prevalence differs insignificantly
in individu& with colorectal cancer compared to unaffected controls.
Because colorectal cancers aise from a recognizable precursor, the adenornatous
polyp, and because both these lesions occur commonly in Western society, case and control
colorectal tumor counts offer a powerful method for evaluating relative risk. The
"amplification" of both cases and controls offered by this detection method may be used to
overcome difficulties in detecting modestly increased rïsks imparted by relatively infiequent
deles. We have previously utilized colorectd tumor counts in order to delineate a significant
odds ratio of Iess than two in APC Il3O7K carriers (370). In the current study, APC EI3I 7Q
carriers were observed to harbor significantly fewer colorectal tumors than non-carriers.
Furthermore, while a previous report has suggcsted that the E1317Q polymorphism may
contribute towards hyperplastic polyp formation (359). no such lesions were identified in the
histopathoIogy of segmental coionic resections h m the eleven M C EI3I7Q carriers in this
series.
Cancers in genetically predisposed individuals are typicaily diagnosed at a younger
age than their sporadic counterpart. In the current study. we have demonstrated a trend
towards an older age of diagnosis in APC El31 7Q camers cornpared to non-carrier cases.
Although, this does not disprove a causative role of the APC E1317Q allele. such a
predisposition would have to somehow be linked to a factor associated with advancing age.
One APC van'ant, II307K, has k e n shown to contribute towards colorectal
carcinogenesis by the introduction of a mutational hot spot (295,358). In order to investigate
whether the M C El31 7Q sequence may have a similar effect, we have analyzed the
polymorphism and its surrounding sequences for somatic mutation. While no such mutations
were observeci, three of six informative tumors h m APC E1317Q camers were found to
have undergone allelic loss of the wild-type APC allele. Loss of the E1317Q allele was not
observed This result most likely represents normal variation from the expected equai
distribution of wild-type and El31 7Q delic loss. However, it may hint at subtie hinctional
consequences of the El31 7Q substitution. Similar wild-type ailelic loss has previously been
reported in three APC EI3I7Q carrier tumors and retention of full-length APC EI3I 7Q
protein product has ken observed in a carrier colorectai cancer xenograft (37 1). The APC
EI3I7Q polymorphism substitutes an uncharged hydrophilic amino acid for an acidic
hydrophilic amino acid Taken in the coatext of al1 our results, subtie APC EI3I7Q
functioaal impairment appears unlikely. However, a direct functiond assay of the
polymorphic polypeptide would be required to defi~tively mess this possibility.
PoIymorphic variants of known cancer gatekeeper genes provide attractive candidates
as Iow penetrant disease causing alleles. Despite their modest phenotypic effect, such genetic
variants may conûiiute considerably more to disease burden than much mer, highly
penetrant alleles. Identifying and estimating the risk of these polymorphisms lemains
difficult using current epidemiological rnethods. We have screened a large, unselected
population of colorectal tumor patients in order to investigate a possible causative d e for
APC EI317Q in colorectal carcinogenesis. Carriers of this M C variant appeared
phenotypically, to be no more susceptible to colorectal tumor formation than non-carriers.
Furthemore, somatic mutational analysis of tumors from carriers provided no definitive
evidence of direct involvement of this amino acid substitution in carcinogenesis. Aithough a
causative role of the APC EHI 7Q polymorphism cannot be fully excluded on this bais, Our
data make such involvement unlikely.
C hapter Six
Colorectal cancer microsatelIite instability,
conclusions and future directions
Summary
Colorectal cancer is the third rnost cornmon cancer and the second leading cause of
cancer-related deaths in western societies including Canada Through molecdar genetic
dissection of the adenorna to carcinoma sequence, two geneticaïiy distinct mutational
pathways have been elucidated The more common of these pathways involves chromosomal
instability, and may arise h m deficiencies in mitotic spindle checkpoints. This pathway
appears to target the APC gene for tumor initiation. Additionaily, K-ms activation, p53
mutation, chromosome 18q l o s and COX-2 ovemxpression are commonly observed in
colorectd cancer with chromosomai instability. A second pathway featuring microsatellite
instability due to deficiencies in post-replication DNA mismatch repair is present in 1045%
of sporadic colorectai cancers and the majority of tumors arising in individuals with a farnily
history of hereditary nonpolyposis colorectal cancer. h contrat to colorectal cancers arising
due to chromosomai instability, colorectaf tumors with microsatellite instability appear to be
initiated by either inactivating mutations of the APC gene or stabilizing mutations of the B-
catenin gene. These cancers a l s ~ appear to have other distinct genetic targets including the
TGF-PHI and BAX genes. While commonly regarded as a single disease, the work in
chapter two, 'Tumor microsatellite instabiiity and clinical outcome in young patients with
colonctal cancer" (374), shows that colorectal cancers arising from diffetent mutational
pathways are significandy diffemt with respect to tumor site, histology, metastatic potentid
and prognosis. Chapters three and four, "Somatic instability of the APC 11307K aiiele in
colorectd neoplasia" (358) and 'rilhented colorectal polyposis and cancer risk of the APC
II3WK polyrnorphism" (370) characterize the neoplastic risk of the common Ashkenazi
Jewish M C II3Màr polymorphism- Additionally, this work elucidates the high fhquency
somatic instability of this polymotphism and establishes that it is not due to generahzed
microsatellite instability. FinaUy, chapter five 'The APC E1327Q polymorphism does not
predispose carriers to coionctai adenornatous or hyperplastic polyps" investigates the
relationship of a second APC polyrnorphism with colorectal neoplasia and fin& that this
variant does not likely carry significant tumorigenic nsk.
The clinical phenotype of MSI-H colorectal cancer
Cancer is clinically defined by its ability to invade locaily, metastasize distantiy and
ultimately, kill its host. It is for these reasons that there is no difficulty in clinically
differentiating basal ce11 skin cancer h m adenocarcinorna of the head of the panmas.
Distinguishing between these malignancies is made clearer because they originate in
different organs and are characterized by diflerent histopathologies. Molecular genetic
abnomalities in human cancer have begun to make us appreciate that cancer types once
considered a single entity, may possess subtypes as clinically different as tumors that
originate in differing organs or those displaying differing histopathologies. Perhaps the best-
elucidated example of this is adenocarcinorna of the colorectum. This cancer rnay mise either
due to a chromosomal instability mutator pheno type or a microsatellite instability mutator
phenotype. Both the genetic causes and the genetic effects of these pathways are quite
distinct from one another and have been reviewed extensively in this thesis.
Through the work desctl'bed in chapter two, "Tumor microsatellite instability and
clinical outcome in young patients with colorectal cancer>. it is apparent that MSI-H
co10fectal cancer invades, metastasizes and kiDs differently than colorectal cancers arising
h m the MSS (chromosomal instability) pathway. While we currently regard co1orecta.I
cancer as a single cancer type* these significantly different naturd histories based on
underlying genetic difietences demonstrate that colorectal cancer is at Ieast two different
diseases. These nsults offer both immediate prognostic potentid and raise many research
challenges for the hture. One such challenge is determining the genetic cause of these
differences. Colorectal cancers characterized by MSI-H are known to &se due to
deficiencies in post-repiication DNA mismatch repair. The majority of sporadic MSI-H
coIorectal cancers display MLHI silencing secondary to promoter hypermethylation
(210.21 1). Similady, most genetically characterized hereditary nonpolyposis colorectal
cancer kindreds carry germ-line mutations in either MLHI or MSH2 (188). A number of
other human mismatch repair genes have been identified and more are iikely to be found
(224). The d e played by these genes in colorectal carcinogenesis nmains to be elucidated.
WhiIe no phenotypic differences have yet been noted differentiating cancers arising due to
MLHl compared to MSHZ deficiency or mutational compared to hypermethylation
mechanisms, it is possible that differentiating these carcinogenic origins may explain the
improved prognosis evident in patients with MSI-H colorectal cancers.
Another potentiai explmation for diffkring phenotypes may lie in the genetic targets
of the chromosomal and microsatellite instability pathways. We have recentIy shown that
tmor initiation by stabilizing B-catenin mutation is unique to MSI-H colorectal cancer
compared to MSS cancers that appear to be exclusively initiated by loss of APC gene
fimction (293). While MSI-H colorectal cancers may possess either batenin or APC
mutation (287,293)). these mutations appear to be m u W y exclusive in any particuiar cancer
(292). Thus, batenin mediated tumor initiation rnay potentially explain the improved
clinical outcome observeci for MSI-H colorectd cancer patients. Furtherm~re~ MSI-H cancers
have histologically been observed to generate a potentiaiiy significant antitumor immune
response based on the presence of lymphocytic infiltrates (3 173 18). offenng a potential
clinicd rnechanism for why these tumors are less likely to metastasize and kill.
Current therapeutic regimens for colorectal cancer including radiotherapy and 5-
fluomuracil based chemotherapy were tested and validated without differentiating cancers on
the basis of their mutational pathway (22,2435). Given that prognosis differs in MSI-tI and
MSS colorectal cancers independent of pathologie stage and other standard prognostic
factors, it cannot be assumed that the risks and benefits are the same in treating al1 stage HI
cancers, for instance, (regardless of microsatellite status) with the same chemotherapeutic or
radiotherapeutic protocol. Furthemore, most chemotherapeutic agents exert their actions
through DNA damaging rnechanisrns. It therefore cannot be assumed that cancers that arise
due to mismatch repair deficiency will react the same way to a particular agent as cancers
that evolved through spinde checkpoint deficiency or other chromosomaI instabiüty
mechanisms. Tissue culture experiments suggest that MSI-H ce11 lines may be less
susceptible to 5-fluorourad (375). However, ment clinical data indicates that patients with
MSI-H colorectal cancers, rnay in fact have a better tesponse rate to this chernotherapeutic
agent (376). While causing us to nthink cumnt therapcutics, specific ciifferences in cancer
instability mechanisms may offer cancer-ceil specific targets for future therapeutic agents.
APC 11307Kand the risk of colorectal neoplasla
While we tend to think of inhented diseases in the context of highiy penetrant
syndromes such as famiIiaI adenornatous polypusis and hereditary nonpdyposis cofmctal
cancer, these hi@y penetnmt conditions explain a very smalI proportion ofcolorectd
cancer. It is plausible that the majority of most diseases occur due to gene-environment
interactions in individuals who are genetically 66susceptible" to a particular disease, rather
than those genetically "destineci" to acquire a pdcular disease. The work detailed in
chapters two and t h e , "Tumor microsatellite instability and clinical outcome in young
patients with colorectal cancer" and "Somatic instability of the APC Z1307K ailele in
coloreztai neoplasia" serve to characterize the colorectal cancer risk and mechanism of a low
penetrant, high prevalence allele.
Studies published subsequent to ours have confirmed that the Ashkenazi carrier rate
for the APC 11307K is 5-746 worldwide (377,378). The Toronto Ashkenazi Jewish carrier
rate has been measured at 6.5% (40/614; M. Silverberg, personal communications),
confirming assumptions made when we calculated the colorectal cancer risk of the APC
11307K dlele in our unselected Ashkenazi Jewish colorectal tumor population. Additionally,
the II307K allele hm now been obsenred in Jews of both Yemenite and Sephardic origin, but
at Iesser fhquencies than those seen in the Ashkenazi population (377,378). WhiIe the APC
11307K canier frequency has appeared to be elevated in Ashkenazi breast cancer patients, no
phenotypic or additional genetic evidence such as somatic mutation has been observed to
explain thîs association (296,297,379).
Offering specific clinical recommendations to APC II 3O7K carriers is difficult in the
context of current data and traditional genetic approafhes to disease prevention. Whether an
individual is or is not a carrier would make ünle ciifference on clinîcd endoscopic
recommendations if one were fouowing the current American Gastroenterology Association
protocols (13). Unfortunately, it appears that the minonty of the generai population foUow
such scnening regimens since colorectal cancer remains the second most common cause of
cancer-reIated mortaIity (1) despite king Iargely preventable by current methods. A recent
study suggested that polymorphisms and rare variants of the APC gene may be a common
mechanism of colorectal cancer risk (369). This possibility remains to be explored and offers
great challenges for the future. If a number of the susceptibility loci are recognized, it may be
possible to geneticaily divide the general population into three groups:
1) those with rare, highly penetrant aileles (Le. hereditary nonpolyposis colorectal
cancer, familial adenomatous polyposis),
2) those with common, less penetrant alleles (i.e. APC 11307K). and
3) those without genetic predisposition.
Recognition of these risk groups would allow for more effective screening and prevention
programs aimed at those with a measurably increased risk of colorectal cancer, white
dismissing those who do not possess an increased genetic risk of acquiring this rnalignancy.
A PC Elal 79 does not predispose to colorectal neoplasia
While several compiimentary levels of evidence support the risk of the APC 11307K for
colorectal neoplasia, the same cannot be said for the EI317Q polymorphism. Despite this,
one report concludes that this plymorphism is tikely to be an important contributor to
colorectal adenomatous and hyperplastic poIyposis (359). While our evidence does not
conclusively dismiss this possibility. it does make it highiy unlikely.
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