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Chapter 1
General introduction
Chapter 1
Epidemiology
With over 1.2 million newly diagnosed patients and more than 608.000 deaths annually,
colorectal cancer (CRC) is one of the most frequent malignancies and one of the leading
causes of cancer-related deaths in the world1. This also accounts for the Netherlands,
in which each year 12.000 patients are diagnosed with CRC and 6.000 patients die from
this disease2. Several factors influence the incidence of CRC, such as gender, age, and
geographic region. Both men and women are affected at high rates, however, a higher
incidence and mortality can be seen in men3,4. Like for many cancers, age is a significant
risk factor to develop CRC. More than 90% is diagnosed in persons over age 505. Although
CRC occurs in all races, the incidence is highest in economically developed countries,
such as North America, Europe, Australia and New Zealand. The incidence is lowest in
developing countries such as Africa and South-Central Asia5.
Etiology
The etiology of CRC is complex and heterogeneous. Besides environmental factors,
host-related factors, inherited and somatic (epi)genetic alterations contribute to the
development of CRC.
Environmental and host-related factors
CRC incidence varies between economically developed and developing countries, and
increases in populations that migrate from developing to developed countries6. Hence,
environmental factors such as diet and lifestyle play important roles in the development
of CRC. Risk factors include smoking, obesity, high intake of unsaturated fat and red/
processed meat, excessive alcohol consumption and reduced physical activity7,8. Factors
that decrease the risk of CRC include high intake of fruit, vegetables and fibers, the use
of aspirin or non-steroidal anti-inflammatory drugs (NSAID), calcium, folate, and physical
activity7-9. Furthermore, endogenous factors such as estrogens and androgens are
associated with reduced risk of CRC, and insulin-related hormones with increased risk1012
. A recent model proposed inflammation as an underlying factor of the association with
insulin, estrogen, and energy-related factors (obesity, physical activity, and energy intake)
with CRC11. A role for inflammation as risk factor for CRC development is strengthened
by an increased risk of CRC for patients with inflammatory bowel diseases such as longstanding ulcerative colitis and Chron’s disease13.
Hereditary factors
It is estimated that hereditary factors play a role in 15-30% of all CRCs. Most of these
CRCs are considered familial, with low-penetrant genetic variations or single nucleotide
polymorphisms (SNPs) associated with the increased risk14. In 5% of all cases, CRC arises
in the setting of well-defined inherited syndromes, of which Familial Adenomatous
Polyposis (FAP) and Lynch syndrome are most common. FAP is caused by a germline
mutation of the adenomatous polyposis coli (APC) gene. Somatic inactivation of the
second allele by mutation or less frequently by allelic loss (loss of heterozygosity; LOH)
causes polyp formation. Patients with FAP develop hundreds to thousands polyps in the
colon, which can develop into carcinomas if not removed8,14-16. In Lynch syndrome, unlike
10
General introduction
FAP, most patients do not have an unusual number of polyps. Lynch syndrome, previously
known as hereditary nonpolyposis colon cancer (HNPCC), is mainly caused by germline
mutation in the mismatch repair (MMR) genes MLH1, MSH2 and less frequent in MMR
genes MSH6, PMS1 or PMS2. Somatic inactivation of the second allele, mostly by LOH,
subsequently causes the formation of tumors5,8,17. Recently, it has been discovered that
in a sub-part of Lynch tumors MSH2 can be inactivated by another mechanism as well.
Deletion of the last exons of the upstream gene EpCAM leads to gene-fusion with MSH2
and consequently allele-specific epigenetic gene silencing of MSH218. The disturbance of
the MMR machinery causes an accumulation of DNA mutations throughout the genome
(mutator phenotype) that leads to the formation of a tumor cell. Lynch tumors are
mostly located in the proximal colon and characterized by microsatellite instability (MSI).
Microsatellites are repeating units of DNA that occur normally throughout the genome
and are susceptible to mutations due to replication errors by DNA polymerase slippage.
MMR deficient cells fail to repair those errors. MSI is therefore used as a marker for a
defect in the MMR machinery19.
Somatic alterations
Somatic (epi)genetic alterations accumulate in the genome of all dividing cells as a result
of DNA replication errors or exposure to mutagens. Some somatic alterations provide
a selective growth advantage to the cells and can cause cancer (‘driver’ genes). Other
alterations are biologically neutral and are expected not to contribute to tumorigenesis
(‘passenger’ genes)20. The prevalence and signature of somatic alterations are highly
variable per tumor, but irrefutable includes the activation of proto-oncogenes and
inactivation of tumor suppressor genes, providing tumor cells the required biological
capabilities to survive. These biological capabilities can be summarized into the so-called
hallmarks of cancer: self-sufficiency in growth signals, insensitivity to growth-inhibitory
(antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative
potential, sustained angiogenesis, and tissue invasion and metastasis21. Common somatic
(epi)genetic alterations that occur in colorectal cancer are described in more detail in the
section ‘Molecular pathogenesis of sporadic colorectal cancer.’
Histology, pathology and the adenoma-carcinoma sequence
The normal colon mucosa consists of three main components; the epithelium, a single
layer of columnar cells that creates a barrier between the host and the colon lumen;
the lamina propria; and the muscularis mucosae. The epithelial cells make finger-like
invaginations into the underlying tissue, which are called crypts of Lieberkühn. The basis
of a crypt contains ongoing dividing precursor cells that subsequently differentiate and
migrate towards the luminal surface (Figure 1). Eventually the cells are exfoliated in the
colonic lumen. This process of renewing the epithelium takes 3-8 days22.
It is well established that many, if not all, carcinomas are preceded by pre-malignant
precursor lesions called adenomas. The development of carcinomas from adenomas
is referred to as the adenoma-carcinoma sequence23-25. This does not imply that all
adenomas will eventually become a carcinoma. In fact, it is estimated that only ~5% of
all adenomas will ever progress to a carcinoma9,26,27.
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Chapter 1
Figure 1. Tissue anatomy of the colonic epithelium.
Epithelial cells make finger-like invaginations into the underlying tissue; crypts The
basis of a crypt contains ongoing dividing
precursor cells that subsequently differentiate and migrate towards the luminal surface.
Adapted from: Reya T, Clevers H. Wnt signalling
in stem cells and cancer. Nature 2005;434:84385028
Adenomas
A disturbed balance of cell proliferation and differentiation in the crypt epithelial cells
leads to the formation of polyps. Polyps are quite common in persons over age 60 in
countries with a Western lifestyle29-32. The two most common histologic types are
hyperplastic and adenomatous. Hyperplastic polyps, as the name says are hyperplasias,
do not show dysplastic features and are considered to have no malignant potential33.
However, individuals with hyperplastic polyposis syndrome have an increased risk to
develop CRC34. Adenomatous polyps, or simply adenomas, show dysplastic features,
such as nuclear hyperchromatism, stratification, or atypia33.
The macroscopic appearance of adenomas can be grossly divided in polypoid and
nonpolypoid. Polypoid adenomas protrude above the surrounding mucosa, showing
a pedunculated morphology, i.e. with stalk, or sessile morphology. Nonpolypoid
adenomas include lesions that are slightly elevated, completely flat, or depressed
compared to the surrounding mucosa35-37. The prevalence of nonpolypoid adenomas
varies per study (8% to 42%), but is overall lower compared to polypoid adenomas. The
progression to carcinoma is believed to be faster and more frequent in nonpolypoid
adenomas25,36,38. Yet, it is not clear whether these lesions have a more aggressive
biology, or that their progression rate is higher because they are more easily missed by
colonoscopy.
Besides shape, adenomas have more variable characteristics including size, histology,
and dysplasia. These three pathologic characteristics are currently established as the
most important determinants for malignant progression. From autopsies and endoscopic
studies it has been shown that 70-95% of adenomas are less than 1 cm in diameter39-42.
The size of an adenoma has been linked to risk of progression with adenomas ≥ 1 cm
more likely to progress to carcinoma compared to adenomas < 1 cm43. Adenomas can be
classified in 3 major histological types: tubular, villous and tubulovillous. Adenomas with
villous histology have the highest tendency to progress. Dysplasia is used to describe
12
General introduction
structural and cytological alterations of the epithelial cells9. These abnormalities are
classified as low-grade or high-grade dysplasia, of which the latter is linked to increased
malignant potential25. From this knowledge, literature has adopted the term ‘advanced
adenoma’, which describes adenomas of ≥ 1 cm and/or villous histology (tubulovillous
or villous) and/or high-grade dysplasia44, and are considered as having high potential
to progress. This classification, however, does not fully reflect the (epi)genetic
heterogeneity and complexity of the disease. Because progression of colorectal cancer
is driven by an accumulation of (epi)genetic abnormalities, molecular alterations might
be more specific to discriminate adenomas with high-risk to progress. For example,
chromosomal aberrations of progressed adenomas are highly comparable to that of
cancers, independent of degree of dysplasia45,46. The natural history of adenomas remains
uncertain and difficult to study, however, since all polyps detected at colonoscopy are
removed.
Carcinomas
The vast majority (~95%) of colorectal malignancies are epithelial adenocarcinomas,
which develop from the epithelial cells of the colon mucosa9,47. As mentioned above,
it is well established that carcinomas are preceded by an adenoma, which can be any
type of adenoma. It takes, on average, 8 to 12 years for an adenoma to evolve into a
carcinoma48. A lesion is considered malignant when the neoplastic cells pass through
the muscularis mucosae and infiltrate the submucosa. Carcinomas occur throughout
the colon, but about two-third are located in the left-side of the colon, i.e. distal from
the splenic flexure9,33,49-51 (figure 2). Like adenomas, carcinomas are graded based on
architecture and cytologic features, distinguishing low-grade (well and moderately
differentiated) and high-grade (poorly differentiated) tumors37. Besides, 10% to 20% of
adenocarcinomas show an accumulation of mucin9.
Staging and survival
Staging of colorectal cancer knows different systems. The oldest staging system is the
Dukes classification, which was later modified by Astler and Coller. Dukes A tumors have
invaded the submucosa, but do not extent the muscular wall of the colon. Dukes B tumors
have grown through the muscularis propria into the serosa. Dukes C tumors have at
least one regional lymph node metastasis and Dukes D tumors show distant metastasis9.
Although the Dukes classification is still being used, the tumor, node, metastasis (TNM)
staging system of the American Joint Committee on Cancer (AJCC) and the International
Union Against Cancer (UICC) is now the advised standard for colorectal cancer staging.
In the TNM system, the “T” refers to the depth of invasion of the primary tumor at time
of diagnosis. The “N” refers to the presence of local lymph node metastases, and “M”
refers to the presence of distant metastatic disease53. Table 1 shows the different staging
systems and their classifications.
Survival of colorectal cancer is inversely related to the stage at diagnosis, with 5-years
survival rates of more than 90% for stage I colorectal cancer to up to ~20% for stage IV
colorectal cancer1,54,55 (table 1). At time of diagnosis less than 40% of all colorectal cancer
patients present with localized disease (stage I or II), and about 20% of all colorectal
cancer patients have metastatic disease (stage IV)1. A subgroup of stage II colorectal
13
Chapter 1
Figure 2. Anatomy of the human large intestine and the distribution of colorectal cancers.
Percentages indicate the anatomical distribution of colorectal cancers. The splenic flexure is the boundary between the left (distal) side and the right (proximal) side of the colon. Adapted from: Ang and Nice,
The discovery and validation of colorectal cancer biomarkers, BioMed Chromatogr 2011; 25:82-9952 and
Soreide et al, Evolving molecular classification by genomic and proteomic biomarkers in colorectal cancer: potential implications for the surgical oncologist, Surg Oncol 2009 ;18(1):31-5051
cancer patients, which do not have metastasis at time of surgery, eventually relapse
from recurrent tumors after months or even years after treatment. It is on forehand not
predictable which patients will relapse and which will not. Tumor cells mainly spread via
the blood and lymphatic system, with liver as the most common site of metastasis. Other
sites of metastases are the lungs, peritoneum, pelvis and adrenals, which often become
involved only after hepatic or lymphatic metastases33.
Treatment
Treatment is dependent on the tumor stage at time of diagnosis. Surgical resection
is the first treatment option for most tumors, and is the only treatment with curative
intent. Subsequently the resected specimen is used for pathological examination to
determine the stage of the tumor and to assess prognosis. Adjuvant chemotherapy is
used to eradicate potential micrometastasis in patients with stage III tumors and in a
subset of patients with stage II tumors, and thereby reduces the risk of relapse56. In
patients with advanced disease (i.e. stage IV), chemotherapy is mainly palliative, with
the aim to prolong overall survival and maintain quality of life for as long as possible.
However, metastases restricted to the liver are potentially curable with surgery as well57.
Rectal cancers are often treated with neoadjuvant chemoradiation before surgically
resected58.
5-Fluorouracil (5-FU) has been the only chemotherapeutic regimen for years. The
14
General introduction
Table 1: The different classification systems in colorectal cancer and corresponding survival
rates150-52
AJCC/UICC TNM
Stage 0
Stage I
Stage IIA
Stage IIB
Stage IIIA
Stage IIIB
Stage IIIC
Stage IV
Tis
T1
T2
T3
T4
T1, T2
T3, T4
Any T
Any T
N0
N0
N0
N0
N0
N1
N1
N2
Any N
Modified Astler Coller
M0
M0
M0
M0
M0
M0
M0
M0
M1
N/A
Stage A
Stage B1
Stage B2
Stage B3
Stage C1
Stage C2, C3
Stage C1, C2, C3
Stage D
Dukes
N/A
A
A
B
B
C
C
C
N/A
5-year survival rate
96%
97%
97%
88%
72%
88%
65%
55%
11-20%
AJCC/UICC, American Joint Committee on Cancer/ International Union Against Cancer; TNM, tumor, node, metastasis; N/A,
not available
availability of irinotecan and oxaliplatin last decade has improved patients’ outcome
substantially and standard 1st line chemotherapy now includes fluoropyrimidine-based
therapy combined with either irinotecan or oxaliplatin59. Novel targeted therapies against
angiogenesis (bevacizumab; a monoclonal antibody that targets vascular endothelial
growth factor-A (VEGF-A),) and epidermal growth factor receptor (EGFR) (cetuximab
and panitumumab) have further improved response rates. Bevacizumab combined with
fluoropyrimidine-based chemotherapy is frequently used as first-line treatment for
metastatic colorectal cancer60.
Early detection by screening
Patients can be cured from CRC when the tumor is detected and removed in an early
stage. Since CRC does not often cause symptoms before it has reached an advanced
stage, only a minority of patients is diagnosed while the tumor is still localized61. The
only realistic way to decrease the number of deaths from CRC is by means of screening.
Indeed, incidence and mortality rates of CRC have declined in countries where screening
has been introduced7. For screening to be beneficial, a disease should meet certain
criteria as developed by Wilson and Jungner62. Accordingly, CRC is a suitable candidate
for screening since it has a high prevalence, it has a well-defined precursor lesion
(adenoma) and it has good treatment options63. A variety of screening methods are
available. Because screening involves healthy individuals, the screening method should
be simple, safe, precise and validated62.
Colonoscopy is the gold standard for detecting colorectal tumors, with a sensitivity of
>90% for lesions ≥1 cm in size64. During the procedure, adenomas can be removed and
biopsies of larger lesions can be obtained for pathological examination. Colonoscopy is not
without risk of complications, is an expensive procedure, and needs well trained specialists
to be performed, and would therefore not be the first choice for mass-screening. Other
available screening modalities are flexible sigmoïdoscopy, CT-colonography, and fecal
occult blood testing (guaiac-based fecal occult blood test (gFOBT) or immunochemical
fecal immunochemical test (FIT))64. These methods need follow-up by colonoscopy
when a lesion is found or suspected. FIT is currently the most widely used screening
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Chapter 1
tool in practice. Unfortunately, only half of individuals with a positive FIT test actually
have an advanced adenoma (40%) or CRC (8%) in their colon65. Consequently, half of
individuals with a positive FIT test are offered futile colonoscopy. On the other hand,
among individuals with a negative FIT test there is still a significant number of individuals
that do have a colorectal tumor. Especially high-risk precursor lesions, i.e. advanced
adenomas, are mostly left undetected by FIT66-68, but also around 30% of cancers are
missed66,69. Non-invasive stool- and blood-based molecular tests are in development and
are expected to be more sensitive, specific, and informative compared to current noninvasive screening tools, but are not yet used in clinical practice (for a comprehensive
overview see reference 70 and 71 (chapter 2 of this thesis).
The opinion on which screening approach is the best differs between the USA and
Europe. In Europe there is a stronger preference for organized, programmed screening
using a non-invasive test, such as the FOBT, whereas in the USA sporadic screening using
colonoscopy is most applied72. The overall effectiveness of CRC screening will depend
upon the screening test characteristics (sensitivity and specificity), upon the compliance
of individuals to the offered screening and to further evaluations when tested positive,
and upon the quality of care delivered when a lesion is detected72. In the Netherlands,
population-wide screening to CRC will be implemented in 2013, using FIT as pre-selection
for colonoscopy. All individuals between 55 and 75 of age will be invited to participate
once every two years. With a compliance of 60%, the number of prevented deaths from
CRC is estimated to be 1400 per year66.
Molecular pathogenesis of sporadic colorectal cancer
On the molecular level, colorectal cancer should be considered as a collective term
for a heterogeneous group of diseases. Not only do hereditary types form a distinct
class of tumors, sporadic CRC evolve through multiple pathways as well. In general
the molecular pathogenesis of CRC is associated to the accumulation of genetic and
epigenetic alterations, leading to alterations in tumor suppressor genes and oncogenes
that transform cells and promote tumor progression. For CRC, the two main and best
recognized pathways to tumor progression are the chromosomal instability (CIN)
pathway, comprising ~85% of all CRCs, and the microsatellite instability (MSI) pathway,
comprising ~15% of all CRCs17,73,74.
Chromosomal instability
Chromosomal instability (CIN) is characterized by numerical and/or structural
chromosomal aberrations, resulting in losses and gains of whole or parts of chromosomes.
These chromosomal aberrations show a non-random distribution and are associated
to different times of occurrence in colorectal tumorigenesis. Common observed
aberrations are losses of chromosome 1p, 4q, 5q, 8p, 14, 15q, 17p, 18, 20p and 22q
and gains of chromosome 1q, 7, 8q, 13q, 19q and 2045,46,75-78. The observation that these
aberrations are already present in progressed adenomas implicates their involvement
in the progression from adenomas to carcinomas45,46. Losses of 14q and gains of 1q, 11,
12p, and 19 appear to be late events76.
The fact that these chromosomal aberrations show a non-random distribution indicates
16
General introduction
that these changes provide a growth advantage and lead to clonal expansion of the
altered cells. The affected chromosomal regions therefore may harbor genes that play a
key role in colorectal carcinogenesis. Indeed, tumor suppressor genes are often located
in frequently deleted chromosomal regions, such as APC (5q) and SMAD4 (18q) and
chromosomal gains can increase the expression of proto-oncogenes, such as c-MYC (8q)
and EGFR (7p)77-80. Identifying the relevant cancer-related genes in affected chromosomal
regions is not always straightforward because many chromosomal alterations span large
regions that harbor multiple genes and more than one gene may be important. Gain
of chromosome 20q, for example, is strongly associated with colorectal adenoma to
carcinoma progression and is achieved by gain-of-function of multiple cancer-related
genes at chromosome 20q, rather than affecting a single driver gene45,81. On the contrary,
many genes in the same chromosomal region might be affected, but may not be causally
involved in the neoplastic process.
Microsatellite instability
Microsatellite instable (MSI) tumors are characterized by a high mutation rate because of
a defective mismatch repair (MMR) system. In contrast to the germline mutation of the
MMR genes seen in Lynch syndrome, somatic mutations in MMR genes have rarely been
found in sporadic colorectal MSI tumors. Rather, the majority of sporadic MSI tumors
have their MMR system inactivated by somatic epigenetic silencing of MLH178,82. The
consequence is equivalent, however, with many unrepaired replication errors throughout
the genome. Especially repetitive nucleotide sequences, known as microsatellites,
are prone to these mutations and form the markers to measure the defective MMR
machinery83. The actual cause of tumorigenesis is the increased mutation rate of target
genes that function in various critical cell functions including DNA repair (RAD50, MSH3,
MSH6, BLM), apoptosis (APAF1, BAX, BCL10), signal transduction (TGFβRII, ACTRII, IGFIIR,
WISP3), cell cycle regulation (PTEN, RIZ), transcription factors (TCF4), and other putative
‘driver’ genes83,84. These target genes have in common that they have microsatellite
repeat regions in their coding regions, which are prone to mutation in MMR deficient
cells.
MSI tumors are phenotypically and clinically different compared to CIN tumors. MSI
tumors are characterised by proximal location, mucinous histology, poor differentiation,
and lymphocytic infiltration. In addition, patients with MSI tumors have a better
prognosis51.
Genetic alterations and dysregulated pathways
Colorectal cancer is one of the best characterized tumor types in terms of genetics.
Fearon and Vogelstein have established a genetic model for colorectal tumorigenesis
in 1990, describing the adenoma-carcinoma sequence in which genetic changes in APC,
KRAS, DCC, and TP53 occur in a preferred -but not essential- order85. Research has built
upon this model since and has added substantial knowledge to the biology of CRC and
the key cellular signaling pathways that are dysregulated.
APC plays a prominent role in colorectal tumors and is mutated early in tumorigenesis.
Up to 80% of colorectal tumors, both adenomas and carcinomas, have mutations
that lead to a truncated APC protein86. APC is a multi-functional protein, but the most
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Chapter 1
prominent role in colorectal tumorigenesis is involving the Wnt signaling pathway.
The Wnt signaling pathway functions by regulating the amount of the transcriptional
coactivator beta-catenin, which in turn controls gene expression programs involved in
development and homeostasis e.g. cell proliferation and differentiation87. Wnt signaling
is well controlled in normal cells. In the absence of Wnt, cytoplasmic beta-catenin is
continuously targeted for ubiquitin-mediated proteasomal degradation by a complex of
proteins, including AXIN, APC, CK1 and GSK3. When Wnt binds to cell-surface receptors
of the Frizzled and LRP families, AXIN is recruited to the receptor complex leading to
disruption of the beta-catenin degradation complex. As a consequence, beta-catenin
accumulates, enters the nucleus, and forms a complex with TCF to activate transcription
of Wnt target genes. When APC is mutated, the degradation complex cannot form, and
β-catenin accumulates in the absence of Wnt. Beta-catenin subsequently translocates
to the nucleus where it acts as a transcriptional co-activator of target genes including
oncogenes like c-MYC and Cyclin-D, eventually leading to neoplastic transformation87-89.
Besides mutations in APC, allelic loss (LOH) or epigenetic inactivation of APC by DNA
promoter hypermethylation occurs90, as well as mutations in other Wnt signaling genes,
such as β-catenin91 and AXIN1/292-94.
Other pathways commonly affected in CRC are those of TGF-β (mutations in TGF-β receptor
I and II, mutations and deletions of SMAD2, SMAD4), EGF/MAPK (mutations in KRAS,
BRAF, amplification of EGFR), PI3K (mutations in PIK3CA and PTEN), Notch (consitutively
activated, no mutations described in CRC, but cross-talk with RAS signaling exists), and
Hedgehog (enhanced activation, no mutations described in CRC)48,95.
Obviously, these dysregulated pathways lead to altered processes in a cell, but also
cause an accumulation of genetic alterations that do not necessarily all play a role in
the neoplastic process. Systematic analyses of genetic alterations in CRC have identified
recurrent mutations and chromosomal aberrations that are likely to play a functional
important role during colorectal carcinogenesis and considered as ‘driver’ or candidate
cancer (CAN) genes96,97. This does not imply, however, that low-frequency aberrations are
not biologically or clinically important. These low-frequency aberrations may represent
a different mechanism for tumor initiation or progression in a small sub-set of colorectal
cancers98.
Epigenetics
Besides genetic alterations in cancer, it is increasingly clear that epigenetic alterations
play an equal important role as well. Epigenetics refers to changes in gene expression,
which are maintained through cell division, without alterations in the actual genomic
sequence99. Basically, epigenetic mechanisms are responsible for the different
phenotypes of cells throughout our body whereas they all contain the same genetic
information. The three main epigenetic mechanisms are DNA methylation, histone
modifications, and small non-coding RNAs.
DNA methylation
DNA methylation is the covalent binding of a methyl group (CH3) to the 5’-position of
a cytosine in the context of a CpG dinucleotide. Overall, these CpG dinucleotides are
18
General introduction
underrepresented in the genome, but occur in higher density in short stretches of
DNA (500–4000 bp) called CpG islands. These CpG islands only represent ~1% of the
human genome, but cover more than half of all genes100-102. The addition of methyl
groups to the DNA is carried out by a group of enzymes called DNA methyl transferases
(DNMTs). DNMT3a and DNMT3b are responsible for de novo DNA methylation, which
means methylating DNA that is unmethylated for both strands. DNMT1 is responsible
for maintenance DNA methylation, meaning that DNA methylation is copied to the
newly synthesized DNA strand after replication103,104. Established DNA methylation
consequently involves transcriptional repression. This occurs either direct by inhibiting
transcription factors to bind to their binding sites, or indirect by recruiting methylbinding domain proteins (MBDs) and histone modifying enzymes resulting in chromatin
condensation100,105.
DNA methylation is important in several physiological processes, such as imprinting
(parent-of-origin specific allele silencing), X chromosome inactivation in females,
silencing of transposons and repetitive elements, and ageing100,106. In normal cells
the CpGs throughout the genome are methylated, whereas CpG islands remain
unmethylated. In cancer cells DNA methylation patterns are disturbed and the main
observed events are global, genome-wide, hypomethylation and at the same time
regional hypermethylation of CpG islands. Functionally, global hypomethylation can
cause chromosome instability, activation of transposons, and loss of genomic imprinting.
However, it has also been described that proto-oncogenes can become hypomethylated,
leading to increased expression. DNA hypermethylation mainly occurs in promoter
regions of (tumor suppressor) genes, thereby causing gene silencing100,107-109. DNA
methylation thus represents an alternative mechanism for genetic alterations that lead
to chromosomal instability, activation of proto-oncogenes and inactivation of tumor
suppressor genes. Changes in DNA methylation can affect both alleles of a certain gene,
but can also be added to the Knudson’s two-hit model in which DNA hypermethylation
can be seen as the second inactivating change besides DNA mutation or chromosomal
deletion100,110.
Aberrant DNA methylation is a well-recognized phenomenon in colorectal cancer. Many
(tumor suppressor) genes are described to be hypermethylated in CRC and affect the
same signaling pathways that are targeted for mutational events111,112. A good example
is MLH1, which is mutated in hereditary colorectal cancer, but is bi-allelically methylated
in sporadic colorectal cancers and causes MSI in these tumors82. Another example is the
transcriptional inactivation of negative regulators of the Wnt signaling pathway by DNA
methylation, such as SFRP family members 1, 2, 4, and 5113,114, DKK1-4115,116, DACT3117,
AXIN2118,119, APC119,120, SOX7 and -17121,122, WIF-1114 and Wnt5A123.
A subset of CRCs shows a widespread presence of cancer-specific hypermethylated
genes, a phenomenon which is termed CpG Island Methylator Phenotype (CIMP)124,125.
The definition of CIMP, that is, which genes should be methylated and how many
genes should be examined to define CIMP, is still under debate although the existence
of CIMP is widely accepted125,126. CIMP tumors are believed to be molecularly and
biologically distinct. They are associated with older age, female sex, family history of
CRC, proximal location, mucinous cell differentiation, specific precursor lesions (serrated
adenomas), smoking, MSI, BRAF and KRAS mutations112. To make it more complicated,
within CIMP different subclasses have been described, recognizing CIMP-low or
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Chapter 1
CIMP-high, but also distinct methylation patterns that associate to specific molecular
characterisitcs127,128.
Genome-wide methylation analyses are currently adding substantial knowledge on
differential methylated DNA in (colorectal) cancer. The well recognized concept of
linking promoter CpG island methylation to gene silencing needs some adaptations. For
many genes, the location of hypermethylation within the promoter CpG island varies
per gene but also per tissue-type. Hence, the location of DNA methylation is crucial for
the functional effect129. Also DNA methylation outside the classical promoter CpG island
methylation, such as CpG island shores130, first exons131 and throughout a gene (gene-body
methylation)132 have functionally important effect on gene regulation.
Histone modifications
In its natural state DNA is packaged into chromatin, which is a highly structured and
dynamic complex, consisting of histones and non-histone proteins. The fundamental
unit of chromatin is the nucleosome, which is composed of an octamer of four core
histones H2A, H2B, H3, and H4, around which 147 base pairs of DNA are wrapped.
Histone H1, the linker protein, is bound to DNA between nucleosomes. The N-terminal
tails of the core histones protrude out of the nucleosomal core structure and are subject
to posttranslational modifications, including methylation, acetylation, ubiquitination,
phosphorylation, deamination, sumoylation, ADP-ribosylation and proline isomerisation.
Histone modifications regulate chromatin structure and function, such as gene
transcription, DNA repair, DNA replication, and DNA recombination. Hence, altered
histone modification patterns affect gene expression of crucial genes involved in crucial
cellular pathways, as well as genome integrity and/or chromosome segregation, which
eventually may result in neoplastic transformation133-135. Histone (de)acetylation and (de)
methylation are the best characterized histone modifications. Acetylation of histones
is a hallmark of open chromatin structure and transcriptional activity, and takes place
on lysine residues of all four core histones. Methylation of histones are associated with
either transcriptional active or transcription silent genes depending on their position,
and takes place on lysine and arginine residues on histone H3 and H4134. Altered histone
modification patterns in cancer are the consequence of deregulated activity of proteins
responsible for ‘writing’, ‘reading’ and ‘erasing’ histone modifications135,136. In colorectal
cancer, this includes increased expression of lysine acetyltransferases (CBP and P300)137,
increased expression of lysine deacetylases (HDAC1 and -2)137,138, increased expression of
methyltransferase SUV39H1 (responsible for the repressive mark H3K9 methylation)139,140
and dysregulated expression of polycomb group proteins (PcGs) (EzH2, Suz12, BMI1)141143
. Polycomb-group proteins are a family of proteins that interact with chromosomal
elements to repress genes important for developmental pathways by the repressive
mark H3K27 triple methylation142. In addition, by cross-talk to DNMTs, PcG-mediated
transcriptional silencing might predispose their target genes to promoter CpG island
hypermethylation, since many genes reported to be hypermethylated in CRC are PcG
target genes112,144.
MicroRNAs
Besides epigenetic mechanisms involving DNA and histones that modify gene expression
profiles by changing the accessibility for several factors to the DNA, gene expression
20
General introduction
is also post-transcriptionally controlled by small non-coding RNAs, such as microRNAs
(miRNAs)145. MiRNAs are ~22 nt non-coding RNAs that can hybridize to the 3’ untranslated
regions of mRNAs from protein-coding genes and repress their expression. They do so
mainly by mRNA cleavage, when a perfect complementarity exists between the miRNA
and the target sequence and, for a smaller part, by translation inhibition, when no
perfect complementarity exists between the miRNA and the target sequence146,147.
Each miRNA can target multiple different mRNAs and each mRNA can on its turn be
targeted by more than one miRNA148. The regulatory network of miRNAs is therefore
very complex. miRNAs are highly conserved across different species, are highly specific
for tissue and developmental stage, and play crucial functions in the regulation of
important processes, such as development, proliferation, differentiation, apoptosis, and
stress response149.
Aberrant miRNA expressions contribute to the pathogenesis of all cancers, including CRC.
miRNAs can function either as potential oncogenes or tumor suppressor genes, depending
on the target genes they regulate150. Dysregulation of miRNA expression in cancer occurs
through mutations, epigenetic silencing, and dysregulation of transcription factors, and
miRNAs are also frequently located in fragile or amplified chromosomal regions151. As an
example to the latter, the expression of miRNA-17-92 cluster has been shown to have
an increased expression in association with 13q copy number gain, one of the major
chromosomal aberrations in colorectal adenoma to carcinoma progression152. Other
examples of miRNAs involved in CRC are miRNAs involved in epithelial differentiation
(miRNA-141 and miRNA-200c), WNT signaling (miRNA-145, miRNA-135a and miRNA135b), and migration and invasion (miRNA-373 and miRNA-520c)112.
Interplay between genetics and epigenetics
It is obvious that cancer is not only a genetic disease, and that epigenetic alterations
have at least an equal important contribution. It is likely that these alterations do not
occur as independent mechanisms in a tumor cell but rather are influenced by, or
related to one another. They co-evolve in the same cell, accumulating changes in gene
expression during tumorigenesis, thereby creating the ideal balance in different gene
expression networks, so called ‘pathways’, for a cancer cell to survive. Several aspects of
a relation between genetics and epigenetics have already been mentioned above, such
as epigenetically silenced hMLH1 leading to MMR deficiency and accumulation of genetic
mutations, DNA hypomethylation leading to chromosomal instability, the combination
of DNA hypermethylation and mutation or deletion in Knudson’s two-hit model for
inactivating tumor suppressor genes, or the increased expression of miRNAs due to DNA
copy number gain. Other examples are germline sequence variants (single nucleotide
polymorphisms; SNPs) that make gene promoters prone for being methylated in cancer
cells, e.g. MGMT in lung cancer and MLH1 in CRC153,154, or double strand breaks that
initiate gene silencing and DNA methylation155. The emerging field of genome-wide (epi)
genetic and expression profiling, and integration of these different data, will provide
us with a better insight of the complex interactions of CRC genetics and epigenetics,
which will help us to better understand the disease and hence to develop novel clinical
applications112.
21
Chapter 1
From bench to bedside - the use of molecular markers for early detection and predicting therapy response
The increasing knowledge on the molecular characteristics of CRC provides a large
window of opportunities to improve current clinical practice. Molecular changes related
to the neoplastic process can serve as highly specific markers for early detection,
prognosis, therapy response prediction, and surveillance156,157. Molecular markers can
be measured in body excrements and fluids such as stool and blood, as well as in the
tumor tissue itself, depending on the clinical purpose. A well-known example is serum
carcinoembryonic antigen (CEA), which is widely used as surveillance marker following
curative resection for primary CRC158.
Molecular markers for early detection
Molecular markers have potential to improve current screening tests for the early
detection of CRC. The fecal immunochemical test (FIT) is a widely used screening
tool and detects small traces of human blood in stool. FIT is actually an example of a
molecular test, because it detects the human protein hemoglobin or its early degradation
products68. Yet, improvement is expected by testing for molecules in stool or blood that
are more directly related to the disease process. Research in this field mainly focuses
on the development of new biomarkers with better test-characteristics, large-scale
validation of (combinations of) known markers, and optimization of test methods70. A
variety of molecules have been tested as candidate screening markers in stool and blood.
Most emphasis currently is on DNA and protein markers. Among DNA markers there is
trend to move away from mutation markers in favor of methylation markers, and to use
a panel of markers instead of using single markers70,71. The recent boost in proteomics
research leads to many new candidate protein markers. Although in initial studies, some
of these DNA or protein markers showed a better performance than the FIT or gFOBT,
for which large-scale validation studies are required. Chapter 2 in this thesis provides a
comprehensive overview of the performances of DNA, RNA, and protein markers for CRC
detection in stool and blood.
Molecular markers predicting therapy response
Although CRC is a heterogeneous disease and several systemic therapeutic regimens
are available, treatment protocols are mainly based on a one-size-fits-all concept. Only
a subset of patients actually responds, and consequently many patients unnecessarily
suffer from the toxic effect. Hence, there is a need for markers to select patients
for treatment, in order to reduce toxicity as well as costs, and ultimately to develop
individualized-therapy. Markers that correlate with outcome are either prognostic or
predictive. Prognostic markers correlate with outcome independent of treatment, and
predictive markers correlate to the response of a specific treatment159. A good example
of a predictive marker is KRAS mutation, which causes resistance to treatment with
cetuximab or panitumumab (anti-EGFR therapeutic agents)160. Another example is
the lack of benefit from 5-FU for patients with MSI tumors161. Several prognostic and
predictive markers have been described so far, but most are either not validated, or
show inconsistent results among different studies159. Given the molecular heterogeneity
of CRC, it is expected that several sub-groups of patients exist that differ in their
22
General introduction
prognosis and response on certain treatments, depending on their molecular profile.
One single biomarker will therefore not be relevant for all CRC patients. Nevertheless,
the development of biomarkers that result in response prediction for even a small group
of CRC patients may already be of great value.
Aim and outline of the thesis
Survival from colorectal cancer (CRC) has improved over the past few decades, for which
earlier detection and improved treatment options have played major roles. However,
colorectal cancer remains one of the most common types of malignancies and still half of
the patients diagnosed with colorectal cancer will eventually die. The advantages of early
detection clearly highlight the ongoing need to develop cost-effective, highly accurate
methods to detect colorectal lesions in an early stage. For patients that do present with
advanced CRC several treatment options are available. However, most available drugs
are registered as one size fits all whereas response rates vary substantially, highlighting
the need for methods to identify a priori those patients that will benefit from a specific
treatment.
Because colorectal cancer biologically is a heterogeneous disease we hypothesized that
molecular biomarkers can contribute to these two clinical needs, i.e. improved early
detection and improved therapy response prediction of advanced CRC. In this thesis we
therefore aimed to identify molecular biomarkers (i.e. DNA methylation and proteins)
that improve current non-invasive screening methods (chapter 2-5) and molecular
biomarkers (i.e. DNA methylation) that can predict therapy response (chapter 6). In
addition, we investigated genetic and epigenetic events in CRC and aimed to gain more
insight in the complex interactions of CRC genetics and epigenetics, which will help us to
better understand the disease and hence to develop novel clinical applications (chapter
7-8).
Chapter 2 presents an overview of the literature concerning molecular markers in stool
and blood for the early detection of CRC. In chapter 3, the technical possibilities of
detecting DNA methylation markers in stool were explored by investigating the analytical
sensitivity of a stool-based DNA methylation assay and the rate of DNA degradation in
stool over time. In Chapter 4 we describe the identification of a novel hypermethylated
gene, PHACTR3, which has potential to serve as a biomarker for early detection of
colorectal cancer (CRC) in stool. In addition, we evaluated its complementary value
to a Fecal Immunochemical Test (FIT). Chapter 5 presents a large proteome profiling
study on stool samples from CRC patients and control subjects, in order to identify novel
human protein biomarkers in stool that outperform or complement FIT. In chapter 6,
we describe the identification of a novel hypermethylated gene in CRC, DCR1, which
predicts response to treatment with irinotecan in patients with metastatic CRC. In
Chapter 7 we investigated epigenetic silencing of 5 genes, MBD1, CXXC1, SMAD4, DCC
and MBD2, located on a 4 Mb region on chromosome 18q21; a chromosomal region
which becomes frequently lost during colorectal carcinogenesis. Finally, in chapter 8,
we tested the hypothesis that DNA promoter methylation is one of the mechanisms
involved in controlling gene expression in chromosomal gained regions by integrating
genome-wide DNA methylation, DNA copy number and expression data from CRC cell
lines.
23
Chapter 1
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