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very year in the United States, 160,000 cases of colorectal cancer From the Department of Medicine and
are diagnosed, and 57,000 patients die of the disease, making it the second Ireland Cancer Center, Case Western Re-
serve University School of Medicine and
leading cause of death from cancer among adults.1 The disease begins as a Case Medical Center, Cleveland (S.D.M.);
benign adenomatous polyp, which develops into an advanced adenoma with high- the Howard Hughes Medical Institute,
grade dysplasia and then progresses to an invasive cancer.2 Invasive cancers that are Chevy Chase, MD (S.D.M.); and Brigham
and Womens Hospital, Boston (M.M.B.).
confined within the wall of the colon (tumornodemetastasis stages I and II) are Address reprint requests to Dr. Markow-
curable, but if untreated, they spread to regional lymph nodes (stage III) and then itz at the Division of HematologyOncol-
metastasize to distant sites (stage IV).3-5 Stage I and II tumors are curable by surgi- ogy, Case Western Reserve University,
10900 Euclid Ave., Cleveland, OH 44106, or
cal excision, and up to 73% of cases of stage III disease are curable by surgery at sxm10@cwru.edu; or to Dr. Bertagnolli
combined with adjuvant chemotherapy.3,4,6 Recent advances in chemotherapy have at the Division of Surgical Oncology,
improved survival, but stage IV disease is usually incurable.3,4 Brigham and Womens Hospital, 75 Francis
St., Boston, MA 02115, or at mbertagnolli@
The clinical behavior of a colorectal cancer results from interactions at many partners.org.
levels (Fig. 1). The challenges are to understand the molecular basis of individual
susceptibility to colorectal cancer and to determine factors that initiate the develop- N Engl J Med 2009;361:2449-60.
Copyright 2009 Massachusetts Medical Society.
ment of the tumor, drive its progression, and determine its responsiveness or re-
sistance to antitumor agents. This review summarizes areas of current knowledge,
recognizing that the topics presented are only fragments of the total picture.
The loss of genomic stability can drive the development of colorectal cancer by fa-
cilitating the acquisition of multiple tumor-associated mutations. In this disease,
genomic instability takes several forms, each with a different cause (Table 1).7-26
Chromosomal Instability
The most common type of genomic instability in colorectal cancer is chromosomal
instability, which causes numerous changes in chromosomal copy number and
structure.7 Chromosomal instability is an efficient mechanism for causing the
physical loss of a wild-type copy of a tumor-suppressor gene, such as APC, P53, and
SMAD family member 4 (SMAD4), whose normal activities oppose the malignant
phenotype.2,27,28 In colorectal cancer, there are numerous rare inactivating muta-
tions of genes whose normal function is to maintain chromosomal stability during
replication, and in the aggregate, these mutations account for most of the chromo-
somal instability in such tumors.8 In contrast to some other cancers, colorectal
cancer does not commonly involve amplification of gene copy number29 or gene
rearrangement.
DNA-Repair Defects
In a subgroup of patients with colorectal cancer, there is inactivation of genes re-
quired for repair of basebase mismatches in DNA, collectively referred to as mis-
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The n e w e ng l a n d j o u r na l of m e dic i n e
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Molecular Origins of Cancer
Type of
Type of Instability and Syndrome Defect Genes Involved Phenotype
Chromosomal instability loss Somatic Loss of heterozygosity at Characteristic of 80 to 85% of sporadic
of heterozygosity at mul- APC, TP53, SMAD47,8 colorectal cancers, depending on
tiple loci stage
DNA mismatch-repair defects
Hereditary nonpolyposis Germ-line MLH1, MSH2, MSH6 Multiple primary colorectal cancers, ac-
colon cancer germ-line gene muta- celerated tumor progression, and
tions9-14 increased risk of endometrial, gas-
tric, and urothelial tumors
Sporadic colorectal cancer Somatic MLH1 somatic methyla- Colorectal cancer with increased risk of
with mismatch-repair tion15-17 poor differentiation, more com-
deficiency monly located in right colon, less
aggressive clinical behavior than tu-
mors without mismatch-repair defi-
ciency
CpG island methylator pheno- Somatic Target loci MLH1, MINT1, Characteristic of 15% of colorectal can-
type methylation tar- MINT2, MINT318-23 cers, with most showing mismatch-
get loci repair deficiency from loss of tumor
MLH1 expression
Base excision repair defect Germ-line MYH24-26 Development of 15 or more colorectal
MYH-associated adenomas with increased risk of
polyposis colorectal cancer
nism of gene inactivation in patients with colo unknown, but the phenomenon is reproducibly
rectal cancer.18,20 A methylated form of cytosine observed in about 15% of colorectal cancers and
in which a methyl group is attached to carbon 5 is present in nearly all such tumors with aber-
(5-methylcytosine) defines a fifth DNA base, in- rant methylation of MLH118,19,21,38 (Fig. 2 and 3).
troduced by DNA methylases that modify cyto- The pathogenetic consequence of MLH1 silencing
sines within CpG dinucleotides.18 In the normal is well established, but the contribution of other
genome, cytosine methylation occurs in areas of epigenetic silencing events to colorectal carcino-
repetitive DNA sequences outside of exons; it is genesis remains an area of ongoing study. An
largely excluded from the CpG-rich CpG islands intermediate level of aberrant methylation in
in the promoter regions of approximately half of CIMP may define a subtype (i.e., CIMP2 and
all genes.18 By comparison, in the colorectal-can- CIMP-low) that is thought to account for 30% of
cer genome, there is a modest global depletion of CIMP cases.22,23 A third pattern of aberrant
cytosine methylation but considerable acquisition methylation is exemplified by exon 1 of the gene
of aberrant methylation within certain promoter- encoding vimentin. Although this locus is not
associated CpG islands.18 This aberrant promoter- expressed by normal colon mucosa or colorectal
associated methylation can induce epigenetic si- cancer, it is aberrantly methylated in 53 to 83%
lencing of gene expression.18 In sporadic colorectal of patients with colorectal cancer in a pattern
cancer with microsatellite instability, somatic epi- that is independent of CIMP.39,40
genetic silencing blocks the expression of MLH1.18
Among the loci that can undergo aberrant Mu tat iona l Inac t i vat ion
methylation in colorectal cancer, a subgroup of T umor-Suppr e ssor Gene s
seems to become aberrantly methylated as a
group, a phenomenon called the CpG island APC
methylator phenotype (CIMP, or CIMP-high).18,19 Colorectal cancers acquire many genetic changes,
The molecular mechanism for CIMP remains but certain signaling pathways are clearly singled
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The n e w e ng l a n d j o u r na l of m e dic i n e
Figure 2. Genes and Growth Factor Pathways That Drive the Progression of Colorectal Cancer.
In the progression of colon cancer, genetic alterations target the genes that are identified at the top of the diagram. The microsatellite
instability (MSI) pathway is initiated by mismatch-repair (MMR) gene mutation or by aberrant MLH1 methylation and is further associated
with downstream mutations in TGFBR2 and BAX. Aberrant MLH1 methylation and BRAF mutation are each associated with the serrated-
adenoma pathway. The question mark indicates that genetic or epigenetic changes specific to metastatic progression have not been
identified. Key growth factor pathways that are altered during colon neoplasia are shown at the bottom of the diagram. CIN denotes
chromosomal instability, EGFR epidermal growth factor receptor, 15-PGDH 15-prostaglandin dehydrogenase, and TGF- transforming
growth factor .
out as key factors in tumor formation (Fig. 2 and APC mutations give rise to familial adenomatous
Table 2).41-62 One of these changes, the activation polyposis, an inherited cancer-predisposition syn-
of the Wnt signaling pathway, is regarded as the drome in which more than 100 adenomatous
initiating event in colorectal cancer.2,28,43 Wnt polyps can develop; in carriers of the mutant
signaling occurs when the oncoprotein -catenin gene, the risk of colorectal cancer by the age of
binds to nuclear partners (members of the T-cell 40 years is almost 100%.2,30,43 Somatic muta-
factorlymphocyte enhancer factor family) to tions and deletions that inactivate both copies of
create a transcription factor that regulates genes APC are present in most sporadic colorectal ade-
involved in cellular activation.2,28,43 The -cate nomas and cancers.2,43 In a small subgroup of tu-
nin degradation complex controls levels of the mors with wild-type APC, mutations of -catenin
-catenin protein by proteolysis. A component of that render the protein resistant to the -catenin
this complex, APC, not only degrades -catenin degradation complex activate Wnt signaling.2,4143
but also inhibits its nuclear localization.
The most common mutation in colorectal can- TP53
cer inactivates the gene that encodes the APC The inactivation of the p53 pathway by mutation
protein. In the absence of functional APC the of TP53 is the second key genetic step in colorec-
brake on -catenin Wnt signaling is inappro- tal cancer. In most tumors, the two TP53 alleles
priately and constitutively activated. Germ-line are inactivated, usually by a combination of a
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Molecular Origins of Cancer
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The n e w e ng l a n d j o u r na l of m e dic i n e
Table 2. Tumor-Suppressor Genes and Oncogenes Commonly Associated with Colorectal Cancer.*
* ERK denotes extracellular signalregulated kinase, MAPK mitogen-activated protein kinase, MEK MAPK kinase, PDK1 pyruvate dehydroge-
nase kinase isozyme 1, PI3K phosphatidylinositol 3-kinase, and PKB protein kinase B.
members of the Reference Sequence (RefSeq) data The high degree of genetic heterogeneity makes
base of the National Center for Biotechnology it difficult to determine the clinical effect of indi-
Information.65,66 Cancer-associated somatic mu- vidual mutational events. Moreover, these initial
tations were identified in 848 genes. Of these, results are probably conservative, because some
140 were identified as candidate cancer genes that mutations, which were initially labeled as rare
probably contributed to the cancer phenotype be- passengers in colorectal cancer, have subse-
cause they were mutated in at least two colorectal quently emerged as common and are probably
cancers and when corrected for gene size showed pathogenetic in other cancer types (e.g., an IDH1
more mutations than expected by chance. mutation noted initially in one colorectal cancer
The average stage IV colorectal-cancer genome but subsequently in many gliomas).65,66,74
bears 15 mutated candidate cancer genes and 61 High-throughput sequencing of the colorectal-
mutated passenger genes (very-low-frequency mu- cancer genome has identified new common mu-
tational events). The predominance of low-fre- tational targets. These include the ephrin recep-
quency mutations in candidate cancer genes tors EPHA3 and EPHB6 (receptor tyrosine kinases),
implies enormous genetic heterogeneity among which together are mutated in 20% of colorectal
colorectal cancers, which mirrors the heterogene- cancers, and FBXW7, which functions in a protein
ity of the clinical behavior of colorectal cancers. degradation pathway that regulates levels of cy-
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Molecular Origins of Cancer
clin E and is mutated in 14% of colorectal can by nonsteroidal antiinflammatory drugs prevents
cers.65,66,75 An important challenge is to reduce the development of new adenomas83-86 and medi-
the complexity of the 140 candidate cancer genes ates regression of established adenomas.87
by identifying the biologic pathways and pro-
cesses that are common downstream targets of Epidermal Growth Factor Receptor
multiple mutational events. Epidermal growth factor (EGF) is a soluble pro-
tein that has trophic effects on intestinal cells.
Clinical studies have supported an important role
Genomic Ch a nge s a nd T umor
Pro gr e ssion of signaling through the EGF receptor (EGFR) in
a subgroup of colorectal cancers.88-91 EGFR me-
The sequence of transformation from adenoma diates signaling by activating the MAPK and PI3K
to carcinoma, as initially formulated,2,28,43 was a signaling cascades. Recent clinical data have
model of the development of colorectal cancer in shown that advanced colorectal cancer with tumor-
which specific tumor-promoting mutations are promoting mutations of these pathways includ-
progressively acquired. This model predicts the ing activating mutations in KRAS,92-94 BRAF,95,96
presence of mutations that dictate specific tumor and the p110 subunit of PI3K97 do not respond
characteristics, such as the presence of regional to anti-EGFR therapy.
or distant metastases (Fig. 2). Unexpectedly, the
examination of results of full-genome sequenc- Vascular Endothelial Growth Factor
ing from primary colorectal cancers and distant Vascular endothelial growth factor (VEGF) that is
metastases in the same patient showed no new produced in states of injury or during the growth
mutations in the metastases,76 implying that new of normal tissue drives the production of new
mutations are not required to enable a tumor cell stromal blood vessels (angiogenesis). Clinical stud-
to leave the primary tumor and seed a distant site. ies have suggested a role for angiogenic pathways
Because the ongoing generation of mutations in the growth and lethal potential of colorectal
serves as a molecular clock, the finding that all cancer. Treatment with the anti-VEGF antibody
the mutations in a metastasis are also present in bevacizumab added an average of 4.7 months to
the primary tumor implies that metastatic seed- the overall survival of patients with advanced col-
ing is rapid, requiring less than 24 months from orectal cancer (15.6 months with standard thera-
the appearance of the final mutation in the pri- py).98 The identification of molecular distinctions
mary tumor.76 between cancers that benefit from this treatment
and those that do not remains a challenge.
Grow th Fac t or Path wa ys
S tem- Cel l Path wa ys
Aberrant Regulation of Prostaglandin
Signaling Stem cells in colorectal cancers are believed to be
The activation of growth factor pathways is com- uniquely endowed with the capacity to renew
mon in colorectal cancer (Fig. 2). An early and themselves.99-102 Single colorectal-cancer stem
critical step in the development of an adenoma is cells, by definition, can lodge in a permissive
the activation of prostaglandin signaling.77,78 site, such as the liver, and produce a metastasis.
This abnormal response can be induced by in- Currently, it is not possible to isolate individual
flammation or mitogen-associated up-regulation colorectal-cancer stem cells, although certain
of COX-2, an inducible enzyme that mediates the cell-surface proteins (e.g., CD133, CD44, CD166,
synthesis of prostaglandin E2, an agent strongly and aldehyde dehydrogenase 1) are promising
associated with colorectal cancer.78 Prostaglan- markers. Normal stem cells that reside in the co-
din E2 activity can also be increased by the loss of lonic crypt rely on adhesive and soluble stromal
15-prostaglandin dehydrogenase (15-PGDH), the epithelial interactions to maintain division and
rate-limiting enzyme in catalyzing degradation differentiation. The extent of alterations in these
of prostaglandin.79-81 Increased levels of COX-2 regulatory mechanisms in colorectal-cancer stem
are found in approximately two thirds of colorec- cells is a promising area of investigation, since
tal cancers,78,82 and there is loss of 15-PGDH in agents that control the growth of colorectal-
80% of colorectal adenomas and cancers.79 Clin- cancer stem cells could theoretically be used for
ical trials have shown that the inhibition of COX-2 cancer prevention and treatment.
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The n e w e ng l a n d j o u r na l of m e dic i n e
One ongoing challenge is to translate the wealth The development of molecular diagnostics for the
of knowledge regarding colorectal-cancer genom- early detection of colorectal cancer is an impor-
ics into clinically applicable predictive or prog- tant translation of colon-cancer genetics into
nostic tests (Table 3). The relation between mu- clinical practice. One example is the development
tations in EGFR signaling components RAS and of assays to detect mutations that are specific to
BRAF and anti-EGFR therapy is currently the colorectal cancer and cancer-associated aberrant
only application of colorectal-cancer genomics to DNA methylation in fecal DNA from patients with
treatment.92-96 A few genomic markers are useful colorectal cancer or advanced adenomas. These
for prognosis. For example, germ-line mutations assays have a sensitivity of 46 to 77% for detect-
in tumor-suppressor genes, such as APC, MLH1, ing early-stage colorectal cancer, which is superior
and MSH2, indicate a very high risk of colorectal to the sensitivity of testing for fecal occult blood
cancer and guide the frequency of colorectal- although their superiority in preventing death from
cancer surveillance and recommendations for cancer has not been shown.39,110-113 Stool DNA
prophylactic surgery. Other somatic markers have testing for colorectal cancer has been added to the
modest or unconfirmed prognostic value and are cancer-screening guidelines of the American Can-
not currently used to direct care. Sporadic colo cer Society114 and appears to be equally sensitive
rectal cancers with a mismatch-repair deficiency for detecting advanced adenomas.115 Although still
generally have a favorable prognosis35,103,105,108; in the developmental stage, assays for detecting
poor survival in stage II and III colon cancers is plasma cell-free DNA may also be clinically use-
associated with the loss of p27 (a proapoptotic ful,115 and assays for tumor-specific plasma pro-
regulator of the cell cycle109) or the loss of tein or RNA profiles also remain targets of re-
heterozygosity at chromosomal location 18q.105 search. Questions that remain to be resolved are
* BAX denotes BCL2-associated X protein, EGFR epidermal growth factor receptor, MAPK mitogen-activated protein kinase, PI3K phosphati-
dylinositol 3-kinase, and TGFBR2 transforming growth factor receptor type II.
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Molecular Origins of Cancer
the optimal interval between serial tests and the milial forms of the disease, predictive markers
performance and cost-effectiveness of stool DNA for selecting patients for certain classes of drug
testing as compared with those of newer immuno therapies, and molecular diagnostics for the non-
chemical fecal occult-blood tests.116 invasive detection of early cancers. In addition,
biologic pathways that could form the basis of new
therapeutic agents have been identified. Although
Gene t ic Influence s in
P opul at ion Suscep t ibil i t y some high-frequency mutations are attractive tar-
gets for drug development, common signaling
Genetic epidemiology and twin studies indicate pathways downstream from these mutations may
that 35 to 100% of colorectal cancers and ade- also be tractable as therapeutic targets. Recent
nomas develop in persons with an inherited sus- progress in molecular assays for the early detec-
ceptibility to the disease.117-119 In addition, an tion of colorectal cancer indicates that under-
HNPCC-like syndrome occurs in some families standing the genes and pathways that control the
without any evidence of defects in mismatch re- earliest steps of the disease and individual sus-
pair.120 Several genomic loci that could harbor ceptibility can contribute to clinical management
highly penetrant susceptibility genes have been in the near term.
identified with the use of linkage approaches,121123 An understanding of the signals that dictate
but the underlying mutations are unknown. the metastatic phenotype will provide the infor-
Genomewide association studies have also identi- mation necessary to develop drugs to control or
fied germ-line DNA variants that are strongly as- prevent advanced disease. The considerable recent
sociated with susceptibility, but the clinical use of advances encourage us to believe that improve-
these results is probably limited, since the relative ments in our knowledge of the molecular basis
risk associated with these variants is low.124-129 of colorectal cancer will continue to reduce the
burden of this disease.
C onclusions Dr. Markowitz reports being listed on patents licensed to
Exact Sciences and LabCorp and is entitled to receive royalties
Studies that aid in the understanding of colorec- on sales of products related to methylated vimentin DNA, in
accordance with the policies of Case Western Reserve Univer-
tal cancer on a molecular level have provided im- sity. No other potential conflict of interest relevant to this ar-
portant tools for genetic testing for high-risk fa- ticle was reported.
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