REVIEW

Focus on genetic and epigenetic events of colorectal cancerpathogenesis: implications for molecular diagnosis

Federica Zoratto & Luigi Rossi & Monica Verrico & Anselmo Papa & Enrico Basso &

Angelo Zullo & Luigi Tomao & Adriana Romiti & Giuseppe Lo Russo & Silverio Tomao

Received: 6 December 2013 /Accepted: 12 March 2014# International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Originally, colorectal cancer (CRC) tumorigenesiswas understood as a multistep process that involved accumula-tion of tumor suppressor genes and oncogenes mutations, suchasAPC, TP53 andKRAS. However, this assumption proposed arelatively limited repertoire of genetic alterations. In the lastdecade, there have been major advances in knowledge ofmultiple molecular pathways involved in CRC pathogenesis,particularly regarding cytogenetic and epigenetic events. Mi-crosatellite instability, chromosomal instability and CpG islandmethylator phenotype are themost analyzed cytogenetic chang-es, while DNA methylation, modifications in histone proteinsand microRNAs (miRNAs) were analyzed in the field of epi-genetic alterations. Therefore, CRC development results frominteractions at many levels between genetic and epigeneticamendments. Furthermore, hereditary cancer syndrome andindividual or environmental risk factors should not be ignored.The difficulties in this setting are addressed to understand themolecular basis of individual susceptibility to CRC and to

determine the roles of genetic and epigenetic alterations, inorder to yield more effective prevention strategies in CRCpatients and directing their treatment. This review summarizesthe most investigated biomolecular pathways involved in CRCpathogenesis, their role as biomarkers for early CRC diagnosisand their possible use to stratify susceptible patients into appro-priate screening or surveillance programs.

Keywords Colorectal cancer pathogenesis . Genetic events .

Epigenetic events . Biomolecularmarkers

Introduction

Colorectal cancer (CRC) is the third leading cause of worldwidecancer-related deaths in men and women, and the second lead-ing cause when both genders are combined [1]. The AmericanCancer Society estimates that there will be 142,820 new cases of

F. Zoratto (*)Oncology Unit 2, Azienda Ospedaliera–Universitaria Pisana,Ospedale Santa Chiara, Pisa 56126, Italye-mail: federica.z@libero.it

L. Rossi :M. Verrico :A. Papa :G. L. Russo : S. TomaoOncology Unit, ICOT, Department of Medico-Surgical Sciences andBiotechnologies, “Sapienza” University, Latina 04100, Italy

L. Rossie-mail: dr.rossi@ymail.com

M. Verricoe-mail: monica.verrico@virgilio.it

A. Papae-mail: anselmo.papa@libero.it

G. L. Russoe-mail: giuseppe.lorusso82@gmail.com

S. Tomaoe-mail: silverio.tomao@uniroma1.it

E. BassoOncology Unit, Ospedale San Carlo, Potenza 85100, Italye-mail: enrico.basso79@gmail.com

A. ZulloGastroenterology Division, Ospedale Nuovo Regina Margherita,Rome 00161, Italye-mail: zullo66@gmail.com

L. TomaoBiostatistics and Scientific Direction, Regina Elena National CancerInstitute, Rome, Italye-mail: luigi.tomao@gmail.com

A. RomitiOncology Unit, “Sapienza” University, Ospedale Sant’Andrea,Rome 00189, Italye-mail: adriana.romiti@tin.it

Tumor Biol.DOI 10.1007/s13277-014-1845-9

CRC and 50,830 CRC-related deaths in the United States in2013 [1]. By analyzing the 5-year survival rate for early stage itreaches 60–95 %, while it drops dramatically to 35 % withlymph nodes involvement, indicating that early disease detec-tion and treatment are necessary to improve the management ofCRC patients [2]. It is estimated that about 25 % cases of CRChave a family history of recurrent CRC. However, the majorityof CRC cases (75 % of the patients) have sporadic forms of thedisease [3]. Genes identified in familial CRC syndromes are alsofound to be involved in the genesis of the sporadic cancers.Several analyses have led to the concept of “gatekeepers” and“caretakers” [4]. Gatekeepers are genes that directly regulategrowth by inhibiting proliferation or promoting cell death whilecaretaker genes are responsible for maintaining genetic stability.When a caretaker gene function is lost, all genes experienceincreased mutation rates, but ultimately a gatekeeper is mutatedin both alleles, and tumor initiation is greatly enhanced [4, 5]. InCRC sporadic forms, environmental factors (diet, lifestyle,chronic intestinal inflammation, etc.) are primarily responsiblefor the acquisition of alterations in gatekeepers and caretakersgenes involved in CRC pathogenesis [3]. All of these factorshave led to the theory that CRC is a heterogeneousmultifactorialdisease [6, 7]. Based on this theory, CRC emerges through amultistep process in which genetic and epigenetic alterationsaccumulate in a sequential order. Three different pathogeneticpathways have been implicated in the development of thesetumors: chromosomal instability (CIN), microsatellite instability(MSI) and CpG island methylator phenotype (CIMP), whereasthe involved epigenetic events are DNA methylation, modifica-tions in histone proteins and miRNA aberrations [8]. Therefore,cancer develops through multiple and sequential genetic alter-ations [3, 9], and some patients may present synchronous alter-ations in two or three different pathways [10]. Through clonalselections, the cancer stem cell “chooses” the genetic alterationsmost favorable for its growth and development [11]. Aware ofthe fact that a more thorough understanding of these molecularpathways may contribute to improved strategies for prevention,screening, diagnosis, and therapy, we reviewed the most inves-tigated biomolecular pathways involved in CRC tumorigenesis,their role as biomarkers for early CRC diagnosis and theirpossible use to stratify susceptible patients into appropriatescreening or surveillance programs.

Sporadic colorectal cancer

Genetic alterations background: “adenoma–carcinomasequence” model

CRC is believed to be caused by a cascade of genetic mutationsleading to progressively disordered DNA replication and accel-erated colonocyte replication (Table 1). The progressive accu-mulation of multiple genetic mutations results in the transition

from normal mucosa, to benign adenoma, to severe dysplasia tofrank carcinoma [5]. The steps involved in CRC tumorigenesiswere first described in the classic “adenoma–cancer sequence”model proposed by Fearon and Vogelstein [12]. They outlined afour-step sequential pathway for the development of cancer, thatis characterized by both the over-activation of oncogenes andinactivation of tumor suppressor genes: step 1— AdenomatousPolyposis Coli (APC) gene inactivation causes adenoma devel-opment; step 2— Kirsten Rat Sarcoma Viral Oncogene Homo-log (KRAS) mutations promote adenoma growth; step 3— Lossof heterozygosity (LOH) allowed adenoma progression; step 4— Tumor Protein 53 (TP53) inactivation triggers the finaltransition to carcinoma. This sequence occurs in 60 % of CRCcases and generally the activation of an oncogene or inactivationof a oncosuppressor gene, involved in this sequence, is associ-ated with clinicopathological tumor features (Table 2) [12].

Step 1. APC gene inactivation

The APC gene was a gatekeeper for the colorectal adenoma,mapped to chromosome 5q and usually its alteration is associ-ated with familial adenomatous polyposis (FAP) [13]. The APCgene encodes a huge protein consisting of 2,843 amino acids thathas been implicated in various cellular functions. In particular,APC protein is best understood as a negative regulator of thetranscription factor β-catenin, the effector of the Wnt signalingpathway (the name is derived from Drosophila melanogastermutant wingless), that controls the coordinated expansion anddifferentiation of the intestinal crypt stem cells (colonic epithelialhomeostasis) [14].WhenWnt is absentβ-catenin protein is also,because it is phosphorylated and subsequently degraded by atrimeric complex known as “destruction complex”, which con-tains gene products of GSK-3β, APC and AXIN. In the pres-ence of the Wnt signal, the “destruction complex” is inactivatedand intracellular β-catenin levels increase, as a result β-cateninprotein proceeds to the nucleus and stimulates cell proliferations,by transcriptional activation of c-myc, cyclin D1, gene encodedmembrane factors (MMP-7, CD44), growth factors and Wntpathway feedback regulators [15]. Normally, Wnt signal path-way is inactive, thus allowing phosphorylation of β-catenin forubiquitination and proteolytic degradation [14].

The APCgenemutations in colon cancer occur early duringtumorigenesis and the most common mutations that involvedAPC are either point mutations or small deletions and inser-tions leading to stop codons and therefore to truncations of theprotein. In most cases, the mutation of the APC gene results ina C-terminal truncation of the protein. Mutations of the APCgene result in a protein unable both to down-regulate Wntpathway and to induceβ-catenin phosphorylation. In this way,cytoplasmic β-catenin levels increase and cell proliferation isactivated which finally leads to adenoma formation [15]. Thissignal pathway explains how APC gene mutation is sufficientto cause the growth of small intestinal polyps [16].

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Step 2. RAS mutation

RAS oncogene locus was mapped to chromosome 12 and itencodes a protein involved in the Mitogen Activated ProteinKinases (MAPK) cascade, that is a downstream effector of areceptor tyrosine kinase. In CRC, KRAS oncogene is the mostcommonly mutated member of this family and its mutationshave been reported in 50 % of large polyps and CRCs [11];mutations are in general gene activating missense mutations andresult in locking the enzyme bound to ATP, by inhibiting itsGTPase activities, thus up-regulating the Ras function. There-fore it affects downstream signaling cascades including MAPK[15]. Even in this signaling pathway, oncogene alterations in-duce activation of same transcription factors. In particular, whenRAS swaps its GDP for a GTP through BRAF, it activatesMAP2K, which activates MAPK. MAPK can now activate atranscription factor that expresses proteins with a role in cellularproliferation, differentiation, and survival, such as c-myc [17].Missense mutations are found predominantly in codons 12, 13and 61 [11] but recently rare KRASmutations are discovered incodons 146, 117 and 59 [18]. Therefore, early KRAS oncogenemutations transforms extracellular growth signals to the nucleusand influence process of signal transduction which results topromote intestinal adenomatous growth [10].

Step 3. Chromosome 18q loss of heterozygosityand chromosome instability

In 1988, the use of restriction fragment polymorphism (RFLP)analysis allowed to characterize the frequency and location ofchromosomal losses in CRC [19]. It was shown that, in mostcases of sporadic CRC, several chromosomal segments weredeleted and the loss of one chromosomal allele was called“loss of heterozygosity” (LOH). LOH is the genetic alterationresponsible for CIN.

CIN is the most common type of genomic instability,encompassing 50–85 % of CRCs [4, 20]. Different mecha-nisms contribute to CIN: sequence changes; chromosomenumber alterations, chromosome rearrangements, gene ampli-fication, chromosomal segregation defects/microtubule dys-function, abnormal centrosome number, telomere dysfunc-tion, abnormal centrosome number, telomere dysfunction/telomerase overexpression, DNA damage and LOH. Up to70 % of CRC have been shown to carry LOH at chromosome18q called “deleted in colorectal carcinoma” gene (DCC) [12].Mutations of the DCC gene are also found in 50 % of ad-vanced adenomas of the colon [12]. The residual allele of 18qis generally inactivated. The presence of 18q alterationsallowed to adenomas progression and also it has been proven

Table 1 Gene mutations implicated in CRC pathogenesis

Gene Function Mechanism for mutation increasing CRC risk

APC Tumor suppressor gene Inactivating mutation causes loss of regulation of spindle microtubulesduring mitosis

TP53 Tumor suppressor gene Inactivating mutation causes loss of regulation of cell-cycle arrestand cell death

RAS Oncogene Activating mutations drive cell growth through MAPK pathway

BRAF Oncogene Activating mutations drive cell growth through MAPK pathway

PIK3CA Oncogene Activating mutation up-regulates PI3 K pathway, enhancing prostaglandinE2 synthesis and inhibiting apoptosis

MLH1, MSH2, MSH6, PMS2 MMR genes Inactivating mutation impairs ability to repair strand slippage withinnucleotide repeats

EPCAM Codes for transmembrane glycoproteinepithelial cell adhesion molecule

Deletion of 30 end of EPCAM leads to epigenetic silencing of MSH2

MYH Base excision repair gene Germline inactivating mutation of MYH leads to somatic mutation of APC

Table 2 Clinicopathological features and genomic alterations and instability

Primary tumor site Differentiation grading (G) Sex Age Prognosis

BRAF Right G3 Female Younger population Worse

APC Left NA NA NA Worse

CIN Left G1–G2 NA NA Worse

MSI Right G3 Female Both younger and older populations Favorable

CIMP Right G3 NA NA worse

NA not available, G1 well differentiated, G2 moderately differentiated, G3 poorly differentiated

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as a prognostic marker for survival [21]. LOH at chromosome18q seems caused adenoma progression in CRC tumorigene-sis, since genes on this chromosome includes over DCC evenSMAD2 and SMAD4 [21]. Furthermore, in CRC, CIN hasadditionally been associated with loss of chromosome 5q and17p that contribute to the inactivation of tumor suppressorgenes such as APC and TP53, respectively [9, 22].

Step 4. TP53 gene inactivation

TP53 gene is the name of the gene that encodes for p53 tumorprotein (“guardian of the genome”), and it is located on the shortarm of chromosome 17 [13]. The p53 protein is a transcriptionfactor that is activated in response to DNA damage or oncogenestress. Activation of p53 induces the expression of p21 gene thatarrests cell cycle in G1 phase and plays an important role inapoptosis [11]. Normally, p53 is negatively regulated byMDM2, E3-ubiquitin ligase, and MDM4 that targets p53 forubiquitination. In the presence of cellular stress, MDM2 andMDM4 have disrupted interactions with activation and tran-scription of p53. In CRC, most TP53 mutations occur in exons5–8, and are transition at CpG dinucleotide repeats. Thesemutations induce a loss of function of p53 protein to repairDNA alterations. Inactivation of p53 is a key step in the devel-opment of CRC; in fact, mutations and LOH of p53 are impor-tant with the transition from adenoma to carcinoma [11, 23].

Other genetic modifications

The oncosuppressor gene SMAD4, called deleted in pancreaticcancer 4 (DPC4), is associated with juvenile polyposis syn-drome (JPS) that predisposes to CRC. SMAD4 protein is anintracellular mediator that responds to transforming growthfactor- β (TGF-β) [11]. TGF-β binds to TGF-β receptor IIthat then dimerizes with TGF-β receptor I. This receptor thenphosphorylates receptor-regulated SMAD (R-SMAD) whichbinds to SMAD4, forming a complex, that enters into thenucleus and favors apoptosis and cell cycle regulation [11].The incidence of SMAD4 mutations in CRC is about 30 %,will the incidence of SMAD2 mutations is less frequent.Usually, SMAD4 mutations caused the progression of adeno-ma to carcinoma [11]. Moreover, the phosphatidylinositol-3-kinase (PI3K) signaling pathway plays an essential role inCRC tumorigenesis. PI3K initiates its signaling pathwaythrough the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphos-phate (PIP3). PIP3 then activates the serine/threonine kinaseAKT, which phosphorylates multiple downstream targets re-sponsible for a wide variety of vital cellular functions [6].Mutations of the PIK3C gene are present in 20–30 % ofCRCs. Three mutations are common — E542K, E545K, andH1047R— and result in a dominantly active form of the PI3Kprotein, that promotes cancer cell growth, survival and

metabolism, which further keenly involved in protecting thecells from undergoing apoptotic mode of cell death [6]. Thesystem is restored by Phosphatase and Tensing Homolog(PTEN), which dephosphorylates PIP3 and inhibits AKTsignaling. In CRC, the PTEN gene may be inactivated bysomatic mutations, allelic loss or hypermethylation of theenhancer region [17]. Mutation of PTEN is a later event incarcinogenesis that is correlated with advanced and metastatictumors [17].

Genetic instability

Genetic instability is characterized by the accumulation within acell of a several numbers of genetic alterations (Table 3). Twotypes of genetic instability have been described. One occurs atthe level of chromosomal (as above); while the second is a formof instability occurring at the level of DNA caused by inactiva-tion of mismatch repair (MMR) genes (also defined as genomicinstability) [24]. Recently, the development of CRC has beenconsidered from a different point of view [23, 25, 26]. Geneticalterations at the level of chromosomal are, in fact, only a pieceof a more complex puzzle [27]; genomic and epigenetic varia-tions in cancer-related genes play also a role contributing to themalignant status [28–30]. Genomic instability is emerging as afundamental process in colorectal tumorigenesis and carcinogen-esis is now viewed as an imbalance between mutation develop-ment and cell-cycle control mechanisms. Two separate pathwayshave been identified that contribute to this imbalance: (1) MSIand (2) CIMP [23–29]; finally, genomic instabilities are alsoassociated with clinicopathological tumor features (Table 1).

Microsatellite instability

Amicrosatellites are single sequence repeats of one to six nucle-otides, that are distributed evenly through at the genome every25–50 kb, and theymay be classified as monomorphic (the samenumber of repeats in all individuals) or polymorphic (variednumber of repeats among individuals) [31–33]. In general,microsatellites have a higher mutation rate than other sequencesbecause DNA polymerases slip on these sequences and is par-ticularly susceptible to making mistakes; therefore, when MMRis inactivated and cannot correct these mistakes, MSI is the result[5, 7, 34]. MSI is a cellular state, in which microsatellites gain orlose repeat units at a higher rate than normal and occur in 15–20 % of sporadic CRC. CRC usually are associated with muta-tions in TGF-β receptor II, epidermal growth factor receptor andBAXgenes. Additional genes affected byMSI include regulatorsof proliferation, the cell cycle or apoptosis and DNA repair [22].MMR inactivation may be due to either an inherited germ linemutation to one allele with somatic inactivation of the other orsomatic inactivation of both alleles. The most common mecha-nism ofMMR inactivation is through an acquired methylation ofthe MLK1, MSH2, MSH6 and PMS2 genes promoter [35], and

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in particular hMLH1 was not expressed in most of sporadicCRCs. On this basis, of how many genes promoter are mutatedby the degree of MSI can be categorized as high MSI (MSI-H)when two or more genes are involved, lowMSI (MSI-L) if onlyone marker is involved, and MSS if none [36].

CpG islands methylation phenotype (CIMP)

DNA hypermethylation at specific regulatory sites, enrichedin CpG (cytosine–phosphate–guanine) motifs (CpG islands)in the promoter regions of tumor suppressor genes, has beenlinked to transcription repression in human tumors [28]; there-fore, methylation leads to transcriptional silencing of genesinvolved in tumor suppression, cell cycle control, DNA repair,apoptosis, and invasion. CIMP positivity is found in 35–40 %of CRC [35]. Through hypermethylation of histone CpGislands, the chromatin closes into a compact structure suchthat the gene promoter is inaccessible to transcription factors,thereby inactivating gene transcription [6]. When this occursin oncosuppressor genes involved in CRC tumorigenesis,CRC can develop [11].

Epigenetic instability

In addition to genomic DNA instability being a commonphenomenon in CRC, epigenetic instability also appears tobe common in colorectal neoplasms [37]. Epigenetic eventsplay a role in CRC tumorigenesis as they activate oncogenesand inactivate tumor suppressor genes according to the mul-tistep origin of the process [27, 28]. They do not involvechanges in DNA sequences but rather are self-propagatingand potentially confer reversible molecular signatures [27,28] that was conveyed during cellular division. These changesare detected in approximately 40 % of CRC and they can besub-classified into three categories: (1) miRNA, (2) histonemodification and (3) DNA methylation [31, 38].

MicroRNA

miRNAs are small non-coding 18–25 nucleotides long RNAsthat down-regulate gene expression through binding to the 3′UTR and either degrading the mRNA or inhibiting the mRNA

translation [39]. miRNAs play a critical role in the patho-genesis of CRC because they induce silencing or overex-pression of tumor suppressor genes or oncogenes [40].Usually, overexpression of miR-31, miR-183, miR-17-5,miR-18a, miR-20a and miR-92 and underexpression ofmiR-143 and miR-145 are common in CRC [41], but howmiRNA changes are caused and how they are a result ofcarcinogenesis, remain unclear [11]. The main pathwaysinvolved in CRC tumorigenesis and affected by miRNAinclude: (1) APC—miR-135a and miR-135b that can causedecreased translation; (2) PI3K—miR-126 stabilizes PI3Ksignal and it is lost in CRC; (3) PTEN — miR-21 is re-pressed; (4) KRAS — miR-143 causes decreases expres-sion; (5) p53 — miR-34a induces apoptosis and it is de-creased in 36 % of primary CRCs, miR-192, miR-194-2,and miR-215 are involved in cell cycle arrest and are alsodown-regulated in CRC; (6) EMT—miR-200c overexpres-sion causes inhibition of ZEB1 and induces MET in cellsthat previously underwent EMT [42, 43].

Histone modification

Another epigenetic change is chromatin modification,specifically covalent modifications of the histone proteins.Histone acetylation is a hallmark of active regions whilehypoacetylated histone tails are found in transcriptionallyinactive euchromatic or heterochromatic regions [44]. Theacetylation/deacetylation is performed by histonedeacetylases (HDACs) and histone acetyl transfers [8].Histones are proteins that play an important role in generegulation. Histone modifications, including acetylation,methylation, phosphorylation, and ubiquitinylation of the-se proteins cause their inactivation [45]. Hypoacetylationand hypermethylation are characteristic of transcriptional-ly repressed chromatin regions. Mutations in histones aremost common at lysine and arginine residues [46]. Ifacetylation occurs at this position, the gene is expressedwhereas if it is methylated, the gene is silenced [46]. Themost important histone modifications that occur in CRCtumorigenesis are deacetylation and methylation of lysine9 in histone H3, monoacetylation of lysine 16 andtrimethylation of lysine 20 on histone H4 [44, 45].

Table 3 Genomic instability in CRC pathogenesis

Genomic instability Example gene mutations Syndrome associated with germline mutation Notes

Chromosomal instability Loss of function mutation ofAPC gene

Familial adenomatous polyposis Somatic APC mutation found in 85 %of sporadic CRC

Microsatellite instability Mismatch repair genes MLH1,MSH2, MSH6 and PMS2

Lynch syndrome Somatic inactivation of mismatch repairgenes found in 15 % sporadic CRC

DNA base excision repairdefect

MYH gene MYH-associated polyposis No recognized somatic equivalent

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DNA methylation and CpG islands

One of the epigenetic processes influencing gene expression isDNA methylation, a post-replicative DNA modification thatoccurs in genome regions rich in cytosine and guanosine (CG)dinucleotides, so called CpG islands. Modification of bases byaddition of a methyl group can physically inhibit binding oftranscription factors, and also permits a recruitment group thatcan physically inhibit binding of transcription factors and like-wise permits recruitment of the methyl-CpG-binding domainproteins to promote regions, which can repress transcriptioninitiation [47]. Hypermethylation-mediated changes in generegulation play a key role during development of several tumortypes, including CRC cancer. Over 40 % of human DNAcontains short interspersed transposable elements (SINEs) andlong interspersed transposable elements (LINEs) that are meth-ylated in normal colic cell but become hypomethylated in CRCdevelopment [44]. This epigenetic change increases chromo-somal susceptibility to breakage, thus creating genomic insta-bility, reactivating retrotransposons that disrupt gene structureand function, or activating oncogenes involved in CRC tumor-igenesis [45, 48–50]. These changes are believed to occur earlyin carcinogenesis, as hypomethylation is detected in benignpolyps but is not changed once they become malignant; there-fore, determination of DNA segments methylation can be use-ful in screening for CRC tumors [47].

Molecular classification of sporadic CRC

Clinically distinct subtypes of CRC are starting to emergethrough extensive molecular profiling studies [51].Sadanandam et al. [52] carried out clustering analysis on pub-lished gene expression data from 445 human primary CRCsamples. They identified five subtypes based on the expressionof 786 genes and named these according to their prominentgene expression signature: goblet-like, enterocyte, transit-amplifying, inflammatory and stem cells [52]. De Sosa E Meloet al. [53] also carried out gene expression profiling of humanCRC samples and they have recognized three CRC subtypes:subtypes 1 and 2 in their study showed a much strongerconcordance with the more traditional subtypes of CIN andMSI CRCs, respectively. However subtype 3, is a novel CRCsubtype that is mostly MSS, and shows the signature of epithe-lial mesenchymal transitions and extracellular matrix remodel-ing [53]. Moreover, recently Ogino et al. [54–56] have recog-nized that traditional CRC may be subdivided in differentdistinct subclasses based on epigenetic and genetic profiles:(1) CIMP 1 (intense methylation of multiple genes), MSI andBRAF mutations; (2) CIMP 2 (methylation of a limited groupof genes, increased methylation level for age-related genes) andKRAS mutations; (3) CIMP negative (rare methylation) andTP53 mutations. These three groups are relatively homoge-neous on a molecular level and likely representative of three

different subclasses of disease [57, 58]. The three CRC groupsalso differ clinically; CIMP1 and CIMP2 are more often prox-imal [59]; CIMP 1 has a good prognosis because it consistsmostly of MSI cancers; whereas CIMP2 has a poor prognosis[60]. Moreover, they may have distinct precancerous lesionssuch as serrated adenomas for CIMP1 and villous adenoma forCIMP2 [61, 62]. In this regard, approximately 35 % of CRCarise from the serrated pathway, developing from precursorlesions often referred to as the “serrated polyp” [63]. Serratedpolyps are lesions composed of epithelial infoldings creating aserrated appearance. These lesions include typical hyperplasticpolyps (Hps), sessile serrated adenomas (SSAs), and dysplasticserrated polyps (SSADSs) [22]. There are also two separatemolecular pathways involved: (a) BRAF mutation with CIMP1 is seen in the majority of syndromic, nonsyndromic cancers,andMSI cancers. Overall, 12%–15% ofMSI cancers occur byepigenetic silencing of the promoter methylation of DNAMMR gene hMLH-1 as the key step leading to MSI with rapidprogression from low to high grade dysplasia to invasive can-cer. (b) KRAS mutations are CIMP 2 or 3, no hMLH-1 activa-tion, and are MSS with many of them harboring TP53 muta-tions like conventional CRCs. CIMP 1 cancers are seen in theproximal colon, in females, have prominent glandular serrationswith mucinous differentiation or poorly differentiated glandswith intratumoral lymphocytes and Crohn’s-like nodularperitumoral infiltrates [22]. Finally four molecular subtypes ofCRCs and their precursor lesions are identified on the basis ofboth CIN and MSI status: (a) conventional adenomas CIMP−/MSI−or CIMP−/MSI+; (b) sessile serrated adenomas CIMP+/MSI+; serrated adenomas CIMP+/MSI−[22]. In summary, rec-ognition of genomic instability and the subtype is important toguide systemic therapy and affects outcome.

Hereditary CRC syndromes and risk factors

Approximately 5 %–6 % of CRC cases are associated with aknown hereditary CRC syndrome; however, in some cases,diagnosing a genetic susceptibility might be a challenge forthe clinician due to the similarity in clinical presentation withsporadic CRC. Nevertheless, its identification allows the cli-nician to tailor cancer treatment, follow up recommendations,and prevention strategies for the patient and their familymembers at highest risk. Therefore, in our efforts towardsCRC prevention, it is crucial to identify those patients associ-ated to genetic germline susceptibility.

Usually, when family history includes two or more relativeswith CRC, the possibility of a genetic syndrome is increasedsubstantially [64, 65]. Other CRC risk factors include thepresence of large serrated polyps (serrated adenomas and hy-perplastic polyps), a diet rich in total fat and meat, cigarettesmoking, male gender, the use of nonsteroidal anti-inflammatory drugs, alcohol intake, low folate intake and

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sedentary lifestyle, high bodymass index (BMI) and abdominalobesity [66–71]. High intakes of folate, vitamins and dietaryfiber, colonoscopy with removal of adenomatous polyps, andpostmenopausal hormone use have been associated with de-creased CRC risk [72–75]. Some of these risk factors areassociated with the development of genetic or epigenetic alter-ations that predispose to occurrence of CRC: obesity is associ-ated with CIMP, and for MSI [76–78] also there are data onFASN expression or CTNNB1 expression [79, 80], whereassmoking is associated with BRAF mutation, MSI and CIMP[81–83]. On the other hand, aspirin is protective against CRCdevelopment reducing BRAF mutation and PTGS2 expression[84, 85]. Also, inflammatory bowel disease (IBD) represents amajor risk factor for the development of CRC. Clinical andexperimental data indicate that chronic colitis increases the riskof developing tumors. However, it is now clear that tumor-promoting inflammation and anti-tumor immunity coexist incolitis-associated CRC. Although mechanisms underlying thisneoplastic transformation are not fully understood, some stud-ies suggest that inflammatory cell-derived cytokines could di-rectly or indirectly stimulate the uncontrolled growth of cancercells. Mucosal inflammatory cell types, such as regulatory Tcells (Tregs), Type 2 macrophages, CD4+ T helper (Th)-17cells, can promote CRC cells growth and the expansion of othercells, such as CD8+ T cells, natural killer (NK) cells, thusinhibiting carcinogenesis [86, 87].Moreover, inflammatorycells may modulate the process of colon carcinogenesis bystimulating stromal cells to synthesize a large population ofmolecules with mitogenic effects on CRC cells [88]. The risk ofIBD-associated CRC could also strictly linked to the durationand extension of inflammation [79–102].

Lynch syndrome is the most prevalent hereditary CRC syn-drome, firstly reported around a century ago. Since CRC is themain cause of death among individuals with Lynch syndrome,and CRC incidence can be reduced with adequate surveillance,it is crucial to identify those people at risk of having this geneticsusceptibility. In fact, with a 95 % compliance of the screeningrecommendations, CRC incidence has shown to decrease by62 %, with respect to those who do not adhere to screeningrecommendations. Lynch syndrome is caused by a germlinemutation in one of four genes associated with the DNA MMRsystem: MLH1, MSH2, MSH6, or PMS2 [103, 104].

Since more than 90 % of Lynch syndrome CRC cases showMSI and/or loss of the corresponding protein by immunohisto-chemistry (IHC), upfront molecular screening might be a goodstrategy to identify candidates for germline genetic testing[105]. With a similar diagnostic yield, the strategy of selectingpatients younger than 70 years, along with those older than 70fulfilling one of the Bethesda guidelines, allows researchers todecrease the number of patients undergoing germline genetictesting and can be considered a good strategy for clinicalpurposes. Diagnostic algorithms to rule out sporadic cases withloss of expression of MLH1 can be followed [106].

Once a germline susceptibility has been found, cancer treat-ment for the patient and screening recommendations for familymembers can be personalized. Adequate surveillance must beadvised according to their cancer risk and family communica-tion about the inherited condition should be encouraged. Indi-viduals with this genetic susceptibility have an increased risk ofCRC (lifetime risk 30 %–70 %), endometrial cancer (lifetimerisk 30%–60%), and others such as cancers of the urinary tract(8 %–11 %), ovary (4 %–12 %), pancreas (4 %), and biliarytract, brain and skin. Surveillance should be initiated at youngerages and be repeated at more frequent intervals due to a fasteradenoma–carcinoma sequence. Chemoprevention with aspirinand the role of prophylactic surgery are other attractive optionsto discuss with the patient [86, 107, 108].

Among polyposis syndromes associated with an increasedrisk of CRC, FAP is the most easily identified because it ischaracterized by hundred of adenomatous polyps that developearly in life. It is associated with mutations in the APC geneand inherited with an autosomal dominant pattern [109, 110].Attenuated manifestations of AFP might be related with APCmutations or be associated with biallelic mutY Homolog(MYH) gene mutations. MYH-associated polyposis followsan autosomal recessive inheritance and both alleles must havea mutation to cause the polyposis. Implications on otherfamily members will likely to be less relevant than in the caseof APC polyposis due to the type of inheritance. These syn-dromes are associated with a high risk of CRC, and bothintensive surveillance and prophylactic colectomy must bediscussed when appropriate [111, 112].

Finally, other intestinal hamartomatous polyposes, associ-ated with inherited conditions, are the Peutz–Jeghers syn-drome, juvenile polyposis or Cowden syndrome [111, 112].

Serrated neoplastic pathway

It has been recognized that about 20 % of CRCs arise from adistinct pathway so-called “serrated pathway”, which is asso-ciated with a sequence of genetic and epigenetic alterations[113]. Activating mutations of the BRAF gene are an earlyevent, and the anti-apoptotic BRAF function plays a crucialrule in the establishment of serration. The BRAF mutation isfollowed by up-regulation of p16INK4a and an increase se-cretion of an insulin-like growth factor binding protein 7(IGFBP7). Silencing of either p16INK4a or IGFBP7 CIMPsensible cells via methylation is proposed as essential in theprogression to sessile serrated adenoma/polyp (SSA/P) [114].SSA/P constitutes about 20 % of all serrated polyps and ismorphologically defined by the elongation of serrated cryptsand distortion of the proliferative zone. Progression of SSA/Pis associated with the occurrence of cytological dysplasia anddevelopment of invasive adenocarcinoma that are preferen-tially found in the right colon.

Tumor Biol.

Traditional serrated (TAS) are morphological variants ofserrated adenomas and show considerable differences fromSSA/P, concerning mutations (KRAS mutation is about25 %), localization (left sided) and methylation status (in-creased methylation status) [115]. In terms of histomorphology,TAS display a filiform configuration, eosinophilic cytoplasm,hyperchromatic nucleus and the formation of ectopic crypts.Given the malignant potential of serrated polyps, two importantserrated pathways of CRC carcinogenesis were characterized:sessile serrated pathway and TAS pathway. These CRCs canhave MSI-L or MSI-L, BRAF or KRAS mutations and CIMPbut given the heterogeneity of serrated adenocarcinomas astrong genotype to phenotype relation is not well establishedat the present. On the other hand molecular characterization ofserrated lesions is needed to improve screening practice andpoints of therapeutic intervention.

Colorectal cancer stem cells (CSC)

The CSC model of tumorigenesis postulates that tumors arenot cellularly homogeneous but display a hierarchical struc-ture and contain a rare population of cells, the CSC, thatdisplay the same self-renewal and proliferative potentials asa normal stem cell associated with the capacity to give riseto tumors [116].

It has been hypothesized that CSCs may derive from trans-formation of quiescent, normal long term stem cells or couldresult from the de-differentiation of more malignant cells. InCRC, the first hypothesis is supported by the observation thatnormal and cancer stem cells show similar properties andsurface markers. However, it cannot be excluded that CSCmight derive from cells that at same specific stages of differ-entiation undergo malignant transformation acquiring newproperties including stem-like features and this hypothesismight explain the different aggressiveness of tumors [116].The CSCmodel of tumorigenesis has the potential to radicallyrevolutionize the way how we look at malignant diseases aswell as the clinical management of CRC patients. To achievethis aim, it is essential that a definitive assessment is maderegarding the roles that CRCs play in the development of CRCand in particular aspects of malignancy.

Role of genetic and epigenetic events in CRC screening

As we mentioned in the Introduction section of this review,CRC is one of the most frequent malignant diseases in theworldwide [1] and for this reason its early diagnosis is re-quired to increase the survival rates of patients [117]. Current-ly, colonoscopy is the standard tool for CRC diagnosis, butendoscopic examination is invasive, unpleasant and carries anumber of associated risks [118].

As a rule, fecal occult blood test (FOBT) is available as apre-colonoscopy examination and although it can significant-ly reduce mortality due to CRC, FOBT is subject to high ratesof false negative and false positive results [119]. In this con-text, new specific CRC markers for diagnosis of CRC areneeded. Lately, there has been a surging interest in the detec-tion of earlier molecular markers in fecal and in blood samplesas a screening tool for CRC [120, 121] (Table 4).

Much focus been placed on molecular DNA-based stooltests, which promise a much more accurate alternative con-ventional methods of CRC screening. The earliest of suchstudies concentrated on the detection of KRAS mutations.Another single gene study tested APC mutations in fecalDNA from patients with CRC. The same laboratory studiedthe microsatellite marker BAT26 in the feces of patients withsporadic CRC. However, genetic alterations in CRC are high-ly heterogeneous and multiple rather than single genes shouldbe targeted to design a highly sensitive assay [120–123]. Donget al. [124], used a panel of three genetic targets (p53, KRASand microsatellite marker BAT26) to detect tumor associatedalterations in the feces from patients with CRC; and the threealterations were able to detect 36 out of 51 (71 %) patientswith CRC.

A study conducted by Alquist et al. [125] targeted muta-tions on KRAS, APC and p53 as well as MSI marker BAT-26.The sensitivity of this study was 91 % for CRC and 82 % foradenomas. Specificity was 93 % increasing to 100 % whenKRAS was excluded. Also, Calistri et al. [126] have investi-gated p53, KRAS, APC andmicrosatellite loci in the feces andtumors of patients with CRC and healthy subjects, respective-ly. The most frequent alterations in tumors were KRAS(34 %), p53 (34 %), APC (13 %) and MSI (13 %) while thesame alterations were not observed in the stool of healthyindividuals. Roperch et al. [127] identified novel epigeneticmarkers as Wnt inhibitor factor 1 (WIF1), Neuropeptide Y(NPY), proenkephalin (PENK) for CRC diagnosis. Authors

Table 4 Putative biomarkers for CRC detection

Gene Specimen

APC Stool DNA

KRAS Stool DNA, tissue

p53 Stool DNA, colonic effluent,colonic DNA

MSI Stool DNA, tissue

DCC Tissue

SMAD2/4 Tissue

WIF1 Stool DNA, serum

NPY Stool DNA, serum

PENK stool DNA, serum

miRNA-21, miRNA-29a, miRNA-72a,miRNA-760

stool DNA, serum

Tumor Biol.

showed that combining the methylation values of NPY,PENK, and WIF1 is potentially useful as a sensitive andspecific blood test for identifying among individuals withdigestive symptoms, those individuals for whom colonoscopyis recommended. Many studies have evaluated the feasibilityof circulating miRNA for detecting early CRC stage: miRNA29a, miRNA92a and miRNA760 are the most investigatedand better related with early CRC diagnosis [128], but theyhave not been validated prospectively. Also, miRNA 21 is apromising biomarker for the early detection of CRC in factserum miR-21 levels robustly distinguished adenoma andCRC patients [129]. Nowadays, molecular characterizationof CRC tumorigenesis has opened a wide spectrum of screen-ing tools for early diagnosis and prevention of CRC and a lotof molecular markers are currently under investigation in thissetting. Research on biomarkers in liquid biopsies (plasma,serum samples) or in fecal samples shows that this approach issimple, non-invasive and feasible; at the same time, molecularscreening tests have to be compared with conventional screen-ing methods and validated in prospective studies.

Conclusion

In hereditary cancers, genomic instability can be attributedto mutations in DNA repair genes; however, the relationshipbetween DNA repair systems, chromatin-remodeling com-plexes, and molecular basis of genomic instability in spo-radic cancers remains unclear. The multistep model of co-lorectal tumorigenesis has been seminal and paradigmatic incancer biology. One of the most intriguing but still unan-swered questions is to understand the precise molecularevents and their temporal occurrences that lead to tumorinitiation, abnormal cellular expansion, and phenotypicchanges. The three distinct pathways involving genomicinstability (MSI, CIN, and CIMP) appear to enhance thediversity of gene expression and phenotypic changes inCRC [130, 131]. Based on the stringent link between mo-lecular, pathological, and clinical features, have been pro-posed five molecular CRC subgroups, stratified on the basisof genomic instability [132]: CIMP−/CIN; CIMP low/MSS/KRAS+; CIMP+/MSS/BRAF+; CIMP+/CIN−/BRAF+;CIMP−/MSI+. In addition to this classification, it shouldbe noted that a tumor could take different paths; therefore, itis important to obtain data about age, sex, tumor site,stadiation, and diet when investigating genetic and epige-netic risk factors for CRC. Elucidation of the link betweenage, environmental risk, and carcinogenesis will help todefine the impact of epigenomic/genomic instability onmultiple CRC pathways. These findings may have broadimplications for cancer prevention, risk prediction, andprognosis. The goal for the future lies in the developmentof research tools that can target early detection of many of

the ongoing well recognized subcellular molecular events toimprove the burden of mortality and morbidity related tocolorectal carcinoma.

Finally, the genotype-to-phenotype relation is assumed tobe the great challenge in the field of cancer research and thedevelopment of effective targeted therapies. At present, astrong genotype-to-phenotype relation is characterized onlyfor a minority of CRCs. Consequently, the molecular charac-terization of CRCs is essential to interpret the histologicalpatterns and identify prognostic groups well as patients fortargeted therapies.

Conflicts of interest None

References

1. Siegel R, Naishadham D, Jemal A. Cancer statistics 2012. CACancer J Clin. 2012;62(1):10–29.

2. Sengupta N, Gill KA,MacFie TS, Lai CS, Suraweera N,McDonaldS, et al. Management of colorectal cancer: a role for genetics inprevention and treatment? Pathol Res Pract. 2008;204(7):469–77.

3. Migliore L, Migheli F, Spisni R, Coppedè F. Genetics, cytogeneticsand epigenetics of colorectal cancer. J Biomed Biotechnol.2011;2011:792362.

4. Kinzler KW, Vogelstein B. Cancer susceptibility genes.Gatekeepers and caretakers. Nature. 1997;386(6627):761–3.

5. Karen E. Kim; Early detection and prevention of colorectal cancer.Slack Incorporated 2009.

6. Deschoolmeester V, Baay M, Specenier P, Lardon F, VermorkenJB. A review of the most promising biomarkers in colorectalcancer: one step closer to targeted therapy. Oncologist.2010;15(7):699–731.

7. Kahng LS. Genetic aspects of non-polypoid colorectal neoplasms.Gastrointest Endosc Clin N Am. 2010;20(3):573–8.

8. Pancione M, Remo A, Colantuoni V. Genetic and epigenetic eventsgenerate multiple pathways in colorectal cancer progression. PatholRes Int. 2012;2012:509348.

9. Worthley DL, Leggett BA. Colorectal cancer: molecular featuresand clinical opportunities. Clin Biochem Rev. 2010;31(2):31–8.

10. Costedio M, Church J. Pathways of carcinogenesis are reflected inpatterns of polyp pathology in patients screened for colorectalcancer. Dis Colon Rectum. 2011;54(10):1224–8.

11. Fearon ER. Molecular genetics of colorectal cancer. Annu RevPathol. 2011;6:479–507.

12. Fearon ER, Vogelstein B. A genetic model for colorectal tumori-genesis. Cell. 1990;61(5):759–67.

13. Pino MS, Chung DC. The chromosomal instability pathway incolon cancer. Gastroenterology. 2010;138(6):2059–72.

14. Huang D, Du X. Crosstalk between tumor cells and microenviron-ment via Wnt pathway in colorectal cancer dissemination. World JGastroenterol. 2008;14(12):1823–7.

15. Booth RA.Minimally invasive biomarkers for detection and stagingof colorectal cancer. Cancer Lett. 2007;249(1):87–96.

16. De Roock W, Biesmans B, De Schutter J, Tejpar S. Clinical bio-markers in oncology: focus on colorectal cancer. Mol Diag Ther.2009;13(2):103–14.

17. Lievre A, Blons H, Laurent-Puig P. Oncogenic mutations as predic-tive factors in colorectal cancer. Oncogene. 2010;29(21):3033–43.

18. Douillard YJ, Oliner KS, Siena S, et al. N Engl J Med.2013;369(11):1023–34.

Tumor Biol.

19. Vogelstein B, Fearon ER, Hamilta SR, et al. N Engl J Med.1988;319:525–32.

20. Deschoolmeester V, BaayM, Specenier P, Lardon F, Vermorken JB. Areview of themost promising biomarkers in colorectal cancer: one stepcloser to targeted therapy. Oncologist. 2010;15(7):699–731.

21. Jen J, Kim H, Piantadosi S, et al. N Engl J Med. 1994;331:213–21.22. Kanthan R, Senger AL, Kanthan C. Molecular events in primary

and metastatic colorectal carcinoma: a review. Pathol Res Int.2012;2012:597497.

23. Markowitz SD, Bertagnolli MM. Molecular basis of colorectalcancer. N Engl J Med. 2009;361(25):2404–60.

24. Cahill DP, Lengauer C, Yu J, et al. Nature. 1998;392(6673):300–3.25. Ogino S, Goel A. Molecular classification and correlates in colo-

rectal cancer. J Mol Diagn. 2008;10(1):13–27.26. Jass JR. Classification of colorectal cancer based on correlation of

clinical, morphological and molecular features. Histopathology.2007;50(1):113–30.

27. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer.Carcinogenesis. 2009;31(1):27–36.

28. Esteller M. Molecular origins of cancer: epigenetics in cancer. NEngl J Med. 2008;358(11):1148–096.

29. Hanahan D, Weinberg RA. Hallmarks of cancer: the next genera-tion. Cell. 2011;144:646–74.

30. Grady WM, Carethers JM. Genomic and epigenetic instability incolorectal cancer pathogenesis. Gastroenterology. 2008;135(4):1079–99.

31. Boland CR, Goel A. Microsatellite instability in colorectal cancer.Gastroenterology. 2010;138(6):2073–87.

32. Iacopetta B, Grieu F, Amanuel B. Microsatellite instability in colo-rectal cancer. Asia-Pac J Clin Oncol. 2010;6(4):260–9.

33. De la Chapelle A, Hampel H. Clinical relevance of microsatelliteinstability in colorectal cancer. J Clin Oncol. 2010;28(20):3380–7.

34. De la Chapelle A. Microsatellite instability. N Engl J Med.2003;349(3):209–2010.

35. Goel A, Nagasaka T, Arnold CN, Inoue T, Hamilton C, NiedzwieckiD, et al. The CpG island methylator phenotype and chromosomalinstability are inversely correlated in sporadic colorectal cancer.Gastroenterology. 2007;132(1):127–38.

36. Yim KL. Microsatellite instability in metastatic colorectal cancer: areview of pathology, response to chemotherapy and clinical out-come. Med Oncol. 2012;29(3):1796–801.

37. Grady WM, Carethers JM. Genomic and epigenetic instability incolorectal cancer. Gastroenterology. 2008;135(4):1079–99.

38. Yamauchi M, Lochhead P, Morikawa T, Huttenhower C, Chan AT,Giovannucci E, et al. Colorectal cancer: a tale of two sides, or acontinuum? Gut. 2012;61(6):794–7.

39. Garzon R, Calin GA, Croce CM. MicroRNAs in cancer. Annu RevMed. 2009;60:167–79.

40. Slaby O, Svoboda M, Michalek J, Vyzula R. MicroRNAs in colo-rectal cancer: translation of molecular biology into clinical applica-tion. Mol Cancer. 2009;8:102.

41. Goel A, Boland CR. Recent insights into the pathogenesis ofcolorectal cancer. Curr Opin Gastroenterol. 2010;26(1):47–52.

42. de Krijger I, Mekenkamp LJM, Punt CJA, Nagtegaal ID. MicroRNAsin colorectal cancer metastasis. J Pathol. 2011;224:438–47.

43. Yang L, Belaguli N, Berger DH. MicroRNA and colorectal cancer.World J Surg. 2009;33(4):638–46.

44. Portela A, Esteller M. Epigenetic modifications and human disease.Nat Biotechnol. 2010;28(10):1057–68.

45. Wong JJL, Hawkins NJ, Ward RL. Colorectal cancer: a model forepigenetic tumorigenesis. Gut. 2007;56(1):140–8.

46. Venkatachalam R, Ligtenberg MJ, Hoogerbrugge N, de Bruijn DR,Kuiper RP. Geurts van Kessel, A. The epigenetics of (hereditary)colorectal cancer. Cancer Genet Cytogenet. 2010;203(1):1–6.

47. Kim YS, Deng G. Epigenetic changes (aberrant DNA methylation)in colorectal neoplasia. Gut Liver. 2007;1(1):1–11.

48. Ogino S, Hazra A, Tranah GJ, Kirkner GJ, Kawasaki T, Nosho K,et al. MGMT germline polymorphism is associated with somaticMGMT promoter methylation and gene silencing in colorectalcancer. Carcinogenesis. 2007;28(9):1985–90.

49. Raptis S,MrkonjicM,GreenRC, PetheVV,MongaN,ChanYM, et al.MLH1-93G>A promoter polymorphism and the risk of microsatellite-unstable colorectal cancer. J Natl Cancer Inst. 2007;99(6):463–74.

50. SamowitzWS, Curtin K, Wolff RK, Albertsen H, Sweeney C, CaanBJ, et al. The MLH1-93 G>A promoter polymorphism and geneticand epigenetic alterations in colon cancer. Genes ChromosomeCancer. 2008;47(10):835–44.

51. Burgess DJ. Gene expression: colorectal cancer classification. NatRev Cancer. 2013;13(6):380–1.

52. Sadanandam A, Lyssiotis CA, Homicsko H, et al. A colorectalcancer classification system that associates cellular phenotype andresponses to therapy. Nat Med. 2013;19(5):619–25.

53. De Sosa E, Melo F, Wang X, Jansen M, et al. Poor-prognosis coloncancer is defined by a molecularly distinct subtype and developsfrom serrated precursor lesions. Nat Med. 2013;19(5):614–8.

54. Ogino S, Stampfer M. Lifestyle factors and microsatellite instabilityin colorectal cancer: the evolving field of molecular pathologicalepidemiology. J Natl Cancer Inst. 2010;102(6):365–7.

55. Ogino S, Chan AT, Fuchs S, Giovannucci E. Molecular Pathologicepidemiology of colorectal neoplasia: an emerging transdisciplinaryand interdisciplinary field. Gut. 2011;60(3):397–411.

56. Ogino S, Kawasaki T, Kirkner GJ, Loda M, Fuchs CS. CpG islandmethylator phenotype-low (CIMP-Low) in colorectal cancer: pos-sible associations with male sex and KRASmutations. J Mol Diagn.2006;8(5):582–8.

57. Hinoue T, Weisenberger JD, Lange CPE, Shen H, Byun HM, vanden Berg D, et al. Genome-scale analysis of aberrant DNA methyl-ation in colorectal cancer. Genome Res. 2012;22(2):271–82.

58. Shen L, Toyota M, Kondo Y, Lin E, Zhang L, Guo Y, et al.Integrated genetic and epigenetic analysis identifies three differentsubclasses of colon cancer. Proc Natl Acad Sci U S A.2007;104(47):18654–9.

59. Issa JP. Methylation and prognosis: of molecular clocks andhypermethylator phenotypes. Clin Cancer Res. 2003;9(8):2879–81.

60. Shen L, Catalano PJ, Benson AB, O’Dwyer P, Hamilton SR, IssaJP. Association between DNAmethylation and shortened surviv-al in patients with advanced colorectal cancer treated with 5-f luorourac i l based chemotherapy. Cl in Cancer Res .2007;13(20):6093–8.

61. Minoo P, Baker K, Goswami R, Chong G, Foulkes WD,Ruszkiewicz AR, et al. Extensive DNA methylation in normalcolorectal mucosa in hyperplastic polyposis. Gut. 2006;55(10):1467–74.

62. Chirieac LR, Shen L, Catalano PJ, Issa JP, Hamilton SR. Phenotypeof microsatellite-stable colorectal carcinomas with CpG islandmethylation. Am J Surg Pathol. 2005;29:429–36.

63. Snover DC. Update on the serrated pathway to colorectal carcino-ma. Hum Pathol. 2011;42:1–10.

64. Jasperson KW, Tuohy TM, Neklason DW, Burt RW. Hereditary andfamilial colon cancer. Gastroenterology. 2010;138:2044–58.

65. Gonzalez CA, Riboli E. Diet and cancer prevention: contributionsfrom the European Prospective Investigation into Cancer andNutrition (EPIC) study. Eur J Cancer. 2010;46:2555–62.

66. Hiraoka S, Kato J, Fujiki S, Kaji E, Morikami T, Nawa T, et al. Thepresence of large serrated polyps increases risk for colorectal cancer.Gastroenterology. 2010;139:1503–10.

67. Hoffmeister M, Schmitz S, Karmrodt E, Stegmaier C, Haug U,Arndt V, et al. Male sex and smoking have a larger impact on theprevalence of colorectal neoplasia than family history of colorectalcancer. Clin Gastroenterol Hepatol. 2010;8:870–6.

68. Huang L, Wang X, Gong W, Huang Y, Jiang B. The comparison ofthe clinical manifestations and risk factors of colorectal cancer and

Tumor Biol.

adenomas: results from a colonoscopy-based study in southernChinese. Int J Color Dis. 2010;25:1343–51.

69. Larsson SC, Wolk A. Meat consumption and risk of colorectalcancer: a meta-analysis of prospective studies. Int J Cancer.2006;119:2657–64.

70. Bretthauer M. Evidence for colorectal cancer screening. Best PractRes Clin Gastroenterol. 2010;24:417–25.

71. Schernhammer ES, Giovannucci E, Kawasaki T, Rosner R, FuchsCS, Ogino S. Dietary folate, alcohol, and B vitamins in relation toline-1 hypomethylation in colon cancer. Gut. 2010;59:794–9.

72. Dahm CC, Keogh RH, Spencer EA, Greenwood DC, Key TJ,Fentiman IS, et al. Dietary fiber and colorectal cancer risk: a nestedcase-control study using food diaries. J Natl Cancer Inst. 2010;102:614–26.

73. Hildebrand JS, Jacobs EJ, Campbell PT, McCullough ML, TerasLR, ThunMJ, et al. Colorectal cancer incidence and postmenopaus-al hormone use by type, recency, and duration in cancer preventionstudy II. Cancer Epidemiol Biomarkers Prev. 2009;18:2835–41.

74. Kim DH, Smith-Warner SA, Spiegelman D, Yaun SS, Colditz GA,Freudenheim JL, et al. Pooled analyses of 13 prospective cohortstudies on folate intake and colon cancer. Cancer Causes Control.2010;21:1919–30.

75. Burt RW, Leppert MF, Slattery ML, Samowitz WS, Spirio LN,Kerber RA, et al. Genetic testing and phenotype in a large kindredwith attenuated familial adenomatous polyposis. Gastroenterology.2004;127:44–51.

76. Slattery ML, Curtin K, Sweeney C, Levin TR, Potter J, Wolff RK,et al. Diet and lifestyle factor associations with CpG island methyl-ator phenotype and BRAF mutations in colon cancer. Int J Cancer.2007;120(3):656–63.

77. Satia JA, Keku T, Galanko JA, Martin C, Doctolero RT, Tajima A,et al. Diet, lifestyle, and genomic instability in the North CarolinaColon Cancer Study. Cancer Epidemiol Biomarkers Prev. 2005;14:429–36.

78. Campbell PT, Jacobs ET, Ulrich CM, Figueiredo JC, Poynter JN,McLaughlin JR, et al. Case-control study of overweight, obesity,and colorectal cancer risk, overall and by tumor microsatelliteinstability status. J Natl Cancer Inst. 2010;17:391–400.

79. Kuchiba A, Morikawa T, Yamauchi M, Imamura Y, Liao X, ChanAT, et al. Bodymass index and risk of colorectal cancer according tofatty acid synthase expression in the nurses’ health study. J NatlCancer Inst. 2012;104:415–20.

80. Morikawa T, Kuchiba A, Lochhead P, Nishihara R, Yamauchi M,Imamura Y, et al. Prospective analysis of body mass index, physicalactivity, and colorectal cancer risk associated with b-catenin(CTNNB1) status. Cancer Res. 2013;73(5):1600–10.

81. Samowitz WS, Albertsen H, Sweeney C, Herrick J, Caan BJ,Anderson KE, et al. Association of smoking, CpG island methylatorphenotype, and V600E BRAF mutations in colon cancer. J NatlCancer Inst. 2006;98(23):1731–8.

82. Limsui D, Vierkant RA, Tillmans LS, Wang AH, Weisenberger DJ,Laird PW, et al. Cigarette smoking and colorectal cancer risk by molec-ularly defined subtypes. J Natl Cancer Inst. 2010;21(102):1012–22.

83. Nishihara R, Morikawa T, Kuchiba A, Lochhead P, Yamauchi M,Liao X, et al. A prospective study of duration of smoking cessationand colorectal cancer risk by epigenetics-related tumor classifica-tion. Am J Epidemiol. 2013;1(178):84–100.

84. Nishihara R, Lochhead P, Kuchiba A, Jung S, Yamauchi M, Liao X,et al. Aspirin use and risk of colorectal cancer according to BRAFmutation status. JAMA. 2013;309(24):2563–71.

85. Chan AT, Ogino S, Fuchs CS. Aspirin and the risk of colorectalcancer in relation to the expression of COX-2. N Engl J Med.2007;356(21):2131–42.

86. Rizzo A, Pallone F, Monteleone G, Fantini MC. Intestinal inflam-mation and colorectal cancer: a double-edged sword? World JGastroenterol. 2011;17:3092–100.

87. Erreni M, Mantovani A, Allavena P. Tumor-associated macro-phages (tam) and inflammation in colorectal cancer. CancerMicroenviron. 2011;4:141–54.

88. Mueller MM, Fusenig NE. Friends or foes—Bipolar effects of thetumour stroma in cancer. Nat Rev Cancer. 2004;4:839–49.

89. Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancerin ulcerative colitis: a meta-analysis. Gut. 2001;48:526–35.

90. Ekbom A, Helmick C, Zack M, Adami HO. Ulcerative colitis andcolorectal cancer. A population-based study. N Engl J Med.1990;323:1228–33.

91. Curtin K, Slattery ML, Ulrich CM, Bigler J, Levin TR, Wolff RK,et al. Genetic polymorphisms in one-carbon metabolism: associa-tions with CpG island methylator phenotype (CIMP) in coloncancer and the modifying effects of diet. Carcinogenesis.2007;28(8):1672–9.

92. Keku T, Millikan R, Worley K,Winkel S, Eaton A, Biscoco L, et al.5,10-Methylenetetrahydrofolate reductase codon 677 and 1298polymorphisms and colon cancer in African Americans and whites.Cancer Epidemiol Biomarkers Prev. 2002;11(12):1611–21.

93. Levine AJ, Siegmund KD, Ervin CM, Diep A, Lee ER, Frankl HD,et al. The methylenetetrahydrofolate reductase 677C→T polymor-phism and distal colorectal adenoma risk. Cancer EpidemiolBiomarkers Prev. 2000;9(7):657–63.

94. Yamaji T, Iwasaki M, Sasazuki S, Sakamoto H, Yoshida T, TsuganeS. Methionine synthase A2756G polymorphism interacts with al-cohol and folate intake to influence the risk of colorectal adenoma.Cancer Epidemiol Biomarkers Prev. 2009;18(1):267–74.

95. Iacopetta B, Heyworth J, Girschik J, Grieu F, Clayforth C, FritschiL. The MTHFR C677T and ΔDNMT3B C-149 T polymorphismsconfer different risks for right- and left-sided colorectal cancer. Int JCancer. 2009;125(1):84–90.

96. Mokarram P, Kumar K, Brim H, Naghibaldhossaini F, Saberi-frooziM, Nouraie M, et al. Distinct high-profile methylated genes incolorectal cancer. PLoS One. 2009;4(9):e7012.

97. Zeisel SH. Gene response elements, genetic polymorphisms andepigenetics influence the human dietary requirement for choline.IUBMB Life. 2007;59(6):380–7.

98. Van Engeland M, Weijenberg MP, Roemen GMJM, Brink M, DeBruine AP, Goldbohm RA, et al. Effects of dietary folate andalcohol intake on promoter methylation in sporadic colorectal can-cer: the Netherlands cohort study on diet and cancer. Cancer Res.2003;63(12):3133–7.

99. Hamid A, Kiran M, Rana S, Kaur J. Low folate transport acrossintestinal basolateral surface is associated with down-regulation ofreduced folate carrier in in vivo model of folate malabsorption.IUBMB Life. 2009;61(3):236–43.

100. Paun BC, Kukuruga D, Jin Z, Mori Y, Cheng Y, Duncan M, et al.Relation between normal rectal methylation, smoking status, andthe presence or absence of colorectal adenomas. Cancer.2010;116(19):4495–501.

101. Slattery ML, Curtin K, Sweeney C, Levin TR, Potter J, Wolff RK,et al. Diet and lifestyle factor associations with CpG island methyl-ator phenotype and BRAF mutations in colon cancer. Int J Cancer.2007;120(3):656–63.

102. Hughes LAE, van den Brandt PA, de Bruïne AP, Wouters KA,Hulsmans S, Spiertz A, et al. Early life exposure to famine andcolorectal cancer risk: a role for epigenetic mechanisms. PLoS One.2009;4(11):e7951.

103. Lynch HT, Lynch PM, Lanspa SJ, Snyder CL, Lynch JF, BolandCR. Review of the Lynch syndrome: history, molecular genetics,screening, differential diagnosis, and medicolegal ramifications.Clin Genet. 2009;76(1):1–18.

104. Plaschke J, Engel C, Kruger S, Holinski-Feder E, Pagenstecher C,Mangold E, et al. Lower incidence of colorectal cancer and later ageof disease onset in 27 families with pathogenic msh6 germlinemutations compared with families with MLH1 orMSH2mutations:

Tumor Biol.

the German Hereditary Nonpolyposis Colorectal CancerConsortium. J Clin Oncol. 2004;22:4486–94.

105. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ,Goldberg RM, et al. Tumor microsatellite-instability status as apredictor of benefit from fluorouracil-based adjuvant chemotherapyfor colon cancer. N Engl J Med. 2003;349:247–57.

106. Järvinen HJ, Aarnio M, Mustonen H, Aktan-Collan K, AaltonenLA, Peltomäki P, et al. Controlled 15-year trial on screening forcolorectal cancer in families with hereditary nonpolyposis colorectalcancer. Gastroenterology. 2000;118:829–34.

107. Burn J. Aspirin prevents cancer in Lynch syndrome. Eur J Cancer.2009;7:320–1.

108. Schmeler KM, Lynch HT, Chen LM, Munsell MF, Soliman PT,Clark MB, et al. Prophylactic surgery to reduce the risk ofgynecologic cancers in the Lynch syndrome. N Engl J Med.2006;354:261–9.

109. Juhn E, Khachemoune A. Gardner syndrome: skin manifestations,differential diagnosis and management. Am J Clin Dermatol.2010;11(2):117–22.

110. Nieuwenhuis MH, Vasen HF. Correlations between mutation site inAPC and phenotype of familial adenomatous polyposis (FAP): areview of the literature. Crit Rev Oncol Hematol. 2007;61:153–61.

111. McGarrity TJ, Amos C. Peutz–Jeghers syndrome: clinicopathologyand molecular alterations. Cell Mol Life Sci. 2006;63(18):2135–44.

112. Calva D, Howe JR. Hamartomatous polyposis syndromes. SurgClin N Am. 2008;88(4):779–817.

113. Kaemmerer E, Klaus C, Jeon MK, Gassler N. Molecular classifica-tion of colorectal carcinomas: the genotype to phenotype relation.World J Gastroenterol. 2013;19(45):8163–7.

114. Leggett B, Whitehall V. Role of the serrated pathway in colorectalcancer pathogenesis. Gastroenterology. 2010;138:2088–100.

115. East JE, Saunders BP, Jass JR. Sporadic and syndromic hyperplasticpolyps and serrated adenomas of the colon: classification, moleculargenetics, natural history, and clinical management. GastroenterolClin N Am. 2008;37:25–46.

116. Fanali C, Lucchetti D, Farina M, Corbi M, Cufino V, Cittadini A,et al. Cancer stem cells in colorectal cancer from pathogenesis totherapy: controversies and perspectives. World J Gastroenterol.2014;20(4):923–42.

117. Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, et al.Colorectal cancer screening and surveillance: clinical guidelines andrationale-update based on new evidence. Gastroenterology.2003;124(2):544–60.

118. Osborn NK, Ahlquist DA. Stool screening for colorectal cancer:molecular approaches. Gastroenterology. 2005;128(1):192–206.

119. Hardcastle JD, Thomas WM, Chamberlain J, Pye G, Sheffield J,James PD, et al. Randomised, controlled trial of faecal occult bloodscreening for colorectal cancer: results for first 107,349 subjects.Lancet. 1989;1(8648):1160–4.

120. Brenner DE, Rennert G. Fecal DNA biomarkers for the detection ofcolorectal neoplasia: attractive, but is it feasible? J Natl Cancer Inst.2005;97(15):1107–9.

121. Creeden J, Junker F, Vogel-Ziebolz S, Rex D. Serum tests forcolorectal cancer screening. Mol Diagn Ther. 2011;15(3):129–41.

122. Kim MS, Lee J, Sidransky D. DNA methylation markers in colo-rectal cancer. Cancer Metastasis Rev. 2010;29(1):181–206.

123. Wong JJL, Hawkins NJ, Ward RL, Hitchins MP. Methylation of the3p22 region encompassing MLH1 is representative of the CpGisland methylator phenotype in colorectal cancer. Mod Pathol.2011;24(3):396–411.

124. Dong SM, Traverso G, Johnson C, et al. Detecting colorectal cancerin stool with the use of multiple genetic targets. J Natl Cancer Inst.2001;93:851–65.

125. Alquist DA, Skoletsky JE, Boynton KA, et al. Colorectal cancerscreening]by detection of altered human DNA in stool; feasibility ofa mutitarget assay panel. Gastroenterology. 2000;119:1219–27.

126. Calistri D, Rengucci C, Bocchini R, et al. Fecal multiple moleculartests to detect colorectal cancer in stool. Clin Gastroenterol Hepatol.2003;1:377–83.

127. Roperch JP, Incitti R, Forbin F, et al. Aberrant methylation of NPY,PENK, and WIF1 as a promising marker for blood-based diagnosisof colorectal cancer. BMC Cancer. 2013;13:566.

128. Madhavan D, Cuk K, Burwinkel B, Yang R. Cancer diagnosis andprognosis decoded by blood-based circulating microRNA signa-tures. Front Genet. 2013;4:116.

129. Toiyama Y, Takahashi M, Hur K, et al. Serum miR-21 as a diag-nostic and prognostic biomarker in colorectal cancer. J Natl CancerInst. 2013;105(12):849–59.

130. Kim MS, Lee J, Sidransky D. DNA methylation markers in colo-rectal cancer. Cancer Metastasis Rev. 2010;29(1):181–206.

131. Grady WM, Carethers JM. Genomic and epigenetic instability incolorectal cancer pathogenesis. Gastroenterology. 2008;135(4):1079–99.

132. Jass JR. Classification of colorectal cancer based on correlation ofclinical, morphological and molecular features. Histopathology.2007;50(1):113–30.

Tumor Biol.