**3.2 Chromosomal instability (CIN) phenotype**

The acquisition of genomic or chromosomal instability is a key feature in CRC development [39]. In fact, CIN has been found in approximately 85% of CRC cases and is characterized by increased chromosomal losses and gains as well as increased loss of heterozygosity [13, 14]. Although the exact mechanisms underlying CIN remain incompletely understood, it has been attributed to defects in genes related to the DNA damage response, telomere stability, and chromosomal segregation [39]. Unfortunately, standardizing the precise quantitative criteria that define a "CINpositive" tumor has been challenging due to difficulties in the detection approaches of chromosomal instability [39]. The approaches currently in use include cytometry, karyotyping, and loss of heterozygosity analyses [76].

Along with the typical chromosomal abnormalities, accumulation of a characteristic set of mutations in specific tumor suppressor genes and oncogenes is

also a prominent feature observed in CIN tumors [39]. These include mutations in *APC*, *p53*, Cyclooxygenase-2 (*COX-2*), and *KRAS* as well as 18q alterations [39]. Interestingly, many studies have sought to determine the prognostic value of *KRAS*, *TP53*, or 18q alterations. So far, evidence of increased risk of relapse or mortality in CRC patients with *KRAS* mutations has been presented, but other studies have failed to confirm this correlation [77]. Consequently, some of these putative individual prognostic markers are still undergoing rigorous study. However, several compelling studies indicate that the overall CIN phenotype is associated with a less favorable outcome in patients than those with the MSI phenotype, and unlike MSI tumors, it is not significantly influenced by adjuvant therapy in patients with stage II–III CRC [78].

Several ongoing phase I and II clinical trials are underway to therapeutically target pathways that directly or indirectly initiate and perpetuate CIN. Some of these include small-molecule inhibitors of *COX-2*, *Polo-Like Kinases* (*Plks*), *Eg5*, and *Centromere protein E* (*CENP-E*) [39]. Swanton et al. also showed that CINpositive tumors are intrinsically resistant to taxanes due to the similarity between pathways that regulate the chromosomal segregation and those implicated in the taxane response [79]. These and other studies have collectively prompted the Chromosomal Instability and Anti-Tubulin Response Assessment (CINATRA) trial to assess whether patients with MSI-positive solid tumors derive benefit from EPO906 (new microtubule stabilizer) compared to patients with CIN-positive cancers [80]. Overall, phase I trials showed encouraging tumor control and response rates in patients with metastatic CRC (mCRC), although the trial was prematurely closed due to toxicity issues [80]. In summary, these data support the role of the CIN pathway in guiding patient stratification and the clinical management of CRC. However, more studies to better define the mechanisms underlying CIN and determine how CIN influences progression will be critical to advance our understanding of the most common form of genetic instability in CRC. Moreover, the feasibility of standardizing detection of CIN-positive tumors and thus be able to target chromosomally unstable cells, will be critical.

#### **3.3 CpG island methylator phenotype (CIMP)**

The term "CpG island methylator phenotype," or CIMP, was coined in 1999 by Toyota and Issa to denote the CRC tumor subtypes characterized by widespread promoter DNA hypermethylation at certain tumor suppressor genes [81]. More than 50% of genes have promoters found within CpG islands [44]. Hence, the frequency of CIMP CRCs depends on which promoters are examined for methylation, with some promoters being more beneficial than others for identifying CIMP. Several studies have revealed that this methylation is common at the promoters of a diverse spectrum of genes, including *Phosphatase and tensin homolog* (*PTEN*), *RUNX3*, and *Unc-5 netrin receptor C* (*UNC5C*), making these key genes part of the expression signature profile in the evolution of CIMP CRCs [44]. Moreover, based on a panel of CIMP-specific markers coupled with the *B-Raf proto-oncogene* (*BRAF*) mutational status, CIMP tumors may be further classified according to the fraction of promoters that exceed a certain threshold of DNA methylation as being CIMP high, low, or negative [44]. Although CIMP-high and CIMP-low CRCs are significantly associated with biological sex as well as BRAF and KRAS mutational status, these classifications need additional refinement [44]. Nonetheless, it is reminiscent of the classification of CRCs based on degrees of MSI [82].

Notably, several of the clinicopathological characteristics of CIMP-high tumors have also been correlated to MSI tumors [82, 83]. For example, like MSI, CIMP tumors also represent a clinically distinct group characterized by epigenetic instability, distinct histological and pathological features, and discrete precursor lesions [84].

**87**

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer*

Pathologically, CIMP tumors also originate similarly to MSI tumors in the proximal colon, with a mucinous and poorly differentiated histological type and are frequently diagnosed in elderly and female patients [84, 85]. However, the determination of which specific methylated loci should be used to define CIMP remains a major challenge in the evaluation of CIMP tumors. Currently, several studies have proposed the classic panel containing the genes *hMLH1* and *p16* as well as Munc-18-interacting (MINT) proteins *MINT1*, *MINT2*, and *MINT31*. This panel has been further developed to contain the genes *Calcium voltage-gated channel subunit alpha1 G* (*CACNA1G*), *Cellular retinoic acid binding protein 1* (*CRABP1*), *IGF2*, *Neurogenin 1* (*NEUROG1*), *RUNX3*, *Suppressor of cytokine signaling 1* (*SOCS1*), *Hypermethylated in cancer 1* (*HIC1*), *IGF-binding protein 3* (*IGFBP3*), and *Werner syndrome ATP-*

In recent years, the use of DNA hypermethylation of specific genes to predict CRC patient outcome and therapeutic approach has received much attention. Although further validation is warranted, many studies have found a correlation between MSS CIMP+ CRC patients and poor prognosis [87]. Furthermore, the correlation between CIMP status and response to chemotherapy has been investigated. CIMP status predicts poor survival in metastatic MSS CRC patients treated with chemotherapeutic agent 5-FU [88]. Overall, patients who did not receive chemotherapy treatment but had tumors identified as MSS and CIMP had a worse survival outcome [89]. In contrast, two separate studies reported better outcomes for patients with CIMP tumors, a conflict that could be attributed to differences in the criteria used across the studies to define CIMP status. Additionally, this also suggests that the heterogeneous nature of CIMP tumors may warrant further classification [44].

Intriguingly, many studies have also found an association between CIMP status and other important epidemiological factors [90]. For instance, reports of an association between cigarette smoking, obesity, and CIMP showed that the number of cigarettes smoked as well as body mass index (BMI) had a significant relationship to CIMP tumor development [90, 91]. Furthermore, associations of CIMP status with smoking and obesity were evident only for females [90]. Taken together, the above evidences support a critical role of the CIMP pathway in the pathogenesis of CRC, which has also become a significant part of the current management of CRC. In the future, it will also be essential to have a consensus on a standardized panel of loci to

The three molecular pathways described so far also fall within several consensus classification systems for CRC. These systems vary in terms of the number of proposed subtypes, which can range from three to six depending on the combinations of genetic, epigenetic, clinical, and histopathological parameters used as well as the extent of the overlap between the three molecular pathways. For instance, the Consensus Molecular Subtypes (CMS) consortium has been suggested as one of the most robust classification systems and describes four groups (CMS1–4) based on expression profiling data from multiple studies [92]. While the majority of MSI-H CRCs fall into the CMS1 category, CMS2–4 display higher CIN. However, CMS3 samples have a distinctive profile compared with other CIN tumors. They tend to have lower CIN, higher prevalence of CIMP and close to 30% of the tumors are hypermutated which confers significant overlap with MSI status tumors [93]. Additionally, the Cancer Genome Atlas study also demonstrated that CIMP overlaps with the MSI pathway because of the fact that sporadic MSI-H CRCs usually harbor CIMP-high clinicopathological features [94]. Meanwhile, CMS4 are defined as CIN-heterogeneous tumors with mesenchymal characteristics that occur in later disease stages [93].

define CIMP, similar to that utilized to identify MSI CRCs.

**3.4 Consensus molecular subtypes**

*DOI: http://dx.doi.org/10.5772/intechopen.82572*

*dependent helicase* (*WRN*) [86].

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.82572*

Pathologically, CIMP tumors also originate similarly to MSI tumors in the proximal colon, with a mucinous and poorly differentiated histological type and are frequently diagnosed in elderly and female patients [84, 85]. However, the determination of which specific methylated loci should be used to define CIMP remains a major challenge in the evaluation of CIMP tumors. Currently, several studies have proposed the classic panel containing the genes *hMLH1* and *p16* as well as Munc-18-interacting (MINT) proteins *MINT1*, *MINT2*, and *MINT31*. This panel has been further developed to contain the genes *Calcium voltage-gated channel subunit alpha1 G* (*CACNA1G*), *Cellular retinoic acid binding protein 1* (*CRABP1*), *IGF2*, *Neurogenin 1* (*NEUROG1*), *RUNX3*, *Suppressor of cytokine signaling 1* (*SOCS1*), *Hypermethylated in cancer 1* (*HIC1*), *IGF-binding protein 3* (*IGFBP3*), and *Werner syndrome ATPdependent helicase* (*WRN*) [86].

In recent years, the use of DNA hypermethylation of specific genes to predict CRC patient outcome and therapeutic approach has received much attention. Although further validation is warranted, many studies have found a correlation between MSS CIMP+ CRC patients and poor prognosis [87]. Furthermore, the correlation between CIMP status and response to chemotherapy has been investigated. CIMP status predicts poor survival in metastatic MSS CRC patients treated with chemotherapeutic agent 5-FU [88]. Overall, patients who did not receive chemotherapy treatment but had tumors identified as MSS and CIMP had a worse survival outcome [89]. In contrast, two separate studies reported better outcomes for patients with CIMP tumors, a conflict that could be attributed to differences in the criteria used across the studies to define CIMP status. Additionally, this also suggests that the heterogeneous nature of CIMP tumors may warrant further classification [44].

Intriguingly, many studies have also found an association between CIMP status and other important epidemiological factors [90]. For instance, reports of an association between cigarette smoking, obesity, and CIMP showed that the number of cigarettes smoked as well as body mass index (BMI) had a significant relationship to CIMP tumor development [90, 91]. Furthermore, associations of CIMP status with smoking and obesity were evident only for females [90]. Taken together, the above evidences support a critical role of the CIMP pathway in the pathogenesis of CRC, which has also become a significant part of the current management of CRC. In the future, it will also be essential to have a consensus on a standardized panel of loci to define CIMP, similar to that utilized to identify MSI CRCs.

### **3.4 Consensus molecular subtypes**

The three molecular pathways described so far also fall within several consensus classification systems for CRC. These systems vary in terms of the number of proposed subtypes, which can range from three to six depending on the combinations of genetic, epigenetic, clinical, and histopathological parameters used as well as the extent of the overlap between the three molecular pathways. For instance, the Consensus Molecular Subtypes (CMS) consortium has been suggested as one of the most robust classification systems and describes four groups (CMS1–4) based on expression profiling data from multiple studies [92]. While the majority of MSI-H CRCs fall into the CMS1 category, CMS2–4 display higher CIN. However, CMS3 samples have a distinctive profile compared with other CIN tumors. They tend to have lower CIN, higher prevalence of CIMP and close to 30% of the tumors are hypermutated which confers significant overlap with MSI status tumors [93]. Additionally, the Cancer Genome Atlas study also demonstrated that CIMP overlaps with the MSI pathway because of the fact that sporadic MSI-H CRCs usually harbor CIMP-high clinicopathological features [94]. Meanwhile, CMS4 are defined as CIN-heterogeneous tumors with mesenchymal characteristics that occur in later disease stages [93].

*Advances in the Molecular Understanding of Colorectal Cancer*

target chromosomally unstable cells, will be critical.

classification of CRCs based on degrees of MSI [82].

The term "CpG island methylator phenotype," or CIMP, was coined in 1999 by Toyota and Issa to denote the CRC tumor subtypes characterized by widespread promoter DNA hypermethylation at certain tumor suppressor genes [81]. More than 50% of genes have promoters found within CpG islands [44]. Hence, the frequency of CIMP CRCs depends on which promoters are examined for methylation, with some promoters being more beneficial than others for identifying CIMP. Several studies have revealed that this methylation is common at the promoters of a diverse spectrum of genes, including *Phosphatase and tensin homolog* (*PTEN*), *RUNX3*, and *Unc-5 netrin receptor C* (*UNC5C*), making these key genes part of the expression signature profile in the evolution of CIMP CRCs [44]. Moreover, based on a panel of CIMP-specific markers coupled with the *B-Raf proto-oncogene* (*BRAF*) mutational status, CIMP tumors may be further classified according to the fraction of promoters that exceed a certain threshold of DNA methylation as being CIMP high, low, or negative [44]. Although CIMP-high and CIMP-low CRCs are significantly associated with biological sex as well as BRAF and KRAS mutational status, these classifications need additional refinement [44]. Nonetheless, it is reminiscent of the

Notably, several of the clinicopathological characteristics of CIMP-high tumors have also been correlated to MSI tumors [82, 83]. For example, like MSI, CIMP tumors also represent a clinically distinct group characterized by epigenetic instability, distinct histological and pathological features, and discrete precursor lesions [84].

**3.3 CpG island methylator phenotype (CIMP)**

also a prominent feature observed in CIN tumors [39]. These include mutations in *APC*, *p53*, Cyclooxygenase-2 (*COX-2*), and *KRAS* as well as 18q alterations [39]. Interestingly, many studies have sought to determine the prognostic value of *KRAS*, *TP53*, or 18q alterations. So far, evidence of increased risk of relapse or mortality in CRC patients with *KRAS* mutations has been presented, but other studies have failed to confirm this correlation [77]. Consequently, some of these putative individual prognostic markers are still undergoing rigorous study. However, several compelling studies indicate that the overall CIN phenotype is associated with a less favorable outcome in patients than those with the MSI phenotype, and unlike MSI tumors, it is not significantly influenced by adjuvant therapy in patients with stage II–III CRC [78]. Several ongoing phase I and II clinical trials are underway to therapeutically target pathways that directly or indirectly initiate and perpetuate CIN. Some of these include small-molecule inhibitors of *COX-2*, *Polo-Like Kinases* (*Plks*), *Eg5*, and *Centromere protein E* (*CENP-E*) [39]. Swanton et al. also showed that CINpositive tumors are intrinsically resistant to taxanes due to the similarity between pathways that regulate the chromosomal segregation and those implicated in the taxane response [79]. These and other studies have collectively prompted the Chromosomal Instability and Anti-Tubulin Response Assessment (CINATRA) trial to assess whether patients with MSI-positive solid tumors derive benefit from EPO906 (new microtubule stabilizer) compared to patients with CIN-positive cancers [80]. Overall, phase I trials showed encouraging tumor control and response rates in patients with metastatic CRC (mCRC), although the trial was prematurely closed due to toxicity issues [80]. In summary, these data support the role of the CIN pathway in guiding patient stratification and the clinical management of CRC. However, more studies to better define the mechanisms underlying CIN and determine how CIN influences progression will be critical to advance our understanding of the most common form of genetic instability in CRC. Moreover, the feasibility of standardizing detection of CIN-positive tumors and thus be able to

**86**

It is also noteworthy that many CRC tumors demonstrate mixed characteristics compatible with two or more of these subtypes, which may represent a transition phenotype or intratumoral heterogeneity, while others cannot be precisely classified into any of these pre-defined subgroups [95]. Furthermore, these classifications often lack incorporation of the molecular markers used for traditional TNM staging of CRC [96]. Taken together, these challenges as well as the existing incongruity between the various systems illustrate the need to further refine these consensus classifications by developing more progressive and integrated approaches.
