**3.1 Microsatellite instability phenotype (MSI)**

*Advances in the Molecular Understanding of Colorectal Cancer*

**features**

CRC subtypes.

**3. Classification of CRC pathways and subtypes using epigenetic** 

The development of CRC occurs via aberrations in multiple genetic and epigenetic pathways. These pathways can be defined by three principal molecular phenotypes (**Figure 1**). As aforementioned in Section 1, these include the MSI phenotype, which is characterized by mutations in DNA MMR genes; the CIN phenotype characterized by mutations in *APC*/*Wnt*/*β-catenin* pathway; and a third CIMP, defined by global CpG island hypermethylation, which results in widespread silencing of tumor suppressor genes [9, 71]. Notably, each pathway is characterized by distinct epigenetically related pathological features that drive the process of tumor initiation and development. In this section, we will describe various epigenetic aspects of these CRC phenotypes as well as how the molecular aspects of each pathway have been employed as useful diagnostic and prognostic tools to guide the clinical management of CRC patients. Finally, we also briefly acknowledge how these pathways may overlap within broader systems of subtype classification and highlight some of the current challenges in precisely defining

*Schematic depicting epigenetic alterations in CRC and their association with CRC molecular subtypes. The CRC "epigenome" harbors alterations in their histone modification states, particularly regarding aberrant histone methylation (blue dot) and acetylation (yellow dot). These global histone aberrations serve as biomarkers to predict the clinical outcome in CRC patients. DNA methylation is another major epigenetic alteration seen in CRC. Usually, global DNA hypomethylation occurs gradually and early in the process of CRC carcinogenesis. This occurs in concert with systematic and discrete CpG island hypermethylation events at the promoters of genes involved in DNA repair, apoptosis, proliferation, angiogenesis, adhesion, and invasion. This generally results in transcriptional silencing. Aberrations of micro-RNAs (miRNAs) expression, a major class of ncRNAs, are also often observed in CRC and are considered to play a major role in tumorigenesis and CRC progression. This figure also illustrates three main epigenetic-related molecular subtypes of CRC, namely the microsatellite instability (MSI) phenotype, the chromosomal instability (CIN) phenotype, and the CpG island methylator phenotype (CIMP). MSI CRCs arise from a defective DNA mismatch repair (MMR) system, which is associated with frameshift mutations and base pair substitutions in genes. CIMP CRCs are characterized by widespread promoter CpG island hypermethylation, whereas the CIN CRCs arise through widespread imbalances in chromosome number (aneuploidy), global hypomethylation, and loss of heterozygosity (LOH).*

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**Figure 1.**

Approximately 15–20% of all CRC cases harbor MSI [72]. Microsatellites are defined as repetitive one to six base pair DNA sequences distributed along coding and noncoding regions of the genome. Importantly, the repetitive nature of these regions makes them particularly susceptible to mismatch errors [72]. Tumors with the MSI phenotype are therefore driven by the inactivation of MMR genes, which are involved in repairing DNA recombination and replication errors as well cellular responses to DNA damage [12]. The net effect of defective MMR machinery is accumulation of single base-pair mismatches, which results in a hypermutable cellular state [72].

MSI tumors are typically classified as MSI-high (MSI-H) or MSI-low (MSI-L). In MSI-H CRCs, usually two or more of the five microsatellite markers show instability, whereas in MSI-L tumors, only one of the five markers shows instability [12]. If none of the markers show instability, however, these are classified as microsatellite stable (MSS) CRC tumors, which account for approximately 80–85% of CRC patients [12]. Although majority of MSI-H tumors sporadically arise, a few are also linked to a familial hereditary syndrome known as Lynch syndrome or hereditary non-polyposis CRC (HNPCC) which account for about 3–5% of all CRC cases [12]. Sporadic MSI tumors are generally affected by epigenetic inactivation at the *MLH1* promoter via CpG island hypermethylation, whereas Lynch syndrome is caused by germline mutations in *MLH1*, *PMS2*, *MSH6*, or *MSH2* [12].

Importantly, unlike MSS, MSI tumors are poorly differentiated and are more often located in the proximal colon. MSI tumors also harbor a mucinous or signet ring type histology and increased number of tumor-infiltrating lymphocytes [72]. Additionally, when MSI was first identified in CRC patients, it was shown that MSI-H patients tended to have a better patient prognosis compared with MSS tumors and had an overall lower tumor stage at diagnosis [12]. Moreover, randomized phase III clinical trials along with several prospective studies have shown that the MMR status of these patients is also predictive of response to adjuvant 5-FU-based chemotherapy [12, 73]. The consensus was that patients with MSI-H tumors did not benefit from adjuvant 5-FU therapy compared to their non-MSI-H counterparts [73]. Furthermore, these data are consistent with other studies that revealed that human CRC cell lines with MSI-H phenotypes displayed resistance to DNA damaging agents, such as 5-FU, which could be overcome by the restoration of normal MMR function [74, 75].
