**3. Integrative molecular analysis of colorectal cancer**

It is clear that anatomical factors and common DNA alterations are helpful in identifying subtype characteristics in CRC, but they alone are inadequate to define the boundaries between the different molecular entities that comprise CRC. In recent years, many studies have begun to exploit microarray technology to investigate gene expression profiles (GEPs) in CRC; however, no single signature

**5**

**Table 1.**

*Advances in Molecular Subclassification of Colorectal Cancer*

has proven clinically meaningful, especially in regard to predicting prognosis, and studies have been poorly reproducible due to the high molecular heterogeneity that

**3.1 The Cancer Genome Atlas (TCGA) comprehensive analysis of** 

In 2012, The Cancer Genome Atlas (TCGA) Research Network produced a comprehensive integrative analysis of 224 colorectal cancer tumour samples with paired normal samples in order to improve our understanding of the biology of this disease and identify potential therapeutic targets [8]. In addition, independent scientific groups also attempted to define intrinsic subtypes of CRC using GEPs in the hope that this will refine the molecular classification of CRC and facilitate clinical translation [9–14]. The findings of all of these independent analyses are discussed below.

The comprehensive analysis of CRC undertaken by TCGA Research Network included tumours whose clinical and pathological characteristics reflected the usual breadth of features of CRC patients. Tumours were split into two main groups by mutation rate: those that were hypermutated (16%) and those that were non-hypermutated (84%) which seems to match the previously described MSI and CIN groups. The hypermutated group was then subdivided into those caused by defective MMR (dMMR) with a mutation rate of 12–40 mutations/Mb (approximately 13%) and those with an extremely high mutation rate of >40 mutations/Mb

Initially, TCGA researchers considered colon and rectal tumours as separate entities due to their known anatomical and therapeutic differences. However, it was found that similar patterns of genomic alteration (copy number, expression profile, DNA methylation and miRNA changes) were seen in both types of tumours, so they

Thirty-two genes were identified to be recurrently mutated, and, after removal of non-expressed genes, the hypermutated and non-hypermutated groups had 15

It was found that the tumour suppressor genes ATM and ARID1A displayed a disproportionately high number of frameshift or nonsense mutations. As expected, KRAS and NRAS mutations were activating oncogenic mutations at codons 12, 13 or 61, whereas the other genes had inactivating mutations. BRAF mutations were the classical V600E-activating mutations [8]. Given the differences in recurrently

**Hypermutated group Non-hypermutated group**

EDNRB (3%)

ACVR2A (63%) CASP8 (29%) APC (81%) TCF7L2 (9%) APC (51%) CDC27 (29%) TP53 (60%) FAM123B (7%) TGFBR2 (51%) FZD3 (29%) KRAS (43%) SMAD2 (6%) BRAF (46%) MIER3 (29%) TTN (31%) CTNNB1 (5%) MSH3 (40%) TCERG1 (29%) PIK3CA (18%) KIAA1804 (4%) MSH6 (40%) MAP7 (26%) FBXW7 (11%) SOX9 (4%) MYOB1 (31%) PTPN12 (26%) SMAD4 (10%) ACVR1B (4%) TCF7L2 (31%) NRAS (9%) GP6C (4%)

were subsequently analysed together within the non-hypermutated group.

and 17 recurrently mutated genes, respectively (see **Table 1**).

*Significantly mutated genes in non-hypermutated and hypermutated groups.*

*DOI: http://dx.doi.org/10.5772/intechopen.80679*

exists in this disease.

**colorectal cancer**

(approximately 3%)—the ultramutated group.

*Advances in Molecular Subclassification of Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.80679*

*Advances in the Molecular Understanding of Colorectal Cancer*

**2. Early molecular characterisation of colorectal cancer**

(distal) and have higher rates of KRAS mutation [5].

free survival (PFS) than MMR-proficient tumours [7].

**3. Integrative molecular analysis of colorectal cancer**

in CRC.

PMS2 and MSH6.

through the analysis of responders and nonresponders to targeted agents and the subsequent discovery of RAS mutations conferring resistance to anti-epidermal growth factor receptor (EGFR) therapies. More recently, our deeper understanding of the underlying biology of CRC has also revealed that clonal, stromal and immune characteristics of tumours are important when considering therapeutic targets. The ongoing need to accurately define molecularly distinct subgroups and identify the underlying genetic drivers as well as novel therapeutic targets within each subgroup in order to rationalise drug development continues to be of paramount importance

It is now well established that the majority of sporadic CRC cases (85%) exhibit chromosomal instability (CIN) with changes in chromosome number and structure such as deletions, gains, translocations and amplifications. CIN is associated with inactivating mutations or losses in the APC tumour suppressor gene which occurs early in the adenoma-carcinoma sequence [3]. The remaining 15% of sporadic CRCs demonstrate microsatellite instability (MSI) through changes in the number of repeats or length of microsatellites. MSI arises through defective DNA mismatch repair (MMR) mechanisms caused by epigenetic silencing of the MLH1 gene by promotor hypermethylation [4]. Epigenomic studies have shown that MSI tumours have a high CpG island methylator phenotype (CIMP-H) which involves aberrant methylation of CpG-rich gene promoter regions. This leads to silencing of expression of critical tumour suppressor genes such as MLH1, thereby leading to the development of CRC [5]. Familial syndromes, such as Lynch syndrome/hereditary non-polyposis colorectal cancer syndrome (HNPCC), occur through germline mutational inactivation of genes encoding MMR proteins, namely, MLH1, MSH2,

Clinicopathological features and the mutational status of CRC tumours differ according to the above classification. Sporadic MSI-high (MSI-H) tumours are more likely to be right-sided (proximal), poorly differentiated, mucinous and associated with tumour-infiltrating lymphocytes (TILs) and have higher rates of BRAF mutation, whereas microsatellite-stable (MSS) tumours are more frequently left-sided

It has been shown that MSI status has both a prognostic and a predictive role in CRC. MSI-H tumours have better stage-adjusted survival (in stages I–III) when treated with surgery alone and do not derive as much benefit from adjuvant fluorouracil-based chemotherapy as MSS tumours do [4]. In advanced disease, MSI-H tumours are associated with a worse prognosis, and this is due to their association with activating BRAF mutations [6]. It has more recently been shown that MSI status also predicts for significant response and benefit from anti-PD1 antibodies with MMR-deficient tumours exhibiting higher response rates and longer progression-

It is clear that anatomical factors and common DNA alterations are helpful in identifying subtype characteristics in CRC, but they alone are inadequate to define the boundaries between the different molecular entities that comprise CRC. In recent years, many studies have begun to exploit microarray technology to investigate gene expression profiles (GEPs) in CRC; however, no single signature

**4**

has proven clinically meaningful, especially in regard to predicting prognosis, and studies have been poorly reproducible due to the high molecular heterogeneity that exists in this disease.

In 2012, The Cancer Genome Atlas (TCGA) Research Network produced a comprehensive integrative analysis of 224 colorectal cancer tumour samples with paired normal samples in order to improve our understanding of the biology of this disease and identify potential therapeutic targets [8]. In addition, independent scientific groups also attempted to define intrinsic subtypes of CRC using GEPs in the hope that this will refine the molecular classification of CRC and facilitate clinical translation [9–14]. The findings of all of these independent analyses are discussed below.
