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

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 (approximately 3%)—the ultramutated group.

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 were subsequently analysed together within the non-hypermutated group.

Thirty-two genes were identified to be recurrently mutated, and, after removal of non-expressed genes, the hypermutated and non-hypermutated groups had 15 and 17 recurrently mutated genes, respectively (see **Table 1**).

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


**Table 1.**

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

mutated genes between hypermutated and non-hypermutated cancers, it appears that these tumours progress through different sequences of genetic events.

Interestingly, the recent data published by Jones et al. has identified that non-V600 BRAF-mutated advanced CRC represents a molecular subtype with distinct characteristics (which are different to BRAFV600E-mutated CRC) and an excellent prognosis [15]. These patients may not require the aggressive chemotherapy treatment that is beneficial to classical BRAF-mutated patients. It is not yet clear whether non-V600 BRAF-mutated cancers harbour the same resistance to anti-EGFR therapies as cancers with BRAFV600E mutations, but higher frequency of concomitant RAS mutations in this subgroup will have to be taken into account.

TCGA analysis provided further confirmation on the pathways previously known to be deregulated in CRC. The vast majority of tumours in both groups (93% of non-hypermutated and 97% of mutated tumours) had deregulated Wnt signalling, predominantly via inactivation of APC. The MAPK signalling pathway was also commonly activated, as was the PI3K signalling pathway. Inactivation of the TGF-β inhibitory pathway was also seen, resulting in increased activity of MYC. Almost all of the analysed tumours, irrespective of location or mutation levels, exhibited changes in MYC transcriptional targets, highlighting the important role of MYC in CRC development. New findings identified by TCGA included recurrent mutations in FAM123B, ARID1A and SOX9 and very high levels of overexpression of the Wnt ligand-receptor gene FZD10. The SOX9 gene is associated with intestinal stem cell differentiation and has not previously been shown to be implicated in CRC. It has been shown to facilitate β-catenin degradation [16], and its transcription is suppressed by Wnt signalling which is activated by extrinsic Wnt ligands. These findings suggest a number of potential therapeutic targets in CRC, namely, Wnt signalling inhibitors and small molecule β-catenin inhibitors, which are beginning to show initial promise [17–19]. In addition, overexpression of the genes ERBB2 and IGF2, which are involved in regulating cell proliferation, were identified thus indicating potential therapeutic opportunities of inhibiting the products of these genes.

mRNA expression profiles of a subset of 189 TCGA samples separated the colorectal tumours into three clusters. One significantly overlapped with CIMP-H tumours and was enriched for hypermutated tumours, thereby representing a MSI/ CIMP subgroup. The two other groups were representative of a CIN and an invasive phenotype subgroup.

#### **3.2 Intrinsic subtypes of colorectal cancer identified by independent groups**

Three molecular CRC subtypes were also identified by Roepman and colleagues ((A) MMR-deficient epithelial, (B) proliferative epithelial and (C) mesenchymal) using unsupervised clustering of whole genome data from 188 CRC tumour samples [9]. These intrinsic subtypes were subsequently validated in a cohort of 543 patients with stage II–III disease. In addition to identifying these subtypes with phenotypes matching those identified via TCGA, prognostic features and chemotherapy benefit characteristics were also investigated in this study. The dMMR subtype A (22%) was found to be epithelial-like and displayed a strong MSI phenotype linked to dMMR and a high mutational rate including activating BRAF mutations. Type A patients exhibited the best prognosis with minimal benefit from adjuvant 5-FU chemotherapy. The mesenchymal subtype C (16%) tumours exhibit epithelial-tomesenchymal transition and show dMMR characteristics. These patients showed a poor baseline prognosis and no benefit from adjuvant 5-FU chemotherapy which is probably linked to their mesenchymal phenotype and low proliferative activity. The proliferative epithelial subtype B (62%) is almost exclusively MSS, BRAF wild type

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*Advances in Molecular Subclassification of Colorectal Cancer*

relevance on a larger set of stage IV tumours is warranted.

mutant subtype (C3) and a cancer stem cell subtype (C4).

pathway, GSK3β, PI3K and TOR than subtype 2.1 [12].

and MMR proficient. They exhibit a relatively poor baseline prognosis but receive the most benefit from adjuvant chemotherapy. This study focused on stages II and III CRC; therefore, further validation of the subtype classification and its clinical

In addition, De Sousa E Melo and colleagues also identified three similar subtypes using over 1100 CRC tumour samples: chromosomal instable (subtype A), microsatellite instable (subtype B) and a third subtype (subtype C) which is largely microsatellite stable and contains relatively more CIMP-H carcinomas but cannot be identified on the basis of characteristic mutations [10]. This third subtype is therefore similar to the third subtype described in the studies above. This subtype was found to be associated with a very unfavourable prognosis as well as resistance to anti-EGFR targeted therapy. It is thought to relate to sessile-serrated adenomas due to a very similar GEP involving upregulation of genes involved in matrix remodelling and epithelial-mesenchymal transition (EMT) which was seen in both. This study therefore suggests that sessile-serrated adenomas and tumours belonging to subtype C possess high malignant potential and need to be clinically managed as such [10]. Further groups have also used GEPs to identify more than three intrinsic subtypes

of CRC using large numbers of tumour samples. The biological relevance of the subtypes has been investigated in regard to treatment response and prognosis. Marisa and colleagues utilised a large multicentre cohort of tumour samples from patients with stage I–IV CRC, of which 556 fulfilled RNA quality requirements for GEP analysis [11]. These samples were split into a discovery set (n = 443) and a validation set (n = 1029) which also included 906 samples from eight public datasets. Unsupervised hierarchical clustering was applied to gene expression data which form the discovery subset to identify six molecular subtypes (C1–C6) with distinct clinicopathological features, molecular alterations, enrichments of supervised gene expression signatures and deregulated signalling pathways. In addition to identifying a deficient MMR subtype (C2), three CIN subtypes were shown (C1, C5 and C6): one with downregulated immune pathways (C1), one with upregulation of Wnt pathway (C5) and one displaying a normal-like GEP (C6). The remaining two were comprised of a KRAS

As expected, BRAF mutation was associated with the C2 subtype but was also frequent in the C4 CIMP-H, poor prognosis subtype. Although TP53 and KRAS mutations were found in all subtypes, the C3 subtype was highly enriched for KRAS mutant tumours suggesting a specific role for this mutation in this subtype of CRC. The biological relevance of these six subtypes is highlighted by their differing prognoses with the C4 and C6 subtypes being independently associated with the shortest relapsefree survival (RFS). However, the robustness of this gene signature as a prognostic classification requires further confirmation as some established prognostic factors in CRC, such as tumour grade and number of nodes examined, were not available for a

Schlicker et al. performed genome-wide mRNA expression profiling on 62 primary CRC samples using an unsupervised iterative approach [12]. Two main groups were identified (type 1 mesenchymal and type 2 epithelial) which were then split into five subtypes which were validated in independent published datasets comprising over 1600 samples. This subtype stratification was successfully aligned to several CRC cell line panels, and it was found that the GEPs defining the subtypes were well represented in these cell lines. Pharmacological response data showed that type 2 cell lines were more sensitive to treatment with aurora kinase inhibitors in keeping with the high levels of expression of aurora kinase A seen in the samples of this subtype. Additional data suggested that subtype 1.2 cell lines were most sensitive to inhibition of Src and also showed a higher sensitivity to inhibition of proteins on the PI3K

significant proportion of cases and thus were not included in the analysis.

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

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

*Advances in the Molecular Understanding of Colorectal Cancer*

mutated genes between hypermutated and non-hypermutated cancers, it appears

Interestingly, the recent data published by Jones et al. has identified that non-V600 BRAF-mutated advanced CRC represents a molecular subtype with distinct characteristics (which are different to BRAFV600E-mutated CRC) and an excellent prognosis [15]. These patients may not require the aggressive chemotherapy treatment that is beneficial to classical BRAF-mutated patients. It is not yet clear whether non-V600 BRAF-mutated cancers harbour the same resistance to anti-EGFR therapies as cancers with BRAFV600E mutations, but higher frequency of concomitant RAS mutations in this subgroup will have to be taken into account. TCGA analysis provided further confirmation on the pathways previously known to be deregulated in CRC. The vast majority of tumours in both groups (93% of non-hypermutated and 97% of mutated tumours) had deregulated Wnt signalling, predominantly via inactivation of APC. The MAPK signalling pathway was also commonly activated, as was the PI3K signalling pathway. Inactivation of the TGF-β inhibitory pathway was also seen, resulting in increased activity of MYC. Almost all of the analysed tumours, irrespective of location or mutation levels, exhibited changes in MYC transcriptional targets, highlighting the important role of MYC in CRC development. New findings identified by TCGA included recurrent mutations in FAM123B, ARID1A and SOX9 and very high levels of overexpression of the Wnt ligand-receptor gene FZD10. The SOX9 gene is associated with intestinal stem cell differentiation and has not previously been shown to be implicated in CRC. It has been shown to facilitate β-catenin degradation [16], and its transcription is suppressed by Wnt signalling which is activated by extrinsic Wnt ligands. These findings suggest a number of potential therapeutic targets in CRC, namely, Wnt signalling inhibitors and small molecule β-catenin inhibitors, which are beginning to show initial promise [17–19]. In addition, overexpression of the genes ERBB2 and IGF2, which are involved in regulating cell proliferation, were identified thus indicating potential therapeutic opportunities of inhibiting the

mRNA expression profiles of a subset of 189 TCGA samples separated the colorectal tumours into three clusters. One significantly overlapped with CIMP-H tumours and was enriched for hypermutated tumours, thereby representing a MSI/ CIMP subgroup. The two other groups were representative of a CIN and an invasive

**3.2 Intrinsic subtypes of colorectal cancer identified by independent groups**

Three molecular CRC subtypes were also identified by Roepman and colleagues ((A) MMR-deficient epithelial, (B) proliferative epithelial and (C) mesenchymal) using unsupervised clustering of whole genome data from 188 CRC tumour samples [9]. These intrinsic subtypes were subsequently validated in a cohort of 543 patients with stage II–III disease. In addition to identifying these subtypes with phenotypes matching those identified via TCGA, prognostic features and chemotherapy benefit characteristics were also investigated in this study. The dMMR subtype A (22%) was found to be epithelial-like and displayed a strong MSI phenotype linked to dMMR and a high mutational rate including activating BRAF mutations. Type A patients exhibited the best prognosis with minimal benefit from adjuvant 5-FU chemotherapy. The mesenchymal subtype C (16%) tumours exhibit epithelial-tomesenchymal transition and show dMMR characteristics. These patients showed a poor baseline prognosis and no benefit from adjuvant 5-FU chemotherapy which is probably linked to their mesenchymal phenotype and low proliferative activity. The proliferative epithelial subtype B (62%) is almost exclusively MSS, BRAF wild type

that these tumours progress through different sequences of genetic events.

**6**

products of these genes.

phenotype subgroup.

and MMR proficient. They exhibit a relatively poor baseline prognosis but receive the most benefit from adjuvant chemotherapy. This study focused on stages II and III CRC; therefore, further validation of the subtype classification and its clinical relevance on a larger set of stage IV tumours is warranted.

In addition, De Sousa E Melo and colleagues also identified three similar subtypes using over 1100 CRC tumour samples: chromosomal instable (subtype A), microsatellite instable (subtype B) and a third subtype (subtype C) which is largely microsatellite stable and contains relatively more CIMP-H carcinomas but cannot be identified on the basis of characteristic mutations [10]. This third subtype is therefore similar to the third subtype described in the studies above. This subtype was found to be associated with a very unfavourable prognosis as well as resistance to anti-EGFR targeted therapy. It is thought to relate to sessile-serrated adenomas due to a very similar GEP involving upregulation of genes involved in matrix remodelling and epithelial-mesenchymal transition (EMT) which was seen in both. This study therefore suggests that sessile-serrated adenomas and tumours belonging to subtype C possess high malignant potential and need to be clinically managed as such [10].

Further groups have also used GEPs to identify more than three intrinsic subtypes of CRC using large numbers of tumour samples. The biological relevance of the subtypes has been investigated in regard to treatment response and prognosis. Marisa and colleagues utilised a large multicentre cohort of tumour samples from patients with stage I–IV CRC, of which 556 fulfilled RNA quality requirements for GEP analysis [11]. These samples were split into a discovery set (n = 443) and a validation set (n = 1029) which also included 906 samples from eight public datasets. Unsupervised hierarchical clustering was applied to gene expression data which form the discovery subset to identify six molecular subtypes (C1–C6) with distinct clinicopathological features, molecular alterations, enrichments of supervised gene expression signatures and deregulated signalling pathways. In addition to identifying a deficient MMR subtype (C2), three CIN subtypes were shown (C1, C5 and C6): one with downregulated immune pathways (C1), one with upregulation of Wnt pathway (C5) and one displaying a normal-like GEP (C6). The remaining two were comprised of a KRAS mutant subtype (C3) and a cancer stem cell subtype (C4).

As expected, BRAF mutation was associated with the C2 subtype but was also frequent in the C4 CIMP-H, poor prognosis subtype. Although TP53 and KRAS mutations were found in all subtypes, the C3 subtype was highly enriched for KRAS mutant tumours suggesting a specific role for this mutation in this subtype of CRC. The biological relevance of these six subtypes is highlighted by their differing prognoses with the C4 and C6 subtypes being independently associated with the shortest relapsefree survival (RFS). However, the robustness of this gene signature as a prognostic classification requires further confirmation as some established prognostic factors in CRC, such as tumour grade and number of nodes examined, were not available for a significant proportion of cases and thus were not included in the analysis.

Schlicker et al. performed genome-wide mRNA expression profiling on 62 primary CRC samples using an unsupervised iterative approach [12]. Two main groups were identified (type 1 mesenchymal and type 2 epithelial) which were then split into five subtypes which were validated in independent published datasets comprising over 1600 samples. This subtype stratification was successfully aligned to several CRC cell line panels, and it was found that the GEPs defining the subtypes were well represented in these cell lines. Pharmacological response data showed that type 2 cell lines were more sensitive to treatment with aurora kinase inhibitors in keeping with the high levels of expression of aurora kinase A seen in the samples of this subtype. Additional data suggested that subtype 1.2 cell lines were most sensitive to inhibition of Src and also showed a higher sensitivity to inhibition of proteins on the PI3K pathway, GSK3β, PI3K and TOR than subtype 2.1 [12].

Budinska et al. performed unsupervised clustering of 1113 CRC samples based on gene models and distinguished at least five different gene expression CRC subtypes which they called surface crypt-like (A), lower crypt-like (B), CIMP-Hlike (C), mesenchymal (D) and mixed (E) [13]. These subtypes showed distinct biological motifs and morphological features as well as differences in prognosis. The subtypes were validated in an independent dataset of 720 CRC expression profiles. Subtype C was enriched for both MSI and BRAF mutations, and its characteristics were in keeping with the described CIMP-H phenotype and hypermutated tumours found in TCGA analysis. This subtype had one of the best outcomes for RFS but the worst outcome in survival after relapse (SAR). Once again, KRAS mutations were found in all subtypes, and this supports the emerging theory that KRAS mutant CRCs are highly heterogeneous and that the oncogenic role of KRAS varies with the specific mutation and molecular background of the tumour in which it occurs [20]. Subtypes C and D were associated with the worst overall survival (OS)—for subtype D this was primarily due to early relapse associated with high EMT gene expression and low proliferation-associated gene expression, and for subtype C, it was the result of short SAR.

Subtypes B and E highly expressed canonical Wnt signalling target signatures, whereas subtypes A and D and normal samples expressed low levels of this signature. This was in concordance with the corresponding high percentages of β-catenin-positive nuclei seen in subtypes B and E and converse low percentages seen in subtypes A and D. This analysis is in support of the data suggesting that the colon stem cell signature, under the condition of silenced canonical Wnt target genes, is associated with a higher risk of recurrence (subtype D) [21].

Sadanandam and colleagues performed an analysis of GEPs from 1290 CRC samples using consensus-based unsupervised clustering. The resultant clusters were then correlated with response to cetuximab using a dataset annotated with therapeutic response to cetuximab in 80 patients [14]. The results of these studies identified five clinically relevant CRC subtypes which were named according to genes preferentially expressed in each. The transit-amplifying subgroup was found to contain two groups which differed in cetuximab sensitivity, so it was split into cetuximab-sensitive and cetuximab-resistant, thereby making six subgroups in total. These sub-subtypes showed the best response to cetuximab and increased sensitivity to cMET inhibition, respectively.

Additionally, response to standard chemotherapy with FOLFIRI (5-FU and irinotecan) was also investigated, and the analyses suggested that stem-like subtype tumours, both in the adjuvant and metastatic settings, and inflammatory-subtype tumours in the adjuvant setting may best be treated with FOLFIRI [14]. The transitamplifying sub-subtypes and the goblet-like subtype were not likely to respond to FOLFIRI in the adjuvant setting, thereby potentially sparing some patients from toxicity of futile treatment. These findings obviously warrant further retrospective and prospective validation, but in unselected CRC patients, FOLFIRI chemotherapy has not shown a survival benefit in the adjuvant setting.

#### **3.3 Outcomes of integrative molecular analysis in CRC**

As is evidenced above, up to six molecular subtypes of CRC have been identified by these independent groups, but only superficial similarities exist between the studies. The main characteristics of these subtypes are summarised in **Table 2**. Two subtypes have been repeatedly identified (microsatellite instability enriched and high expression of mesenchymal genes), but full consistency amongst the others has not been achieved probably due to the underlying biological complexity of this cancer and the significant

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*Advances in Molecular Subclassification of Colorectal Cancer*

A-type MSI Hypermutated, dMMR

B-type Epithelial MSS, BRAF WT, pMMR

C-type Mesenchymal Undergone EMT

CCS1 Epithelial Mainly left sided

CCS2 MSI Mainly right sided

CCS3 Mesenchymal BRAF and KRAS++

C2: dMMR MSI dMMR/CIMP+++

C5: CIN Wnt up Epithelial CIN+++

C6: CIN normal Mesenchymal CIN+++

Epithelial CIN+++

Epithelial KRAS+++

Mesenchymal KRAS++

1.1 Mesenchymal Activation of MAPK, TGFβ and

1.2 MSI Activation of immune system-

2.1 Epithelial Activation of immune system-

2.2 Epithelial High expression of genes on

C1: CIN immune down

C3: KRAS mutated

C4: Cancer stem cell

**Subtype characteristics Prevalence Ref**

22% [9]

49% [10]

21% [11]

62%

16%

24%

27%

19%

13%

10%

27%

10%

15%

23%

32%

19% [12]

MSI/CIMP-H MSI Enriched for hypermutated tumours 30% [8]

High proliferative activity Relatively poor baseline prognosis Most benefit from adjuvant

Low proliferative activity Poor baseline prognosis No benefit from adjuvant

CIN Epithelial 30% Invasive Mesenchymal 40%

Good prognosis

chemotherapy

chemotherapy

dMMR/MSI-H

Poor prognosis

KRAS and TP53++

BRAF++, KRAS++

EMT upregulated

KRAS and TP53++ Wnt pathway upregulated

EMT upregulated

calcium signalling

related pathways

related pathways

chromosomes 13q and 20q

1.3 Mesenchymal High expression of transporter genes 11%

regulated

upregulated

regulated

Upregulation of genes involved in matrix remodelling and EMT

Immune system and EMT down

Immune system and proliferation

Immune system and EMT down

Proliferation down regulated

Proliferation down regulated

Highly enriched for MSI-H tumours

KRAS and TP53++

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

**category**

**Subtype Major subtype** 


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

*Advances in the Molecular Understanding of Colorectal Cancer*

was the result of short SAR.

sensitivity to cMET inhibition, respectively.

has not shown a survival benefit in the adjuvant setting.

**3.3 Outcomes of integrative molecular analysis in CRC**

Budinska et al. performed unsupervised clustering of 1113 CRC samples based

Subtypes B and E highly expressed canonical Wnt signalling target signatures,

Sadanandam and colleagues performed an analysis of GEPs from 1290 CRC samples using consensus-based unsupervised clustering. The resultant clusters were then correlated with response to cetuximab using a dataset annotated with therapeutic response to cetuximab in 80 patients [14]. The results of these studies identified five clinically relevant CRC subtypes which were named according to genes preferentially expressed in each. The transit-amplifying subgroup was found to contain two groups which differed in cetuximab sensitivity, so it was split into cetuximab-sensitive and cetuximab-resistant, thereby making six subgroups in total. These sub-subtypes showed the best response to cetuximab and increased

Additionally, response to standard chemotherapy with FOLFIRI (5-FU and irinotecan) was also investigated, and the analyses suggested that stem-like subtype tumours, both in the adjuvant and metastatic settings, and inflammatory-subtype tumours in the adjuvant setting may best be treated with FOLFIRI [14]. The transitamplifying sub-subtypes and the goblet-like subtype were not likely to respond to FOLFIRI in the adjuvant setting, thereby potentially sparing some patients from toxicity of futile treatment. These findings obviously warrant further retrospective and prospective validation, but in unselected CRC patients, FOLFIRI chemotherapy

As is evidenced above, up to six molecular subtypes of CRC have been identified by these independent groups, but only superficial similarities exist between the studies. The main characteristics of these subtypes are summarised in **Table 2**. Two subtypes have been repeatedly identified (microsatellite instability enriched and high expression of mesenchymal genes), but full consistency amongst the others has not been achieved probably due to the underlying biological complexity of this cancer and the significant

whereas subtypes A and D and normal samples expressed low levels of this signature. This was in concordance with the corresponding high percentages of β-catenin-positive nuclei seen in subtypes B and E and converse low percentages seen in subtypes A and D. This analysis is in support of the data suggesting that the colon stem cell signature, under the condition of silenced canonical Wnt target

genes, is associated with a higher risk of recurrence (subtype D) [21].

on gene models and distinguished at least five different gene expression CRC subtypes which they called surface crypt-like (A), lower crypt-like (B), CIMP-Hlike (C), mesenchymal (D) and mixed (E) [13]. These subtypes showed distinct biological motifs and morphological features as well as differences in prognosis. The subtypes were validated in an independent dataset of 720 CRC expression profiles. Subtype C was enriched for both MSI and BRAF mutations, and its characteristics were in keeping with the described CIMP-H phenotype and hypermutated tumours found in TCGA analysis. This subtype had one of the best outcomes for RFS but the worst outcome in survival after relapse (SAR). Once again, KRAS mutations were found in all subtypes, and this supports the emerging theory that KRAS mutant CRCs are highly heterogeneous and that the oncogenic role of KRAS varies with the specific mutation and molecular background of the tumour in which it occurs [20]. Subtypes C and D were associated with the worst overall survival (OS)—for subtype D this was primarily due to early relapse associated with high EMT gene expression and low proliferation-associated gene expression, and for subtype C, it

**8**


**Table 2.**

*Intrinsic molecular subtypes of CRC based on gene expression profiles.*

overlap of features between subgroups. Methodological differences in the processing and analysing of samples have also contributed to these inconsistencies.

In addition, the majority of samples from these datasets have been derived from primary tumours, so their applicability to advanced disease also needs to be considered as the molecular makeup of primary tumours versus metastases may vary, especially in response to the tumour microenvironment and immune cell infiltrate. Altogether, this has meant that the usefulness of these subclassification systems in clinical practice has been limited.

**11**

**Table 3.**

*Advances in Molecular Subclassification of Colorectal Cancer*

**4. The consensus molecular subtypes of colorectal cancer**

More recently, in order to resolve inconsistencies in subclassification systems and to aid clinical translation, the CRC research community formed an international consortium dedicated to large-scale data sharing and analytics [22]. After analysing the independent transcriptomic-based classification systems (which comprised 18 CRC datasets and 4151 patients in total) and using unsupervised clustering techniques, four robust consensus molecular subtypes (CMSs) with distinguishing features were proposed. Tumours with mixed features (approximately 13%) were thought to represent a transition phenotype or intratumoural heterogeneity. **Table 3** summarises the main biological, molecular, clinical and prognostic

In regard to genomic aberrations, CMS1 samples were hypermutated and encompassed the majority of MSI-H tumours. This group also displayed widespread hypermethylation and low prevalence of SCNAs. CMS2 and CMS4 subgroups displayed higher CIN via high SCNA counts. CMS3 samples consisted of fewer SCNAs than other CIN tumours, a significant proportion (30%) of hypermutated tumours

> **CMS2 Canonical**

Higher chromosomal instability

Wnt and Myc activation

Left sided tumours

Better survival after relapse

**14 37 13 23**

**CMS3 Metabolic**

Mixed MSI status SCNA low CIMP low

Intermediate levels of gene hypermethylation

KRAS mutations Activation of RTK and MAPK pathways

Metabolic deregulation

SCNA high Distinctive profile:

**CMS4 Mesenchymal**

SCNA high

Higher chromosomal instability

Stromal infiltration TGF-β activation Angiogenesis

More advanced stages

Worse relapsefree and overall survival

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

associations of the four consensus subtypes.

**CMS1 MSI immune**

MSI high CIMP high Hypermutation SCNA low

Overexpression of proteins involved in DNA damage repair

Widespread hypermethylation

BRAF mutations Activation of RTK and MAPK pathways

Immune infiltration and activation Strong activation of immune evasion pathways

Right sided tumours

Better relapse-free survival

Worse survival after

status

Females

relapse

*The four consensus molecular subtypes of CRC.*

Higher grade

**Percentage of samples**

Biological characteristics

Molecular features

Clinical features

Prognostic features

*Advances in the Molecular Understanding of Colorectal Cancer*

Lower crypt Epithelial Upregulated top colon crypt,

Mesenchymal Mesenchymal Upregulated EMT/stroma, CSC,

Inflammatory MSI Comparatively high expression of

Goblet Epithelial High mRNA expression of goblet-

Enterocyte Epithelial High expression of enterocyte-

Stem-like Mesenchymal High expression of Wnt signalling

**Subtype characteristics Prevalence Ref**

26% [13]

30%

11%

19%

14%

14%

18%

32%

18%

18% [14]

Upregulated top colon crypt, secretory cell and metallothioneins

Upregulated proliferation, immune,

Upregulated EMT/stroma, immune, top colon crypt, Chr20q, CSC

chemokines and interferon-related

specific MUC2 and TFF3, Good

May not benefit from adjuvant

known to predict cetuximab

Overexpressed FLNA (regulates expression and signalling of cMET receptor), cell lines more sensitive to

targets plus stem cell, myoepithelial and mesenchymal genes and low expression of differentiation markers

Intermediate prognosis

proliferation, Wnt Longest SAR

metallothioneins Shortest SAR

immune

genes

prognosis

chemotherapy

specific genes Intermediate prognosis

Epithelial Higher levels of EGFR ligands

response Good prognosis

cMET inhibition Good prognosis

Worst prognosis May benefit from adjuvant

chemotherapy

**Subtype Major subtype** 

**category**

CIMP-H MSI MSI+, BRAF+

Mixed Mesenchymal P53+

Cetuximabsensitive transit amplifying

Cetuximabresistant transit amplifying

Surface crypt Epithelial KRAS+

overlap of features between subgroups. Methodological differences in the processing

In addition, the majority of samples from these datasets have been derived from primary tumours, so their applicability to advanced disease also needs to be considered as the molecular makeup of primary tumours versus metastases may vary, especially in response to the tumour microenvironment and immune cell infiltrate. Altogether, this has meant that the usefulness of these subclassification systems in

Most benefit from FOLFIRI

and analysing of samples have also contributed to these inconsistencies.

**10**

**Table 2.**

clinical practice has been limited.

*Intrinsic molecular subtypes of CRC based on gene expression profiles.*
