With an estimated overall incidence in Europe of more than 400/100.000 new cases, colorectal cancer (CRC) is the second leading cause of cancer death, even though new and improved treatments have considerably reduced mortality in the last years(1).
Generally, most of CRCs are sporadic, and 5-10% of cases are considered to be the result of an obvious heritable component; despite the absence of an identified germline defect, up to 30% of cases might have a familial predisposition. Genome-wide association studies (GWAS) have revealed that about 5% of the CRC cases may be explained by a cumulative effect of low-penetrant risk factors(2). Aproximatively 30% of patients with sporadic CRC are diagnosed in advanced stages, and relapse occurs in almost 50% of those diagnosed in early stages. The use of different chemotherapeutic strategies, combined with targeted biologic therapies, has considerably improved the median overall survival (OS) of patients with metastatic CRC. Nevertheless, the majority of these patients have to receive second- and third-line treatments, resulting in a 5-year survival of less than 10%(3).
Despite the encouraging progresses such as the usefulness of KRAS mutation testing in predicting treatment response, and the fact that many other molecular alterations have been proved promising biomarkers in treatment selection, prognosis and early detection of CRC, the tumors with similar histological features and/or tumor stage are characterized by varied clinical outcomes and drug response, and also high mortality. These differences can be explained, partly, by tumor heterogeneity and by CRC initiating molecular events such as MSI, CIMP, CIN, and KRAS and BRAF mutations(4). Nevertheless, the clinicopathological staging and histological assessment of tumor tissue still remains the milestone of CRC prognosis and treatment selection. The updated Tumor Node Metastasis (TNM) classification system is used in clinical practice, although the original Duke’s staging system is still used by some clinicians and researchers. Molecular testing is usually required for accurate assessment of those alterations that provide prognostic and predictive information beyond clinicopathologic features. Although much effort has been made to identify novel prognostic biomarkers, only few are currently used in clinical practice. The goal of this article is to provide a summarized review of the molecular genetics basis of CRC and how these alterations contribute to a better molecular classification of the disease in order to improve prognosis and treatment response.
Major molecular classifiers of colorectal cancer
CRC carcinogenesis is the result of gradual accumulation of the acquired genetic and epigenetic alterations that transform normal epithelial cells into invasive adenocarcinomas, but is considered that genetic instability is more significant in most aggressive and metastatic forms of the disease.
Three main instabilities have been described, which may result in proliferative advantages of the tumor, affecting various tumor-related factors, such as invasive ability and tumor aggressiveness:
Chromosomal instability (CIN)
Microsatellite instability (MSI)
CpG island methylator phenotype (CIMP).
Some superposable characteristics of these categories and imprecise use of these terms generated confusions and haziness in the interpretation of literature(5).
Major genetic mutations identified in CRC are acting as key events, and affects genes involved in WNT (APC, CTNNB1), MAPK (KRAS, BRAF), PI3K, PTEN-mTOR, TGF-b/SMAD and TP53 pathways(6). The patterns of mutated genes vary according to the type of genomic instability. Thus, BRAF mutations are prevalent in CRC with high microsatellite instability, while TP53 mutations are more frequent in CIN tumors. Also, during CRC carcinogenesis, DNA repair, DNA damage checkpoint and DNA replication gene expressions are mostly down-regulated in tumor cells compared to proliferating normal adjacent tissues, and a widespread of CNV can be observed, some of which are highly recurrent(7).
1. Chromosomal instability
Chromosome instability (CIN) is the most common form of genomic instability, and it is characterized by a widespread imbalance in chromosome number (aneuploidy) and loss of heterozygosity (LOH). Although CIN can result from various defects in mitotic checkpoint, chromosomal segregation, telomere function, centrosome number and function, DNA damage response or defects in oncogenes and tumor suppressor genes, and despite its high frequency in CRCs, the mechanisms that give rise to this form of genomic instability and the role of aneuploidy in tumor progression remain poorly understood(8).
A number of heterogeneous key events related to CIN colorectal tumors have been identified and include mutations in tumor suppressor genes and oncogenes such as: APC, TP53, K/N-RAS, CTNNB1, PIK3CA, BUBR1, AURKA or FBXW7(9). Although KRAS mutational status guides the administration of anti-EGFR targeted therapy in advanced CRC, due to the well-known heterogeneity of CRC, only 20-30% of the CRC patients with wild type RAS respond to therapy. Many potential biomarkers have been studied in the last decade, but they haven’t been incorporated into clinical practice due to inconclusive results. Studies have shown that CIN is a marker of poor prognosis in CRCs and an independent predictor of early relapse and death among stage II patients, but lacking standardized markers and criteria for CIN status in CRC has limited its use in clinical practice(10-12).
2. Microsatellite instability
Microsatellite instability (MSI) involves inactivation of DNA Mismatch Repair (MMR) family genes, either by aberrant DNA methylation, or by somatic mutation and alteration of lengths of short nucleotide repeat sequences named microsatellites. Approximately 90% of individuals with Lynch syndrome develop MSI colorectal cancers because they harbor germline mutations in MMR genes: MLH1, MSH2, MSH6, PMS2, thus routine clinical MSI testing is performed as a screening test to identify individuals with this disease. About 10-15% of the sporadic CRC exhibit high MSI(13). The sporadic deficient MMR tumors carry somatic mutations in the BRAF gene in about half of cases. Although BRAF mutational status confers prognostic information, its value in predicting anti-EGFR therapy resistance remains to be more detailed.
Currently, MSI is used to guide clinical management. Compared with stable microsatellite (MSS) neoplasms, high MSI tumors are associated with low rates of recurrence and metastasis, longer overall and cancer-specific survival, poor differentiation and serrated neoplasia, BRAF mutations, diploid DNA content, exhibits abundant tumor-infiltrating lymphocytes, and are commonly located on the proximal side of the colon(14). Generally, patients with sporadic CRC and high instability of microsatellites have a better prognosis compared with stable tumors, but they have limited benefit to 5-fluorouracil (5-FU) chemotherapy, particularly stage II patients(15). According to the National Comprehensive Cancer Network guidelines, it is recommended MSI testing of all stage II CRC patients, and genetic screening for Lynch syndrome in patients younger than 70 years of age at diagnosis(14,15).
3. CpG Island Methylator Phenotype
The epigenetic instability observed in tumors is the result of hypermethylation of CpG islands (CIMP) located in gene promoters, global DNA hypomethylation and histone modification. Aberrant DNA methylation is present in all CRCs, but only about 20% of the tumors exhibit high proportion of methylated CpG loci that are described as being CIMP+. Methylator phenotype discovery and the classification of CIMP-based tumors have improved our understanding of the molecular pathology of CRC. CIMP+ tumors have a distinct clinical, pathological and molecular profile which includes associations with proximal colon tumor location, higher occurrence in females, older patients and early stages of tumor progression, poor differentiation, mucinos histology, MSI, high BRAF and KRAS rates and wild type p53(16). The mechanism underlying the methylator phenotype is poorly understood(17). Even though several of the clinical and pathological features correlated with CIMP are also related to MSI, studies have shown that the associations between high methylator phenotype and a right-sided location, mucinos histological type and BRAF mutation were confirmed in analyses of MSI and MSS tumors separately. The results suggest that these characteristics are associated to CIMP independently of MSI status(18,19). According to many research studies, patients with CIMP+ colorectal tumors have worse prognosis, but adjuvant treatments seem to be more efficient. Instead, CIMP+/high-MSI tumors have a better prognostic(19-21).
Despite these promising results, the prognostic and predictive value of CIMP status remains controversial; at this very moment the existing data are not sufficient to sustain the clinical utility of this tumor subgroup, mainly due to the lacking of a standardized criterion for determining CIMP.
Colorectal cancer molecular subtypes
A large amount of studies revealed that groups of CRC patients defined by various and numerous molecular events, but with similar histological features and/or tumor stage, show different biological traits, clinical outcome and sensitivity to chemotherapy, targeted agents and immunotherapy. Consequently, several molecular approaches to stratify CRC have been proposed(3).
1. Colon cancer subtype system (CCS)
De Sousa E. Melo and collaborators identified three CRC subtypes based on their genetic instabilities and clinico-pathological futures(3,22):
CCS1 (CIN subtype), accounting for 49% of the cases which are characterized by high mutational rate of KRAS and TP53 genes, strong CIN, hyperactivity of the WNT signaling and good disease free survival (DFS).
CCS2 (MSI subtype) comprises 24% of tumors, are predominately located in the proximal colon, have abundant inflammatory cell infiltration, are high MSI/CIMP+ and intermediate DSF.
CCS3 (serrated subtype) accounts for 27% of all CRC and it is characterized by overexpression of EMT-related and cell migration genes, high frequency of BRAF and PIK3CA gene mutations, TGF-b pathway activation, high MSI, intermediate CIMP, sessile-serrated histology, being refractory to EGFR-targeted therapy, poor prognosis and high rate of recurrence.
2. Colorectal cancer assigner system (CRCA)
Based on the differential gene expression among CRCs, Sadanandam et al. stratified tumors into five subtypes that reflect different stages of differentiation of the intestinal cells and have distinctive characteristics of potential clinical significance(3,23):
Stem-like tumor subtype is characterized by the overexpression of WNT signaling pathway transcripts, mesenchymal stem-cell features, poor prognostic and shorter survival.
Goblet-like subtype includes tumors with high mRNA expression of goblet-specific genes MUC2 and TFF3, and has a favorable prognosis.
Transit-amplifying (TA) subtype includes lesions with variable expression of stem cell related genes and WNT pathway. This subgroup can be subdivided into two groups, based on different responses to EGFR-targeted therapy: CR-TA (resistant to targeted therapy) and CS-TA (sensitive to targeted therapy).
Inflammatory subtype includes colorectal neoplasms characterized by the overexpression of genes related to cytokines and interferon. The patients included in this subcategory have moderate DFS.
Enterocyte subtype shows overexpression of specific genes of enterocytes cells, increased chemokines and intermediate DFS of CRC patients.
3. Colon cancer molecular subtype system (CCMS)
CCMS is a robust molecular classification proposed by Marisa and co-authors, and it is based on genome-wide mRNA expression profile analyses and clinico-pathological features of CRC(3,24):
C1 subtype (CIN immune down) accounts for 21% of CRCs and it is characterized by CIN+, frequent mutations in KRAS and TP53 genes, downregulation of the tumor immune and EMT pathways and frequently distal colon localization of the tumor.
C2 subtype (MMR deficient) includes 19% of colorectal neoplasms with high MSI, CIMP+, frequent mutations of MMR and BRAF genes, activation of the proliferative and immune pathways, suppression of the WNT signaling, proximal colon localization of the neoplasm and mucinous histology.
C3 subtype (KRAS mutant) comprises 13% of patients with CRC characterized by MSS, KRAS mutation, inactivation of the immune system and EMT pathways, proximal localization of the tumor, and serrated phenotype.
C4 subtype (cancer stem cell - CSC) accounts for 10% of the cases which show CIN+/CIMP+, frequent mutations of KRAS, BRAF and TP53 genes, overexpression of EMT pathway genes, high incidence of metastases, downregulation of cell growth pathways and apoptosis, up-regulation of the EMT/motility pathways, proximal localization of the lesion, serrated phenotype, high risk of recurrence and worse prognosis.
C5 subtype (CIN WNT up) covers 27% of CRCs and have marked CIN, high rate of KRAS and TP53 genes mutations, overexpression of the WNT and EMT pathway genes, and distal localization of the tumor.
C6 subtype (CIN normal) accounts for 10% of CRCs which show frequent mutations in KRAS and TP53 genes, downregulation of cell growth and death pathways, up-regulation of the EMT/motility pathways, distal localization of the tumor, serrated phenotype and worse prognosis.
4. CRC intrinsic subtypes
Another classification system was described by Roepman’s team. They considered three major intrinsic subtypes of CRC based on three major molecular events of the tumor: EMT, deficiency in MMR genes and cellular proliferation(3,25):
Subtype A (MMR deficient, epithelial) was observed in 22% up to 35% of the CRC patients and it is characterized by high MSI, frequent MMR and BRAF genes mutations and good prognosis.
Subtype B (epithelial proliferative), that accounts for 52% up to 62% of CRC cases which shows high proliferative index, low overall mutation frequency, MSS, BRAF wild type and absence of MMR deficiency. Patients of this group have poor prognosis, but could benefit from treatment with adjuvant chemotherapy.
Subtype C (mesenchymal) represents 13% up to 16% of the CRC cases which are characterized by the overexpression of EMT genes and low proliferative index. Patients in this group have a poor prognosis and do not benefit from adjuvant therapy.
5. Colorectal cancer subtyping consortium (CRCSC)
Sadanandam’s research team classified CRC into four molecular subtypes that, currently, represent the complex description of CRC heterogeneity at the gene expression level(26). They also included in the classification a fifth group that does not have a clear assignment(3,26,27):
CMS1 (MSI, strong immune) comprises 14% of CRCs with hypermutability (KRAS, TP53, PIK3CA), strong immune infiltration (CD8+ cytotoxic T lymphocytes, CD4+ T helper 1 cells and natural killer cells), JAK-STAT activation, low prevalence of CNV, high MSI, CIMP+, frequent BRAF mutations, preferably diagnosed in: right-sided lesions, females and older age patients. Patients show good prognosis and the recurrences rate is low.
CMS2 (canonical, epithelial) affects 37% of CRC cases which describe marked upregulation of WNT and MYC downstream targets, MSS, CIN+, overexpression of EGFR, ERBB2, IGF2, IRS2, HNF4A and cyclins, activation of MYC, SRC, VGFR, TGF-b and integrins, TP53 gene mutations, preferably affecting the left colon and rectum and with better overall survival.
CMS3 (metabolic, epithelial) represents 13% of the tumors characterized by low CIN, metabolic dysregulation, including activation of glutaminolysis and lipidogenesis, DNA damage repair, intermediate levels of gene hypermethylation, overexpression of IGFBP2, moderate activation of WNT and MYC pathways, KRAS and PIK3CA gene mutations, and intermediate survival rate.
CMS4 (mesenchymal) comprises 23% of sporadic CRCs. These tumors show heterogeneous CIN and MSI, activation of pathways related to EMT and stemness, strong stromal infiltration, immunosuppression and complement-mediated inflammation, active angiogenesis, overexpression of NOTCH3/VEGFR2 and of proteins implicated in extracellular matrix remodeling, a tendency to occur at later disease-stages and to recur, and are associated with younger age at diagnosis and worse survival.
The remaining 13% of samples did not have a consensus allotment. This group shows similar features with two or more subtypes, probably due to the intratumoral heterogeneity and/or variable EMT activation reflected in a transient phenotype(3,27).
Progresses made so far in molecular biology have enabled precision medicine to become a clinical reality, with molecular testing at the forefront of this major advance in medicine.
In sporadic CRC, the most clear examples of the molecular alterations role in improving clinical care are the acquired KRAS mutations, routinely used as a negative predictive marker to avoid treatment with anti-EGFR antibody in patients with mCRC, and MMR status that guide adjuvant chemotherapy in patients with early stage colon cancer. Numerous other genetic and epigenetic biomarkers showed encouraging results towards early cancer detection, risk stratification, prognosis and treatment selection, but the limited amount of samples included in studies, the intratumoral heterogeneity and the lack of some standardized assays have led to inconsistent results.
Clinical and pathological staging and histological assessment of CRC remain the cornerstone of prognostic and treatment selection, but the new molecular biomarkers have to complete the clinical picture of the tumor.
The progresses made in understanding the genetic and epigenetic alterations of CRC have facilitated and improved classification systems of this disease; the molecular subtypes better inform clinicians regarding prognosis, therapeutic prediction and novel strategies, and are expected to become part of the routinely managing of these patients.