Adjuvant and Neoadjuvant Therapy for Colorectal Cancer: Molecular-Based Therapy

Chromosomal Instability Pathway of Colorectal Cancer

Fearon and Vogelstein originally theorized that CRC formation was a multistep process and that most, if not all, CRCs arose from benign tumors or adenomas. During this multistep process, initially mutational inactivation of tumor suppressor genes occurred followed by mutational activation of oncogenes that result in CIN and eventual creation of CRC. Fearon and Vogelstein also theorized that mutations in at least four to five genes are required for the formation of a tumor. The development of CRC via the CIN pathway has been referred to as the classic adenoma-carcinoma sequence. CRC often progresses over 10 years, beginning with dysplastic adenomas or polyps as the most common premalignant precursor lesion. A key step in this process is the early mutation of the adenomatous polyposis coli (APC) gene. APC is a tumor suppressor gene involved in both sporadic CIN and, when germ line mutated, in all persons with familial adenomatous polyposis (FAP). Sporadic CIN occurs in approximately 60% to 70% of adenomas that progress to carcinomas. The adenoma-carcinoma process is enhanced further by mutations that activate the Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene and inactivate the tumor protein 53 (TP53) tumor suppressor gene. These gene mutations are associated with CIN and promote tumor cell proliferation and invasiveness. Other gene alterations found in the CIN pathway affect CIN, such as loss of heterozygosity (LOH) of the long arm of chromosome 18 (18q), which inactivates tumor suppressor genes. Other genetic changes include extreme hypomethylation of the LINE-1 (long interspersed nucleotide element-1) gene, changes in the kinetochore, a multiprotein complex essential for normal segregation during mitosis, and overexpression of hypoxia-inducible factor 1α (HIF1α), which regulates the HIF1 and HIF2 genes mediating the cellular response to hypoxia. These genetic and epigenetic changes in the genome increase expression of genes involved in angiogenesis, cell survival, and glucose metabolism, as well as influencing different pathways to promote cellular growth of the tumor.

Microsatellite Instability Pathway

The MSI pathway represents a form of genomic instability that is found in approximately 15% of sporadic CRCs and in almost all cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, which represents 2% to 5% of all CRC. MSI is caused by inactivity of the DNA mismatch repair (MMR) system. When the DNA MMR system is not active, there is a 100-fold increase in the mutation rate in colorectal mucosa cells. In cases of sporadic CRC the MMR defect is caused primarily by hypermethylation of the MutL homolog 1 (MLH1) gene promoter due to senescence, which results in altered gene expression. In CRC occurring in Lynch syndrome patients, the MMR defect is caused primarily by germ-line mutations in one of the MMR genes ( MLH , MutS protein homolog 2 [MSH2] , MSH6 , and PSM1 homolog 2 [PMS2] ).

The MMR system is highly conserved and involves a number of genes, MLH1 , MSH2 , MSH6 , and PMS2 , whose products interact and are actively engaged in identifying and correcting DNA mismatches created during DNA replication. Microsatellites are short tandem sequences of nucleotides (one to six base pairs [bps]) that are repeated from 5 to 50 times in regions within the DNA sequence. They are distributed throughout the genome, and they are especially prone to replication errors. If DNA replication errors occur and are not repaired due to a deficient MMR system, protein synthesis is disrupted due to frameshift mutations as a consequence of insertions and deletions of nucleotides in the DNA sequence. Therefore an accumulation of frameshift mutations in microsatellites results in genetic instability. However, most importantly, a deficient MMR system has tumorigenic potential, particularly when it alters the function of key genes that regulate cellular growth and apoptosis.

Immunohistochemical (IHC) staining of tissues is a simple, fast, and inexpensive method to detect high-level MSI (MSI-H), which is defined as the absence of expression in one or more of the MMR proteins (MLH1, MSH2, PMS2, and MSH6). IHC in the case of a normal pattern of MMR protein staining, provides good sensitivity (>90%), excellent specificity (100%), and a 96.7% predictive value of a microsatellite stable (MSS) phenotype. In the case of an abnormal pattern of protein expression, there is a 100% predictive value of MSI phenotype. However, normal IHC staining may not entirely exclude all MSI cases because certain mutations in the MMR genes can lead to the production of a nonfunctional protein that retains antigenicity (results in normal IHC staining but does not function). The polymerase chain reaction (PCR)–based method has become the gold standard for MSI testing. Currently, the PCR-based method uses a five quasimonomorphic mononucleotide (one base) repeats: BAT-25, BAT-26, NR21, NR24, and NR27 as a panel to identify MSI. Contrary to most microsatellites that are polymorphic (more than one base), these mononucleotides are called quasimonomorphic because they are characterized in normal DNA by a one nucleotide repeat of 20 to 30 bps that is variable or polymorphic in the population but is almost identical in size between individuals. This means that one can determine MSI in tumor tissue without having to analyze matched DNA from normal tissues. The MSI-H phenotype is defined by the presence of at least two unstable markers among the five analyzed (or >30% of unstable markers if a larger panel is used), whereas those with instability at one marker or showing no instability are defined as MSI-low (MSI-L) and MSS tumors, respectively.

In sporadic settings, MSI-H CRCs are due to the epigenetic silencing of the hMLH1 gene promoter. The resulting mutant phenotype, as in the case of HNPCC, leads to inactivation of target genes, in particular those that regulate cellular growth and apoptosis. Most MSI-H CRC cases harbor the V600E mutation of the B-RAF protooncogene. BRAF , a member of the RAF gene family, makes a serine/threonine kinase called B-Raf, which is involved in the RAS/MAPK pathway in the cell. B-Raf is involved in cell growth and division, apoptosis, and cell migration. In the case of the V600E mutation, the BRAF gene is constitutively active, meaning tumor cells undergo dysregulated growth and division. MSI-H sporadic CRCs also display CIMP features, and this will be described in the CIMP pathway of CRC section.

CRC that develops as a consequence of the MSI pathway (hereditary and sporadic) has distinct clinical features; the malignancy is often located in the proximal colon, with a poorly differentiated and mucinous or medullary histologic subtype, and frequently includes intense peritumoral and intratumoral lymphocytic infiltrations. It is thought that the high density of tumor-infiltrating lymphocytes is due to a heightened host immune system response that not only recognizes tumor antigens but also novel tumor antigens that arise due to the frameshift mutations in the DNA sequence as a consequence of MSI. In general, the prognosis and survival of patients affected by MSI-H CRC is better and longer than survival of patients with CRC derived from CIN.

Cpg Island Methylator Phenotype and Serrated Pathway of Colorectal Cancer

A third pathway responsible for carcinogenesis in the colon is the CIMP. The CIMP phenotype is due to the aberrant hypermethylation of CpG islands, regions with a high incidence of CpG sites located in the promoter regions of genes involved in cell cycle regulation, apoptosis, angiogenesis, DNA repair, cell invasion, and adhesion. CpG islands are at least 200 bps in length, with a GC percentage higher than 50%. Hypermethylation of these CpG islands in the promoter region of genes causes loss of gene expression and thereby loss of function. CIMP is found in 20% to 30% of CRC cases, and the clinical features of CIMP CRCs are similar to CRCs that are MSI-H.

Based on the numbers of methylated markers, CRC with the CIMP phenotype can be divided into CIMP-high and CIMP-low. A mutation in the BRAF protooncogene often is identified in CIMP-high CRCs. CRCs containing BRAF mutations are associated with increased cell growth, progression of carcinogenesis, and high CRC-specific mortality. CRCs with mutations in the BRAF gene are a negative prognostic factor for overall survival (OS) in early-stage CRC due to its association with poor survival after relapse. It is important to determine the tumor MSI status in a BRAF -mutated CRC and the anatomic location of the tumor. As mentioned previously, MSI-H sporadic CRCs are a result of epigenetic silencing of the hMLH1 gene promoter in the MSI pathway and early-stage MSI-H right-sided CRCs containing BRAF mutations have a more favorable prognosis than the MSS left-sided colon tumors.

Mutations in the BRAF gene are found in 90% of CRC cases with sessile serrated adenomas (SSAs), but they are never present in conventional adenomas. BRAF mutations are an early event in the serrated pathway that leads to cancer. These mutations are present in early hyperplastic (serrated precursors) polyps and in advanced dysplastic serrated polyps, confirming its role in CRC neoplastic progression. SSA polyps with BRAF mutations frequently have CIMP-high and MSI-H features; thus researchers have surmised that in sporadic settings CIMP-high, MSI-H CRCs arise from the serrated pathway of carcinonogenesis.

BRAF and KRAS gene mutations are considered mutually exclusive in CRC. Researchers have found that when a KRAS mutation is present in a CRC, this cancer is CIMP-negative. KRAS mutation–positive, CIMP-low CRCs are also frequently associated with mutations in the DNA repair gene, methylguanine methyltransferase (MGMT) that likely contributes to the development of mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene. Therefore CIMP-low CRCs appear to have a different phenotype than CIMP-high CRCs. An “alternative serrated pathway” has been described, in which CIMP-low CRCs are the result of a hybrid carcinogenesis pattern observed for both adenomatous and serrated polyps. It has been hypothesized that CRC derived from these types of polyps that carry the KRAS mutation represent only 2% of CRC cases, and they have the potential to be extremely aggressive due to the inactivation of the MGMT gene.

Inflammatory Pathway

Patients with IBD are at an increased risk for developing CRC. The high risk for acquiring CRC in the setting of IBD increases with length of duration of IBD, as well as the extent and degree of tissue inflammation, family history of CRC, and the coexistence of primary sclerosing cholangitis. The pathogenesis of IBD-associated CRC is poorly understood. Many of the genetic changes associated with the initiation of sporadic CRC (such as CIN, MSI, and CIMP) also play roles in the incidence of IBD-related CRC. However, unlike sporadic CRC, which develops from colon tissue dysplasia in one or two foci of the colon or rectum, cancer that arises from chronically inflamed mucosa usually develops from multifocal areas of dysplasia, indicating that a “field cancerization defect” may come into play. The result of chronic inflammation, such as the induction of cyclooxygenase (COX)-2 gene expression and increased levels of inflammatory cytokines and chemokines, may play a role in the pathogenesis of IBD-related CRC. Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to decrease the risk of CRC in IBD patients by 40% to 50%. NSAIDs exert their effects through inhibition of the activity of COX enzymes. Among the three isoforms of the COX enzyme, COX-1, -2, and -3, COX-2 expression is enhanced by inflammation and its levels are elevated in 50% of colorectal adenomas and in 85% of colorectal adenocarcinomas. COX-2 protein overexpression occurs earlier in colorectal neoplasia originating in IBD, which may explain why CRC develops in patients with IBD at a younger age. In addition, oxidative stress develops in inflammatory stales where inflammatory cells, activated neutrophils, and macrophages produce large amounts of reactive oxygen and nitrogen species (RONs). Active IBD-affected mucosa shows increased expression of nitric oxide synthase and RONs. RONs can promote mutations in genes that are key in carcinogenic pathways associated with CRC, such as the TP53 and MMR genes.

Conclusions

Identifying the different molecular pathways that cause the development of CRC has helped us to better understand how CRC initiates and progresses. However, more importantly, a better understanding of the pathogenesis of CRC gives us a guide for identifying the molecular markers that predominantly influence this disease's behavior, as well as its prognosis and response to treatments.