Etiopathogenesis and Treatment of Colorectal Cancer

Abstract

Human colorectal cancer (CRC) encompasses tumors affecting a segment of the large intestine (colon) and rectum. It is the third most commonly diagnosed malignancy and the second leading cause of cancer deaths worldwide. It is a multifactorial disease, whose carcinogenesis process involves genetic and epigenetic alterations in oncogenes and tumor suppressor genes, including genes related to DNA repair. The pathogenic mechanisms are described based on the pathways of chromosomal instability, microsatellite instability, and CpG island methylator phenotype. When detected early, CRC is potentially curable, and its treatment is based on the pathological characteristics of the tumor and factors related to the patient, as well as on drug efficacy and toxicity studies. Therefore, the aim of this study was to review the pathogenesis and molecular subtypes of CRC and to describe the main targets of disease-directed therapy used in patients refractory to current treatments.

1. Introduction

Human colorectal cancer (CRC) encompasses tumors that affect a segment of the large intestine (the colon) and the rectum [1]. The most recent global estimate shows around 1.066 million new cases of CRC in men, being the third most common tumor among all cancers, with an estimated risk of 23.4/100 thousand. For women, there were around 865 thousand new cases, being the second most common tumor, with an incidence rate of 16.2/100 thousand [2].

The main risk factors associated with the development of the disease include a family history of CRC, age, sedentary lifestyle habits, obesity, and the presence of some inflammatory bowel diseases, such as Crohn’s disease and chronic ulcerative colitis, as well as hereditary intestinal diseases, such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) [3].

CRC carcinogenesis occurs through genetic and epigenetic alterations in oncogenes and tumor suppressor genes, including genes related to DNA repair mechanisms. The pathogenic mechanisms involve chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP) [4] and, depending on the origin of the mutation, colorectal carcinomas can be classified as sporadic, hereditary, and familial [5].

CRC is treatable and, in most cases, is curable when detected early [1]. Assessment of the anatomical extent of the disease through clinicopathological staging is an important prognostic factor for defining CRC treatment, which involves surgical resection for initial tumors, radiotherapy, and chemotherapy [6]. Furthermore, new therapies have been introduced, such as the use of monoclonal antibodies and immunotherapy, aiming at a more targeted treatment, with an improvement in the prognosis and overall survival of patients with CRC [7]. Therefore, the aim of the present study was to review the pathogenesis and molecular subtypes of CRC and describe the main treatments used in the disease.

1.1. Etiology

CRC exhibits diverse etiologies, broadly categorized by mutation origin into sporadic, hereditary, and familial forms [8]. The majority, approximately 70%, are sporadic, emerging in individuals lacking a familial disease history or inherited genetic mutations. Their molecular pathogenesis is complex and multifactorial, stemming from somatic genetic mutations and epigenetic alterations, often linked to modifiable risk factors. Sporadic CRC disproportionately affects older adults, a phenomenon likely attributed to extended temporal exposure to environmental, dietary, and aging-related influences [4,9].

The consumption of foods rich in saturated fats and refined sugars, for instance, predisposes individuals to the development of CRC. This association has been observed as visceral fat induces inflammation in the colorectal region, stimulated by pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which are released by adipocytes. This inflammatory environment leads to DNA damage, increased cell proliferation, angiogenesis, and immunosuppression. Furthermore, the intake of ultra-processed foods triggers the release of reactive oxygen and nitrogen species in response to inflammation, also culminating in DNA damage to intestinal epithelial cells and neurons of the enteric nervous system [10,11,12].

Comprising around 5% of all CRC cases, hereditary tumors arise from an inherited mutated allele, with a subsequent point mutation in the second allele being critical for carcinoma initiation [5]. These neoplasms are further differentiated based on the presence or absence of associated polyposis. CRC linked to polyposis is predominantly exemplified by FAP, a condition marked by the proliferation of numerous potentially malignant polyps within the colon. FAP is an autosomal dominant disorder, triggered by a mutation in the adenomatous polyposis coli (APC) tumor suppressor gene, situated on chromosome 5q21. Such mutations impair APC protein function, culminating in overactivation of the Wnt/β-catenin signaling pathway, which in turn promotes heightened cell growth, differentiation, dissemination, and adhesion in colorectal cells [13].

Concerning HNPCC, Lynch syndrome is the primary underlying cause, resulting from germline mutations in one of several mismatch repair (MMR) genes (e.g., MSH2, MLH1, MSH6, and PSM2). The inactivation of these genes compromises DNA repair mechanisms, leading to alterations in DNA’s tandem repetitive sequences, specifically microsatellites. This process gives rise to a phenotype termed MSI [14]. It is crucial to note that while characteristic of Lynch syndrome, MSI is not exclusive to it, as approximately 12% of sporadic CRC cases also exhibit this instability [15].

The third category is familial CRC, accounting for an estimated 20–30% of all disease occurrences. The etiology of this CRC form is inherently diverse, with multiple genetic contributors implicated in its development. This is presumed to stem from alterations in a spectrum of less penetrant susceptibility genes, distinct from those underlying established hereditary syndromes [16].

1.2. Pathophysiology of CRC

Colorectal carcinogenesis initiates within the stem cells residing in colonic crypts. This process involves the sequential accumulation of genetic and epigenetic alterations, which progressively inactivate tumor suppressor genes and activate oncogenes. Consequently, these stem cells transform into malignant entities, leading to the formation of an aberrant crypt. This aberrant structure then evolves into a neoplastic precursor lesion, commonly known as a polyp, eventually progressing to an invasive carcinoma [17].

Two principal pathways describe the development of precursor lesions in CRC: the adenoma–carcinoma sequence (also termed the classical or chromosomal instability pathway), responsible for 70–90% of CRC, and the serrated neoplasia pathway, accounting for 10–20% of reported cases [8].

Initially elucidated by Fearon and Vogelstein in 1990, the adenoma–carcinoma sequence (Figure 1) establishes the adenoma as a direct precursor to CRC. Within this pathway, an early adenomatous polyp (typically less than 1 cm in size and exhibiting tubular or tubulovillous histology) undergoes gradual progression. This evolution leads to an advanced adenoma (defined by a size exceeding 1 cm and/or the presence of villous histology), culminating in the development of carcinoma. This progression is fundamentally driven by the cumulative acquisition of genetic and epigenetic alterations [5,18].

The etiology of this pathway is complex and multifaceted, involving the contribution of several genes to disease progression. Key genetic alterations include loss-of-function mutations in the APC tumor suppressor gene, gain-of-function mutations in the Kirsten rat sarcoma oncogene (KRAS), and inactivation of other tumor suppressor genes like deleted in colorectal cancer (DCC) and tumor protein p53 (TP53). These cumulative changes facilitate the crucial transition from advanced adenoma to adenocarcinoma [18,19].

Beyond the adenoma–carcinoma sequence, an estimated 10–20% of colorectal tumors originate through the serrated pathway [20]. Serrated polyps represent a diverse collection of colorectal lesions, encompassing hyperplastic polyps (HPs), sessile serrated adenomas/polyps (SSA/P), traditional serrated adenomas (TSA), and formerly categorized mixed polyps (MP), which exhibit a combination of two or more of these features [21].

Serrated CRC develops via two distinct molecular routes: the sessile serrated pathway and the traditional serrated pathway (Figure 2). The sessile serrated pathway predominantly occurs in the proximal colon and is often initiated by a BRAF gene mutation [22]. This mutation drives the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, leading to uncontrolled cellular proliferation. Consequently, normal mucosa undergoes transformation into HPs. Further hypermethylation of the p16 tumor suppressor gene and the gene encoding insulin-like growth factor binding protein 7 (IGFBP7) then facilitates their evolution into SSA/P [23].

The progression to sessile serrated adenoma with high-grade dysplasia (SSAD) is primarily driven by epigenetic alterations in the MLH1 gene, leading to subsequent microsatellite instability-high (MSI-high) amplification. Less frequently, this transformation can result from hypermethylation in other genes, such as O(6)-Methylguanine-DNA Methyltransferase (MGMT), maintaining a microsatellite-stable (MSS) profile. Tumors exhibiting MLH1 methylation typically present with a phenotype characterized by BRAF gene mutations, high CpG island methylator phenotype (CIMP-high), and MSI-high. Conversely, when hypermethylation affects other gene promoters, the tumor is generally marked by BRAF mutations, CIMP-high, and an MSS status [24].

The traditional serrated pathway typically manifests in the distal colon and rectum. Its carcinogenic mechanism, leading to malignant transformation, involves mutations in KRAS or BRAF genes within the normal colonic epithelium. These genetic alterations, in conjunction with MGMT or other epigenetic methylation changes, drive the malignant progression to traditional serrated adenoma (TSA), high-grade dysplasia, and ultimately, serrated adenocarcinoma [25].

Beyond the well-established adenoma–carcinoma sequence and the serrated pathway, some researchers suggest an alternative mechanism for MSI [8]. As described by Raskov et al. [26], this pathway is characterized by germline mutations in MMR genes such as MLH1, MSH2, MSH6, and PMS2. It also involves epigenetic silencing, primarily via MLH1 gene hypermethylation, and somatic mutations in the BRAF gene.

1.3. CRC Molecular Pathways

The process of colorectal carcinogenesis involves genetic alterations, described through the CIN and MSI pathways, and epigenetic alterations, such as the CIMP pathway [27]. Initially, these pathways were described as independent mechanisms, however, recent evidence indicates a crosstalk between them [28].

1.3.1. Chromosomal Instability (CIN)

The CIN pathway is defined by alterations in chromosome copy numbers, which, in turn, lead to the activation of pro-growth signaling and/or a reduction in apoptotic pathway activity [29]. This genomic imbalance promotes the development of tumors exhibiting aneuploidy (an abnormal chromosome number) and loss of heterozygosity (LOH) [30]. Key mechanisms within this pathway encompass defects in chromosomal segregation, telomere dysfunction, and altered DNA damage response, all of which can inactivate tumor suppressor genes and activate oncogenes [28].

Inactivating mutations in the APC tumor suppressor gene are commonly observed, leading to constitutive hyperactivation of the Wnt/β-catenin signaling pathway. This pathway plays a pivotal role in stem cell differentiation and cellular proliferation; consequently, its dysregulation can drive uncontrolled cell growth and tumor initiation [31]. Another significant mutation involves the KRAS oncogene. This gene encodes small (21 kDa) guanosine triphosphate (GTP)-binding proteins; when mutated, KRAS becomes constitutively active, promoting unchecked cellular proliferation [32,33].

LOH at chromosome 18q is detected in over 70% of advanced-stage colorectal tumors. This alteration primarily affects the DCC gene and genes encoding proteins involved in the transforming growth factor-beta (TGF-β) pathway, notably SMAD2/4. Under normal physiological conditions, TGF-β signaling inhibits the cell cycle, influencing processes like cell growth, differentiation, and apoptosis. Hence, the inactivation or loss of these genes can result in unconstrained cellular proliferation and evasion of programmed cell death [34]. TGF-β signaling exhibits a paradoxical role in CRC. While it functions as a tumor suppressor in the early stages of tumorigenesis—primarily by inducing cytostasis and apoptosis—in later stages, TGF-β switches to a pro-tumorigenic role. This includes the promotion of epithelial-to-mesenchymal transition (EMT), angiogenesis, immunosuppression, and maintenance of cancer stemness [35,36]. Notably, the immunomodulatory effects of TGF-β within the tumor microenvironment have been implicated in resistance to immunotherapy, and recent findings suggest its activity may serve as a biomarker for therapeutic responsiveness, particularly in immune checkpoint blockade therapies [37].

Additionally, the inactivation of the TP53 tumor suppressor gene frequently contributes to CRC progression [38]. Located on the short arm of chromosome 17 (17p13.1), this gene encodes a 53 kDa phosphoprotein critical for cell cycle regulation, either by inducing DNA repair or initiating apoptosis [39]. Mutations in TP53 compromise DNA repair mechanisms, thereby permitting uncontrolled proliferation of tumor cells and the accumulation of further genetic alterations [40].

From a clinical perspective, colorectal tumors exhibiting CIN are typically linked to a distal anatomical location and are associated with a poorer prognosis [41]. While CIN-positive CRC is predominantly connected to the adenoma–carcinoma sequence, the precise causal relationship remains undefined: it is currently unclear whether CIN drives the accumulation of mutations in key tumor suppressor genes and oncogenes, or if the reverse is true [42].

1.3.2. Microsatellite Instability (MSI)

CRC carcinogenesis is intricately linked to the DNA repair system. This vital system functions during DNA replication, tasked with correcting incorrectly paired nucleotides. When this system is inactivated, it can result in the erroneous accumulation of mismatched nucleotides, thereby predisposing cells to carcinogenesis. Specifically, mutations in MMR genes lead to a deficiency in this critical system, culminating in MSI [43].

Microsatellites are short, repetitive nucleotide sequences (typically 1 to 6 base pairs in length) dispersed across the genome. MSI is defined as any discrepancy in the number of these nucleotide repeats when compared to normal tissue. This instability arises from a failure of the mismatch repair mechanism to accurately correct DNA base errors [26].

MSI is observed in approximately 15% of CRC patients. Of these, 3% are linked to Lynch syndrome, recognized as the most prevalent form of hereditary CRC. Lynch syndrome is an autosomal dominant condition, displaying incomplete yet high penetrance, and is caused by germline mutations in MLH1, MSH2, MSH6, and PMS2 genes [15]. The remaining 12% of patients present with sporadic CRC exhibiting MSI, typically characterized by BRAF gene mutations and MLH1 gene hypermethylation, often associated with the CIMP [44].

The National Comprehensive Cancer Network (NCCN) guidelines for colon cancer recommend assessing MSI or MMR status for all newly diagnosed patients with CRC [45]. The European Society for Medical Oncology (ESMO) guidelines recommend determining MSI status for all patients at the time of diagnosis of metastatic CRC [46]. This assessment can be performed using immunohistochemistry, next-generation sequencing (NGS), or polymerase chain reaction (PCR) techniques. The panel used in the PCR technique comprises five mononucleotide markers: BAT-25, BAT-26, MONO-27, NR-21, and NR-24 [47]. Based on this analysis, MSI status in CRCs is characterized as MSI-high when instability is blocked in at least two markers, MSI-low when instability is present in a single marker, and MSS when no marker instability is observed [28].

Studies have elucidated that the microsatellite-stable (MSS)/MSI-low and MSI-high groups exhibit distinct genomic profiles, accompanied by varying phenotypes and prognoses. Clinically, the MSI-high group typically presents with a predominance in the proximal colon, often characterized by poor histological differentiation and/or mucin production, along with an increase in tumor-infiltrating lymphocytes [48]. Moreover, MSI-high tumors are frequently diagnosed at a younger age, commonly originate from sessile serrated adenomas, and are identified at an earlier stage compared to MSS CRCs, most frequently at stage II [49].

In addition to the analysis of MSI status, the NCCN and ESMO recommend determining tumor gene status for KRAS/NRAS and BRAF mutations, as well as HER2 amplifications, for all patients with metastatic CRC, due to their relevance in prognosis and selection of targeted therapy [45,46].

1.3.3. CpG Island Methylation Phenotype (CIMP)

Epigenetic instability, which underlies the CIMP, is a notable feature in CRC. This pathway is defined by molecular epigenetic alterations that profoundly impact gene expression or function [50]. DNA methylation stands as one of the most prevalent modifications, involving the enzymatic addition of a methyl group (CH3) to the C5 position of the cytosine ring by DNA methyltransferases (DNMTs), yielding 5-methylcytosine. This specific modification typically occurs at CpG dinucleotides, which are DNA regions where a cytosine residue is immediately followed by a guanine residue, linked by a phosphodiester bond [29].

DNA hypermethylation frequently targets CpG islands located within gene promoters. Consequently, this promoter CpG hypermethylation is intrinsically linked to the transcriptional silencing of tumor suppressor genes in malignant cells. In the context of CRC, this phenomenon has been identified within the promoter regions of critical tumor suppressor genes, such as APC and MLH1 [51].

CIMP is identified in approximately 35% of colorectal tumors, often demonstrating associations with proximal anatomical location, female gender, advanced age, mucinous histological features, and an advanced disease stage [29]. Moreover, CIMP exhibits a strong correlation with the serrated pathway of colorectal carcinogenesis and with mutations in the BRAF gene [52]. The mutation of this oncogene is considered a foundational event in the genesis of hyperplastic polyps, as it drives uncontrolled cellular proliferation through the constitutive activation of the MAPK pathway. Following the formation of hyperplastic polyps, CIMP then facilitates their progression to sessile serrated adenomas and subsequent malignant transformation [53].

1.4. Key Molecular Targets for CRC Targeted Therapy

1.4.1. RAS/MAPK/MEK Signaling

• EGFR and KRAS:

The epidermal growth factor receptor (EGFR), initially isolated and sequenced by S. Cohen in 1980, functions as a transmembrane receptor tyrosine kinase (RTK) within the ErB family. This receptor is encoded by a gene located on the short arm of chromosome 7 and comprises 28 exons [54]. Structurally, EGFR consists of an N-terminal extracellular ligand-binding domain, a lipophilic transmembrane segment, and a C-terminal intracellular tyrosine kinase domain [55]. Upon specific ligand binding to its extracellular domain, EGFR undergoes homo- or heterodimerization. This dimerization induces a conformational change in the receptor, activating the tyrosine kinase domain and leading to the transphosphorylation of tyrosine residues within the C-terminal region [56].

Activation of EGFR initiates a signal transduction cascade, engaging critical pathways such as Ras/MAPK, PI3K/Akt, and JAK/STAT, which ultimately promote cell growth and proliferation. Genetic mutations impacting these pivotal signaling molecules can disrupt normal cellular processes, contributing to carcinogenesis by fostering uncontrolled proliferation, angiogenesis, apoptosis inhibition, cellular invasion, and metastasis [55].

Within the EGFR gene, the most prevalent somatic mutations are deletions in exon 19 (e.g., dels746-750) or a leucine-to-arginine substitution at codon 858 in exon 21 (L858R). Both types of mutations reside within the ATP-binding domain of the tyrosine kinase and are classified as EGFR-activating alterations. These changes frequently co-occur with enhanced production of EGFR ligands, often mediated by autocrine or paracrine feedback loops. Additionally, EGFR mutations can lead to aberrant receptor trafficking, further augmenting signaling and promoting tumor progression [57].

Recent advancements in antineoplastic drug development have focused on agents targeting specific receptors and oncogenic signaling pathways, demonstrating considerable efficacy in CRC management. Therapeutic strategies can involve monoclonal antibodies (mAbs) directed against the extracellular domain of EGFR, exemplified by panitumumab or cetuximab [58].

Cetuximab, a chimeric IgG1 monoclonal antibody, exerts its action by binding to the extracellular domain of EGFR. This interaction blocks receptor phosphorylation and the subsequent biochemical cascade that would otherwise stimulate cell proliferation [59]. For patients with metastatic CRC refractory to prior treatments and irinotecan, the inclusion of cetuximab has shown potential to overcome resistance to this chemotherapeutic agent [60]. Panitumumab, a humanized antibody, similarly targets EGFR and is a therapeutic option for patients with metastatic CRC who have become refractory to chemotherapy [58]. Notably, both these drugs have proven effective in treating CRC patients who lack a mutation in the KRAS gene [61].

The KRAS gene was first described by Scolnick et al. as part of a cancer-inducing virus, the Kirsten sarcoma virus [62]. By the 1980s, it was firmly established as an oncogene with a significant role in the pathogenesis of numerous cancers. KRAS is a member of the Ras protein superfamily, and alongside neuroblastoma rat sarcoma viral oncogene homolog (NRAS) and Harvey rat sarcoma viral oncogene homolog (HRAS) genes, it functions downstream of receptor tyrosine kinases, including EGFR. These proteins collectively regulate vital cellular processes such as proliferation, differentiation, adhesion, apoptosis, and migration [44].

Ras proteins inherently possess GTPase activity, meaning their activation and regulation hinge on guanosine triphosphate (GTP) binding and hydrolysis. In its wild-type state, KRAS protein activation is typically triggered by EGFR binding, leading to enzymatic events that ultimately support the neoplastic phenotype [63]. Deactivation of KRAS occurs when the GTP molecule is hydrolyzed to guanosine diphosphate (GDP) [64]. However, the presence of somatic mutations in this gene renders protein activation independent of growth factor stimulation, resulting in the constitutive activation of downstream signaling cascades, notably the RAF-MEK-ERK and PI3K pathways, thereby contributing to uncontrolled cellular proliferation [65].

Mutations within the KRAS gene are identified in approximately 30% to 40% of CRC patients [66], with the highest frequency observed in codons 12 and 13. Key mutations at codon 12 include glycine-to-aspartic acid (p.G12D), glycine-to-valine (p.G12V), or glycine-to-cysteine (p.G12C) substitutions. At codon 13, the most common mutation is c.38G > C, leading to a glycine-to-aspartic acid change (p.G13D) [67].

The glycine-to-cysteine mutation at position 12 (p.G12C) is located near a crucial region known as the switch-II pocket (S-IIP). This mutation stabilizes the active, GTP-bound form of the KRAS protein, promoting enhanced proliferation and survival in tumor cells. The identification of this allosteric site paved the way for the development of sotorasib, the first inhibitor specifically targeting KRAS p.G12C, approved by the Food and Drug Administration (FDA) in 2021. Sotorasib selectively and irreversibly binds to cysteine 12 (Cys12) within the S-IIP pocket, effectively locking the KRAS p.G12C protein into an inactive conformation [68,69].

According to studies, the use of sotorasib produces considerable clinical benefits and lower gastrointestinal and hepatic adverse effects. Furthermore, the majority of CRC patients presented disease control, with a median duration of stable disease of 5.4 months and a median progression-free survival of 4 months, with conventional therapies having a median progression-free survival rate of 1.9 to 2.1 months [28].

Two novel KRAS p.G12C inhibitors have been proposed in combination with cetuximab. In a phase 1b trial, divarasib showed an overall response rate of 62.5% and a median progression-free survival of 8.1 months in 24 patients with CRC harboring the KRAS p.G12C mutation [70]. Adagrasib (MRTX849) showed an overall response rate of 46% and a median progression-free survival of 6.9 months with combination therapy [71].

• BRAF:

The BRAF gene encodes the B-Raf protein, a cytoplasmic serine-threonine kinase that plays a pivotal role in activating the MAPK pathway by acting downstream of the Ras protein [72]. Activation of this critical signaling cascade contributes to fundamental cellular processes, including growth, proliferation, differentiation, migration, survival, and angiogenesis [44]. Over 40 distinct missense mutations in BRAF, spanning 24 different codons, have been identified across various human cancers [73]. Approximately 90% of BRAF mutations result from a thymine-to-adenosine transversion at nucleotide T1799A in exon 15, leading to a valine-to-glutamate substitution at codon 600, designated as V600E [74]. This specific mutation drives the constitutive activation of the pro-proliferative MAPK pathway [75].

Approximately 10% of all CRC patients harbor a mutation in the BRAF gene [76], and research indicates a strong association between this mutation and several clinicopathological parameters in metastatic CRC. Tumors carrying BRAF mutations commonly originate from serrated adenomas, are predominantly located in the right colon, and exhibit a higher incidence in women over 60 years of age. Histologically, these tumors are frequently characterized by mucinous features and poor differentiation [77].

In recent years, BRAF V600E-mutated CRC has been recognized as a distinct biological entity. This subtype is typically refractory to standard chemotherapy regimens approved for metastatic CRC, and is consistently associated with a poorer prognosis [78]. Patients with BRAF V600E-mutated tumors demonstrate reduced overall survival and shorter progression-free survival when compared to those with BRAF wild-type CRC [79].

Consequently, targeted therapies against the BRAF gene mutation, including agents like vemurafenib, dabrafenib, and encorafenib, have been proposed. However, preclinical studies involving BRAF V600E-mutated CRC models revealed a rapid feedback activation via EGFR upon BRAF inhibition. This phenomenon indicates that treatment with BRAF inhibitors alone is insufficient to adequately suppress pathway signaling, thereby explaining the limited clinical efficacy observed with single-agent BRAF inhibition in this tumor type [80,81].

To circumvent this paradoxical activation, a combination approach has been proposed, integrating an EGFR inhibitor (e.g., cetuximab, panitumumab) with a MEK inhibitor (e.g., binimetinib, trametinib) [82]. The BEACON study [83] evaluated 665 patients with metastatic CRC with BRAF V600E mutation. Participants were assigned to receive the triple combination (encorafenib, cetuximab and binimetinib), the double combination (encorafenib and cetuximab), or one of the control regimens (irotecan or FOLFIRI).

The results demonstrated that the median overall survival was 9.3 months (95% CI, 8.2 to 10.8) for the triple and double doses and 5.9 months for the control. The confirmed objective response rate was 26.8% for the triple group, 19.5% for the double group, and 1.8% (95% CI, 0.5% to 4.6%) for the control group. Notably, the BEACON study demonstrated that a triplet regimen of encorafenib, cetuximab, and binimetinib yielded significantly prolonged overall survival and a superior response rate compared to standard therapy in patients with metastatic BRAF V600E-mutated CRC [83].

1.4.2. PD-1 and CTLA-4

Approximately 15% of CRC present MSI, a molecular indicator of defective DNA mismatch repair. The high tumor mutational burden and neoantigen load in MSI tumors favor the infiltration of immune effector cells, such as CD8+ tumor-infiltrating lymphocytes, CD4+ T helper 1 cells, and macrophages, resulting in antitumor immune responses. In order to promote immune escape, MSI tumor cells upregulate T cell inhibitory ligands, such as programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (Figure 3) [84,85].

Immune checkpoint molecules regulate T cell function by activating (costimulatory molecules) or inhibiting signals (coinhibitory molecules). Costimulatory molecules, such as CD28 and CD40, can regulate multiple T cell functions, including activation, specialization, differentiation, and survival, through binding to their B7 ligands (B7-1/CD80 and B7-2/CD86) and CD40L. Coinhibitory molecules, such as programmed death 1 (PD-1) and CTLA-4, can attenuate or suppress the T cell response after initial activation, preventing excessive immune responses and tissue damage [86,87].

PD-1 is an immune checkpoint receptor expressed primarily on T cells, B cells, natural killer (NK) cells, and myeloid-derived suppressor cells (MDSCs). Its primary function involves regulating immune responses and preventing unwarranted immune reactions [88]. PD-1 binds to its ligand PD-L1, expressed on the outer membrane of numerous normal cells, such as antigen-presenting cells (APCs), as well as certain cancer cells. The interaction between PD-L1 on cancer cells and PD-1 on T cells transmits inhibitory signals to T cells, resulting in decreased activity and compromised antitumor immune responses, culminating in tumor immune evasion [89,90].

CTLA-4, a cell surface immunoglobulin receptor, is expressed on FoxP3+ Tregs and conventional T cells following activation by the T cell receptor (TCR). As a negative regulator of T cells, it structurally resembles the CD28 receptor and shares the ability to bind B7 ligands on APCs [91]. In the tumor environment, CTLA-4 higher affinity for B7 competes with and depletes CD28 at the immune synapse. Subsequent loss of the B7-CD28 costimulatory signal leads to T cell inactivation and hyporesponsiveness [88].

In this sense, blocking the PD-1/PD-L1 pathway and CTLA-4 may increase the antitumor activity of T cells and, thus, immune control and the ability to kill cancer cells. The presence of immune cells in MSI CRC supports the notion that a treatment based on immunotherapies should provide clinical benefits for this group of patients. Thus, the use of immunomodulatory monoclonal antibodies targeting immune control points has been studied and, in recent years, PD-1 inhibitors, such as pembrolizumab or nivolumab, associated or not with a CTLA-4 inhibitor, such as ipilimumab, were approved by the FDA for the treatment of patients with refractory or metastatic CRC with MSI, increasing progression-free survival and overall survival [92,93].

André et al. [94] reported the efficacy and safety of pembrolizumab versus chemotherapy in patients with MSI-high metastatic CRC from the KEYNOTE-177 trial after 5 years of follow-up. According to the authors, median progression-free survival was 16.5 months with pembrolizumab and 8.2 months with chemotherapy. The 5-year overall survival rates were 55% for the immunotherapy agent versus 44% for chemotherapy. Furthermore, median survival was improved with pembrolizumab (77.5 versus 36.7 months; HR, 0.73; 95% CI 0.53–0.99).

A randomized phase III clinical trial demonstrated that treatment of patients with MSI-high metastatic CRC with nivolumab together with ipilimumab resulted in improved progression-free survival compared to nivolumab alone (hazard ratio 0.62, 95% CI 0.48–0.81; p = 0.0003) [95]. Dual treatment with anti-PD-1/anti-PD-L1 and anti-CTLA-4 appears to reverse the upregulation of other immune checkpoints in T cells, which are induced as a compensatory effect by either drug alone [88].

1.5. Treatment

CRC treatment is based on factors related to the pathological characteristics of the tumor, such as disease staging and the presence of markers such as MSI, KRAS, and BRAF, as well as factors related to the patient (age and presence of comorbidities) and the efficacy and toxicity profiles of the medications. All of these factors help in decision-making regarding the use of adjuvant therapy for these patients [96].

Non-metastatic CRC is initially treated with surgery and, depending on the stage of the disease, adjuvant chemotherapy is recommended [5]. The treatment of high-risk stage II and III patients, i.e., patients with T4 tumors, poorly differentiated histology, lymphovascular and/or perineural invasion, the presence of intestinal obstruction or perforation, positive or indeterminate margins, and inadequate lymph node sampling (<12 dissected lymph nodes), is performed with combined regimens of 5-fluorouracil (5-FU), capecitabine, leucovorin (LV), oxaliplatin and irinotecan, such as the FOLFOX (LV + 5-FU + oxaplatin) and XELOX (capecitabine + oxaplatin) regimens [97].

Approximately 50 to 60% of patients diagnosed with CRC develop metastases [98]. Treatment of metastatic disease involves the administration of single or combined drugs, considering the goals of therapy, the type and timing of prior therapy, the mutational profile of the tumor, and the different toxicity profiles of the drugs. The different strategies are based on the use of chemotherapy regimens such as FOLFOX, XELOX, FOLFIRI (LV + 5-FU + irinotecan), FOLFIRINOX (5-FU + LV + oxaplatin + irinotecan) or trifluridine-tipiracil in combination with anti-EGFR (cetuximab and panitumunab), anti-VEGF (bevacizumab, aflibercept, ramucirumab, regorafenib and fruquintinib) and anti-HER2 (trastuzumab, tucatinib and trastuzumab deruxtecan) agents, KRAS p.G12C inhibitors (sotorasib and adagrasib), BRAF and MEK inhibitors (encorafenib and brinimetinid) and immune checkpoint inhibitors (pembrolizumab, nivolumab and ipilimumab) [6,45,46,99].

Table 1. Treatment for unresectable stage IV metastatic CRC.

Treatment Line	Tumor Characteristics	Treatment
First-Line Treatment	RAS-wt, BRAF-wt, MSS:
	Left colon	FOLFOX or FOLFIRI + Cetuximab or Panitumumab
	Right colon	FOLFOX, FOLFIRI or FOLFOXIRI + Bevacizumab
	RAS-mut	FOLFOX, FOLFIRI or FOLFOXIRI + Bevacizumab
	BRAF-mut	FOLFOX + Cetuximab + Encorafenib
	MMR/MSI-high or MMR/MSI-high + BRAF-mut	Pembrolizumab Nivolumab + Ipilimumab
Second-Line Treatment	RAS-mut, Anti-EGFR naïve:
	Left colon	FOLFIRI or Irinotecan + Cetuximab or Panitumumab
	Right colon	5-FU or Capecitabine-based ChT + Bevacizumab, Ramucirumabe or Aflibercept
	BRAF V600E-mut	Encorafenib + Cetuximab If not previously used
	MMR/MSI-high	Nivolumab + Ipilimumab (Patients not receiving immunotherapy) Pembrolizumab
	KRAS p.G12C	Cetuximab + Adagradib Panitumumab + Sotorasib
Third-Line Treatment	RAS-wt, BRAF-wt, HER2+	Trastuzumab + Tucatinib Trastuzumab deruxtecan
	RAS-wt, BRAF-wt	Trifluridine/Tipiracil + Bevacizumab Cetuximab or Panitumumab (If not previously used) Irinotecan + Cetuximab Regorafenib Trifluridine/Tipiracil or Fruquintinib
	RAS-mut	Trifluridine/Tipiracil + Bevacizumab Regorafenib Trifluridine/Tipiracil or Fruquintinib
	KRAS p.G12C	Cetuximab + Adagradib Panitumumab + Sotorasib If not previously used
	BRAF-mut	Encorafenib + Cetuximab (If not previously used) Trifluridine/Tipiracil + Bevacizumab Regorafenib Trifluridine/Tipiracil Fruquintinib

Legend: mut—mutant; wt—wild type.

demonstrates the treatment for unresectable stage IV metastatic CRC.

As can be seen, in addition to classic therapeutic regimens, immunotherapy and targeted therapy regimens are becoming an increasingly important part of the treatment landscape for metastatic CRC [100]. In this sense, a deeper understanding of cancer biology will reveal new targets, develop new therapies, and improve outcomes for patients with the disease. Figure 4 demonstrates the clinical applicability of the molecular classification of CRC in the selection of targeted therapies and prescription of immunotherapy.

2. Conclusions

CRC results from uncontrolled inspection of cells due to factors, whether environmental or genetic, which can culminate in tissue invasion close to the primary tumor or even the development of several metastases. Therefore, the research and review of the genetic and immunological aspects of molecules involved in this disease contribute to better understanding of the pathogenesis and evolution of CRC, providing clinically relevant data that could be of great value in evaluating the prognosis and in designing therapy for patients. Although research related to CRC has advanced, there remain significant gaps that need to be addressed in order to understand the etiology of the disease, improve treatment responses, and prolong patient survival while preserving their quality of life.