The Kirsten Ras (KRAS) oncogene encodes for a guanosine triphosphate (GTP)/guanosine diphosphate binding protein downstream of the extracellular epidermal growth factor receptor in the RAS/RAF/MAPK signaling pathway. Activating KRAS exon 2 mutations occur in up to 45% of all CRC, and are involved in initiation, proliferation and progression of CRC[6-10]. While initial studies suggested a possible clinical benefit from anti-epidermal growth factor receptor (EGFR) therapy for patients whose tumors express KRAS codon 13 (G13D) mutations, a meta-analysis comprised of several large phase III trials failed to demonstrate benefit from panitumumab, an anti-EGFR monoclonal antibody, in CRC patients whose tumor harbored a KRAS codon 13 mutation[11]. In addition to KRAS exon 2, an approximate additional 10% of patients with other RAS mutations have been identified in CRC, including NRAS or non-exon 2 KRAS mutations[6]. In patients who exhibited activating non-exon 2 KRAS and NRAS mutations, an absence of clinical benefit, and perhaps a negative effect, was seen from the addition of anti-EGFR therapy in combination with several chemotherapy regimens in various treatment settings[6,12,13]. On this basis, anti-EGFR therapy should not be given to any patient with CRC exhibiting a RAS mutation.

While mutations in RAS as a predictive biomarker to anti-EGFR therapy has been recognized, its relevance as a therapeutic target is unknown. Given the high incidence of RAS in CRC and its importance as an oncogene, targeting RAS represents an ideal and promising strategy. Developing strategies to directly block oncogenic RAS activity has remained a challenge due to several factors, including the high binding affinity of the oncoprotein to the GTP-bound “on” state, as well as the lack of accessibility to active sites within KRAS to bind[14]. One alternative approach includes targeting pathways and its effectors downstream of RAS. The clinical benefit from targeting single pathways is often limited due to mechanisms of resistance including communication between signaling pathways and its resulting downstream effector activation and inhibition through a feedback loop mechanism[15,16]. Alternatively, secondary treatment resistance to anti-EGFR treatment may result from the development of RAS mutations during a course of anti-EGFR therapy. Several studies have demonstrated up to 96% of patients who initially had RAS and BRAF wild-type CRC were later identified to have acquired activating RAS (KRAS or NRAS) or BRAF mutations on repeat tumor genomic assessment at the time of disease progression on anti-EGFR therapy[17-19].

Alternative treatment strategies in RAS mutant CRC include the combination of various therapeutic agents targeting several genes involved in the MAPK pathway that would cause sufficient suppression of activated RAS activity. Combining small molecule inhibitors of MEK to anti-EGFR therapies have demonstrated the reversal of acquired anti-EGFR therapy resistance, prompting ongoing clinical trials investigating the clinical utility of the combination of multiple signaling pathway inhibitors in the first-line setting, as well as in salvage therapy for refractory disease (Table 1). Targeting multiple signaling pathways may also be effective in overcoming resistance from secondary activation of parallel signaling pathways[20]. Alternatively, administering anti-EGFR therapies in a pulsatile manner instead of to the point of clinical progression may prolong anti-EGFR therapy efficacy. A recent study that performed dynamic monitoring of tumor somatic variants through circulating tumor DNA demonstrated a decrease in drug resistant KRAS clones upon withdraw of cetuximab, allowing for the reemergence of drug sensitivity to anti-EGFR therapy[21]. This suggests a rationale for intermittent EGFR blockade, and explains for the seldom efficacy seen with re-challenging anti-EGFR therapies. Lastly, inhibitors of the MAPK and PI3K/Akt/mTOR pathway are considered to cause G1 cell cycle arrest through the suppression of D-type cyclins and the upregulation of cell cycle inhibitors[22,23]. Pre-clinical studies have demonstrated potent inhibition of G1/S transition and phosphorylation of retinoblastoma (Rb) protein with the inhibition of the MAPK and PI3K/Akt pathway[24]. The combination of MEK and CDK inhibitors may be considered a potential strategy in treating RAS activated CRC or in tumors harboring mutations in CDK.

BRAF V600E mutations occur in up to 5% of metastatic CRC[25,26] and are often associated with a more aggressive phenotype (with the exception in microsatellite instability high CRC, where the effect is attenuated). Patients with BRAF V600E mutations tend to respond poorly to conventional chemotherapy and have worse outcomes[6,27-31]. The constitutive activation of BRAF evading any upstream inhibition of EGFR may explain the limited clinical benefit seen with anti-EGFR therapies in BRAF mutant metastatic CRC[6,32,33].

In metastatic melanoma, inhibition of BRAF with small molecule inhibitors has led to improvement in clinical outcomes in patients whose tumors exhibit mutations in BRAF[34-37]. However, the clinical efficacy from single agent BRAF inhibition has not translated to patients with BRAF mutant metastatic CRC[36,38]. The lack of anti-tumor activity may be a result of insufficient inhibition of the MAPK pathway as a result of a feedback loop mechanism, resulting in the persistent activation of the MAPK signaling pathway[16,39] (Figure 1). To overcome this compensatory mechanism, the combination of tyrosine kinase inhibitors aimed at inhibiting the MAPK pathway has resulted in an improvement in treatment efficacy in comparison to single-agent BRAF small molecule inhibitor in metastatic melanoma[40]. Based on these results, a phase II study investigating the clinical activity from the combination of BRAF and MEK inhibition with dabrafenib and trametinib was conducted in patients with BRAF mutant metastatic CRC[41]. While modest clinical activity was observed with the combination, with 12% of patients experiencing either a partial or complete response, correlative laboratory studies suggest an inhibition in MAPK signaling in patients receiving the combination[37]. The absence of significant anti-tumor activity may be attributed to insufficient suppression of MAPK signaling, which may be due to upstream activation of EGFR, leading to reactivation of MAPK and other integral signaling pathways[15]. Based on this rationale, ongoing studies are investigating the combination of anti-EGFR therapies with BRAF tyrosine kinase inhibitors in patients with BRAF mutant mCRC (Table 1)[42-45].

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Figure 1 RAS/RAF/MEK/ERK pathway and mechanisms of resistance to BRAF inhibition. The figure above shows the MAPK pathway and the compensatory feedback loop activation (arrow) despite BRAF inhibition, resulting in the upstream reactivation of the MAPK pathway.

The fibroblast growth factor receptors (FGFR) comprise a group of highly conserved tyrosine kinase receptors consisting of four members (FGFR1, 2, 3 and 4). These receptors bind to one of 18 secreted glycoprotein ligands, or fibroblast growth factors (FGFs), to their extracellular domain[48]. Binding of the appropriate ligand results in FGFR dimerization, autophosphorylation and activation of downstream signaling pathways that include the MAPK, PI3K/Akt and signaling transducer and activator of transcription or STAT pathway, inducing cell differentiation, growth and survival[49]. FGFR overexpression has been identified in CRC samples, where the presence of FGFR signaling has been identified as playing an important role in the tumor microenvironment, with a correlation in FGFR overexpression with tumor invasion, advanced stage disease and chemotherapy resistance[50-53]. Pre-clinical studies have demonstrated the reversal of chemotherapy resistance by combining small molecule FGFR inhibitors with chemotherapy in CRC cell lines, confirming the importance of targeting this receptor and representing a potential strategy in overcoming treatment resistance in CRC[54].

Hepatocyte growth factor receptor, also known as c-MET, is a proto-oncogene that encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF). Abnormal expression of c-MET through somatic mutations or overexpression has been identified in up to 66.7% of CRC samples and its microenvironment and is a negative prognostic marker related to tumor oncogenesis, invasiveness, local recurrence, and chemotherapy resistance[55-58]. MET activation confers acquired anti-EGFR therapy resistance, through re-activation of anti-apoptotic signaling pathways, including the PI3K and MAPK pathways[59-62]. Thus, inhibiting c-MET represents an emerging target of interest in the development of novel agents in CRC. Pre-clinical studies have demonstrated that the blockade of c-MET inhibits tumor growth in CRC cell lines[63,64], thus agents targeting MET, including small molecule multi-kinase inhibitors (e.g., crizotinib, tivantinib), HGF inhibitors (e.g., AMG102, AV299) and immunotherapeutic agents are of interest and under investigation in the treatment of CRC.

RET is located on chromosome 10q11.2 and encodes a transmembrane receptor tyrosine kinase that has three unique isoforms[65]. Four ligands can bind and activate RET, leading to the aberrant activity of several signaling pathways including PI3K/Akt and MAPK pathway[66]. While the aberrant expression of RET may function as an oncogene in certain solid tumor malignancies including papillary and medullary thyroid cancers[67], in colorectal cancer RET has been identified as a tumor suppressor and as an oncogene. Studies have shown that the hypermethylation and mutational inactivation of RET, as well as RET fusions, promote colorectal cancer formation[68-70]. In several studies, RET mutations were identified in up to 7% of mCRC samples[46,71,72]. Regorafenib, an approved multi-target small molecule inhibitor in metastatic CRC[73], has demonstrated tumor growth inhibition in RET mutant cancer cells lines[70,74], and may explain part of its efficacy in this disease. Further studies investigating its activity in RET mutant CRC is warranted as it may provide further benefit in this specific cohort of CRC patients.

Human growth factor receptor 2, also known as HER-2 or HER-2/neu, is part of the human epidermal growth factor receptor family and has been identified as an oncogene in several solid tumor malignancies, including CRC. Its overexpression has been a poor prognostic biomarker in breast cancer and is an effective therapeutic target in breast and gastric cancer[75-77]. Studies evaluating HER-2 overexpression in CRC have identified HER-2 somatic mutations and amplification in 7% of patients with CRC[46], where pre-clinical studies have demonstrated its role in inducing resistance to anti-EGFR therapy, and its inhibition showing durable tumor regression with anti-HER-2 therapy[78]. Based on these findings, the HERACLES trial, a phase II study investigated dual anti-HER-2 therapy blockade in patients with HER-2 amplified (immunohistochemistry staining 3+ or 2+ with FISH positive (HER2:CEP17 ratio > 2) in > 50% of tumor cells) mCRC with previous anti-EGFR therapy. Patients received the combination of trastuzumab, an anti HER-2 monoclonal antibody with either lapatinib, a small molecule inhibitor of HER-2 or pertuzumab, an anti HER-2 monoclonal antibody. Early results from the lapatinib and trastuzumab arm demonstrated a 35% response rate and median progression free survival of 5.5 mo, despite being heavily pre-treated after failing multiple lines of therapy[79]. Based on these findings, anti HER-2 therapy may be an effective treatment option in a pre-selected patient population with mCRC.

Mismatch repair (MMR) genes function to remove erroneous DNA nucleotides during mitosis. With deficient MMR activity, altered DNA nucleotides are incorporated into cells that increase their risk in forming into a neoplastic, hypermutated makeup[80], resulting in microsatellite instability. Deficient MMR activity is found in approximately 15% of CRC, in which 3% is attributed to Lynch Syndrome through germline mutations in MLH1, MSH2, MSH6, PMS2 or EPCAM3 genes[81,82]. The remaining 12% is due to sporadic inactivation of MLH1. While microsatellite instability (MSI) is a driver for tumor formation and proliferation in CRC, recent studies have demonstrated its relevance for potential novel therapeutic agents in this cohort of CRC. Tumors expressing MSI have been characterized by an intense immune infiltration, likely related to a high density of mutations, creating numerous neo-antigens and targets for immune therapies. Based on this rationale, a single-arm phase II study was conducted to assess the clinical efficacy of pembrolizumab, a programmed cell death protein (PD)-1 inhibitor, in patients with treatment resistant, metastatic cancer that did or did not express mismatch-repair deficiency[83]. Individuals with deficient MMR colorectal cancer experienced a response rate of 40% and immune-related PFS of 78% at 20 wk. Interestingly, a lack of activity was associated with mismatch repair-proficient CRC patients, confirming that immunotherapeutic agents may be beneficial in only certain cohorts of CRC, unless alternative approaches can be developed to transform tumors into an immune-responsive phenotype[83]. Based on these findings, several ongoing clinical trials are investigating various novel, immunotherapeutic agents in the treatment of CRC that include studies in patients with MSI high tumors and those with positive PDL-1 expression (Table 3). While current trials will substantiate the role of immune therapy in CRC, a better understanding of mechanisms of acquired resistance, optimal duration of necessary treatment and predictive biomarkers associated with treatment efficacy are paramount.

While anti-tumor activity from targeted therapies can lead to dramatic responses, the clinical benefit is often limited due to inherent and acquired resistance through the acquisition of new tumor genomic alternations, including the oncogenic activation of MET or acquiring RAS mutations, as described above. While tumor genomic alterations are usually assessed through tumor samples, obtaining tissue can be challenging due to insufficient material from tumors only accessible through fine-needle aspirates, as well as the invasive nature of such procedures.

Tumor circulating free DNA can be non-invasively assessed in peripheral blood, a “liquid biopsy,” through the assessment of circulating tumor DNA (ctDNA) and tumor cells (CTCs) and are present in advanced malignancies[84,85]. CtDNA and CTCs can provide dynamic assessments of tumor specific mutations that may arise during the course of therapy. Although previous methodologies demonstrate low rates of sensitivity and concordance, the incorporation of new technology, including real time digital PCR has increased sensitivity (87.2%) and specificity (99.2%) in identifying tumor specific mutations responsible for treatment resistance in patients who initially responded to targeted therapies[18]. While there are limitations, including false negative results as well as the inability to identify genomic alterations from central nervous system (CNS) lesions, this non-invasive tool can monitor patients for resistance-conferring mutations as well as assessing all tumors concurrently, as heterogeneity can exist between different foci of disease.