**5.1 Immune checkpoint inhibitors**

exist for CRC patients, basket trials (as the TAPUR trial) may give valuable insights

RET is a proto-oncogene encoding a transmembrane tyrosine kinase receptor for

RET fusions occur in 0.2% of solid tumors, being very typical in specific tumor types, such as thyroid carcinomas [119]. The effect of RET activation is less clear in CRC, but several studies suggest that it might be associated with worse prognosis, poor treatment response, and reduced OS. Due to rarity of this aberration, clinical trials in CRC are not easy to conduct, with data derived mainly from early trials or case reports [110]. Clinicopathological factors associated with RET fusions include right colon location, older age, RAS and BRAF wild-type status, and MSI-H status [119].

Fibroblast growth factor receptors (FGFRs) are a subfamily of RTKs occurring in approximately 3–5% of CRC patients [112]. Initial evidence shows poor outcomes associated with FGFR3 alterations [120]. There is no evidence of clinicopathological

Regorafenib, a multi-kinase inhibitor also targeting FGFR, is currently approved by the FDA for metastatic CRC patients who progressed on frontline therapies. This agent can be considered in CRC patients with FGFR alterations while novel FGFR inhibitors are not available [121]. Newly developed, more potent FGFR inhibitors

Microsatellite instability (MSI) is currently a key biomarker in CRC, with diagnostic, prognostic, and therapeutic implications. For these reasons, MSI analysis is becoming increasingly important and testing for deficient mismatch repair (d-MMR)/MSI is recommended, both for hereditary syndrome screening and due to

Inactivation of a DNA mismatch repair (MMR) gene (MLH1, MSH2, MSH6, or PMS2) by mutation or transcriptional silencing results in deficient function of the MMR system, responsible for excising DNA mismatches introduced by DNA polymerase during cell division. This activity loss translates in an accumulation of DNA replication errors and mismatches in repeated sequences, leading to hypermutated tumors [123]. In most cases, d-MMR and MSI arise due to sporadic somatic

hypermethylation of MLH1 and other genes, but they can also result from germline mutations in MMR genes and from Lynch syndrome in approximately 3% of all

The MMR system can be assessed through different approaches, as IHC, polymerase chain reaction (PCR)-based assays, and more recently NGS. IHC looks at MLH1, MSH2, MSH6, and PMS2 staining in tumor samples to identify the protein expression loss that characterizes d-MMR [125]. PCR amplification requires both tumor and matched normal samples. Five microsatellite loci have been PCRamplified and analyzed by capillary electrophoresis. Instability at more than one locus was defined as MSI-high (MSI-H), at a single locus as MSI-low (MSI-L), and absence of instability at any locus as microsatellite stable (MSS), proficient MMR

in this setting [112].

the glial-derived neurotrophic factor family [110].

characteristics related to these alterations [120].

prognostic and treatment implications [122].

are currently being investigated in multiple solid tumors [112].

**5. Microsatellite instability and immune checkpoints inhibitors**

*4.3.2 RET fusions*

*Colorectal Cancer*

*4.3.3 FGFR*

CRCs [124].

**146**

The success of immune checkpoint inhibitors (ICI) in d-MMR over the last years has disclosed a new therapeutic scenario. Endogenous peptides are processed and presented on major histocompatibility complex (MHC) class I molecules on the surface of all cells, being recognized by T cell receptors (TCRs). TCR–MHC signaling pathways are modulated by co-stimulatory or co-inhibitory signals. ICI target coinhibitory receptors, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) on T cells, or their ligands, as programmed cell death ligand 1 (PDL-1), on tumor and various immune cells [129]. ICI are approved in several malignancies. In mCRC, phase I trials reported response to immune checkpoint therapy in a subgroup of patients with MSI-H, d-MMR, or high TMB [130].

Pembrolizumab is a humanized IgG4 antibody and was the first anti-PD-1 to show efficacy in d-MMR mCRC (**Table 5**). In the phase II KEYNOTE-016 trial, patients with d-MMR tumors responded better to pembrolizumab (RR of 40%, 20-week PFS of 78%) than MSS tumors (RR of 0%, 20-week PFS of 11%) [131]. In the updated analysis, an ORR of 52%, 2-year PFS of 59%, and OS of 72% was reported for MSI-H CRC [132]. The phase II KEYNOTE-164 trial confirmed the efficacy of pembrolizumab in second-line setting of MSI-H CRC, with an ORR of 33%, median PFS of 2.3 months, and median OS of 31.4 months [133]. Based on these results, pembrolizumab was approved by the FDA for MSI-H/d-MMR unresectable or metastatic CRC after progression on CT. In the phase III KEY-NOTE-177 trial, first-line treatment with pembrolizumab in monotherapy significantly reduced the risk of disease progression or death by 40% (HR 0.60; 95% CI 0.45–0.80; p = 0.0004), with a median PFS of 16.5 months versus 8.2 months with CT in MSI-H CRC. The study is ongoing, and OS data will be presented later this year [134]. This led to FDA approval of pembrolizumab in first-line treatment of unresectable or metastatic MSI-H/dMMR CRC.

Nivolumab, a humanized monoclonal IgG4-based PD-1 antibody, showed activity in MSI-H/d-MMR refractory CRC in the phase II CheckMate-142 trial, with an ORR of 31.1% regardless of tumor PD-L1 expression, 1-year PFS of 50%, and OS of 73% [135]. This trial included a cohort of nivolumab in combination with the CTLA-4 inhibitor ipilimumab, which showed a 55% ORR, 71% PFS, and 85% OS. Both nivolumab and the combination of nivolumab plus ipilimumab were approved by the FDA for CT-refractory MSI-H/dMMR mCRC. The immunotherapy doublet was also evaluated in first line in the CheckMate-142 trial, with 1-year PFS and OS of 77% and 83%, respectively, ORR of 60%, and DCR of 84% [136].

Following these studies, MSI status has become a crucial biomarker to define therapeutic options for patients in the metastatic setting.

Other PD-1/PD-L1 inhibitors are under investigation, like atezolizumab, avelumab, and durvalumab, and new immune checkpoint targets are in phase I trials, such as tumor-overexpressed T cell Ig and mucin domain-containing protein 3 (TIM-3), T cell Ig, and T cell-derived lymphocyte activation gene 3 (LAG-3). [137].


PD-L1 expression level is an established biomarker in some malignancies, but the relationship between PD-L1 positivity and response has not been proven in CRC [144]. TMB has emerged as a marker of response to immunotherapy in some tumors, suggesting that tumor cells with high mutational burden generate and present more peptide neoantigens on their MHC class I molecules, increasing T cell infiltration [145]. In CRC, dMMR/MSI-H tumors have a high mutational burden, as well as some pMMR/MSS, which may present an ultramutated phenotype as DNA polymerase epsilon (POLE) mutations, found in 1–2% of pMMR CRC. POLE mutations cause an increased immunogenicity and upregulation of immune checkpoint genes, such as PD-1/PD-L1 and CTLA-4, which result in similar clinical responses to dMMR tumors and may predict response to anti-PD-1 therapy [146]. Some case reports link POLE mutations with efficacy to PD-1 blockade, and phase II

*Landscape of Current Targeted Therapies for Advanced Colorectal Cancer*

The interaction between tumor and microenvironment led to the development of an immunoscore based on calculation of two lymphocytic populations (CD3/ CD45-CD8 or CD8/CD45) in the centre and invasive margins of the tumor, which may predict ICI response [147]. Other lines of investigation are being explored, including the study of factors that indicate cytotoxic T cell activity, such as

CRC is one of the tumor types for which immunotherapy has been less effective.

CRC treatment has changed over the last decades, not only by including differ-

In metastatic setting, anti-EGFR and anti-VEGF drugs are widely used and have shown gains in survival and response rate, an important marker in CRC potentially resectable liver metastases. In contrast, several trials with targeted agents have been conducted in the adjuvant setting, without survival benefit. Immunotherapy emerged as a new treatment option with survival benefit, but at the moment it is only effective in a small portion of patients. Several other agents targeting other pathways are emerging, such as NTRK, c-MET, ALK, ROS1, and FGFR inhibitors, with promising results.

In conclusion, patients with CRC are living longer with targeted treatments, but

The authors gracefully acknowledge Joana Cavaco-Silva (jo.cvsilva@gmail.com) for manuscript English language revision and Inês Gomes (ines.gomes@medicina.

more information about resistance mechanisms and biomarkers is necessary to

L. Costa performed consulting activities for Amgen, Novartis and Servier outside the scope of this manuscript. The remaining authors declare no conflicts

ent chemotherapy agents and combinations, but mainly because new targeted

Better knowledge of the molecular immune mechanisms is required to develop predictive biomarkers and effective therapeutic combination strategies, converting "cold" tumors, immune-desert and immunotherapy-resistant, in "hot" tumors, inflamed, infiltrated by the immune system, and immunotherapy responsive.

studies are ongoing in this setting.

*DOI: http://dx.doi.org/10.5772/intechopen.93978*

granzymes, perforins, and IFN-γ levels.

extend even more their survival gains.

ulisboa.pt) for **Figure 1** graphic drawing.

**6. Conclusions**

agents have emerged.

**Acknowledgements**

**Conflict of interest**

of interest.

**149**

*CT, chemotherapy; dMMR, deficient mismatch repair, FDA, Food and Drug Administration; mCRC, metastatic colorectal cancer; MSI-H, microsatellite instability-high; NR, not reached; OS, overall survival; PFS, progression-free survival; RR, response rate.*

### **Table 5.**

*Immune checkpoint inhibitors in mCRC.*

### **5.2 Immunotherapy resistance**

Most mCRC patients are MSS/p-MMR and results with ICI have been unsatisfactory, with immune resistance mechanisms not clearly elucidated yet. Several trials have been developed exploring ways to overcome this resistance, including by modulating tumor microenvironment, reducing tumor-specific antigen expression, altering immunosuppressive pathways, and activating other immune checkpoint pathways, immune regulatory cells, and cytokines [138]. Combining immunotherapy with CT, radiotherapy, bispecific antibody therapy, other immune checkpoint modulators, and other targeted agents are among strategies explored. The rationale behind this multimodal approach is the potential synergistic effect of targeting different immune escape pathways, resulting in improved response to ICI and patient outcomes [139].

CT has anti-tumor activity due to the direct cytotoxic effect on cancer cells and to stimulating host immune response, and several clinical trials are ongoing investigating the combination of immunotherapy with CT and targeted agents [140]. Radiotherapy can activate the host immune response by upregulating expression of tumorspecific neoantigens through cell damage and increasing membrane MHC class I expression, and several studies are ongoing in CRC combining radiotherapy with ICI. Another combined strategy is ICI and MEK blockers, considering that MEK blockade seems to increase T cell response via upregulation of PD-L1 expression [141]. Following a phase Ib trial of atezolizumab and the MEK inhibitor cobimetinib in MSS CRC, other trials were conducted, with no significant survival improvement [142]. The CEA CD3 TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, which displays anti-tumor activity, leading to increased intra-tumoral T cell infiltration and activation and PD-1/PD-L1 upregulation. CEA-TCB antibody was tested in phase I trials of MSS CRC plus atezolizumab, showing antitumor activity with acceptable toxicity [143].

### **5.3 Biomarkers**

Considering immune side effects associated with ICI and their variable efficacy, it is important to identify biomarkers that help predict response to ICI and select potentially sensitive patients that can be candidates for these agents.

### *Landscape of Current Targeted Therapies for Advanced Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.93978*

PD-L1 expression level is an established biomarker in some malignancies, but the relationship between PD-L1 positivity and response has not been proven in CRC [144]. TMB has emerged as a marker of response to immunotherapy in some tumors, suggesting that tumor cells with high mutational burden generate and present more peptide neoantigens on their MHC class I molecules, increasing T cell infiltration [145]. In CRC, dMMR/MSI-H tumors have a high mutational burden, as well as some pMMR/MSS, which may present an ultramutated phenotype as DNA polymerase epsilon (POLE) mutations, found in 1–2% of pMMR CRC. POLE mutations cause an increased immunogenicity and upregulation of immune checkpoint genes, such as PD-1/PD-L1 and CTLA-4, which result in similar clinical responses to dMMR tumors and may predict response to anti-PD-1 therapy [146]. Some case reports link POLE mutations with efficacy to PD-1 blockade, and phase II studies are ongoing in this setting.

The interaction between tumor and microenvironment led to the development of an immunoscore based on calculation of two lymphocytic populations (CD3/ CD45-CD8 or CD8/CD45) in the centre and invasive margins of the tumor, which may predict ICI response [147]. Other lines of investigation are being explored, including the study of factors that indicate cytotoxic T cell activity, such as granzymes, perforins, and IFN-γ levels.

CRC is one of the tumor types for which immunotherapy has been less effective. Better knowledge of the molecular immune mechanisms is required to develop predictive biomarkers and effective therapeutic combination strategies, converting "cold" tumors, immune-desert and immunotherapy-resistant, in "hot" tumors, inflamed, infiltrated by the immune system, and immunotherapy responsive.
