**4.1 NTRK fusions**

The constitutive activation of RTKs promoted by genomic translocations play an important role in tumorigenesis across different malignancies, including CRC. Examples include ALK, ROS1, and NTRK1–2-3 (NTRK), which altogether occur in 0.2–2.4% of CRCs and may represent new therapeutic targets (**Table 4**) [107].

The NTRK (neurotrophic tropomyosin receptor kinase) 1, 2, and 3 genes encode three tropomyosin receptor kinase (TRK) receptors —TrkA, TrkB, and TrkC which are transmembrane proteins [2, 108, 109]. Gene fusions involving those genes lead to constitutively activated NTRK proteins and, consequently, tumorigenesis [107]. The prevalence of NTRK fusions in mCRC is estimated to be 0.5–2.0% [110], but increases to 4% in microsatellite instability-high (MSI-H) mCRC [2].

NTRK gene rearrangements are more commonly detected in non-Lynch syndrome MSI-H/ deficient mismatch repair (dMMR) tumors with MLH1 promoter hypermethylation and wild-type BRAF/KRAS/NRAS, and define a molecular subgroup associated with poor prognosis [111]. They are also more frequent in elderly females with right-sided tumors [107, 109, 112].

Fusion-detection options include targeted DNA and RNA panels, RNA sequencing, FISH, and IHQ [2]. Recent ESMO recommendations for NTRK fusion detection state that, in tumors with low NTRK fusion frequency, as mCRC, detection can be done via one-step next-generation sequencing (NGS) or via IHQ followed by NGS (if IHQ positive) [113].

Larotrectinib and entrectinib are TRK inhibitors approved by the FDA and EMA in more than 10 tumor types. Larotrectinib, a small-molecule inhibitor targeting all three TRK proteins, has been tested in the multicenter single-arm LOXO-TRK-14001, SCOUT, and NAVIGATE clinical trials [111]. Larotrectinib at the dose of 100 mg twice daily showed a good safety profile and good responses (75% of ORR, 1-year PFS of 55%) [114]. In November 2018, the FDA granted accelerated tissueagnostic approval to larotrectinib for solid tumors with NTRK gene fusions [2, 111, 112] Entrectinib is an oral pan-TRK, -ROS1, and -ALK inhibitor that is clinically active in patients with NTRK-rearranged tumors and is able to penetrate the blood–brain barrier [107]. Three clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2) have investigated this agent [107]. Pooled analyses of the three trials presented at the ESMO 2018 Congress and ASCO 2019 Meeting showed that entrectinib induced clinically meaningful durable responses in patients with solid tumors with or without metastatic central nervous systemic disease harboring NTRK fusions [111].

The second-generation TRK inhibitor BAY2731954 (formerly known as Loxo-195) and the next-generation ROS1, pan-TRK, and ALK inhibitor repotrectinib are being tested, with promising results [111].

As already shown with BRAF V600E mutations, patients with ALK-, ROS-, and NTRK-rearranged tumors seem to derive no benefit from treatment with antiEGFR monoclonal antibodies [107]. Additionally, the high prevalence of MSI-H status in rearranged tumors opens the way for evaluation of new combination approaches including targeted (ALK, ROS1, TrkA-B-C) and immunotherapy agents [107].

Regarding resistance mechanisms, a dose-dependent effect seems to affect mutation emergence. Two mutations have been associated with entrectinib resistance: NTRK1 p. G667C and NTRK1 p.G595R [108]. For larotrectinib, three different mutational categories have been described: solvent front mutations (NTRK1 p. G595R, NTRK3 p.G623R); gatekeeper mutations (NTRK1 p.F589L); and xDFG mutations (NTRK1 p.G667S, NTRK3 p.G696A). Novel agents under development intend to overcome NTRK1 p.G595R-mediated resistance to TRK inhibitors [115].

### **4.2 MET alterations**

The mesenchymal-epithelial transition (MET) protooncogene (also known as Nmethyl-N<sup>0</sup> -nitroso-guanidine human osteosarcoma transforming gene) encodes for c-MET, a receptor with tyrosine kinase activity targeting HGF. Activation of this pathway has been implicated in CRC metastatic progression [2].

MET receptor tyrosine kinase can be overexpressed in 50–60%, amplified in 10%, and mutated in 5% of CRCs [2]. In a study by Lee et al., c-MET overexpression showed no correlation with primary tumor site, histological type, or molecular aberrations, but correlated with shorter OS and was a predictive biomarker of shorter PFS in bevacizumab-treated patients [3].

EGFR and MET are co-expressed in CRC and MET activation has been implicated in resistance to the anti-EGFR therapy [2, 116]. Inhibition of the HGF/c-Met pathway may improve response to EGFR inhibitors in CRC and combination therapy should be further investigated [116]. This supports the hypothesis that anti-EGFR therapy selects MET-amplified (cetuximab- and panitumumab-resistant) preexisting clones, eventually limiting the efficacy of further anti-EGFR therapies [117].

Multiple clinical trials have evaluated MET inhibition, but several of those conducted in mCRC have been unsuccessful [2]. Treatment strategies targeting HGF and c-Met include HGF antagonists, c-Met and HGF-blocking antibodies, and small-molecule c-Met inhibitors [118].

Although MET genomic aberrations are commonly observed in mCRC, these remain in the research setting [2].

### **4.3 Other rearrangements**

### *4.3.1 ALK/ROS1 translocations*

The EML4-ALK fusion gene is produced by inversion in the short arm of chromosome 2, where anaplastic large-cell lymphoma kinase (ALK) joins echinoderm microtubule-associated protein-like 4 (EML4), resulting in a chimeric protein with constitutive ALK activity. ROS1 is an orphan receptor tyrosine kinase phylogenetically related to ALK [110].

ALK and ROS1 gene rearrangements have not been extensively studied in CRC. Around 0.8–2.5% of patients with mCRC have been reported to have either ALK or ROS1 rearrangements [110]. ALK, ROS1, and NTRK fusions occur more frequently in elderly patients with right-sided, RAS wild-type, MSI-H mCRC, and are associated with shorter OS and poor prognosis [107, 110]. The small patient numbers make it challenging to develop a clinical trial of targeted therapies for this patient population [110]. As no FDA-approved agents targeting these genomic alterations

**Target**

**145**

NTRK genes

0.5–2.0% in mCRC (4% in MSI-H)

 Associated with

RAS, elderly females and right sided tumors;

associated with poor prognosis; resistance to

anti-EGFR monoclonal

 antibodies

MSI-H/dMMR;

 wt BRAF/

IHC and

Larotrectinib

Small molecule inhibitor

Approved by the FDA and

EMA

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

targeting TRK proteins

Entrectinib

NGS

(NTRK 1,

NTRK 2,

NTRK 3)

fusions

ALK/ROS1

FGFR c-Met

Overexpressed

 in 50–60%,

Shorter OS, shorter PFS with

treatment; poor prognosis; resistance to

anti-EGFR monoclonal

 antibodies IHC and

Vandetanib,

cabozantinib

 Under

investigation

 Under investigation

*microsatellite*

FISH

*hybridization;*

*progression-free*

 *survival; RT-PCR, reverse transcription*

 *IHC,* 

*immunohistochemistry;*

 *polymerase chain reaction; TKI, tyrosine kinase inhibitor; wt, wild-type.*

 *mCRC, metastatic colorectal cancer; MSI-H,* 

bevacizumab

IHC

Clinical trials

 Under

investigation

 Under investigation

overexpression

RET fusions *dMMR, deficient mismatch repair; FGFR, fibroblast growth factor receptor; FISH, fluorescent* in situ

*instability-high;*

**Table 4.** *Summary of new targeted therapies in mCRC.*

 *NGS,* 

*next-generation*

 *sequencing; OS, overall survival; PFS,* 

amplified in 10% and mutated in

5% of CRCs

0.2%

Worse prognosis, poor treatment response,

and reduced OS

3–5%

0.8–2.5%

Associated with

RAS, elderly females and right-sided tumors;

associated with poor prognosis; resistance to

anti-EGFR monoclonal FGFR3 related with worse prognosis

 antibodies

 NGS plus

Regorafenib

 and newly

Small-molecule

targeting FGFR

signaling

 inhibitor

Under

investigation

FISH

developing

FGFR-specific

TKIs

MSI-H/dMMR;

 wt BRAF/

FISH,

Clinical trials; Ceritinib

Small-molecule

targeting ALK/ROS1

 inhibitor

Under

investigation

*Landscape of Current Targeted Therapies for Advanced Colorectal Cancer*

RT-PCR,

NGS

**Frequency**

**Clinicopathological**

 **features**

 **Testing**

**Agent**

**Mechanism**

 **of action**

 **Current status**

**methods**


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

**Table 4.**

*Summary of new targeted therapies in mCRC.*

EGFR monoclonal antibodies [107]. Additionally, the high prevalence of MSI-H status in rearranged tumors opens the way for evaluation of new combination approaches including targeted (ALK, ROS1, TrkA-B-C) and immunotherapy

Regarding resistance mechanisms, a dose-dependent effect seems to affect mutation emergence. Two mutations have been associated with entrectinib resistance: NTRK1 p. G667C and NTRK1 p.G595R [108]. For larotrectinib, three different mutational categories have been described: solvent front mutations (NTRK1 p. G595R, NTRK3 p.G623R); gatekeeper mutations (NTRK1 p.F589L); and xDFG mutations (NTRK1 p.G667S, NTRK3 p.G696A). Novel agents under development intend to overcome NTRK1 p.G595R-mediated resistance to TRK inhibitors [115].

The mesenchymal-epithelial transition (MET) protooncogene (also known as N-

c-MET, a receptor with tyrosine kinase activity targeting HGF. Activation of this

MET receptor tyrosine kinase can be overexpressed in 50–60%, amplified in 10%, and mutated in 5% of CRCs [2]. In a study by Lee et al., c-MET overexpression showed no correlation with primary tumor site, histological type, or molecular aberrations, but correlated with shorter OS and was a predictive biomarker of

EGFR and MET are co-expressed in CRC and MET activation has been implicated in resistance to the anti-EGFR therapy [2, 116]. Inhibition of the HGF/c-Met pathway may improve response to EGFR inhibitors in CRC and combination therapy should be further investigated [116]. This supports the hypothesis that anti-EGFR therapy selects MET-amplified (cetuximab- and panitumumab-resistant) preexisting clones,

Multiple clinical trials have evaluated MET inhibition, but several of those conducted in mCRC have been unsuccessful [2]. Treatment strategies targeting HGF and c-Met include HGF antagonists, c-Met and HGF-blocking antibodies, and

Although MET genomic aberrations are commonly observed in mCRC, these

The EML4-ALK fusion gene is produced by inversion in the short arm of chromosome 2, where anaplastic large-cell lymphoma kinase (ALK) joins echinoderm microtubule-associated protein-like 4 (EML4), resulting in a chimeric protein with constitutive ALK activity. ROS1 is an orphan receptor tyrosine kinase phylogeneti-

ALK and ROS1 gene rearrangements have not been extensively studied in CRC. Around 0.8–2.5% of patients with mCRC have been reported to have either ALK or ROS1 rearrangements [110]. ALK, ROS1, and NTRK fusions occur more frequently in elderly patients with right-sided, RAS wild-type, MSI-H mCRC, and are associated with shorter OS and poor prognosis [107, 110]. The small patient numbers make it challenging to develop a clinical trial of targeted therapies for this patient population [110]. As no FDA-approved agents targeting these genomic alterations

pathway has been implicated in CRC metastatic progression [2].

eventually limiting the efficacy of further anti-EGFR therapies [117].

shorter PFS in bevacizumab-treated patients [3].

small-molecule c-Met inhibitors [118].

remain in the research setting [2].

**4.3 Other rearrangements**

cally related to ALK [110].

**144**

*4.3.1 ALK/ROS1 translocations*


agents [107].

*Colorectal Cancer*

**4.2 MET alterations**

methyl-N<sup>0</sup>

exist for CRC patients, basket trials (as the TAPUR trial) may give valuable insights in this setting [112].

(p-MMR) [126]. NGS detection directly targets certain genes, which are genome sequenced to retrieve information on MSI and MMR and tumor mutational burden (TMB), integrating all information in the same test. NGS requires a smaller sample and is more accurate than PCR. Ethical issues may arise with the use of this technique regarding counseling and consent for additional genetic testing [127]. In CRC, MSI varies according to tumor stage, with higher incidence reported in early stages (20% in stages I-II, 12% in stage III) and lower incidence reported in the metastatic

*Landscape of Current Targeted Therapies for Advanced Colorectal Cancer*

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

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

Following these studies, MSI status has become a crucial biomarker to define

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].

setting (4–5%) [128].

**5.1 Immune checkpoint inhibitors**

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

unresectable or metastatic MSI-H/dMMR CRC.

77% and 83%, respectively, ORR of 60%, and DCR of 84% [136].

therapeutic options for patients in the metastatic setting.

**147**
