The Programmed Cell Death Protein 1 (PD-1), a crucial member of the checkpoint proteins and a member of the CD28 family (23), plays an important role in immune regulation. Its ligand, Programmed Death-Ligand 1 (PDL-1), is a type 1 transmembrane glycoprotein belonging to the B7 ligand family and is primarily expressed on the surface of tumour cells (24). The PD-1/PDL-1 pathway is activated in response to infections, serving to restrain the elimination of host cells and mitigate the risk of autoimmune diseases (25). In the context of malignancies, the PD-1/PDL-1 signal transduction pathway assumes one of the central roles in the mechanisms of tumour-mediated immunosuppression which facilitates immune evasion by suppressing the anti-tumour immune response (26, 27).

The FDA has approved several monoclonal antibodies targeting PD-1, including nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab. Additionally, three PDL-1 inhibitors, namely atezolizumab, durvalumab, and avelumab, have also received FDA approval. Immunotherapy has durably demonstrated efficacy in the treatment of various haematological and solid malignancies such as melanoma, non–small cell lung cancer, renal cell carcinoma, urothelial carcinoma, gastroesophageal carcinoma, and hepatocellular carcinoma have seen notable clinical benefits from PD-1/PDL-1 blockade (See Table 1) (28, 29). In case of mCRC, pembrolizumab and nivolumab are among the FDA-approved PD-1/PDL-1 inhibitors (30).

Cytotoxic T-lymphocyte antigen-4 (CTLA-4), predominantly expressed on the surface of activated T cells, serves as an immune checkpoint involved in the early-stage response of T cells. In contrast to the stimulatory signal mediated by CD28, CTLA-4 transduces an inhibitory signal to T cells, regulating the delicate balance of immune responses (31). The dynamic interplay between CTLA-4 and CD28 occurs through their interaction with B7 ligands expressed on the surface of antigen-presenting cells. Notably, CTLA-4 exhibits a 20-fold higher affinity for the B7 ligand compared to CD28, resulting in a potent inhibitory effect on T cells which contributes to the suppression of T cell activation and proliferation (32). The competition for B7 ligands between CD28 and CTLA-4 establishes a regulatory mechanism in which CTLA-4 dampens T-cell responses. The mediatory role of CTLA-4 extends to the inhibition of T-lymphocyte responses, leading to the curtailment of T lymphocyte proliferation. Furthermore, CTLA-4 promotes the heightened activity of regulatory T cells (Tregs), further contributing to immune tolerance (33).

FDA-approved therapies targeting CTLA-4 in combination with PD-1/PDL-1 inhibitors have shown promise in the treatment of mCRC. Combining anti-CTLA-4 therapy with PD-1/PDL-1 blockade represents a synergistic approach to enhance the anti-tumor immune response. While combination therapy has gained regulatory approval, the efficacy of monotherapy with CTLA-4 inhibitors is still under evaluation (34).

The erythroblastosis oncogene B (ERB) family consists of four transmembrane tyrosine kinase receptors: EGFR (ErbB1 or HER1), HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4) (58). Overexpression of EGFR is one of the potential pathophysiological components of CRC as HER2 (1.3%–47.7%) and EGFR (25%–82%) overexpression have been widely reported among patients with CRC (57, 59, 60). Additionally, mutations within ERB may be accompanied by the increased tyrosine kinase activity existing separately or induced by the therapy (60). Clinically wise, overexpression of the receptors is considered to be correlated with poor prognosis and lower survival rate, metastases, drug resistance (61).

The chain of reaction of EGFR, HER3, and HER4 is induced by binding a ligand to their extracellular domains which allows the formation of active hetero-oligomers and phosphorylation of the tyrosine kinase domain (58). Activation of HER2 is still a matter of discussion as currently, the scientific community has not elucidated any ligand family that stimulates its extracellular domain. The more commonly accepted theory involves the formation of heterodimers with any of the other three receptors from the ERB family which creates a high-affinity complex for the ligands (62, 63). Particularly, HER3 receptor must undergo dimerization with the other ERB receptor due to lack of its intrinsic kinase activity. Regardless of ERB-receptor activation pathways, the initiated signalling pathway promotes proliferation, angiogenesis, continued existence, migration, and adhesion of the stimulated cells (56). Since cancer cells depend on those pathways, ERB family receptors play a crucial role in target treatment.

A list of typical adverse effects of anti-EGFR drugs includes: papulopustular rash, mucositis, xerosis, hypermagnesemia, diarrhoea, hypersensitivity/allergic reaction, skin fissuring, and paronychia infection (56, 64).

So far two antibodies have been registered for targeting EGFR: cetuximab and panitumumab (IgG2 antibody) as both of them are based on a similar mechanism—attachment to the extracellular domain of EGFR inhibiting binding of ligands which leads to tyrosine kinase internalization and degradation. Additionally, cetuximab binds to NK cells leading to antibody-dependent cellular and complement-mediated cytotoxicity (56, 60).

Panitumumab was registered based on a PRIME trial in which it was added to FOLFOX4 in first-line treatment markedly improving PFS (10 months vs. 8.6 months) in patients with wild-type KRAS CRC (65). Median OS was 23.9 months vs. 197 months. The acceptance of cetuximab was based on a CRYSTAL trial in combination with the FOLFIRI regimen (66). The previously untreated patients had 0.68 HR (95% CI, 0.50–0.94) compared to FOLFIRI alone (1.07). Median PFS was 9.9 months vs. 8.7 months in favour of the combination. It is essential to highlight that positive effects were observed only in patients with the KRAS wild-type variant.

In ASPECCT—a phase III RCT—panitumumab was found non-inferior to cetuximab as no differences in terms of OS, PFS, and ORR between these two drugs were found. As far as ADRs are concerned, the incidence of skin adverse effects was comparable. However, grade 3–4 hypomagnesemia was more frequent among patients treated with panitumumab, but grade 3–4 infusion reactions were more common in cetuximab. In other studies, patients with therapeutic plans based on panitumumab more often developed mutations in the EGFR extracellular domain than those treated with cetuximab but lower risk of developing hypersensitivity reactions (0.6%–3% vs. 3.5%–7.5%) (64, 67).

Left-sided CRC is characterized by higher expression of EGFR than their right-sided counterparts which has its clinical implications as shown during the Fire-3 trial which proved cetuximab is a superior treatment option for left-side CRC compared to bevacizumab when added to FOLFIRI (68). It depicted higher ORR (77% vs. 65%) and longer median OS (33 vs. 26 months) but PFS was comparable between the two groups. It is important to note that this advantage was observed only in left-side tumours.

EGFR inhibitors do not typically present a response in patients with BRAFV600E mutations when used as single agents or combined with cytotoxic chemotherapy. However, there is evidence that simultaneous inhibition of EGFR and BRAF can be beneficial in patients who underwent prior one or two regimens in refractory CRC. Kopetz et al. in the SWOG S1406 trial on 106 patients with BRAFV600E demonstrated the efficacy of a combination of irinotecan and cetuximab with the addition of Vemurafenib (69). The observed PFS of the group with all three medications vs. the group without vemurafenib was 17% vs. 4%. The DCR was 65% vs. 21% but OS was comparable between the two groups. Simultaneous blockade of EGFR and BRAF results in downregulation of mismatch-repair (70).

The combined treatment represented a more successful approach also in the study by (71) in which 44 patients received therapy with adagrasib and 32 adagrasib combined with cetuximab. The ORR was 19% vs. 46%, the median response duration was 4.3 months vs. 7.6 months and the PFS was 5.6 vs. 6.9 months—all in favour of combined therapy. Also, the grade 3 or 4 adverse effects rate was lower among the adagrasib-cetuximab group (34% vs. 16%).

Cetuximab monotherapy was also shown to be effective but, unsurprisingly, combined treatment has better clinical outcomes such as in the trial by (72). PFS of a group receiving cetuximab plus irinotecan was 4.1 vs. 1.5 months compared with a group receiving monotherapy, ORR 22.9% vs. 10.8% and median OS was 8.6 months vs. 6.9 months. A similar outcome was achieved during the ERMES phase 3 study (73) which resulted in reporting a lack of non-inferiority of cetuximab maintenance therapy compared to the FOLFIRI/Cet regimen. However, treatment with cetuximab alone presented a lower risk of grade 3 adverse effects rate.

Maintenance therapy is another field in which panitumumab has found its application (74). Currently, the main purpose in the development of maintenance therapy is the reduction or replacement of oxaliplatin-based chemotherapeutics due to high toxicity and tolerance issues. In the PANAMA trial, panitumumab in combination with folinic acid and fluorouracil was confronted with fluorouracil and folinic acid alone in patients with RAS wild-type CRC. There was noted a significant improvement in terms of PFS (8.8 months vs. 5.7 months) and also better outcomes in OS (28.7 months vs. 25.7 months), ORR (40.8% vs. 26%). These data suggest that adding panitumumab after induction to standard maintenance therapy is a viable clinical option. However, in the phase 2 VALENTINO trial panitumumab alone did not prove superior as a maintenance therapy over the panitumumab plus fluorouracil-leucovorin in terms of 10 months PFS (75).

Overall, cetuximab and panitumumab were shown as effective in first-line therapy of left-sided CRC and are recommended to be included in the treatment of RAS, BRAFV600E, and ERBB2 wild-type variants (57, 70). The major concern of anti-EGFR inhibitors is unsatisfying outcomes in the management of patients with mutated variants of CRC. However, some patients with KRAS-mutation G13-D mutation benefited from administering anti-EGFR therapy, suggesting that not all cases present resistance to this line of drugs (8, 76).

In second-line therapy and above, anti-EGFR agents are not typically among the top choices for the further management of mCRC as many studies failed to reach statistically significant results of their benefit (57). Nevertheless, some trials demonstrated their usefulness in particular clinical scenarios (66). In a phase III trial presented evidence for the efficacy of panitumumab in refractory CRC in a group of 463 patients with 1% or more EGFR tumour cell membrane staining. The outcomes of panitumumab with best supportive care vs. best supportive care alone were as follows: PFS (8 weeks vs. 7.3 weeks), ORR (10% vs. 0%) with equal OS.

Due to unsatisfactory available methods for third-line treatment of CRC, there are attempts to evaluate the effectiveness of panitumumab in rechallenge therapy of refractory RAS wild-type CRC. According to (77), mCRC cells develop resistance mechanisms, notably through RAS and EGFR ectodomain mutations, under sustained anti-EGFR treatment. However, upon cessation of this treatment, mutated malignant cells lose their competitive advantage over non-mutated malignant cells, leading to a decline in their numbers over time. Parseghian et al. elucidate that the relative mutant allele frequency of RAS and EGFR undergoes exponential decay (r2 = 0.93 for RAS; r2 = 0.94 for EGFR) with a cumulative half-life of 4.4 months which potentially rationalizes rechallenge and intermittent anti-EGFR therapy. In a trial by (78) 62 patients were divided into two groups and tested with tifluridine-tipiracil with or without panitumumab. Median PFS was noted as 4 months compared to 2.5 months. Furthermore, in patients with pretreatment plasma RAS/BRAF wild-type ctDNA, there was observed even better response to treatment. PFS of the group with this pattern of DNA and simultaneously receiving panitumumab was 4.5 months and PFS rates at 6 months were 38.5% vs. 13% (compared to a group that did not present RAS/BRAF wild-type ctDNA and received panitumumab).

As far as the intermittent therapy with an anti-EGFR agent is concerned, during the phase 2 trial IMPROVE (79) patients treated as a first-line treatment with FOLFIRI/PANI continuously not only had lower PFSot compared to the intermittent regimen (12.6 months (95% CI: 9.0–16.1) vs. 17.6 months (95% CI: 7.5–27.8) respectively) but also higher risk of grade 3 or 4 toxicities. Nevertheless, further evaluations of the results are required for a better understanding of intermittent therapy with anti-EGFR agents.

The uncontrolled proliferation of cancer cells results in inadequate perfusion of tumour cells, giving rise to local acidosis and hypoxia which leads to the promotion of the transcription of hypoxia-associated factors, specifically HIF-1a and HIF-2a which subsequently initiates the transcription of several key ligands for angiogenesis e.g., VEGF (vascular endothelial growth factor), angiopoietin, Notch and integrins (89) hence angiogenesis is inherently linked with the process of carcinogenesis and creation of metastases.

The pathway of paramount clinical relevance involves the VEGF-A ligand and VEGFR-2 receptor, facilitating endothelial cell migration and proliferation, elevating vascular permeability, and inducing alterations in gene expression (90). Given its important contribution to the pathophysiology of carcinogenesis, numerous anti-VEGF agents have been developed, exhibiting notable success in the management of mCRC e.g., Bevacizumab, Ramucirumab, Aflibercept, and Regorafenib.

The NTRK genes (NTRK1/2/3) encode tropomyosin receptor kinase proteins (TRK A/B/C) primarily involved in neural development and cellular homeostasis (117). NTRK fusions are a rare occurrence in CRC patients, accounting for around 1% of cases (118–120). Previous reports have indicated that CRC characterized by NTRK positivity may constitute a distinctive subset exhibiting specific features e.g., high TMB and a higher likelihood of MSI (121).

Recent trials—ALKA-372-001 and STARTRK, evaluating TRK inhibitors, such as larotrectinib and entrectinib, are prompting clinicians to consider NTRK testing in mCRC patients, particularly in later lines of therapy (122, 123). Larotrectinib is a first-in-class, highly selective TRK inhibitor, which produces durable responses in patients with NTRK fusion–positive, according to long-term data from the phase II basket of NAVIGATE trial (NCT02576431). Out of 34 patients treated with larotrectinib, 33% showed an OR (3% complete response, 30% partial response) (124). Additionally, 45% had stable disease, 12% experienced disease progression, and 9% had an undetermined best overall response. Notably, 9 out of 29 patients with measurable disease experienced tumour shrinkage.

To overcome secondary resistance mechanisms, ongoing clinical evaluations are assessing the efficacy of additional second-generation TRK inhibitors, including repotrectinib and taletrectinib (125).

The FGFR1-4 (fibroblast growth factor receptor) genes encode four closely related receptor tyrosine kinases, which compose the FGFR family. After binding with FGF (fibroblast growth factor) ligands the RAS/MAPK, PI3 K/AKT, and JAK/STAT pathways are activated (126). The FGFR signalling pathway is involved in a range of biological functions spanning from angiogenesis, wound repair, and tissue regeneration to cell proliferation, migration, and anti-apoptotic processes. Among individuals diagnosed with CRC, around a third of them present modifications in FGFR genes, involving specific mutations, amplifications in gene copy numbers, and heightened mRNA expression levels (127). Prior preclinical studies indicated that these genetic modifications were linked to sensitivity to FGFR inhibitors, but predicted a poor prognosis, invasiveness, and the potential for metastases (128).

Over recent years, various targeted FGFR inhibitors have undergone clinical trials to assess their therapeutic impact on patients. These compounds competitively attach to the ATP-binding sites of FGFR interrupting further signal propagation. However, due to their high specificity, broad applicability in treating CRC patients is impossible. Several TKIs have been used to treat tumours with FGFR irregularities. Ponatinib, dovitinib, and lucitanib have undergone assessment for their ability to inhibit FGFR, yet their high toxicity has limited their ability for future development (127). As of late, F1-7, a new FGFR inhibitor has been outlined, which can induce DNA damage. This results in impeding cell growth and metastasis, ultimately causing cellular apoptosis. Experiments conducted on mouse models further validated the effective suppression of tumour growth by inhibiting the FGFR pathway (129).

Pemigatinib, another inhibitor of FGFR1-3, during a phase II single-arm study, showcased improved response rates among patients dealing with resistant metastatic CRC linked to FGF/FGFR alterations. All participants enrolled in the trial and treated with pemigatinib exhibited confirmed mutations in FGFR1-4 and/or amplifications in FGF/FGFR, with no FGFR translocations. The mPFS stood at 9.1 weeks (95% CI, 7.9 to not evaluable [NE), while the mOS reached 7.9 months (95% CI, 3.4 to NE). Severe grade 3 or higher adverse events (AE) were observed in 42.9% of treated patients, including one instance of a grade 5 AE. Among the most frequently occurring AE of any grade were anaemia, hyperphosphatemia, elevated alkaline phosphatase, increased AST levels, and fatigue. Despite a favourable safety profile, pemigatinib demonstrated suboptimal effectiveness in this specific population. Ongoing translational studies aim to unravel the mechanisms contributing to resistance against pemigatinib (130).

The DNA damage response (DDR) pathway plays a vital role in identifying and accurately repairing damaged DNA, crucial for preserving the cell's genetic integrity and preventing issues like cellular ageing, apoptosis, and the onset of cancer. This pathway involves eight distinct processes that cater to different types of DNA damage, including mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), checkpoint factors (CPF) and more (131). DDR alterations can prompt a hyper-mutated state or microsatellite instability-high (MSI-H), leading to increased TMB, which acts as a biomarker predicting better responses to immune checkpoint inhibitor (ICI) therapy (132).

Recent studies have unveiled germline and/or DDR defects in a subset of CRC cases, ranging from 13.8% to 36% prevalence (133). These alterations, irrespective of microsatellite instability status, were linked to a higher median tumour mutation burden in CRC, along with increased positivity for PD-L1. Moreover, DDR mutations have shown an association with enhanced overall survival (OS) in CRC patients undergoing treatment with immune checkpoint inhibitors (ICIs). Notably, investigations suggest that DDR-related ATM or BRCA2 somatic mutations hold promise as biomarkers for evaluating the response of stage III CRC patients to oxaliplatin-based chemotherapy (134). In a study performed by (135), DNA DDR mutations were identified in all MSI-H CRCs and 83.77% of MSS CRC cases. The most frequently mutated DDR genes included ARID1A (7.5%), ATM (5.7%), and BRCA2 (2.6%).

The predominant mutation type seen in ARID1A, characterized as a truncating mutation, induces deficiencies in DNA damage repair mechanisms within tumour cells (136). Preclinical research has demonstrated that this deficiency in ARID1A makes CRC cells more responsive to PARP inhibitors (like olaparib, rucaparib or veliparib) both in laboratory experiments and in animal models (137).

Comparison between right- and left-sided CRCs revealed no notable variance in DDR genes and associated pathways. Survival analysis indicated that DDR mutations did not correlate with OS in MSS CRCs. However, left-sided CRC patients with mutations in the homologous recombination repair (HRR) pathway displayed notably prolonged OS compared to right-sided CRCs. Additionally, mutations in the DDR pathway, including those within the homologous recombination repair (HRR) pathway, didn't significantly correlate with improved OS in MSS CRC patients (135). Conversely (133), reported a strong association between DDR mutations and MSI, noting a favourable mOS in CRC patients treated with immune checkpoint inhibitors (ICI). However, in their study, conventional treatments did not display a significant difference in prognosis for patients with DDR mutations, suggesting that DDR mutations might specifically serve as a predictive biomarker for the effectiveness of ICI immunotherapy in CRCs. Consequently, for MSS CRC, it appears that DDR mutations are not notably linked to a better prognosis.

Marks et al. (138) study aimed to explore the potential impact of DDR mutations on the response to first-line treatments containing oxaliplatin (FOLFOX/XELOX) or irinotecan (FOLFIRI) in mCRC. DDR mutations were identified in 11 out of 49 patients (22%). Among these cases, patients treated with a first-line oxaliplatin-based regimen exhibited a statistically significant improvement in mOS (3.4 vs. 1.8 years; P = 0.042) compared to those receiving irinotecan. Additionally, this group showed a numerically higher response rate (50% vs. 33%; P = 0.58). However, in patients without DDR mutations, no significant differences in OS (2.4 vs. 2.5 years; P = 0.42), response rate, or disease control rate were observed between the two regimens.

The POLE gene encodes an enzyme called DNA polymerase epsilon, which holds an important role in DNA replication fidelity. Responsible for synthesizing the leading DNA strand at the replication fork, POLE encompasses a 3′-5′ exonuclease domain crucial for enhancing the precision of replication. This domain functions by identifying and excising mismatched base pairs, thereby promoting accurate DNA replication. Somatic and heritable deficiencies in POLE's proofreading mechanism, notably mutations occurring within its exonuclease domain, are more prevalent in tumours proficient in mismatch repair and cause a rise in the rate of mutations, which ultimately results in the development of tumours (139).

The occurrence rate of acquired mutations within the exonuclease domain of POLE in CRC stands at around 3%, surpassing the frequency observed in inherited mutations (“Comprehensive Molecular Characterization of Human Colon and Rectal Cancer,” 2012). While the prevalence of inherited mutations within the exonuclease domain of POLE in familial colorectal adenoma or CRC is merely 0.1%–0.25%, the presence of these germline mutations substantially amplifies the likelihood of developing CRC (140).

POLE somatic mutations are linked to a favourable prognosis and are more prevalent in men, the right colon, and individuals in the early stages of the disease (141). CRC patients carrying POLE mutations often showcase elevated TMB and infiltration of immune cells within their tumours, thereby amplifying their responsiveness to immune checkpoint inhibitors (ICI) (142).

In a study performed by (139) among 14,229 next-generation sequencing reports, 458 patient tumours exhibited POLE mutations. Of these mutations, 15.0% were categorized as pathogenic, 15.9% as benign, and 69.1% as variants of unknown significance. Among 82 patients who received programmed death 1 or programmed death ligand-1 inhibitors either individually or alongside cytotoxic T-cell lymphocyte-4 inhibitors, those with pathogenic POLE mutations demonstrated substantial improvements. They exhibited enhanced clinical benefit rates (82.4% vs. 30.0%; P = .013), a longer median progression-free survival (15.1 vs. 2.2 months; P < .001), increased overall survival (29.5 vs. 6.8 months; P < .001), and a prolonged treatment duration (median 15.5 vs. 2.5 months; P < .001) in comparison to individuals harbouring benign variants.

The role of immunotherapy among patients with POLE mutation remains unclear, however, in 2020, a prospective, open-label, multicenter phase 2 experiment was carried out by (143) to evaluate the safety and efficacy of avelumab in 30 patients with dMMR/MSI-H and 3 patients with POLE mutations. Even though no patients with POLE mutations responded to avelumab, it is important to consider the constraints of the small sample size and the variance in mutation locations.

RET is a proto-oncogene that produces the receptor for growth factors from the glial-derived neurotrophic factor family (144). Abnormal chromosomal rearrangements can result in RET fusion, which is most often observed in thyroid and lung cancers, however, in rare instances, RET gene fusion can also occur in mCRC cancer, with a frequency of less than 1% (145). mCRC tumours with in-frame RET display unique characteristics, such as a preference for the right colon, diagnosis at an older age, and predominantly MSI-h phenotype (146).

One oral RET inhibitor, selpercatinib, was approved by the FDA in 2020 to treat RET fusion-positive non-small cell lung cancer and thyroid cancer (147). In the phase I/II LIBRETTO-001 study, which included 41 patients with various solid tumours (excluding lung and thyroid cancer) (148), 10 patients (24%) had CRC. The ORR for these malignancies was 20% (95% CI, 2.5–56), while the OR in the entire group was 44% (95% CI, 28–60). People with solid tumours studied had a 5-year survival rate ranging from 3% to 40%. Another RET inhibitor, pralsetinib, was examined in the phase I/II ARROW trial. In this cohort of 23 participants, two had CRC, but no response was observed in these patients (149).

The RSPO family consists of four genes RSPO1-4, which encode signalling proteins, known to play a significant role in activating the Wnt/β-catenin signalling pathway (150). This pathway is crucial for controlling important biological functions like cell growth, stem cell management, and maintaining tissue balance and regeneration. RNF43 is recognized as a negative modulator of WNT signalling and acts as a tumour suppressor (151). When RNF43 is lost, there is a reduction or absence of frizzled receptor degradation, leading to heightened WNT signalling. In cancer cells, the inactivation of RNF43 due to mutations is among the factors contributing to the sustained activation of the WNT signalling pathway.

The RSPO family is observed in up to 8% of CRC, and studies indicate that similar to RNF43 mutations, RSPO translocations, even in isolation, are sufficient to initiate carcinogenesis. RNF43 mutations, occurring in various malignancies including colorectal and gastric cancers, can be observed with a frequency of up to 20% (152). Notably, the frequency of RNF43 mutations is higher in MSI cancers. Neoplastic cells with loss-of-function mutations in RNF43 result in a higher quantity of the Wnt receptor Frizzled which, consequently, exhibits heightened sensitivity to the inhibition of the porcupine homolog (PORCN) protein, known for its role in posttranslational modification of the Wnt protein (153).

In a study performed by (154) the initial group of 7,245 CRC samples was examined. Among these, RSPO gene fusions (RSPOfp) were found in 1.3% of cases, while RNF43 mutations were present in 6.1%. Tumours with RNF43 mutations were notably linked to tumours located on the right side. Interestingly, none of the RSPOfp tumours displayed RNF43 mutations. RNF43-mutated tumours tended to have a higher occurrence of MSI-H/dMMR in 64.3% of cases and a tumour mutation burden of ≥10 mutations per megabase (mt/Mb) in 65.8%. In contrast, RSPOfp did not exhibit any correlation with MSI-H/dMMR.

The outcomes in the (155) study imply that RNF43 mutations are associated with better OS in CRC patients treated with ICBs, likely due to increased TMB and the presence of gene signatures related to the immune system.

The changes observed in RSPO2 expression and rearrangements offer a potential avenue for creating targeted treatments in tumours reliant on the Wnt pathway. Despite significant efforts, the availability of FDA-approved drugs and inhibitors for routine inclusion in clinical trials aiming at the Wnt pathway in CRC remains limited. Developing precise and effective inhibitors to target Wnt signalling in CRC continues to present significant obstacles (156).

Multiple ongoing clinical trials aim to validate the effectiveness of diverse inhibitors targeting the Wnt/β-catenin signalling pathway in RNF43/RSPO-positive tumours. Additionally, the identification of MSI-h/dMMR traits in a subset of RNF43-mutated tumours suggests that combining immune checkpoint inhibition with and without Wnt/β-catenin signalling inhibitors might represent a viable therapeutic strategy that should be assessed in future prospective trials (154).

NKG2D is a type of immunoreceptor present in several subtypes of lymphocytes. It was proposed that it could serve as a target in the CAR-T type of therapy called CYAD-01. Physiologically the protein is either absent or its expression is relatively low but during infections or hyper-proliferation, there can be a significant elevation of its expression which allows the immune system to eliminate the affected cells. Its elevation was linked with poor survival in patients with most variants of CRC in contrast to other cancer types where its protective role was observed. This mechanism was used in adoptive cell therapy (166, 167). So far in-vitro and in-vivo experiments demonstrated cytotoxicity against CRC cells and significantly reduced the extent of the tumour and suppression of its growth in the xenograft model (168). The same outcome was observed in human subjects. In a study by Xiao et al., the modified NK cells were used in the therapy of 3 patients with CRC (163). In two patients, the infusion into the peritoneal cavity resulted in a reduction of tumour cells in intraperitoneal fluid and a reduction of ascites generation. In one patient USG-guided intrahepatic infusion allowed rapid regression of metastasis in the liver confirmed in a PET-CT scan.

CEA is an immunoglobulin-glycoprotein involved in cell adhesion (169), frequently found in the epithelium of the embryo which is also associated with overexpression in 98.8% of CRC tissue samples, making CEA the predominant prognostic marker for CRC (170). A pre-clinical study conducted by (171) concerning a novel, chimeric SIRPg-CD28 co-receptor for CRC exhibited anti-tumour in vivo properties. The cytotoxicity effect was specific only to CEA and CRC cells. In mice CRC xenografts models, the tumours were eradicated within 21 days and persisted until the end of the experiment. CAR-T with an “armed” co-receptor demonstrated remarkable anti-tumour efficiency in CRC.

In a phase-I study by (172), 10 patients received CEA-targeted CAR-T with progressing doses (1 × 105–1 × 108 /CAR + /kg cells). 7 of them noted transformation from progressive to stable disease, 2 of them remained stable after 30 weeks and 2 presented tumor shrinkage in imaging studies. No severe adverse effects were observed.

GUCY2C is a cancer mucosa antigen expressed in both human and mouse intestinal mucosa. Similarly, to CEA, its overexpression in CRC implies its distinctive suitability as a potential target for CAR-T cell therapies. An open-label, single-arm, investigator-initiated exploratory trial conducted by (173) examined 13 individuals with GUCY2C-positive mCRC. In the analysis of 10 evaluable patients, there were 6 partial responses, 2 stable diseases, and 2 progressive diseases. Across the dosage levels, ORR = 60%, DCR = 80%. The primary toxicity was decreased lymphocytes in 12/13 pts (92%), leukopenia in 1/13pts and thrombocytopenia in 1/13pts. Overall, the anti-GUCY2C CAR-T cell therapy was tolerated well with mild AE.

Another potential target for CAR-T is HER2 transmembrane glycoprotein which as mentioned above exhibits tyrosine kinase activity playing a crucial role in regulating growth and differentiation of epithelial cells. The heightened expression of HER2 is observed in various types of solid tumours such as breast cancer, ovarian cancer, lung cancer, gastric cancer, and CRC (174), however, a mutation within the gene encoding the HER2 receptor is present only in 3–5% of mCRC (175) thus narrowing usefulness of target and CAR therapies aimed at HER2. The meta-analysis by (176) findings indicates that, on average, participants in the six trials, totalling 238 patients with HER2 + mCRC, had undergone a median of three prior lines of therapy before enrollment with ORR = 31.33% and DCR = 74.37%, PFS = 6.2 months. The outcomes suggest a significant response to the therapy, leading to meaningful enhancement in survival rates. Regarding CAR-T therapy for HER2 + mCRC, additional studies are required to elucidate its potential role in the future (177) observed that HER2 CAR-T cells demonstrate anti-tumour activity in CRC xenograft models. However, clinical trials are fraught with uncertainties. Phase I/II NCT00924287 clinical trial was terminated due to the first patient death because of treatment. However, TAC T-Cell therapy for HER-2 + solid tumours (NCT04727151) has been reported to be well tolerated without reported neurotoxicity in phase I, the only AE were haematological (178).

EpCAM, a prominent tumor-associated antigen found on the surface of CRC, has also been considered a potential target in CAR-T cell therapy which led to the development of clinical trials (NCT02915445, NCT03013712, NCT05028933, NCT03563326). First-in-human trial by (179) IMC001 designed to target EpCAM-positive gastrointestinal tumours with CAR-T has already exhibited promising anti-tumour capabilities and a favourable safety profile. Moreover, downsizing the tumour creates additional opportunities for surgical resection.