The assessment of RAS and BRAF genes and mismatch repair (MMR)/microsatellite status should be evaluated as molecular panel upfront in all cases of advanced colorectal cancer (CRC) to drive patients’ selection toward biological approved treatments, although the standard cytotoxic drugs (fluoropyrimidines, oxaliplatin, and irinotecan), remain the backbone in most of the cases without specific targets [1]. However, in the early stages of CRC (I–III), the choice of adjuvant treatment is not based on molecular targets, but pweople know that in stage II CRC, microsatellite instability high (MSI-H)/MMR deficiency is associated with a lower recurrence rate than MSI-low/MMR proficient tumors ( hazard ratio (HR)HR 0.65; 95% confidence interval (CI)CI 0.59–0.71) [2].

In this regard, there is a clinical need to improve molecular targets, regardless of those having received strict histology-agnostic approval.

In the last few decades, one of the most studied mechanisms involved in cancer progression concerns the immune system and how it interacts with cancer cells. This research led to revolutionary approaches with the immune checkpoint inhibitors (ICIs) by blocking the immune checkpoint proteins or their ligands to reactivate the antitumor immune response ^[3][4][3,4].

Plenty of biomarkers are appearing on the scientific landscape in order to identify those patients who could really benefit from immunotherapy. Among these, certainly, the most validated in CRC is microsatellite status, which occurs as MSI in 5% of sporadic CRC, while the remaining 95% are classified as stable (MSS) or MSI-low, and the efficacy of immunotherapy is yet to be defined [3].

Further biomarkers involved in immune system regulation should be identified.

Considering the role of immune checkpoints in tumor development and immune escape, the use of anti-programmed cell death protein 1 (PD-1)/PD-Ligand 1(PD-L1) monoclonal antibodies, stimulating the immune response against the tumor, has routinely become part of clinical practice [4].

The prognostic and/or predictive role of PD-1/PD-L1 and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4)LA-4 expression was recognized in various types of cancer, such as melanoma, lung, head and neck, urothelial cancer, and others [4]. ICIs are used in these tumors in different settings, often according to different cut-off values of PD-L1 expression evaluated by immunohistochemistry (IHC) and established by different scores [5].

Most equivocal is the role of the tumor mutational burden (TMB) due to massive molecular heterogeneity resulting in dissimilar correlations between TMB levels and prognostic outcomes such as overall survival (OS) and progression-free survival (PFS) ^[6][7][8][9][6,7,8,9].

The attention over other immune checkpoints increased significantly in the last years, and new potential targets were identified, such as Lymphocyte-activation gene 3 (LAG3), which is highly expressed on tumor-infiltrating lymphocytes (TILs) [10]. In some tumors, such as ovarian, melanoma, non-small-cell lung cancer (NSCLC), and gastrointestinal cancers, PD-1 was usually co-expressed with LAG3 ^{[11][12][13][14]}[11,12,13,14]. Simultaneous activation of the LAG3 and PD-1 pathways in TILs results in greater T-cell exhaustion, and restraining both pathways may improve T-cell activity and immune response [15]. Elevated expression of LAG3 has seen positive impacts on PFS in estrogen receptor-negative breast cancer [16]. Hald et al. [17] reported that intraepithelial-LAG3 and stromal-LAG3 were both associated with improved disease-specific survival (DSS) and OS in NSCLC. Additionally, in esophageal squamous cell carcinoma, higher LAG3 expression was positively correlated with a better OS and PFS, especially in the patients at early stage I–II [14].

In this scenario, recognizing unresponsive patients before starting the immunotherapy would help the potential development of personalized treatment and allow patients to avoid unnecessary ICIs and toxicities. Nevertheless, the prognostic significance of all these immune checkpoint molecules remains controversial.

In CRC, the RAS gene is mutated in kirsten rat sarcoma viral oncogene homolog (KRAS)KRAS in up to 40% of cases, mostly in exon 2 codons 12 and 13, and in NRAS gene in approximately 3–5% of CRCs in exon 3 (codon 61) and exon 2 (codons 12 and 13) [18]. Identification of RAS status is mandatory in clinical practice due to its role as a negative predictive factor in anti- epidermal growth factor receptor (EGFR) antibody response (cetuximab, panitumumab) ^[19][20][21][19,20,21]. Compared to wild-type tumors, RAS mutant patients have a worse prognosis, both in the adjuvant and metastatic settings ^[22][23][22,23]. Regardless of RAS status, anti-VEGF (vascular endothelial growth factor)/VEGFR (vascular endothelial growth factor receptor) -2/PDGF (Placental-Derived Growth Factor) aagents such as bevacizumab, ramucirumab, aflibercept, and regorafenib demonstrated efficacy in further lines of treatment ^{[24][25][26][27]}[24,25,26,27].

A dynamic process has been identified regarding further KRAS mutations leaving the opportunity to develop new treatment strategies. For example, the KRAS G12C codon mutation, which represents 3% of all CRC cases, is now susceptible to inhibition by small molecule inhibitors that specifically and irreversibly block KRAS G12C by locking it in an inactive GDP-bound site ^[28][29][28,29]. Two basket phase I/II studies proved that sotorasib (CodeBreaK100) and adagrasib (KRYSTAL-1) are both active in patients with advanced solid tumors harboring a KRASG12C mutation, among which mCRC ^[30][31][32][30,31,32]. However, EGFR signaling mutations were identified as the dominant mechanism of CRC resistance to KRAS G12C inhibitors [33]. Indeed, the combination of adagrasib and cetuximab improved objective response rate (ORR) to 43% and disease control rate (DCR)DCR to 100% [34], leading to a possible novel histology-tuned targeted treatment for mCRC, also being tested as sotorasib plus panitumumab [35]. A randomized phase III trial (KRYSTAL-10) [36] is ongoing and evaluates second-line agadrasib plus cetuximab versus standard chemotherapy.

Additionally, several other combinations of sotorasib or adagrasib with diverse compounds as well as newer anti-RAS strategies are ongoing.

The second genetic mutation is represented by BRAF V600E, accounting for 8–10% of mCRC cases, and is nearly always mutually exclusive with KRAS, resulting in RAS-independent oncogenic signaling through the mitogen-activated protein kinase (MAPK) pathway (RAS-RAF-MEK-ERK) [37]. This alteration is associated with a worse prognosis and could predict a poorer response to anti-EGFR treatment [38]. Based on the breathtaking results achieved in other solid tumors such as melanoma, several trials explored the use of BRAF inhibitors as single agents or in combination with MEK inhibitors in the mCRC. However, the inhibition in the MAPK/ERK pathway with a BRAF inhibitor results in adaptive feedback reactivation of MAPK signaling mediated by rapid feedback EGFR reactivation ^[39][40][39,40]. In order to overcome primary resistance, anti-EGFR monoclonal antibodies were tested with BRAF V600E inhibitors, with modest activity for this combination ^[41][42][41,42]. Recently, the BEACON phase III trial compared the combination of anti-BRAF V600E (encorafenib), anti-MEK (binimetinib), and anti-EGFR (cetuximab) inhibitors as a triplet or doublet (encorafenib and cetuximab) scheme versus investigator’s choice (FOLFIRI or irinotecan and cetuximab) in pre-treated BRAF V600E mutant mCRC patients [43]. Long-term follow-up updates showed that both double and triple combinations achieved a median OS of 9.3 months, compared with 5.9 months in the control arm. The overall response rate (ORR) was 26.8% with the triplet, 19.5% with the doublet, and 1.8% in the control arm, with prolonged maintenance of the quality of life (QoL) and reducing the risk of QoL deterioration by more than 40% [43]. The phase II ANCHOR-CRC trial is testing the triplet regimen (encorafenib, binimetinib, and cetuximab) in the first-line setting [44], showing an ORR of 47.8% and DCR of 88%; median PFS was 5.8 months (95% CI, 4.6–6.4) and mOS was 17.2 months (95% CI, 14.1-NE).

Furthermore, approximately 20% of BRAFV600E mCRC have high-level MSI (MSI-H). Currently, an anti-PD-1 antibody, pembrolizumab, represents the standard of care for BRAFV600E mutant MSI-H mCRC [45]. In a phase I/II clinical trial, patients with treatment-refractory BRAFV600E MSS mCRC received encorafenib, cetuximab, and nivolumab, showing effective activity in terms of ORR (45%), DCR (95%), mPFS (7.3 months; 95% CI, 5.5-NA), and mOS of 11.4 months (95% CI, 7.6-NA) [46].

Although the best treatment has not yet been identified, an aggressive strategy involving triplet chemotherapy and targeted therapy is currently the standard of care for fit patients. BRAF- therapies combined with other anti-EGFRs, MEK inhibitors, or PI3K inhibitors seem promising ^[47][48][47,48].

Further developments have identified another molecular biomarker in mCRC, as the prevalence of receptor tyrosine-protein kinase erbB-2 (ERBB2) or frequently called human epidermal growth factor receptor 2 (HER2) amplification, in approximately 3–8%, especially in KRAS wild-type CRC [49]. Although retrospective data have demonstrated that ERBB2 amplification represents a negative predictive biomarker for anti-EGFR therapies, prospective data are needed to define the relationship between ERBB2 and anti-EGFR responsiveness ^[50][51][50,51]. Indeed, RAS/BRAF wild-type mCRC should be screened for ERBB2 amplification before treatment with anti-EGFR therapies [52]. The activity of an anti- ERBB2-targeted therapy was demonstrated in the phase II HERACLES-A trial [53], in which a double signaling blockade by trastuzumab and lapatinib achieved an ORR of 30% in KRAS wild-type chemo refractory tumors. ERBB2 positivity was defined as tumors with a 3 + score in more than 50% of cells by immunohistochemistry or a 2 + score and a HER2:CEP17 ratio > 2 in more than 50% of cells by fluorescent in situ hybridization (FISH). Similar results were confirmed in the MyPathway phase II basket trial (trastuzumab plus pertuzumab) [54], and HER2 positivity was defined as amplification (FISH or HER2/CEP17 ratio 2.0 or copy number > 6), overexpression (IHC 3 +), or activating HER2 mutations. Emergent ERBB2 target agents are evolving in mCRC, such as the tyrosine kinase inhibitors, tucatinib, and an antibody-drug conjugate, trastuzumab-deruxtecan, that in the phase II DESTINY-CRC01 trial [55] achieved in the ERBB2 positive RAS wild-type mCRC an ORR of 45.3%. The preliminary results of the ongoing phase II MOUNTAINEER trial [56] showed 55% ORR with tucatinib combined with trastuzumab.

Remains to be clarified the heterogeneity in HER2/ERBB2 assessment in clinical trials and even across different tumor types [49].