Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene encoding for a transmembrane glycoprotein with a tyrosine kinase activity, a member of the ErbB receptor tyrosine kinases family, and one of the epidermal growth factor receptors (EGFRs) ^[1][2][1,2]. HER2 plays an essential role in normal biological and oncogenic processes, regulating cell proliferation, differentiation, and migration via numerous signaling pathways, such as mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) and phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) ^[1][2][1,2]. The schematic overview of HER2 pathways is represented in Supplementary Figure S1. Alterations of HER2 include gene amplification and missense mutations and often lead to protein overexpression [3]. These types of molecular aberrations are considered founder events in tumorigenesis and tumor progression because of uncontrolled cell proliferation, inhibition of apoptosis, and migration ^[1][4][5][6][1,4,5,6].

In the current clinical practice, HER2 status is tested in breast and gastroesophageal cancers to select patients eligible for anti-HER2 treatment [5]. However, alterations in HER2 have been documented in a plethora of other solid tumors, including colorectal cancer (CRC) ^{[2][3][4][5][6][7]}[2,3,4,5,6,7]. This tumor type is the second leading cause of cancer-associated deaths worldwide in both sexes [8]. In CRC, the frequency of HER2 overexpression is 5–6% with somatic HER2 gene alterations, including amplifications, reported in ~7% of patients ^{[4][6][9][10][11]}[4,6,9,10,11]. HER2 mutations in colonic epithelial cells have been shown to be indicative of HER2 signaling pathway activations, promote independent cell growth, and potentially acquire resistance to EGFR-targeted therapies, subjecting patients to a worse prognosis ^{[2][4][6][9][10][11][12]}[2,4,6,9,10,11,12]. There are currently no approved HER2-targeted therapy guidelines for CRC; however, several studies have shown that HER2 can be effectively targeted in metastatic CRC settings ^{[2][12][13][14]}[2,12,13,14].

2. HER2 Targeting in Metastatic Colorectal Cancer

The administration of anti-HER2 drugs (e.g., trastuzumab, pertuzumab) is presently a standard of care in HER2-positive (i.e., score 3+ by immunohistochemistry (IHC) or 2+/in situ hybridization (ISH)-positive) breast and gastric cancer ^[15][16][15,16]. For CRC, HER2 first emerged as a negative predictive biomarker. Amplification or overexpression of HER2 was associated with a lack of response to anti-EGFR treatment [17]. The novel antibody–drug conjugate trastuzumab deruxtecan (T-DXd) has been approved by the United States Food and Drug Administration (FDA) for HER2-low (i.e., score 1+ or 2+/ISH-negative) breast cancers [18], and it is currently under investigation in histology-agnostic settings in the DESTINY-PanTumor02 study (NCT04482309). The first clinical trials using trastuzumab in mCRC evaluated the combination of this monoclonal antibody with chemotherapy. Clark et al. assessed the association of FOLFOX + trastuzumab in the second- or third-line treatment of HER2-positive mCRC. A total of 5 patients out of 21 (24%) had a partial (PR) or complete response (CR), with a median duration of response of 4.5 months [19]. Another phase II study assessed the combination of trastuzumab and irinotecan in HER2-positive mCRC pretreated with one line of therapy. Objective responses were recorded in five patients (71%), and these were maintained for at least 6 months [20]. Subsequent studies evaluated different strategies of an HER2 dual blockade with significant results. HERACLES-A tested the combination of trastuzumab and lapatinib in patients with KRAS exon-2 wild-type (WT) mCRC refractory to standard treatment and with HER2 amplification and/or overexpression. A total of 914 patients were screened, of whom 48 (5%) had HER2-positive diseases ^[14][21][14,21]. The long-term clinical results at a follow-up of 6.7 years reported an overall response rate (ORR) of 28% with one CR and eight PRs, a disease control rate (DCR) of 69%, median progression-free survival (mPFS) of 4.7 months, and median overall survival (OS) of 10 months for 32 treated patients. Two of them (6%) reported a grade 3 decrease in left ventricular ejection fraction, while fatigue was registered in five cases (16%) [22]. Differently, HERACLES-B tested the combination of pertuzumab and the antibody–drug conjugate trastuzumab emtansine (TDM-1) in RAS and BRAF WT and HER2-positive mCRC refractory to standard therapies. The primary endpoint was not reached, with ORR below the expected rate ≥30% (9.7%). Stable disease (SD) was seen in 21 patients (67.7%), and the DCR rate was 77.4%. Treatment was well-tolerated, with two patients suffering from grade (G) 3 thrombocytopenia [23]. A median PFS of 4.2 months was similar to the HERACLES-A study, and patients with a HER2 3+ score at immunohistochemistry (IHC) had a significantly higher mPFS compared to HER2 IHC and fluorescent in situ hybridization (FISH)-amplified tumors (HR: 0.20; 95% confidence interval (CI): 0.07–0.56; p = 0.0008) [17]. The MyPathway phase II trial enrolled 57 patients with HER2-amplified mCRC receiving a combination of trastuzumab and pertuzumab. One patient had a CR while seventeen (30%) benefited from a PR. On the whole, 18 patients (32%) achieved an objective response, and, in 4 cases, it was longer than 12 months. Treatment was well-tolerated, with the most common adverse events being G 1 or 2 diarrhea, fatigue, and nausea. Patients harboring a KRAS mutation had a significantly shorter PFS and OS compared to the KRAS WT population (PFS: 1.4 months; 95% CI, 1.2–2.8 months versus 5.3 months; 95% CI: 2.7–6.1 months for mutated and WT, respectively; OS: 8.5 months; 95% CI: 3.9–not estimable versus 14.0 months; 95% CI: 8.0–not estimable for mutated and WT, respectively) [24]. Two other phase II studies (TRIUMPH and TAPUR) evaluated the combination of trastuzumab + pertuzumab. In the first study, 19 patients with RAS wt mCRC and HER2 amplification in tissue samples achieved an ORR of 35% with a complete response and five partial responses. Interestingly, the TRIUMPH trial evaluated HER2 status on circulating tumor DNA (ctDNA). Similarly, to the patients with HER2 amplification detected on tissue, 15 patients with ctDNA positivity for HER2 amplification had an ORR of 33% with one CR and four PRs. With a median follow-up of 5.4 months, mPFS was 4 months [25]. In the TAPUR basket trial, a cohort of 28 patients with HER2 amplified mCRC was treated; ORR was 14% and DCR for at least 16 weeks was 50% with an mPFS of 3.8 months [26]. A new anti-HER2 agent, trastuzumab deruxtecan, was tested in the DESTINY-CRC01 phase II trial. This antibody–drug conjugate of a humanized anti-HER2 antibody with a topoisomerase I inhibitor was tested in HER2-positive RAS-BRAF WT mCRC progressed on two or more lines of treatment, with the possibility to include patients pretreated with different anti-HER2 agents. A total of 78 patients were enrolled with 53 placed in cohort A (HER2 IHC 3+ or 2+ and positive in situ hybridization), a total of 7 in cohort B (IHC 2+ and negative in situ hybridization), and 18 in cohort C (IHC 1+). After a median follow-up of 27.1 weeks, the ORR in group A was 45.3% (95% CI, 31.6–59.6), and patients pretreated with anti-HER2 agents obtained a high ORR of 43%, as well. Differently, no responses were seen in groups B and C of treatment [12]. At a longer median follow-up of 62.4 weeks with 86 patients treated, the ORR of the group A was confirmed (45.3%). Moreover, DCR was 83%, mPFS 6.9 months, and mOS 15.5 months. Pulmonary toxicity in terms of interstitial lung disease and pneumonitis was recorded in eight patients (9.3%). Two patients died because of grade 5 lung toxicity. Interestingly, trastuzumab deruxtecan was also effective in the group of patients with RAS mutation-positive ctDNA [27]. In the ongoing MOUNTAINEER trial, trastuzumab is associated with tucatinib, a tyrosine kinase inhibitor (TKI) of the HER2 protein. Twenty-six patients with chemorefractory, RAS WT, and HER2-positive tumors have been treated so far with an ORR of 52.2%, twelve PRs, and six cases of SD. Patients experienced a prolonged median response of 10.4 months, an mPFS of 8.1 months (95% CI, 3.8–not estimable), and a median OS of 18.7 months (95% CI, 12.3–not estimable) [28]. The HER2 FUSCC-G trial is testing the combination of trastuzumab + pyrotinib, an irreversible dual pan-ErbB tyrosine kinase inhibitor (TKI) [29]. A cohort of 11 mCRC HER2-positive patients have received this combination so far, with a global ORR of 45.5% and 55.6% in RAS WT tumors. With a median follow-up of 17.7 months, mPFS was 7.8 months, while mOS 14.9 months. Patients harboring KRAS mutation had poorer outcomes compared to the KRAS WT group (PFS, 7.7 versus 9.9 months; p = 0.19; OS, 12.4 versus 20.6 months; p = 0.021) [30]. Differently, the combination of the anti-HER2 TKI neratinib and anti-EGFR agent cetuximab was not effective in terms of objective responses. A total of 16 patients with KRAS, NRAS, BRAF, and PI3KCA WT tumors were treated; 6 of them (44%) had SD with 5 harboring HER2 amplification at baseline. G 3adverse events, such as diarrhea, skin rash, and an increased level of transaminases, were registered in 67% of patients [31]. Table 1 lists the main trials with HER2-targeted therapies in mCRC that have been conducted so far. While anti-HER2 therapy in CRC is awaiting approval, many early trials continue, demonstrating promising results [32]. Supposing that HER2 may be immunogenic and leads to T-cell activation suggests it is targetable for immunotherapy [33]. Some studies demonstrate the induction of HER2 downregulation in HER2-positive cancer cells with the immune effector cells’ engagement, revealing a new function of immune cells in trastuzumab-mediated antitumor efficacy and probably representing a novel mechanism of action of trastuzumab, predicting active immune effector cells’ recruitment in the tumor microenvironment [34]. The acquisition of drug resistance to trastuzumab has been recently explained in HER2-positive gastric cancer by vessel destabilization and activation of the glycolytic pathway inducing 6-phosphofructo-2-kinase (PFKFB3). The inhibition of PFKFB3 in patient-derived xenograft models has significantly diminished tumor proliferation and promoted vessel normalization. It has also been found that PFKFB3 promotes the interleukin-8 coding gene CXCL8 by activating the PI3K/AKT/NFκB p65 pathway, which leads to the idea that PFKFB3 inhibition might be effective in overcoming trastuzumab resistance in HER2-positive cancers [35]. HER2-activating mutations are also known for their association with microsatellite instability-high tumors, which has been observed in CRC [36]. With the evolution of immunotherapy, after the US Food and Drug Administration (FDA) approval of pembrolizumab, it has become a practice-changing treatment option for unresectable or mCRC in patients with high microsatellite instability (MSI-H) or mismatched repair deficiency (dMMR) [37]. In the metastatic settings, however, only a small proportion of CRC patients responded to immune checkpoint therapy, despite positive results in some phase I trials, and all these tumors were MSI-H/dMMR and had a high tumor mutation burden [38]. While tumor mutational burden has been associated with the immune checkpoint response rate in other tumor types, such as melanoma and non-small-cell lung cancer, the underlying mechanism is still unknown, although it might be related to immune cells’ reactivity, increasing T-cell infiltration ^[38][39][40][38,39,40]. An ongoing recruiting four-part, phase 1/2 dose-escalation/expansion study is aiming to evaluate the effects of BDC-1001 (immune stimulating antibody conjugate (ISAC), consisting of an anti-HER2 monoclonal antibody conjugated to a TLR 7/8 dual agonist) in combination with/without PD1 inhibitor pembrolizumab in patients with progressive HER2-expressing solid tumors (NCT04278144). Current results demonstrate BDC-1001 to be well tolerated and clinically efficient also in patients previously treated with anti-HER2 therapy; however, the safety and efficacy of combining with a PD1 inhibitor is yet to be studied ^[6][41][6,41], although the data on PD-L1 expression in CRC with its regards to microsatellite instability remain controversial [38]. Some authors advocate that HER2-targeted therapies may favorably be combined with other emerging therapeutic strategies for advanced CRC, including immune checkpoint inhibitors, increasing the tailored therapeutic approach ^[42][43][42,43].