Therapeutic strategies targeting the epidermal growth factor receptor signaling pathway in metastatic colorectal cancer

Abstract

More than 1.9 million new colorectal cancer (CRC) cases and 935000 deaths were estimated to occur worldwide in 2020, representing about one in ten cancer cases and deaths. Overall, colorectal ranks third in incidence, but second in mortality. More than half of the patients are in advanced stages at diagnosis. Treatment options are complex because of the heterogeneity of the patient population, including different molecular subtypes. Treatments have included conventional fluorouracil-based chemotherapy, targeted therapy, immunotherapy, etc. In recent years, with the development of genetic testing technology, more and more targeted drugs have been applied to the treatment of CRC, which has further prolonged the survival of metastatic CRC patients.

Key Words: Metastatic colorectal cancer, Epidermal growth factor receptor, B-type RAF mutation, Kirsten rat sarcoma viral oncogene wild type, Kirsten rat sarcoma viral oncogene G12C mutation, Human epidermal growth factor receptor 2 overexpression/ amplification

Core Tip: This review focuses on therapies targeting the epidermal growth factor receptor (EGFR) signaling pathway in metastatic colorectal cancer, mainly including: (1) Treatment targeting rat sarcoma (RAS) status, including RAS wild type, maintenance therapy with anti-EGFR monoclonal antibody, anti-EGFR re-challenge therapy, treatment targeting NoeRAS and Kirsten RAS viral oncogene G12C mutations; (2) treatment for patients with B-type RAF mutations; and (3) treatment targeting human EGFR2 amplification or overexpression.

INTRODUCTION

Globally, colorectal cancer (CRC) ranks third in incidence and second in mortality among malignant tumors[1]. In general, about 20% of CRC patients have metastatic disease at the time of diagnosis, and more than 50% of CRC patients will develop metastatic disease later[2]. Targeting the epidermal growth factor receptor (EGFR) pathway is one of the main treatment strategies for metastatic CRC (mCRC). EGFR is a transmembrane tyrosine kinase receptor located on cell membranes and is an important member of the tyrosine kinase receptor family. When EGFR binds to its ligands (e.g., epidermal growth factor), it will activate its downstream signaling pathways, including several signaling pathways such as RAS-RAF-MEK-ERK, PI3K-AKT-mTOR, STAT, etc. (Figure 1), which in turn promote cell proliferation, growth, angiogenesis, and metastasis. EGFR-targeted drugs work primarily by interfering with the activation of the EGFR signaling pathway. This article focuses on therapies targeting the EGFR signaling pathway in mCRC, including therapies based on the genetic status of rat sarcoma (RAS), RAF, and human EGFR 2 (HER-2).

Figure 1 Epidermal growth factor receptor signaling pathway.

TREATMENTS TARGETING RAS STATUS

RAS wild-type mCRC

Approximately 50% of CRC patients have RAS wild-type (WT) disease[3], and the prognosis of RAS WT CRC is usually better than that of RAS mutant type. Numerous studies have been conducted depending on RAS status and treatment timing, providing a survival benefit in metastatic colon cancer (Table 1). It was first demonstrated in the CRYSTAL study[4] that the anti-EGFR antibody cetuximab in combination with chemotherapy (FOLFIRI) was significantly better than chemotherapy alone in RAS WT patients. In that study, 1198 patients were randomized 1:1 to cetuximab plus FOLFIRI and FOFIRI alone. The hazard ratio (HR) for progression-free survival (PFS) was 0.85 [95% confidence interval (CI): 0.72 to 0.99; P = 0.048] in the cetuximab-FOLFIRI group compared with the FOLFIRI group. There was no significant discrepancy in overall survival (OS) between the two groups (HR = 0.93; 95%CI: 0.81 to 1.07; P = 0.31). Subgroup analysis found that patients with Kirsten RAS viral oncogene (KRAS) exon 2 WT tumors treated with FOLFIRI and cetuximab had a longer median PFS (mPFS) (9.9 mo vs 8.7 mo; HR = 0.68; 95%CI: 0.50-0.94; P = 0.02). Several subsequent studies, like the OPUS study[5], a trial comparing cetuximab plus FOLFOX-4 vs FOLFOX-4 alone as first-line therapy for mCRC; the COIN study[6], a phase III trial comparing cetuximab in combination with FOLFOX4 or XELOX vs FOLFOX4 or XELOX alone in first-line treatment of KRAS WT mCRC patients; and the PRIME study[7], a phase III randomized clinical trial of panitumumab plus FOLFOX4 vs FOLFOX4 in the first-line treatment of the mCRC patients, showed that panitumumab combined with FOLFOX4 significantly improved mPFS compared with FOLFOX4 alone in KRAS WT patients (9.6 mo vs 8.0 mo).

Table 1 Results of clinical trials based on RAS status.

Therapy based on RAS status	Study	Design	Result	Ref.
RAS wild-type first-line therapy	CRYSTAL study	Cetuximab plus FOLFIRI vs FOLFIRI alone	The HR for PFS among patients with KRAS wild-type tumors was 0.68 (95%CI: 0.50 to 0.94), favoring the cetuximab-FOLFIRI group	Van Cutsem et al[4]
	OPUS study	Cetuximab plus FOLFOX-4 vs FOLFOX-4 alone	The median survival was 18.3 mo for patients receiving cetuximab plus FOLFOX-4 and 18.0 mo for FOLFOX-4 alone	Bokemeyer et al[5]
	COIN study	Cetuximab combined with the FOLFOX4 or XELOX regimen vs FOLFOX4 or XELOX regimen alone	The median survival was 19.6 mo vs 18.0 mo	Adams et al[6]
	PRIME study	Panitumumab combined with FOLFOX4 vs FOLFOX4 alone	In KRAS wild-type patients, panitumumab combined with FOLFOX4 significantly improved mPFS compared with FOLFOX4 alone, at 9.6 mo and 8.0 mo, respectively	Douillard et al[7]
RAS wild-type maintenance therapy	COIN-B study	Intermittent chemotherapy plus intermittent cetuximab therapy vs intermittent chemotherapy plus cetuximab maintenance therapy	mOS was 16.8 mo (95%CI: 14.5-22.6) in the intermittent cetuximab group vs 22.2 mo (18.4-28.9) in the continuous cetuximab group, and mPFS from week 12 was 3.1 mo (95%CI: 2.80-4.7) in the intermittent cetuximab group and 5.8 mo (4.9-8.6) with continuous cetuximab	Wasan et al[15]
	MACRO 2 TTD study	FOLFOX combined with cetuximab sequential cetuximab maintenance therapy vs FOLFOX combined with cetuximab	PFS at 9 mo was 60% vs 72%, and OS was 23 mo vs 27 mo. The objective response rate was 48% vs 39%	Aranda et al[16]
	MACBETH study	Modified FOLFOXIRI plus cetuximab, followed by cetuximab (arm A) vs bevacizumab maintenance (arm B)	10-mo PFR was 50.8% (90%CI: 39.5%-62.2%) in arm A and 40.4% (90%CI: 29.4%-52.1%) in arm B. The overall response rate was 71.6% (95%CI: 62.4%-79.5%)	Cremolini et al[17]
	VALENTINO study	Panitumumab plus fluorouracil and calcium folinate (arm A) vs panitumumab (arm B)	10-mo PFS rate was significantly higher in group A than in group B (59.9% vs 49.0%, P = 0.01). The mPFS was 12.0 mo and 9.9 mo, respectively. The OS rate at 18 mo was 66.4% and 62.4% (P = 0.60), respectively, with no significant difference	Pietrantonio et al[18]
	PANAMA (AIO KRK 0212) study	Panitumumab in combination with 5-FU vs 5-FU alone	RAS WT mCRC patients treated with 5-FU/LV + panitumumab maintenance therapy after FOLFOX + panitumumab induction therapy could have a superior outcome to 5-FU/LV maintenance therapy alone, with a PFS of 8.8 mo vs 5.7 mo (P = 0.014)	Modest et al[21]
RAS wild-type anti-EGFR-targeted agents rechallenge	CRICKET trial	Irinotecan and cetuximab as a third-line treatment for patients experiencing an initial response and then progression with a first-line irinotecan and cetuximab-containing therapy. Liquid biopsy samples were analyzed	The confirmed PR rate was 57% in patients with no RAS mutations detected in ctDNA. Patients with RAS wild-type ctDNA (n = 13) had significantly longer PFS than those with RAS-mutated ctDNA (n = 12), with a mPFS of 4.0 mo vs 1.9 mo (HR = 0.44, 95%CI: 0.18-0.98, P = 0.03), while no significant differences were reported in terms of OS (mOS: 12.5 mo vs 5.2 mo, HR = 0.58, 95%CI: 0.22-1.52, P = 0.24)	Cremolini et al[25]
	JACCRO CC-08 trial	Irinotecan plus cetuximab rechallenge as third-line treatment in KRAS wild-type mCRC patients who achieved clinical benefit with first-line cetuximab-containing therapy	The 3-mo PFS rate was 44.1%. The mPFS and OS were 2.4 mo and 8.2 mo, respectively. The response and DCR were 2.9% and 55.9%, respectively	Masuishi et al[26]
	CAPRI 2 GOIM trial	FOLFIRI + cetuximab (first line); FOLFOX + cetuximab (second line); irinotecan + cetuximab (third line)	Ongoing	Martini et al[28]
	WJOG8916G trial	TAS-102 in combination with cetuximab	DCR was 54%, PR was 3.6%, and mPFS was 2.4 mo. Patients with wild-type RAS, BRAF, EGFR, PIK3CA, ERBB2, and MET genes had a DCR of 75% vs 39% (P = 0.02), respectively, compared to mutant patients. mPFS was 4.7 mo vs 2.1 mo (P < 0.01)	Izawa et al[29]
	Phase II single-arm study	Cetuximab and palbociclib in KRAS-WT mCRC treated with ≥ 2 prior lines of therapy	The 4-mo DCR was 20%, which did not fulfill the prespecified 4-mo DCR rate. mPFS was 1.8 mo and mOS was 6.6 mo	Sorah et al[32]
	CAVE Trial	Cetuximab plus avelumab as a rechallenge strategy in patients with RAS wild-type metastatic colorectal cancer	The mOS was 17.3 mo for wild-type and 10.4 mo for ctDNA mutant patients (HR = 0.49, P = 0.02), with a PFS of 4.1 mo and 3.0 mo (HR = 0.42, P = 0.004), respectively	Martinelli et al[33]
	CAVE 2 study	Avelumab plus cetuximab as a reinvigorating strategy in pre-treated patients with RAS and BRAF WT mCRC	Ongoing	Napolitano et al[34]
	CRACK study	Cetuximab rechallenge combined with liposomal irinotecan and camrelizumab	The ORR was 25%, and the DCR was 75%. The mPFS and OS were 6.9 mo and 15.1 mo, respectively	Quan et al[36]
NeoRAS wild-type	C-­PROWESS trial	Panitumumab plus irinotecan therapy for patients with NeoRAS mCRC	Ongoing	Osumi et al[40]
KRAS G12C mutation	CodeBreaK100 trial	Sotorasib	The ORR was 9.7%	Fakih et al[44]
	CodeBreaK300 trial	Sotorasib (960 mg daily in arm A and 240 mg daily in arm B) with panitumumab vs chemotherapy	HR = 0.49 (95%CI: 0.30-0.80, P = 0.006) in the sotorasib 960 + panitumumab group and HR = 0.58 (95%CI: 0.36-0.93, P = 0.03) in the sotorasib 240 + panitumumab group, and OS data was immature	Pietrantonio et al[45]
	KRYSTAL-1 study	Adagrasib (MRTX849) alone vs adagrasib plus cetuximab	In the adagrasib monotherapy group, the ORR and DCR were 19% and 86%, respectively, while the mPFS was 5.6 mo. In the cetuximab–adagrasib group, a higher ORR of 46% and DCR of 100% were observed, and the mPFS was 6.9 mo	Yaeger et al[46]

ORR: Overall response rate; mPFS: Median progression-free survival; OS: Overall survival; DCR: Disease control rate; HR: Hazard ratio; PFS: Progression-free survival; RAS: Rat sarcoma; KRAS: Kirsten RAS; ctDNA: Circulating tumor DNA; mCRC: Metastatic colorectal cancer; BRAF: B-type RAF.

All of the above studies have confirmed the efficacy of anti-EGFR monoclonal antibodies in RAS WT patients, while B-type RAF (BRAF) mutations were a negative prognostic factor; they have been approved by the Food and Drug Administration (FDA) for the first-line treatment of patients with RAS WT mCRC. In addition to RAS status as a predictive marker for anti-EGFR therapy, several studies have reported that the primary site of the tumor correlates with the efficacy of anti-EGFR therapy. Researchers from The Ottawa Hospital Research Institute[8] were the first to report the relationship between the primary site of colon tumors and the efficacy of anti-EGFR therapy. They reanalyzed the NCIC CO.17 trial and classified the primary tumor site as RC (cecum to transverse colon) or LC (splenic flexure to rectosigmoid colon). Associations between tumor side and baseline characteristics were assessed. Cox regression models identified factors affecting OS and PFS. The results showed that PFS was significantly improved with cetuximab vs best supportive care in patients with LC (median 5.4 mo vs 1.8 mo, HR = 0.28), whereas there was no improvement in RC patients (median 1.9 mo vs 1.9 mo, HR = 0.73, P = 0.26). In 2017, Arnold et al[9] investigated the prognostic and predictive influence of the localization of the primary tumor in patients with unresectable RAS WT mCRC included in six randomized trials (CRYSTAL, FIRE-3, CALGB 80405, PRIME, PEAK, and 20050181), compared chemotherapy plus EGFR antibody therapy (experimental arm) with chemotherapy or chemotherapy and bevacizumab (control arms). This pooled analysis showed a significant benefit for anti-EGFR antibody plus chemotherapy therapy in patients with left-sided tumors (HRs = 0.75 and 0.78 for OS and PFS, respectively) compared with no significant benefit for those with right-sided tumors (HRs = 1.12 and 1.12 for OS and PFS, respectively). Rossini et al[10] searched for phase II and III trials (PEAK, CALGB/SWOG 80405, FIRE-3, PARADIGM, and CAIRO5), including 2739 patients, 77% with left- and 23% with right-sided tumors. The addition of anti-EGFR therapy was associated with a higher objective response rate (ORR) (74% vs 62%) and longer OS (HR = 0.77, P < 0.0001) in patients with left-sided mCRC. A subgroup analysis confirmed a significant interaction between the primary tumor side and the treatment group in terms of ORR (P = 0.02), PFS (P = 0.0004), and OS (P = 0.001).

Maintenance anti-EGFR therapy

Maintenance therapy for tumors refers to the selective discontinuation of drugs with greater adverse effects in patients who have achieved disease control after standard treatment, and the use of lower-intensity drugs with fewer adverse effects for ongoing treatment. Maintenance therapy generally uses one of the induction chemotherapy drugs, or another drug that is not cross-resistant to the induction drug, to delay tumor recurrence or deterioration and prolong PFS as long as possible by maintaining a relatively small dose[11].

In recent years, a variety of targeted agents have been widely used in the palliative care of patients with advanced CRC with remarkable results. However, there is not yet a clear, unified consensus on which regimen to use for maintenance therapy in patients with mCRC. Generally speaking, in the treatment of advanced mCRC, there are two ways of maintenance therapy: One is to keep the original treatment regimen with accurate efficacy unchanged, reduce the dose, or subtract a certain drug from the original regimen, and the other is to discontinue the original treatment regimen and replace it with a new therapeutic drug. Compared with other tumors, CRC has fewer drugs of choice for treatment, so clinically, the original regimen is usually chosen, with some drugs reduced. Two earlier studies, OPTIMOX-1[12] and OPTIMOX-2[13] trials, explored trends in the efficacy and safety of maintenance chemotherapy regimens with fluorouracil (FU) after intermittent chemotherapy with oxaliplatin in patients with mCRC, laying the groundwork for further studies exploring the optimal strategy for maintenance therapy. Subsequently, studies based on EGFR monoclonal antibody for maintenance therapy in mCRC were conducted.

NORDIC-VII[14] was an open-label, randomized, multicenter phase III trial which reported the efficacy of cetuximab as maintenance therapy for the first time in 2012. Patients were randomly assigned in a 1:1:1 ratio to FLOX alone (Arm A), cetuximab plus FLOX (Arm B), or cetuximab continuously and FLOX intermittently (Arm C). Unfortunately, in this study, not only was it not possible to observe a benefit from cetuximab maintenance therapy, but it was also not possible to observe a benefit from cetuximab in combination with FLOX, even in the KRAS WT group. In the KRAS WT subgroup, PFS was 8.7, 7.9, and 7.5 mo, and OS was 22.0, 20.1, and 21.4 mo in the three groups, respectively. This result was inconsistent with previous studies like CRYSTAL, OPUS, and PRIME. We do not have sufficient evidence to determine whether the reason for this result is related to the different patients enrolled or the combination chemotherapy regimen. In 2014, COIN-B[15] study was published in Lancet Oncology. This study compared the efficacy of intermittent chemotherapy plus intermittent cetuximab therapy vs intermittent chemotherapy plus cetuximab maintenance therapy in the first-line treatment of mCRC. The study recruited 226 patients. At the time of analysis, results for 169 with KRAS WT are reported. The median duration of follow-up in the KRAS WT intention-to-treat (ITT) population was 32.8 mo in the intermittent cetuximab group and 34.2 mo in the continuous cetuximab group. The median OS (mOS) was 16.8 mo in the intermittent cetuximab group vs 22.2 mo in the continuous cetuximab group, the mPFS from week 12 was 3.1 mo in the intermittent cetuximab group and 5.8 mo with continuous cetuximab, and toxic effects and adverse events (AEs) were similar in each treatment group. Although not statistically compared, maintenance cetuximab with intermittent chemotherapy seemed to be slightly more active than intermittent cetuximab with intermittent chemotherapy.

Subsequently, a phase II randomized MACRO2 TTD study[16] explored the non-inferiority of FOLFOX combined with cetuximab followed by sequential cetuximab maintenance therapy compared to FOLFOX combined with cetuximab. The results achieved non-inferiority, but a trend towards decreased OS and ORR was observed in the cetuximab monotherapy maintenance group, pending further validation in phase III clinical studies. The MACBETH study[17] compared the modified FOLFOXIRI combined with cetuximab, followed by cetuximab (arm A) with bevacizumab maintenance (arm B) as first-line treatment for patients with RAS and BRAF WT mCRC. It was also a randomized phase 2 clinical trial, and neither of the two study arms met the primary endpoint of increasing the 10-mo PFS rate from 50% (literature-based) to 70%, so neither of the two investigated strategies appeared worthy of further investigation. However, the findings indicate that a 4-mo induction regimen of mFOLFOXIRI plus cetuximab is feasible and provides relevant activity results, leading to a high surgical resection rate. The study observed some survival advantages with cetuximab maintenance therapy vs switching to bevacizumab maintenance therapy, but no statistical comparisons were made. The VALENTINO study[18] was an open-label, randomized, multicenter phase 2 noninferiority trial. In this study, the efficacy of panitumumab vs panitumumab plus FU and calcium folinate was compared for maintenance therapy in patients with RAS WT mCRC. Patients were randomized (1:1) to first-line panitumumab plus FOLFOX-4 for 8 cycles followed by maintenance therapy with panitumumab plus FU-leucovorin (arm A) or panitumumab (arm B) until progressive disease, unacceptable toxic effects, or consent withdrawal. The primary study endpoint was the 10-mo PFS rate in the ITT population, and the upper limit of non-inferiority for the one-sided 90%CI for HR in arm B compared to arm A was set at 1.515. The PFS results showed that arm B was inferior to arm A. The upper limit of the HR one-sided 90%CI of 1.857 exceeded the preset cut-off value of 1.515. The 10-mo PFS rate was significantly higher in arm A than in arm B, (59.9% vs 49.0%, P = 0.01). The mPFS was 12.0 and 9.9 mo, respectively. The OS rates at 18 mo were 66.4% and 62.4% (P = 0.60), respectively, with no significant differences between the two arms. There were also no between-arm differences in the overall remission rate (66.7% vs 67.0%) and disease control rate (DCR) (82.9% vs 83.9%). During maintenance therapy, the incidence of grade ≥ 3 treatment-related AEs (TRAEs) was higher in arm A than in arm B (42.4% and 20.3%, respectively). The incidence of panitumumab-related AEs was also higher in group A (31.8% and 16.4%, respectively). This phase II study demonstrated that sequential panitumumab monotherapy maintenance therapy after first-line FOLFOX combined with panitumumab in patients with RAS WT mCRC resulted in an inferior PFS to panitumumab combined with FU-calcium folinate, but the toxicity of the combination maintenance therapy was also slightly increased. Earlier results from the Turkish Oncology Group phase III "Stop and Go" clinical study[19] showed that in untreated mCRC patients, first-line treatment with bevacizumab + XELOX followed by maintenance therapy with bevacizumab + capecitabine significantly prolonged mPFS compared to the control group (11.0 mo vs 8.3 mo, P = 0.002). So, does capecitabine in combination with anti-EGFR therapy provide benefits to patients with advanced CRC? In 2020, Wang et al[20] reported in JAMA Netw Open a phase 2 clinical trial aimed to appraise the biological activity and safety of capecitabine combined with cetuximab as a novel maintenance treatment for RAS WT mCRC. This prospective clinical trial was conducted at five centers in China. Patients diagnosed as having RAS WT mCRC were recruited to receive FU-based cytotoxic agents combined with cetuximab followed by capecitabine plus cetuximab for maintenance therapy. Induction therapy was 8 to 12 cycles of FU-based chemotherapy plus cetuximab for patients with RAS WT mCRC. After achieving stable disease (SD) or better status, cetuximab plus reduced-dose capecitabine was administered for maintenance therapy. The primary endpoint was PFS during maintenance therapy. The secondary endpoints were total PFS, OS, quality of life, safety, and adverse reactions to treatment. The results showed that the mPFS was 12.7 mo. The mOS was 27.4 mo. Grades 3 to 4 AEs during induction therapy included neutropenia (4 patients, 9%), diarrhea (4 patients, 9%), nausea or vomiting (3 patients, 6%), rash acneiform (10 patients, 21%), and hand-foot syndrome (8 patients, 17%). Grades 3 to 4 AEs during maintenance therapy included diarrhea (2 patients, 4%), rash acneiform (8 patients, 17%), and hand-foot syndrome (5 patients, 11%). The results of this study indicated that reduced-dose capecitabine combined with cetuximab maintenance therapy is a new option for RAS WT mCRC after induction therapy with tolerable toxicity. The PANAMA (AIO KRK 0212) study[21] was one of the more notable oral presentations in the Bowel Cancer Special Session at the 2021 American Society of Clinical Oncology (ASCO) Annual Meeting. This is a phase II multicentre randomized controlled study first registered in 2013 and the first large-sample clinical study on maintenance therapy with panitumumab in combination with 5-FU vs 5-FU alone against RAS WT (RAS WT) mCRC. After 6 cycles of induction therapy with FOLFOX and panitumumab, patients with RAS WT mCRC were randomized 1:1 to the 5-FU/LV + panitumumab and 5-FU/LV alone maintenance therapy groups, with the primary endpoint being PFS. The results showed that RAS WT mCRC patients treated with 5-FU /LV + panitumumab maintenance therapy after FOLFOX + panitumumab induction therapy could have a superior outcome to 5-FU/LV maintenance therapy alone, with a PFS of 8.8 mo vs 5.7 mo (P = 0.014). The results of the study confirm the feasibility of maintenance therapy with EGFR-targeted agents, with panitumumab in combination with FU and calcium folinate as an effective option. The results of this study change the paradigm of maintenance therapy for advanced CRC, adding EGFR-targeted agents as a new modality for maintenance therapy. Despite the efficacy of anti-EGFR monoclonal antibodies in patients with RAS/BRAF WT, most of them were evaluated phase II clinical studies, which have yet to be confirmed by large, phase III multicentre clinical studies. Therefore, rational anti-EGFR maintenance regimens remain controversial, and long-term anti-EGFR monotherapy is still associated with persistent skin toxicity and acquired resistance. Whereas the mechanisms of acquired anti-EGFR inhibitor resistance are usually related to EGFR dependence, activation by EGFR bypass or downstream signaling pathways, and histological phenotypic transformation[22], hyperactivation of other pathways, such as vascular endothelial growth factor and angiogenesis, could potentially contribute to the emergence of resistant clones. Thus, numerous questions await further study.

Anti-EGFR re-challenge therapy

The combination of anti-EGFR monoclonal antibody (panitumumab or cetuximab) with two-agent chemotherapy is a first-line treatment protocol for patients with RAS and BRAF WT mCRC. Whether anti-EGFR therapy can be reintroduced in subsequent treatment after disease progression has been studied and reported by many authors. Santini et al[23] reported in 2012 on cetuximab rechallenge in mCRC. In this study, the researchers proposed that KRAS mutations had occurred at very early stages of human colorectal carcinogenesis and that KRAS status was not altered by treatment despite different treatments given, yet 5%-10% of mCRC cases exhibited KRAS heterogeneity between primary, lymph node, and distant metastases[24]. Anti-EGFR therapy resulted in the destruction of KRAS WT cells and the proliferation of mutant clones, leading to tumor progression. An alternative line of therapy without cetuximab restores the K-RAS WT clone, which can constitute a major part of the tumor mass during subsequent disease progression. At this point, further shrinkage of the disease can be determined by the rescue with a new line of cetuximab-based therapy. Although this study was a small-sample retrospective study, it provided support for the biology of cetuximab rechallenge in subsequent studies. In recent years, several studies based on circulating tumor DNA (ctDNA) detection to drive cetuximab rechallenge have been reported. The CRICKET trial[25] was designed to prospectively estimate the activity of a rechallenge strategy with irinotecan and cetuximab as a third-line treatment for patients who had experienced an initial response and then progression with irinotecan- and cetuximab-containing first-line therapy and received second-line chemotherapy plus bevacizumab. The prospective collection of liquid biopsy samples from enrolled patients was planned to investigate whether the analysis of potential mechanisms of acquired resistance to anti-EGFR monoclonal antibodies in ctDNA could help judge the benefit of this strategy. The confirmed partial response (PR) rate was 57% in patients with no RAS mutations detected in ctDNA. Patients with RAS WT ctDNA had significantly longer mPFS than those with RAS mutated ctDNA (4.0 mo vs 1.9 mo, HR = 0.44, P = 0.03), while no significant differences were reported in terms of OS (mOS, 12.5 mo vs 5.2 mo, HR = 0.58, P = 0.24). This is the first prospective study of cetuximab and irinotecan rechallenge in RAS and BRAF WT mCRC patients and provides a clear signal of activity for rechallenge with anti-EGFR in the third-line setting. JACCRO CC-08 trial[26] evaluated the efficacy and safety of irinotecan plus cetuximab rechallenge as third-line treatment in KRAS WT mCRC patients who achieved clinical benefit with first-line cetuximab-containing therapy. The primary endpoint was 3-mo PFS rate. The study enrolled 34 patients who had received at least one line of therapy. The 3-mo PFS rate was 44.1%. The mPFS was 2.4 mo and OS was 8.2 mo. The response rate and DCR were 2.9% and 55.9%, respectively. In the study, it was found that the cut-off value for cetuximab-free intervals (CFI) was set at 372 d and that patients with long CFIs had significantly better PFS and OS than those with short CFIs. The mPFS was 4.6 mo in the long CFI group and 2.1 mo in the short CFI group (HR = 0.40, P = 0.020). The mOS was 14.1 and 6.3 mo in the long and short CFI groups, respectively (HR = 0.31, P = 0.008). Schulz et al[27] reported on a real-world study from a large German oncology center. In this single-center retrospective study, 524 patients with CRC were included and patients adopting anti-EGFR-based therapy and anti-EGFR rechallenge (progression on first-line anti-EGFR therapy) or re-introduction (discontinuation due to intolerance, toxicity, and so on) were identified. The novelty of this study is that separate statistics were done for rechallenged and reintroduced patients. The results showed that a total of 143 patients received first- or second-line anti-EGFR-based therapy, and 33 patients met the eligible criteria for anti-EGFR re-exposure and were assigned to either the rechallenge or re-introduction subgroup. Anti-EGFR re-exposure was significantly associated with a trend toward improved outcomes (mOS: 56.0 mo vs 35.4 mo, P = 0.06). In subgroup comparisons, reintroduction was correlated with a trend towards higher OS and PFS compared with rechallenge (mOS: 66 mo vs 52.4 mo; mPFS: 7.33 mo vs 3.68 mo).

The CAPRI 2 GOIM trial[28] assessed the efficacy and safety of a biomarker-driven cetuximab-based treatment regimen over three treatment lines in mCRC patients with RAS/BRAF WT tumors at the start of the first-line treatment. The study is expected to enroll 200 patients with mCRC using ctDNA testing to monitor the genetic status of the patients, who will receive the following treatments at each time point of progression for RAS/BRAF WT status: FOLFIRI + cetuximab (first line); FOLFOX + cetuximab (second line); irinotecan + cetuximab (third line). If, after first-line progression, liquid biopsy assessment indicates RAS and/or BRAF mutation status, patients will receive FOLFOX + bevacizumab as second-line therapy. If, after second-line progression, liquid biopsy detection indicates RAS and/or BRAF mutation, patients will receive the investigator's choice (regorafenib or trifluridine-tipiracil) as third-line therapy. Each treatment will be managed using a standard dose and schedule until disease progression or intolerable toxicity occurs. This study is a truly prospective study based on ctDNA-guided administration of different treatment regimens and may answer the question of whether third-line cetuximab rechallenge in RAS/RAF WT patients is superior to the current standard third-line treatment. This study is currently ongoing and will end in 2026, and we look forward to the days when it will give us a new basis for treatment.

All of the above studies were of third-line rechallenge with cetuximab in combination with standard chemotherapy, and the investigators have also reported several studies of some targeted therapies in combination with cetuximab, which provide a choice of new therapeutic approaches for follow-up treatment. The WJOG8916G trial[29] was a study from Japan. Fifty-six patients were included, with a median age of 60 years, of whom 91% had left-sided tumors, and ORR after prior anti-EGFR antibody therapy was 61%. The patients received TAS-102 in combination with cetuximab, and the primary endpoint was DCR. DCR was 54%, PR was 3.6%, and mPFS was 2.4 mo. ctDNA analysis showed that patients with WT RAS, BRAF, EGFR, PIK3CA, ERBB2, and MET genes had a DCR of 75% vs 39% (P = 0.02) in mutant patients. mPFS (4.7 mo vs 2.1 mo, P < 0.01) was better in patients without variants in any of the six genes (n = 20) than in patients with any of the variants (n = 33). The most common grade 3/4 hematological AE was neutropenia at 55%, and there were no treatment-related death events. In recent years, CDK4/6 inhibitors have attracted much attention due to their unique mechanism of action and have also achieved good efficacy in the treatment of many tumors. Studies[30,31] have reported that anti-EGFR treatment resistance mechanisms are associated with bypass activation, including the MEK-ERK pathway and the PI3K pathway, the activation of which further leads to an increase in cyclin D expression, resulting in cell proliferation. Sorah et al[32] reported the phase II Single-Arm Study about Palbociclib and Cetuximab rechallenge in patients with KRAS/NRAS/BRAF WT CRC treated with ≥ 2 lines of therapy and reported on a group of patients rechallenged with anti-EGFR-based treatment who had disease control for at least 4 mo on anti-EGFR therapy priorly. The 4-mo DCR was 20%, which did not meet the prespecified 4-mo DCR rate. mPFS was 1.8 mo and mOS was 6.6 mo. This finding suggests that we may need to screen for some biomarkers to target treatment.

In recent years, immunotherapy has made rapid progress in several tumor types, but in CRC, the indications for immunotherapy are limited to microsatellite instability-high (MSI-H) mCRC only, and microsatellite-stable mCRC has poor response to immunotherapy, so researchers have carried out many modes of combination therapy, including combining with anti-angiogenic drugs and chemotherapy. In the context of cetuximab rechallenge, combination modalities with immunotherapy have also been explored. The phase 2 Single-Arm Clinical CAVE trial[33] examined the effect of cetuximab plus avelumab as a rechallenge strategy in patients with RAS WT mCRC. It enrolled 77 patients with RAS WT mCRC who benefited from first-line anti-EGFR-containing therapy and were retreated with cetuximab combined with avelumab in third or further lines of therapy as a rechallenge strategy. ctDNA analysis for KRAS, NRAS, BRAF, and EGFR extracellular structural domain S492R mutations was performed at baseline in 67 of 77 patients, 48 of whom were KRAS, NRAS, and BRAF WT. The results showed a mOS of 17.3 mo for WT and 10.4 mo for ctDNA mutant patients (HR = 0.49, P = 0.02), with a PFS of 4.1 mo and 3.0 mo (HR = 0.42, P = 0.004), respectively. This result suggests that pre-treatment baseline assessment of plasma ctDNA RAS and BRAF mutations may allow to select patients who could benefit from the treatment. Based on the results obtained in the CAVE clinical trial, in 2022 the investigators reported the design and rationale for the phase II randomized CAVE 2 clinical study[34], which combined avelumab plus cetuximab as a reinvigorating strategy in patients with RAS and BRAF WT mCRC who had been treated with first-line chemotherapy plus cetuximab and who had obtained a clinical benefit (complete or partial remission). A total of 173 patients will be randomized (2:1) to receive cetuximab + avelumab (115 patients) or cetuximab monotherapy (58 patients). The primary endpoint is OS. Key secondary endpoints include the overall remission rate, PFS, and safety. Because the trial is still ongoing, results have not yet been reported. Martini et al[35] conducted a pooled analysis of two prospective phase II trials, CAVE and CRICKET. Individual data of 33 and 13 patients from CAVE and CRICKET trials that received cetuximab rechallenge as third-line therapy were collected. For the whole 46-patient population, the mPFS was 3.9 mo with a mOS of 16.9 mo. For CRICKET patients, mPFS was 3.9 mo and mOS was 13.1 mo with OS rates at 12, 18, and 24 mo being 62%, 23%, and 0%, respectively. For CAVE patients, mPFS was 4.1 mo and mOS was 18.6 mo with OS rates at 12, 18, and 24 mo being 61%, 52%, and 21%, respectively. Skin rash was more frequently reported in the CAVE trial (87.9% vs 30.8%, P = 0.001), whereas an increased incidence of hematological toxicities was observed in the CRICKET trial (53.8% vs 12.1%; P = 0.003). Third-line cetuximab re-challenge combined with either irinotecan or avelumab in RAS/BRAF WT (ctDNA) mCRC patients represents a promising therapy. The CRACK study[36] is a phase II clinical study of cetuximab rechallenge combined with liposomal irinotecan and camrelizumab therapy in patients with RAS WT mCRC reported by Chinese scholars. Patients with RAS WT mCRC who had received at least two lines of therapies, including anti-EGFR-based treatment in the metastatic or locally advanced stage, were enrolled in cohort B. Patients were treated with cetuximab and camrelizumab plus liposomal irinotecan. As of November 23, 2022, 19 patients were enrolled, and 16 were evaluable for efficacy analyses. The ORR was 25%, and the DCR was 75%. The mPFS and OS were 6.9 mo and 15.1 mo, respectively. Grade 3 TRAEs occurred in 15.8% (3/19) of patients. No grade ≥ 4 TRAEs were found. These studies suggest that anti-EGFR plus immunotherapy may be a promising late-line treatment option with good anti-tumor activity and well-tolerated toxicity in RAS WT mCRC patients.

NeoRAS WT mCRC

Patients with RAS/RAF WT mCRC have derived significant benefit from anti-EGFR therapy, but there has been no significant progress in the treatment of KRAS-mutant mCRC for more than 10 years, except for the KRAS G12C-mutant mCRC. In recent years, the concept of NeoRAS WT has been proposed. Initially genetically tested as KRAS-mutant mCRC, after antitumor therapy and dynamic monitoring of the tumor's KRAS status, the KRAS status of some patients changed to WT. Such cases are called "NeoRAS WT". Klein-Scory et al[37] reported an article on the evolution of RAS mutation status in mCRC after receiving first-line treatment, in which it was found that 10 of 12 patients with RAS mutations were converted to RAS WT by ctDNA monitoring after first-line treatment. All patients with PR or SD showed significantly reduced RAS mutational load at the first follow-up. They concluded that antiproliferative chemotherapy may have a greater effect on KRAS-mutant cell clones and that depletion of KRAS-mutant cell clones can promote the growth of WT clones. Osumi et al[38] reported three cases of RAS-mutant mCRC in which RAS status was re-tested after receiving standard treatment, and two patients were treated with anti-EGFR therapy, which resulted in a PR.

Osumi et al[39] reported a multi-institutional observational study assessing the clinicopathologic features and incidence of NeoRAS WT mCRC. A total of 107 patients with tissue RAS mutation (refractory or intolerant to prior chemotherapy) were enrolled at four institutions. RAS status in ctDNA was evaluated using the OncoBEAM™ RAS CRC assay. The incidence of NeoRAS WT mCRC was 21.5%. In line with the tissue RAS mutation site, the frequency of NeoRAS WT was significantly lower in patients with KRAS exon 2 mutations than in patients with exons 3 and 4 mutations or NRAS mutations (18.2% vs 62.5%, P = 0.011). In terms of clinical background, there was a statistically significant difference in the incidence of NeoRAS WT between male and female patients (30.6% vs 8.9%, P = 0.008), and between patients without and with liver metastases (38.6% vs 9.5%, P < 0.001). Comparing the groups by median value, the NeoRAS WT group had smaller tumor diameters, lower carcinoembryonic antigen levels, and lower carbohydrate antigen 19-9 levels. In the multivariate logistic regression analysis, smaller tumor diameter [odds ratio (OR) = 7.92, P = 0.012], liver metastasis absence (OR = 4.62, P = 0.019), and tissue RAS mutations in other than KRAS exon 2 (OR = 9.04, P = 0.026) were significantly related to the transformation to NeoRAS WT in ctDNA.

The C-PROWESS trial[40] is a single-arm, multicentre, phase II trial to evaluate the efficacy and safety of panitumumab combined with irinotecan therapy for patients with NeoRAS mCRC. The key eligibility criteria include RAS mutant mCRC initially proven in tumor tissue intolerant or refractory to fluoropyrimidine, irinotecan, and oxaliplatin, RAS WT in ctDNA within 28 d before enrolment, and Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2. The primary endpoint is the ORR. Biomarker analyses are planned to be performed using next-generation sequencing-based ctDNA analysis. This study is ongoing, and the main results of the study will be reported in international meetings and medical journals. The above results suggest that NeoRAS WT mCRC patients may benefit from anti-EGFR antibodies, which bring new options for treating RAS-mutant mCRC.

KRAS G12C-mutant mCRC

As mentioned above, KRAS WT CRC has seen breakthroughs in EGFR-targeted therapeutic regimens, which have resulted in significant benefits for patients with KRAS WT mCRC, but approximately half of the other CRCs are RAS-mutant type, and therapeutic agents targeting the KRAS mutation in these populations have not been found until the emergence of drugs targeting the KRAS G12C. KRAS G12C mutation occurred in approximately 3% of CRCs[41,42], and KRAS G12C-mutated CRC has worse outcomes than patients with CRC and other KRAS mutations. The phase 1 CodeBreaK100 trial[43] showed that sotorasib had clinical activity as a monotherapy in KRAS G12C-mutated solid tumors. Subsequently, the single-arm phase 2 CodeBreaK100 clinical trial[44] was done, which reported activity and safety data for 62 patients with previously treated (median three prior treatment lines) advanced KRAS G12C-mutated CRC. Among the 62 patients, the objective response was 9.7%, all of which were PRs. The incidence of TRAEs at grade 3 was 10%, with diarrhea being the most common, and TRAE at grade 4 only occurred in one patient; no fatal events were reported. Treatment-related serious AEs (SAEs) occurred in two patients. Although it failed to meet pre-determined study endpoints, it demonstrated some anti-tumor activity and tolerability and can be evaluated in association with other treatments to enhance potential activity and surmount potential resistance mechanisms. CodeBreaK300 (NCT05198934) comparing the combination of sotorasib (arm A: 960 mg daily and arm B: 240 mg daily) with panitumumab (6 mg/kg every 2 wk) vs standard treatment in the third-line setting is reported on the Congress of the European Society for Medical Oncology (ESMO)[45]. The study met its primary endpoint. Both the sotorasib + panitumumab treatment groups had statistically significant PFS compared with the control group: HR = 0.49 (95%CI: 0.30-0.80; P = 0.006) in the sotorasib 960 + panitumumab group and HR = 0.58 (95%CI: 0.36-0.93; P = 0.03) in the sotorasib 240 + panitumumab group, and OS data was immature. At present, other combination regimens, such as sotorasib in combination with EGFR inhibitor, SHP2 inhibitor, or immunotherapy, aiming to reverse drug resistance are being tested in the ongoing CodeBreaK101 basket trial. The KRYSTAL-1 multicohort phase I/II study[46] evaluated the clinical efficacy of adagrasib (MRTX849) alone or combined with cetuximab in patients with KRAS (G12C)-mutated mCRC. In all, 44 patients were recruited to the adagrasib monotherapy group and 32 patients to the adagrasib plus cetuximab group. Toxicity was tolerated in both groups. The ORR, DCR, and PFS of the combination group vs the monotherapy group were 46% vs 19%, 100% vs 86%, and 6.9 mo vs 5.6 mo, respectively. As the adagrasib-cetuximab regimen has demonstrated promising clinical activity, the combination therapy is currently being evaluated in phase III KRYSTAL-10.

BRAF-MUTANT MCRC

BRAF, known as V-RAF murine sarcoma viral oncogene homolog B, is a member of the serine/threonine protein kinase family and an important effector molecule in the mitogen-activated protein kinase (MAPK) signaling pathway. Mutations in the BRAF gene are the most common mutations in the MAPK signaling pathway downstream of the RAS. About 90% of BRAF mutations occur in exon 15 at the 1799^th nucleotide site, where thymine is mutated to adenine in codon 600, replacing the previously encoded valine with glutamic acid, i.e., the BRAFV600E mutation. The occurrence of this mutation leads to RAS-independent activation of BRAF, which stimulates sustained activation of the MAPK signaling pathway, ultimately leading to tumorigenesis (Figure 1).

According to relevant data[47,48], BRAF mutations are found in about 6%-10% of CRC patients. No obvious lifestyle or dietary risk factors are associated with the development of the BRAF V600E mutation. However, the mutation usually occurs in right colonic cancers with advanced stage, mucinous histological manifestations, and clinical manifestations of atypical metastases (brain metastases, bone metastases, peritoneal metastases, mediastinal lymph node metastases, etc.) and often presents a poorer prognosis. BRAF-mutated tumors tend to show high mutation loads, microsatellite instability, and a CpG island methylation phenotype[49]. Several studies, such as FIRE-3 and PETACC-3, have shown that patients presenting with BRAF mutations have a lower OS and a higher risk of death compared to BRAF WT patients. Some studies have shown that EFGR monoclonal antibody alone or combined with chemotherapy is less effective in patients with BRAF V600E mutation. Chemotherapy has been the basic approach for BRAF V600E-mutant mCRC over the past decades, with first-line regimens including FOLFOX, FOLFIRI, and CAPEOX, but its response to chemotherapy has been poor. The three-drug combination FOLFOXIRI (irinotecan + oxaliplatin + FU + calcium folinate) regimen provided slight clinical benefit. In 2015, results from the BRAF mutation subgroup of the phase III TRIBE study[50] showed that FOLFOXIRI + bevacizumab provided a survival benefit over FOLFIRI + bevacizumab (19.0 mo vs 10.7 mo). Despite the small size of this subgroup and the lack of data support from other large phase III clinical studies, the NCCN guidelines still recommend FOLFOXIRI + bevacizumab as first-line treatment for BRAF V600E-mutated mCRC. However, the results of subsequent clinical studies have been less favorable. Results from the BRAF mutation subgroup of the TRIBE2 study[51] showed that FOLFOXIRI + bevacizumab did not show a survival benefit. A pivotal meta-analysis published in the JCO in 2020 showed that FOLFOXIRI + bevacizumab compared to two drugs (FOLFOX or FOLFIRI) + bevacizumab, did not improve OS in patients with BRAF mutations. The results of the FIRE-4.5 study (AIO KRK-0116)[52], the first phase II clinical trial evaluating the efficacy of FOLFOXIRI in combination with cetuximab or bevacizumab for the first-line treatment of patients with BRAF V600E-mutated mCRC, were presented at the ASCO 2021, and updated in 2023, which enrolled 107 patients randomized 1:2 to FOLFOXIRI + bevacizumab (group A, n = 35) and FOLFOXIRI + cetuximab (group B, n = 72). The primary endpoint was to assess the superiority of group B in terms of overall remission rate according to RECIST 1.1 criteria. Secondary endpoints included PFS, OS, and tolerability. The primary endpoint ORR was 66.7% (group A) and 52.0% (group B), respectively (P = 0.23). The mPFS was significantly longer in group A than in group B (10.7 mo vs 6.7 mo, HR = 1.8, P = 0.06). The mOS analyzed at an event rate of 64.5% showed a tendency toward shorter survival in the cetuximab group (12.9 mo vs 17.1 mo, HR = 1.4, P = 0.20).

In 2011, the FDA approved the first BRAF inhibitor, vemurafenib, marking a breakthrough in melanoma treatment. It gave researchers new hope for the treatment of BRAF-mutated mCRC. However, BRAF inhibitors have not shown the same dramatic therapeutic effects in mCRC as in melanoma[53,54]. Basic studies[55] have shown that CRC negative feedback activates EGFR after BRAF blockade, and the pro-tumor signals will bypass the BRAF pathway to activate the downstream protein kinases MEK and ERK, leading to drug resistance. This has led to the exploration of BRAF inhibitors + EGFR inhibitors or MEK inhibitors. However, no meaningful activity was observed with vemurafenib alone or in combination with anti-EGFR monoclonal antibodies[56,57]. The BRAF inhibitor dabrafenib in combination with trametinib, a MEK inhibitor, only mildly improved the efficacy for BRAF V600E-mutant mCRC[53]. A three-drug regimen of dual-targeting in combination with chemotherapy or dual-targeting in combination with cetuximab has been tried since then, as the efficacy of dual-targeting has not been significant.

In 2016 Hong et al[58] published a phase IB study in Cancer Discovery evaluating the efficacy of vemurafenib + irinotecan + cetuximab (VIC regimen) in the treatment of mCRC patients with BRAF V600E mutation, which initially confirmed the effective activity and tolerability of the VIC regimen. The phase II randomized clinical trial SWOG S1406 published in JCO in 2020 evaluated the efficacy, tolerability, and safety of irinotecan + cetuximab ± vemurafenib (VIC regimen vs IC regimen) in patients with BRAF V600E-mutated mCRC previously treated with one or two regimens[59]. A total of 106 patients were enrolled in the study and 100 were eligible (50 patients/group). The primary endpoint was PFS as assessed by the investigators, and secondary endpoints included toxicity, OS, and ORR, as well as ORR and PFS in patients who transitioned to the experimental regimen after disease progression in the control group. The results demonstrated a significant increase in PFS with the combination of vemurafenib, with an HR of 0.50 (95%CI: 0.32 to 0.76, P = 0.001). The mPFS was 4.2 mo and 2.0 mo in the experimental and control groups, respectively, and 21 patients (42%) in the control group crossed over to the experimental regimen after disease progression. The difference in OS between the two groups was not significant (HR = 0.77, 95%CI: 0.50-1.18, P = 0.23), and the mPFS after crossover was 5.4 mo in this cohort. The remission rate and DCR in the experimental group were 17% and 65%, respectively, compared with 4% and 21% in the control group. In terms of safety, the rates of grade 3 to 4 AEs were higher in the experimental group than in the control group, including neutropenia (30% vs 7%), anemia (13% vs 0%), and nausea (19% vs 2%). Treatment was discontinued due to AEs in 11 cases (22%) in the experimental group compared to 4 cases (8%) in the control patients. The BEACON CRC study[60] was a randomized controlled, open-label, multi-cohort phase III clinical trial designed to evaluate the efficacy and safety of the BRAF inhibitor encorafenib + cetuximab ± the MEK inhibitor binimetinib vs FOLFIRI/IRI + cetuximab in patients with BRAF V600E-mutated mCRC who had been treated with one or two previous regimens for BRAF V600E-mutated mCRC patients. Before initiating the randomized trial, the BEACON CRC study was designed as a safety pilot trial, enrolling 30 patients with BRAF V600E-mutant mCRC to the triple regimen. The primary endpoint was safety, including the incidence of dose-limiting toxic reactions, and the randomized phase of the trial was initiated after reconfirming that safety was manageable. A total of 665 patients were randomly assigned to receive the triple, doublet, or control arm on a 1:1:1 basis. The primary endpoint was OS and was independently reviewed to compare the ORR of the triple vs control group. The secondary endpoint was OS of the doublet vs control group. ASCO 2020 published updated data from the BEACON CRC study with a mOS of 9.3 mo in the triple combination arm and 5.9 mo in the control arm (HR = 0.60, 95%CI: 0.47-0.75). The mOS was 9.3 mo in the doublet arm (HR = 0.61, 95%CI 0.48-0.77). The confirmed ORR (cORR) was 26.8% in the triple combination arm, 19.5% in the doublet arm, and 1.8% in the control arm. AEs conformed with previous primary analyses, with rates of grade ≥ 3 AEs of 65.8%, 57.4%, and 64.2% in the triple, doublet, and control groups, respectively. Based on the results of this trial, the doublet regimen of encorafenib + cetuximab, which was approved by the FDA on August 4, 2020, is considered to be the new standard of care for previously treated patients with BRAF V600E-mutated mCRC. The results of the ANCHOR-CRC study, a single-arm phase II clinical trial of an encorafenib triple regimen, were presented at the WCGI-ESMO Congress 2021 and updated in 2023[61]. The ANCHOR CRC study is the first prospective study of a BRAF inhibitor-based therapy for the first-line treatment of patients with mCRC with the BRAF V600E mutation. The study included 95 mCRC patients with BRAF V600E mutations. The primary endpoint was the cORR based on the local research institute; secondary endpoints included DCR, PFS, OS, and safety. The study met the primary endpoint with a cORR of 47.8% and DCR of 88%. The mPFS was 5.8 mo and the mOS was 17.2 mo. The majority (69.5%) of patients experienced grade 3 or higher AEs and no new safety signals were observed. A prospective study (JapicCTI-205146) of an encorafenib triple combination regimen was presented at ASCO GI 2022, reporting the safety and efficacy of the triple combination as an expanded access program in Japan following the BEACON CRC study. Of the 76 patients with target lesions, 21 were in remission with an ORR of 27.6% and DCR of 82.9%. The ORR was consistent regardless of ECOG performance status, number of prior chemotherapy lines, and number of metastatic lesions. No new safety signals were observed[62]. These results support the triple combination regimen as a new standard treatment option for second- or third-line treatment of mCRC patients with BRAF V600E mutation. Reactivation of EGFR signaling with BRAF inhibition and uninhibited PI3K signaling may explain the limited efficacy of BRAF inhibitor monotherapy in patients with BRAF-mutant mCRC. In 2016, JCO published a phase Ib/II study exploring the efficacy and safety of encorafenib + cetuximab ± the PI3K inhibitor alpelisib in the treatment of patients with BRAF-mutated mCRC who had received at least one prior therapy[63]. A total of 102 patients were enrolled in the study to undergo randomization (triple group, n = 52; dual group, n = 50), with a median number of prior treatments in each group of 2. The primary endpoint was PFS, and the secondary endpoints included ORR and OS. At the time of 73 events, PFS associated with triple vs dual therapy showed an HR of 0.69 (95%CI: 0.43-1.11, P = 0.064), with a mPFS of 5.4 mo and 4.2 mo, and ORR of 27% and 22%, respectively. At the time of 35 events, OS associated with triple vs dual therapy showed an HR of 1.21 (95%CI: 0.61-2.39), with a mOS of 15.2 mo for triple therapy, which was not achieved for dual therapy. Grade 3/4 AEs (without regard to causality) were reported in 79% (triple therapy) and 58% (dual therapy) of patients, respectively. Grade 3/4 AEs that occurred in > 10% of patients in either group were anemia (17% vs 6%), hyperglycemia (13% vs 2%), and elevated lipase (8% vs 18%). Although combination therapy showed some clinical activity, the addition of alpelisib to the dual therapy may add more toxicity.

Existing studies have shown that the BRAF V600E mutation and MSI-H/deficient mismatch repair (dMMR) status coexist and are intertwined. Synergistic effects exist between BRAF-targeted therapies and immune checkpoint inhibitors[64,65]. An early study evaluated the efficacy of the combination of the programmed cell death protein 1 (PD-1) inhibitor spartalizumab (PDR001) + dabrafenib + trametinib in 21 patients with BRAF V600E-mutant mCRC (4 dMMR and 17 microsatellite-stabilized), the majority of whom did not receive previous BRAF inhibition or immunotherapy. Combined therapy produced an ORR of 35% and DCR of 75%[66]. Based on these results, a phase II study of spartalizumab + dabrafenib + trametinib is ongoing, which enrolls patients with BRAF V600E-mutated mCRC that were treatment-naive or pretreated (including prior chemotherapy, anti-EGFR therapy, BRAF or MEK inhibitors, and immunotherapy) (NCT03668431). Additionally, a phase I/II study evaluating the combination of encorafenib + cetuximab + nivolumab in patients with microsatellite-stabilized BRAF V600E-mutated mCRC who have accepted prior first- or second-line systemic therapy, excluding BRAF, MEK, ERK, or PD-1, is underway (NCT04017650).

Overall, BRAF-mutant mCRC is an extremely complex subpopulation of CRC that requires continued in-depth study. Over the past decade, there have been some breakthroughs in understanding and targeting the BRAF-mutated CRC subpopulation, but the optimal first-line therapeutic strategy for patients with BRAF-mutated mCRC remains to be determined. Future therapeutic choices will be highly dependent on further clarifying the mechanisms of primary and acquired drug resistance to targeted therapies in this disease. With deeper biological and translational medicine research on this disease and more and more clinical trials, it is believed that the urgent clinical needs of BRAF-mutated mCRC patients can be better met.

HER-2-AMPLIFIED MCRC

HER-2 is a transmembrane tyrosine kinase receptor protein in the EGFR family. HER-2 overexpression, mostly caused by gene amplification, leads to tumor cell proliferation, aggression, and metastasis[67]. HER-2-targeted therapy is a well-established therapeutic strategy. It was first applied to 20% of breast cancer patients and 10% to 20% of gastric cancer patients with HER-2 amplification. HER-2 amplification occurs in 5% of RAS/BRAF WT mCRC patients; however, it also has been reported in patients with KRAS mutations[68]. HER-2 amplification or overexpression usually occurs in male CRC patients and those with left colon involvement and concurrent lung and/or brain metastases[69]. As mentioned previously, anti-EGFR monoclonal antibodies in combination with chemotherapy are the first-line treatment modality for RAS/RAF WT mCRC; however, various genetic alterations along the intracellular pathways activated by EGFR or other tyrosine kinase receptors impede the efficacy of anti-EGFR monoclonal antibodies[70], with HER-2 amplification or overexpression being one of them. In cetuximab-resistant CRC patients, the occurrence of HER-2 gene amplification increased to 13.6%. In a subgroup of heterozygous patients with cetuximab-refractory KAS/NRAS/BRAF/PIK 3CA WT CRC, the frequency increased to 36%. This suggests that HER2 amplification may be a key driver of anti-EGFR resistance in CRC and that anti-HER2 therapy may be a choice for selected patients[71].

Dual-targeted therapy with anti-HER2 monoclonal antibody

In preclinical models of CRC, monotherapy with HER2 tyrosine kinase inhibitors (TKI) or anti-HER2 antibodies was usually ineffective; however, the combination of an antibody (pertuzumab or trastuzumab) and a TKI (lapatinib) resulted in continuing tumor shrinkage[72].

The HERACLES[73] (HER2 Amplification for CRC Enhanced Stratification) trial aimed to assess the activity of trastuzumab and lapatinib in patients with HER2-positive, KRAS WT mCRC, after the failure of standard therapies. In this study, 27 cases were screened for eventual enrollment in the trial. Objective remission was achieved in 8 (30%) of these patients, with 1 patient (4%) achieving complete remission and 7 (26%) achieving partial remission; 12 (44%) patients had SD. Grade 3 AEs occurred in 6 (22%) of the 27 patients, including fatigue, rash, and elevated bilirubin. No grade 4 or 5 AEs were reported. There were no drug-related SAEs. Based on this result, the findings were followed by the HERACLES-B trial. HERACLES[74], a phase II, single-arm study in which patients with HER2 + RAS/BRAF WT mCRC who had failed normal therapy were given pertuzumab (840 mg intravenously loaded, followed by 420 mg intravenously every 3 wk) and T-DM 1 (3.6 mg/kg every 3 wk) until disease progression or toxicity was intolerable. The primary endpoint was ORR, and the secondary end point was PFS. A total of 311 patients (48% previously treated with ≥ 4 lines of therapy) were treated and assessable. The ORR was 9.7% and the SD was 67.7%. The mPFS was 4.1 mo. This study did not meet the primary endpoint; yet, based on high disease control, PFS similar to that of other anti-HER2 therapeutics, and low side-effects, pertuzumab plus T-DM1 could be considered as a potential regimen for HER2 + mCRC.

MyPathway[75] is a phase IIa multiple-basket study that evaluated the efficacy of targeted therapies in pan-tumors with potentially predictive molecular variations. Fifty-seven patients with treatment-refractory HER2-amplified mCRC were treated with pertuzumab and trastuzumab. The primary endpoint was the ORR. Of the 57 evaluable patients, 1 had a complete response, and 17 had PR, with an ORR of 32%. Grade 3 AEs were observed in 37% (21/57) of patients, most frequently hypokalemia and bellyache (n = 3 each). About 18% (10/57) of patients reported serious post-treatment emergent AEs. There were no treatment-related deaths. However, subgroup analysis showed that those mCRC patients with HER2 amplification and RAS mutations had a PFS of 1.4 mo and OS of 8.5 mo. It was suggested that patients with RAS mutations do not benefit from anti-HER2 therapy. Narita et al[76] compared OS in patients with treatment-refractory, HER2–amplified mCRC receiving pertuzumab combined with trastuzumab (PER-HER) in the phase IIa MyPathway multi-basket study with OS in those receiving routine clinical care in an electronic health record–derived external control arm. Fifty-seven patients were recruited in the PER-HER arm and 18 patients in the external control arm. The approximated HR for OS from the multivariate Cox proportional hazards model in the post-weighting population was 0.729. The results of sensitivity analyses were in accordance with the primary analysis as to the point estimate of HR. Although the sample size is relatively small, these discoveries suggest that PER-HER may have potential OS benefits for this population. The TRIUMPH study[77] is a phase II clinical study evaluating the efficacy of pertuzumab plus trastuzumab for the treatment of mCRC with confirmed HER2 amplification by tumor tissue or ctDNA analysis. The study met the primary endpoint with a cORR of 30% in tissue-positive patients and 28% in ctDNA-positive patients. Exploratory analyses were performed to show that baseline ctDNA genotyping (HER2 copy number) and combined oncogenic alterations (adjusted for tumor fraction), which stratify patients according to efficacy, were similar in accuracy to tissue genotyping. Decreased ctDNA scores at 3 wk after treatment initiation were associated with treatment response. Pertuzumab plus trastuzumab showed similar efficacy in mCRC patients with HER2 amplification in tissue or ctDNA, suggesting that ctDNA genotyping can identify patients who benefit from dual HER2 blockade and monitor treatment response. These findings warrant further use of ctDNA genotyping in clinical trials in HER2-amplified mCRC, which may be of particular benefit to patients on first-line therapy.

The TAPUR Study[78] is a pragmatic phase II basket trial evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancers harboring potentially actionable genomic alterations. Thirty-eight CRC patients with HER2 amplification (n = 28) or HER2 mutation (n = 10) were treated with trastuzumab + pertuzumab. In the HER2 amplification group, the DCR and ORR were 54% and 25%, respectively; the mPFS and mOS were 17.2 wk and 60.0 wk, respectively. In the HER2 mutation group, the DCR and ORR were 10% and 0%, respectively; the mPFS and mOS were 9.6 wk and 28.8 wk. No antitumor activity was observed with the combination of trastuzumab + pertuzumab in the HER2 mutation group, suggesting that the HER2-mutant population may not benefit from anti-HER2 therapy.

Small molecule TKIs

Pyrotinib, an irreversible pan-HER TKI targeting HER2, EGFR, and HER4, was approved in China in 2018 for the treatment of HER2-positive recurrent or metastatic breast cancer and has shown anti-tumor effects in other solid tumors such as lung and gastric cancers[79]. Li et al[80] found that pyrotinib monotherapy could be effectively used for salvage therapy in HER2-amplified mCRC patients who have developed resistance to trastuzumab and lapatinib (Tykerb), based on which Chang et al[81] conducted the HER2-FUSCC-G study. HER2-FUSCC-G is a single-center open-label, non-randomized, ongoing, phase II study with three cohorts to evaluate the efficacy of pyrotinib in patients with HER2-positive gastrointestinal cancer refractory to standard treatment. The cohort included 16 patients with mCRC, including 14 patients with RAS WT status. The ORR was 50.0% for the overall population and 57.1% for RAS WT patients. The median follow-up was 11.2 mo, the mPFS was 7.53 mo, and the OS was 16.8 mo. Survival was prolonged in RAS/BRAF WT patients compared with RAS/BRAF-mutant patients. The most common TEAE was diarrhea. Grade 3 TEAEs were reported in five (31.3%) patients; no deaths were reported. This result shows encouraging antitumor activity that translates into prolonged survival benefits in refractory, HER2-positive, RAS WT mCRC patients with acceptable tolerability.

Tucatinib is an oral, reversible HER2-targeted TKI that selectively inhibits HER2 enzyme activity, and in HER2-amplified CRC, tucatinib has shown considerable anti-tumor efficacy in patient-derived xenograft models. When tucatinib was combined with trastuzumab, its antitumor effect was enhanced[82]. The combination of tucatinib and trastuzumab was further evaluated for its efficacy in HER2-positive mCRC in the phase II MOUNTAINEER trial (NCT03043313)[83]. After a median follow-up of 20.7 mo, 86 HER2-amplified RAS WT mCRC patients treated with the combination therapy achieved an ORR of 38.1% and DCR of 71.4%. Remission lasted longer than one year (12.4 mo), with a mOS of 24 mo, while the cohort of patients receiving tucatinib monotherapy had a response rate of 3.3% at 12 wk and DCR of 80.0%. Therefore, tucatinib in combination with trastuzumab was approved by the FDA for second-line treatment of patients with unresectable, HER2-positive, RAS WT mCRC. Another study evaluating tucatinib/trastuzumab combination therapy is ongoing. MOUNTAINEER-03 (NCT05253651) focuses on exploring the use of trastuzumab in combination with tucatinib for first-line treatment, as well as comparing trastuzumab + tucatinib + standard chemotherapy vs chemotherapy + biologic agent of choice. The phase III MOUNTAINEER-03 trial is expected to be completed in 2028. There are also BDTX-189[84,85], which targets extracellular, transmembrane, and structural domain variant mutations of HER2 and EGFR, and neratinib[86,87], which targets HER2, EGFR, and HER4, and both of them showed some antitumor activity in several tumor types containing HER2 mutations and need to be confirmed by further studies.

Antibody-drug conjugate

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate that is composed of a humanized anti-HER2 monoclonal antibody linked to a topoisomerase inhibitor payload, deruxtecan, through a tetrapeptide-based cleavable linker. The linker is disconnected after internalization by lysosomal enzymes that are upregulated in tumor cells, letting the release of the cytotoxic payload, an exatecan ramification. Since it is penetrable to the cell membrane, a bystander effect on cells near HER2-expressing tumor cells is also achieved. T-DXd has already been approved for the treatment of patients with metastatic HER2-positive breast cancer and gastric cancer in several countries[88,89]. In the open-label, phase II DESTINY-CRC01 trial (NCT03384940)[90], researchers evaluated T-DXd in patients with HER2-amplified mCRC who had received two lines or more prior treatment regimens. Participants received T-DXd 6.4 mg/kg intravenously every 3 mo. After a median follow-up of 27.1 wk, 45.3% of patients with HER2-positive tumors had a confirmed objective remission, and long-term follow-up showed that the median duration of response in the trial group was 7.0 mo, mPFS was 6.9 mo, and mOS was 15.5 mo. However, 3.9% of grade ≥ 3 interstitial pneumonias were reported. On this basis, the investigators designed the DESTINY-CRC02 study[91], which compared the efficacy and safety of 5.4 mg/kg and 6.4 mg/kg T-DXd treatment, and expanded the enrollment criteria based on the CRC01 study to include HER2-overexpressing patients with RAS mutation type. The results of the study suggested that the ORR of the 5.4 mg/kg treatment group was 37.8%, which was significantly higher than the 27.5% of the 6.4 mg/kg treatment group. There was no significant difference in PFS or OS between the two groups. And shifting our attention to the safety concern, among 39 patients in the 6.4 mg/kg treatment group, 1 died due to grade 5 interstitial pneumonitis; among 80 patients in the 5.4 mg/kg treatment group, 7 developed grade 1-2 interstitial pneumonitis with no associated death. This suggests that the safety of the 5.4 mg/kg treatment group has been significantly improved, truly realizing the reduction of dosage and toxicity without reducing the effectiveness. Moreover, subgroup analysis suggests that trastuzumab also has better efficacy in patients with RAS-mutant phenotypes, which is an achievement that previous anti-HER2 treatments have failed to achieve, and this is one of the important breakthroughs. This study is an important one that could change CRC guidelines and clinical practice, and we expect that more excellent treatments will be available for CRC patients.

The drug treatments for CRC mainly include chemotherapy, targeted therapy, and immunotherapy. The application of chemotherapy in CRC, like other tumors, has reached a bottleneck, with an effective rate of 30%-40% for first-line treatment and a survival period between 1-2 years. Immunotherapy has developed rapidly in several tumor types, and it has become an indication for advanced first-line treatment in several tumors, even the adjuvant and neoadjuvant treatments have achieved ideal efficacy. However, in the treatment of CRC, the development has been slow, the approved indications are mainly in about 5% of microsatellite-unstable CRCs, 95% of the patients with CRC are microsatellite-stable, and the main mode of treatment at present is still targeted therapy in combination with chemotherapy. At the beginning of the 21st century, with the application of bevacizumab in mCRC, a new chapter of targeted therapy for CRC was opened. With the development of the concept of precision diagnosis and precision treatment, and the wide application of second-generation sequencing technology, drugs targeting the MAPK pathway have been researched, developed, and applied to the treatment of mCRC, including anti-EGFR monoclonal antibodies, KRAS G12C inhibitors, BRAF inhibitors, anti-HER2 therapy, and so on.

Anti-EGFR monoclonal antibodies applied to RAS/RAF WT mCRC prolonged the survival of such patients by 3-6 mo and became the standard of care for RAS/RAF WT mCRC[4,6,7,21], and further subgroup analyses showed that the benefit was even more pronounced in RAS/RAF WT left colon cancer, and that mutations in either the EGFR pathway and bypass pathway were able to influence the efficacy of anti-EGFR therapy. However, less than half of mCRC patients are WT[48], and about 40% of patients have RAS mutations, including KRAS G12C, KRAS G12D, KARS G12S, etc. Many scholars have been committed to the research and development of drugs targeting the RAS mutations, but have not been able to achieve success, mainly due to the following reasons: (1) The protein molecules are relatively small and the surfaces are smooth, so it is difficult to find the "pockets" where small molecules can be combined with drugs; and (2) since KRAS has a strong affinity for the nucleotide guanosine triphosphate (GTP), it is difficult for drugs to compete with GTP, bind the KRAS protein, and inhibit its activity[92]. In recent years, with the further development of genomics and genetic testing technology, researchers have found that the KRAS G12C mutation is not permanently in the activated state of binding to GTP, but will still rapidly switch between the activated state of binding to GTP, and the inactivated state of binding to GDP. although this "window period" is very short, it still provides an opportunity for covalent inhibitors that bind only to the inactivated KRAS G12C mutant. Therefore, KRAS G12C inhibitors have been developed[43,44]. Translational research targeting KRAS G12D and KARS G12S is currently underway, and whether they can be introduced to the clinic is worthy of anticipation. The rechallenge therapy with anti-EGFR monoclonal antibodies, maintenance therapy, and the concept of NoeRAS in recent years have provided new options for the follow-up treatment of mCRC patients (Table 1).

The incidence of BRAF V600E mutation is about 10%-15% in global CRC patients. mCRC patients with BRAF mutation have a poor prognosis and low response rate to conventional chemotherapy[93,94]. Thus, the treatment progress is highly emphasized. In recent years, mCRC with BRAF V600E mutation has been subjected to dual- or triple-targeted inhibition of the EGFR/RAS/BRAF/MEK signaling pathway. Unlike other tumors, BRAF inhibitors have poor efficacy in monotherapy for BRAF V600E-mutated advanced CRC, so explorations have focused on the entire signaling pathway, including antibody-based closure of the extracellular receptor, as well as multi-targeted inhibition of the RAS/BRAF signaling pathway to overcome feedback activation and thus improve efficacy[58-60]. However, the indications are still mainly limited to patients who failed standard treatment, and whether it can be advanced to first-line therapy is yet to be answered by large phase III clinical studies like the BREAKWATER trial, the results of which are worth waiting for. In addition, there is another population of interest, i.e., dMMR/MSI-H patients with BRAF V600E mutation. From the existing KEYNOTE and CheckMate series of studies, it can be seen that this type of patient receives immunotherapy with good efficacy, so dMMR/MSI-H mCRC patients with BRAF V600E mutation are recommended for preferred immunotherapy.

HER2 is another interesting target of CRC, and HER2 overexpression or mutations, occurring in about 5% or so of CRCs, may be a predictor of poor efficacy of EGFR monotherapy. The best data so far comes from the MOUNTAINEER study of trastuzumab in combination with tucatinib[83], with an ORR of 38.1%, mPFS of 8.2 mo, and mOS of 24.1 mo. Therefore, a multi-agent combination therapy strategy should be used for HER2 targets as well. Last year, the results of an additional analysis of the MOUNTAINEER study were reported in ESMO. The majority of patients treated with tucatinib monotherapy showed sustained antitumor effects, and the therapy was well-tolerated overall. However, the explored efficacy of pertuzumab in combination with other combinations such as T-DM1 was limited. The star anti-HER2 drug that is currently receiving particular attention is DS-8201 (T-DXd). Results from the DESTINY-CRC01 study[90] showed that IHC 3+ or IHC2+/ISH+ patients who have failed standard treatment treated with DS-8201 achieved an ORR of 45.3%, while the ORR for patients with low HER2 expression was 0. The mPFS for IHC 3+ patients was 6.9 mo, and the mOS was 15.5 mo, so DS-8201 treatment of HER2-positive mCRC patients is worth looking forward to. In addition, pyrotinib has been explored in CRC[81]. The retrospective study showed that the ORR of a single agent was 33%, the DCR of combined trastuzumab was 45%, and the efficacy needs to be further improved.

CONCLUSION

To summarize, although the research and development process of target drugs for precision treatment of CRC is not as smooth as that of lung cancer, it is gratifying to see that CRC has gradually entered the era of precision treatment or differentiated treatment in the past two years. It is also expected that under the application of new drug research and the development of ctNDA technology, CRC patients can be brought with a more precise therapeutic strategy and more therapeutic efficacy.