Anti-EGFR treatment resistance in patients with wild-type RAS metastatic colorectal cancer: Three case reports

Abstract

BACKGROUND

Anti-EGFR therapy markedly improves outcomes in advanced colorectal cancer (CRC) patients. Nonetheless, resistance to this treatment is commonly observed even in patients with wild-type RAS tumors.

CASE SUMMARY

In this study, three patients with wild-type RAS metastatic CRC with anti-EGFR resistance were comprehensively assessed for genetic alterations (KRAS, NRAS, BRAF, and PIK3CA mutations; HER2 expression) in primary and metastatic lesions. Among the subjects, two exhibited acquired resistance to anti-EGFR therapy following an initial treatment response. One of these patients developed a KRAS mutation in metastatic lesions, while the other had HER2 overexpression in the primary tumor. The third patient demonstrated primary resistance, attributed to a preexisting KRAS mutation in a metastatic lesion that was not present in the primary tumor.

CONCLUSION

These cases underscore a critical need to understand the diverse mechanisms of resistance to anti-EGFR treatment in patients with wild-type RAS metastatic CRC. Despite the limited sample size, comprehensive genetic profiling of both primary and, when feasible, metastatic lesions is recommended prior to therapy initiation. Furthermore, HER2-targeted therapy could be beneficial for patients with HER2 amplification/overexpression. A multidisciplinary approach and ongoing molecular monitoring remain vital in the management of advanced CRC.

Key Words: Anti-EGFR therapy; Colorectal cancer; Molecular mechanisms; Heterogeneity; Case report

Core Tip: In this study, 3 wild-type RAS metastatic colorectal cancer patients with anti-EGFR resistance were analyzed. Key findings: Acquired resistance is associated with KRAS mutation/HER-2 amplification and primary resistance is associated with metastatic KRAS mutation (tumor heterogeneity). These findings suggest essential genetic profiling of primary/metastatic lesions (pyrosequencing for KRAS/NRAS/BRAF/PIK3CA and IHC+FISH for HER-2) and HER-2-targeted therapy for overexpression.

INTRODUCTION

Anti-EGFR therapy has emerged as a significant advancement in the treatment of advanced colorectal cancer (CRC), leading to improved patient outcomes[1]. Currently, its use is limited to patients with wild-type RAS tumors[2]. However, a substantial proportion of patients with wild-type RAS still develop resistance during anti-EGFR treatment[3]. This resistance can be either primary (innate) or acquired. Through the analysis of three clinical cases, this study aims to enhance our understanding of the underlying mechanisms driving anti-EGFR treatment resistance in patients with wild-type RAS metastatic CRC.

CASE PRESENTATION

Chief complaints

Case 1: A 58-year-old male with persistent abdominal discomfort, diagnosed with left-sided colon cancer and multiple liver metastases.

Case 2: A 48-year-old male with no obvious symptoms, diagnosed with right-sided colon cancer and multiple liver metastases during routine medical examination.

Case 3: A 68-year-old male with intermittent abdominal pain, diagnosed with rectal cancer and synchronous liver metastasis 11 months after radical resection of sigmoid colon cancer.

History of present illness

Case 1: In April 2020, abdominal ultrasound revealed multiple liver metastases, and subsequent colonoscopy confirmed left-sided colon cancer. Pyrosequencing showed wild-type RAS status. The patient received cetuximab combined with mFOLFOX6 chemotherapy. Initial response was observed, with decreased tumor markers (CA199, CA242) and reduced size of liver metastases. However, around the 17^th cycle, tumor markers increased and liver metastatic lesions enlarged, indicating disease progression. The clinical timeline for case 1 is presented in Figure 1A.

Figure 1 Clinical and biomarker dynamics correlating with genomic profiles in anti-EGFR treated colorectal cancer cases. A: Case 1; B: Case 2; C: Case 3.

Case 2: In March 2021, the diagnosis of right-sided colon cancer with multiple liver metastases was confirmed by colonoscopic biopsy and computed tomography (CT). Pyrosequencing of the primary tumor tissue confirmed wild-type RAS status. He was treated with cetuximab combined with FOLFIRI chemotherapy. Initial response was noted, with decreased CEA, CA199, and CA242 levels, and reduced metastatic lesion size. After 7-8 cycles, tumor markers rebounded and lesions enlarged, suggesting acquired resistance. By the 15^th cycle, primary tumor resection was performed. The clinical timeline for case 2 is presented in Figure 1B.

Case 3: Eleven months prior to admission, the patient underwent “radical resection of sigmoid colon cancer” at a local hospital, with pyrosequencing confirming wild-type RAS status of the primary lesion. However, due to its large size and the associated high surgical risk, hepatic metastasis was not resected during the initial surgery. Postoperatively, he received cetuximab plus FOLFOX chemotherapy. Following this, serum tumor markers (CEA, CA199, CA242) remained persistently elevated. He was then admitted to our hospital for further management. During the first two cycles, tumor markers (CEA, CA199, CA242) and lesion sizes fluctuated but remained stable (stable disease). By the fifth cycle, imaging showed no significant reduction in metastatic lesions, indicating primary resistance. The clinical timeline for case 3 is presented in Figure 1C.

History of past illness

Case 1: Had a history of hypertension but was not on regular medication; no history of diabetes or other chronic diseases.

Case 2: No history of chronic diseases or surgical procedures before diagnosis.

Case 3: No significant past medical history or chronic disease.

Personal and family history

The three patients reported no family history of malignancy or liver disease or other significant comorbidities. Case 1 and case 3 had a smoking history, with specific details unknown.

Physical examination

All three patients had normal vital signs. Abdominal examination showed no tenderness, rebound tenderness, or masses. Liver and spleen were not palpable. No abnormalities were found in cardiopulmonary, neurological, or other system examinations.

Laboratory examinations

Case 1: CEA (ng/mL) was initially elevated at 113.26, CA199 (U/mL) at 43.10, and CA242 (U/mL) at 32.16. These tumor markers showed a progressive decrease as the chemotherapy cycles proceeded but began to increase to varying degrees around the 17th cycle of chemotherapy (Figure 1A).

Case 2: The patient’s tumor markers were initially elevated (CEA 105.34 ng/mL, CA199 299.14 U/mL, CA242 153.45 U/mL). They showed an initial decline with chemotherapy but began to rise again from approximately the 7-8 cycle onward (Figure 1B).

Case 3: Despite initial elevations (CEA 46.62 ng/mL, CA199 508.19 U/mL, CA242 > 200.00 U/mL), these tumors markers remained persistently elevated without a clear downward trend across five cycles of chemotherapy (before and after admission) (Figure 1C).

Imaging examinations

Case 1: Before treatment, CT imaging revealed a liver metastasis with a maximum diameter of 39 mm. During the initial phase of chemotherapy, the lesion demonstrated a significant reduction in size. Upon the development of acquired resistance, the maximum diameter of the lesion stabilized at 32 mm (Figure 2).

Figure 2  Computed tomography images of tumor size changes in three cases across different stages.

Case 2: Prior to therapy, the dominant liver metastasis measured 48 mm in maximum diameter. It showed a marked decrease in size during active treatment. However, following the emergence of treatment resistance, the maximum diameter increased to 46 mm, indicating radiographic progression (Figure 2).

Case 3: At diagnosis, a large liver metastasis with a maximum diameter of 68 mm was documented. Throughout the course of chemotherapy, serial imaging showed minimal change in the lesion size. Prior to surgical resection, the final maximum diameter was 70 mm, consistent with stable disease by imaging criteria (Figure 2).

FINAL DIAGNOSIS

Case 1

Wild-type RAS metastatic left-sided colon cancer (liver metastases), anti-EGFR therapy acquired resistance (KRAS exon 2 mutation in metastatic lesions).

Case 2

Wild-type RAS metastatic right-sided colon cancer (liver metastases), anti-EGFR therapy acquired resistance (HER2 overexpression/amplification in primary tumor).

Case 3

Wild-type RAS metastatic rectal cancer (synchronous liver metastasis), anti-EGFR therapy primary resistance (KRAS exon 2 mutation in metastatic lesions, tumor heterogeneity).

TREATMENT

Case 1

A 58-year-old male was diagnosed with leftsided colon cancer and multiple unresectable synchronous liver metastases. A colonoscopic biopsy confirmed wildtype RAS status. Initial treatment was cetuximab plus mFOLFOX6, with regular CEA and imaging surveillance for potential resectability. During the 4^th cycle, he underwent palliative resection of the primary tumor. Pathology showed a pT3N0, welldifferentiated adenocarcinoma. Radiofrequency ablation of liver lesions was performed during the 7^th and 23^rd cycles. After the 12^th cycle of cetuximab plus mFOLFOX6, treatment was modified to two cycles of cetuximab monotherapy followed by one cycle of cetuximab plus FOLFIRI. Following 15 cycles of targeted therapy, rising CEA and imaging progression of the liver metastases prompted an multidisciplinary team (MDT) discussion. Subsequently, during the 17^th cycle, he underwent hepatic resection (segments II/III and V/VI/VII). Pathology of the resected liver confirmed metastatic, moderately differentiated adenocarcinoma. Genetic testing of this metastasis revealed an acquired KRAS exon 2 (codon 12) mutation (Figure 3 and Table 1).

Figure 3 KRAS exon 2 gene phenotypes. A: KRAS exon 2 wild-type; B: KRAS exon 2.

Table 1 Clinical characteristics, anti-EGFR therapy responses, and interventions in 3 cases of metastatic colorectal cancer.

	Case 1	Case 2	Case 3
Clinical baseline
Age	58	48	68
Gender	Male	Male	Male
Primary tumor location	Left-sided colon	Right-sided colon	Rectal
Metastatic site	Liver	Liver	Liver
Anti-EGFR therapy regimen	Cetuximab + mFOLFOX6	Cetuximab + FOLFIRI	Cetuximab + FOLFOX
Cycle of resistance onset	17	7-8	1-2
Summary of resistance mechanism	Acquired resistance	Acquired resistance	Primary resistance
KRAS/NRAS/BRAF/PIK3CA status	KRAS exon 2 mt	-	KRAS exon 2 mt
HER2 expression/amplification	-	+++	-
Clinical interventions	Palliative resection of the primary lesion, followed by resection of the hepatic metastatic lesion 12 months later	Palliative primary tumor resection was performed to resolve intestinal obstruction	Radical resection of sigmoid colon cancer, followed by resection of the hepatic metastatic lesion 14 months later

mt: Mutation.

Case 2

A 48-year-old male with right-sided colon cancer and unresectable synchronous liver metastases, confirmed to have wild-type RAS. Initial treatment comprised cetuximab plus mFOLFOX6, with regular monitoring of CEA and imaging for resectability reassessment. After 7-8 cycles, CEA rose significantly, imaging showed progression of both primary and metastatic lesions, and the patient developed incomplete bowel obstruction. Following MDT review, liver resection was not considered feasible. The patient subsequently underwent palliative right hemicolectomy at 15^th cycles. Immunohistochemical (IHC) analysis of the primary tumor revealed HER2 protein overexpression, scored as 3⁺ (Figure 4). This IHC-positive result was further subjected to genetic testing via fluorescence in situ hybridization, which confirmed HER2 gene amplification (Table 1).

Figure 4 HER-2 immunohistochemistry expression status. A: HER-2 (^-); B: HER-2 (3⁺).

Case 3

A 68-year-old male underwent a radical sigmoidectomy eleven months prior. Pathology confirmed a welldifferentiated adenocarcinoma (pT2, N0, margins negative). Due to the high surgical risk, resection of the synchronous liver metastasis was initially deferred. With a wildtype RAS primary tumor, he began cetuximab plus mFOLFOX6. Over four cycles, monitoring showed no clear decline in CEA and no significant reduction in the liver metastasis on imaging. Following an MDT discussion, he proceeded to hepatic metastasectomy. The liver lesion pathology revealed a moderately differentiated adenocarcinoma, consistent with metastatic disease. Subsequent genetic testing of this metastasis identified a KRAS exon 2 (codon 12) mutation (Table 1).

OUTCOME AND FOLLOW-UP

All three patients underwent their respective surgical procedures without immediate complications and returned for scheduled follow-up evaluations two months postoperatively.

DISCUSSION

Despite the established efficacy of anti-EGFR therapy in patients with RAS wild-type metastatic CRC, resistance remains a major clinical challenge. Our report delves into the underlying mechanisms by discussing three distinct cases of resistance.

Acquired resistance to anti-EGFR treatment

In this series, two patients (cases 1 and 2) developed acquired resistance after an initial objective response (RECIST v.1.1)[4]. The molecular workup revealed two established mechanisms: A KRAS mutation in the progressing metastatic lesion (case 1) and HER2 amplification in the primary tumor (case 2).

Acquired resistance to anti-EGFR antibodies is driven primarily by molecular alterations that reactivate MAPK signaling. These mechanisms can be broadly categorized into two groups: (1) Mutations or amplifications within the EGFR-RAS-RAF-MEK-ERK pathway: This includes secondary mutations in KRAS, NRAS, or BRAF, which are the most common drivers. Amplification of KRAS or specific mutations in the extracellular domain of EGFR (e.g., S492R) also fall into this category; and (2) Activation of alternative receptor tyrosine kinases (RTKs): The amplification of other RTKs, such as HER2 (ERBB2) or MET, can activate downstream signaling (e.g., RAS-RAF-MEK) independent of EGFR, effectively bypassing therapeutic blockade. The prevalence of these mechanisms, as reported in the literature, is summarized in Table 2[5-12].

Table 2 Summary of literature on acquired resistance to anti-EGFR therapy.

Resistance mechanism	Detection source	Number of cases (%)	Ref.
KRAS gene mutation	Tissue	1/10 (10)	Montagut et al[5]
	Serum	9/24 (37.5)	Diaz et al[6]
	Tissue and Serum	5/11 (45.5)	Misale et al[7]
	Tissue and Serum	3/7 (42.9)	Bardelli et al[8]
	Serum	22/24 (91.7)	Bettegowda et al[9]
	Serum	3/4 (75)	Misale et al[10]
NRAS gene mutation	Serum	9/24 (37.5)	Bettegowda et al[9]
	Serum	2/4 (50)	Misale et al[10]
EGFR gene mutation	Tissue	2/10 (20)	Montagut et al[5]
	Serum	2/24 (8.3)	Bettegowda et al[9]
KRAS gene amplification	Tissue and Serum	1/11 (9.1)	Misale et al[7]
	Serum	4/10 (40)	Mohan et al[11]
HER-2 gene amplification	Tissue	2/2 (100)	Yonesaka et al[12]
MET gene amplification	Tissue and Serum	3/7 (42.9)	Bardelli et al[8]
	Serum	1/10 (10)	Mohan et al[11]

Primary resistance to anti-EGFR therapy-tumor heterogeneity

Case 3 demonstrated primary resistance, with the best response being stable disease (per RECIST 1.1)[4]. Subsequent analysis revealed a KRAS mutation in the liver metastasis site that was not detected in the primary tumor. This case highlights the critical issue of tumor heterogeneity. While guidelines recommend RAS testing[13], and many studies have reported high concordance between primary and metastatic sites[5,14,15], significant discordance does occur[16-18]. Our initial assessment, which was based solely on the primary tumor, missed the resistant clone present in the metastasis. This finding suggests the need to genotype metastatic lesions whenever possible before initiating anti-EGFR therapy, especially in patients with equivocal or poor initial response.

Feasibility of combination anti-HER-2-targeted therapy in HER-2-overexpressing resistant CRC patients

Case 2 identifies HER2 amplification as the likely driver of acquired resistance. This is clinically relevant given the established efficacy of HER2-targeted therapies (e.g., trastuzumab) in HER2-positive breast[19] and gastric cancers[20]. A growing body of evidence suggests that dual HER2 blockade can be effective in patients with HER2-amplified, anti-EGFR-resistant CRC, making HER2 testing a crucial part of the resistance workup and opening avenues for subsequent targeted therapy[21].

Mechanisms of resistance and implications for precision medicine

The management of advanced CRC with targeted therapy aims to control disease, convert unresectable metastases to resectable, and guide surgical strategy. Given the complexity of resistance mechanisms and spatial heterogeneity, a MDT involving medical oncologists, liver surgeons, radiologists, and molecular pathologists is essential. This collaboration enables precise and dynamic assessment of treatment response and resistance patterns[6,9], informing critical decisions regarding therapy switches, local interventions (such as resection or ablation), and molecular reprofiling.

The resistance mechanisms observed in this study are consistent with and complement the literature. KRAS mutation resistance due to tumor heterogeneity in case 3, together with the KRAS copy number amplification case reported by Fang et al[22], collectively demonstrate that comprehensive genomic analysis beyond conventional RAS/BRAF point mutation testing, such as next-generation sequencing, is crucial for accurately identifying resistant populations. Conversely, a report by Xiao and Fakih[23] regarding the benefit in a right-sided colon cancer patient, along with the successful experience of Li et al[24] with combination targeted therapy, resonates with the findings of this study: In advanced late-line treatment, a precise molecular profile, particularly the confirmed absence of resistance mutations, is key to identifying potentially benefiting populations and guiding effective combination treatment strategies.

Limitations

This study has a small sample size (3 patients). Although it illustrates the typical molecular mechanisms of anti-EGFR resistance, the generalizability of the conclusions is limited by the single-center, retrospective design. Future multicenter, large-sample studies are needed for further validation. Additionally, analyses covering more molecular targets, larger sample sizes, or more molecular levels (such as genomics, transcriptomics, and proteomics) can be conducted, incorporating detection methods such as circulating tumor DNA (ctDNA) sequencing. For instance, integrating proteomics, tumor immune microenvironment analysis, and ctDNA sequencing may reveal the core mechanisms of drug resistance at the functional level.

CONCLUSION

In conclusion, despite the preliminary nature of findings derived from a small case series, this research emphasizes KRAS mutations as the predominant mechanism, followed by NRAS and BRAF mutations, EGFR gene mutations, and RTK gene amplification. The identified molecular mechanisms contributing to acquired resistance include mutations or amplifications in EGFR-RAS-RAF-MEK-ERK pathway genes and RTKs.

In terms of primary resistance, this study suggests that tumor heterogeneity is a plausible cause. Comprehensive genetic testing of metastatic lesions is hence vital before starting anti-EGFR therapy. Moreover, HER-2 overexpression presents a potential mechanism for primary resistance, with HER-2-targeted therapy, such as trastuzumab, showing promise in the treatment of CRC.

In summary, this research highlights the critical need to understand anti-EGFR treatment resistance mechanisms in patients with wild-type RAS metastatic CRC. It advocates for thorough genetic profiling and consideration of HER-2-targeted therapy as an effective alternative. The role of multidisciplinary collaboration and regular molecular monitoring is underscored in the effective management of advanced CRC.