El impacto de la mutación de APC en la eficacia de la terapia anti-EGFR en el cáncer colorrectal metastásico con RAS tipo salvaje: un análisis retrospectivo.

Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related deaths.^[1] Approximately 20%–25% of patients with CRC present with metastatic disease at diagnosis,^[2] and 20%–30% of those initially diagnosed with localized disease eventually experienced a recurrence, necessitating palliative systemic therapy.^[3] Cetuximab, an immunoglobulin G1 monoclonal antibody against Epidermal Growth Factor Receptor (EGFR), is one of the targeted agents used in the treatment of metastatic CRC.^[4] Cetuximab binds to EGFR and acts by suppressing cancer cell proliferation, promoting cell apoptosis, and reducing matrix metalloproteinases production.^[5] The clinical efficacy of cetuximab has been established through pivotal clinical trials, supporting its incorporation into first-line treatment regimens in combination with 5-Fluorouracil with oxaliplatin (FOLFOX) or 5-fluorouracil with irinotecan (FOLFIRI), specifically in patients with left-sided colorectal tumors and confirmed RAS wild-type status.^[6][]–^[8]

Extensive efforts have been made to identify patient subgroups that derive maximal benefit from cetuximab. Since its initial clinical application, reduced efficacy has been observed in tumors harboring activating mutations in the RAS pathway and in those originating from the right side of the colon.^[4],^[6],^[8] EGFR functions as a membrane-bound receptor tyrosine kinase, initiating downstream signaling pathways such as the RAS-RAF-MEK-ERK cascade. Activating mutations in RAS, particularly in KRAS or NRAS, lead to constitutive activation of this pathway independent of EGFR, rendering anti-EGFR monoclonal antibodies ineffective.^[9] Similarly, tumors harboring BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations, a downstream effector of the MAPK signaling pathway, also show limited responsiveness to anti-EGFR therapy.^[10] As a result, cetuximab is generally not recommended in these settings, with anti-vascular endothelial growth factor (VEGF) antibodies preferentially combined with 5-fluorouracil (5-FU) based chemotherapy. Beyond these established factors, ongoing research has focused on identifying additional biomarkers predictive of optimal response to anti-EGFR antibody therapy.^[11] However, predictive biomarkers for anti-EGFR therapy remain limited.

Mutations in the APC gene play a pivotal role in the early stages of colorectal tumorigenesis and are detected in approximately 80% of sporadic CRC cases.^[12] Also, germline pathogenic mutations in APC are also responsible for familial adenomatous polyposis (FAP). The Wnt signaling pathway, which is essential for embryonic development, becomes dysregulated in the presence of APC mutations. These mutations typically result in premature truncation of the APC protein, leading to impaired regulation of β-catenin. This dysregulation promotes nuclear accumulation of β-catenin and alters T-cell factor 4 (TCF-4) mediated transcriptional activity, ultimately driving the proliferation of intestinal crypt epithelial cells and contributing to tumorigenesis.^[13] Previous studies in vitro have shown that cetuximab suppresses the Wnt/β-catenin pathway by reducing β-catenin nuclear localization and modulating downstream effectors like Phospholipase C Beta3 (PLCB3). PLCB3 is involved in intracellular phosphoinositide signaling and has been implicated in the regulation of Wnt signaling activity. These findings raise the possibility that APC mutation may potentially influence the response to anti-EGFR therapy.^[14]

Herein, we intended to evaluate the impact of APC mutation on the treatment of cetuximab-containing chemotherapy in CRC patients with RAS wild type.

Methods

Study participants

This retrospective study included patients aged 18 years or older with histologically confirmed metastatic CRC and RAS wild-type status. The study population consisted of patients who received cetuximab plus FOLFIRI or FOLFOX as first-line therapy at Samsung Medical Center from February 2021 to June 2024. A key inclusion criteria was the availability of comprehensive genomic profiling via next-generation Sequencing (NGS) to evaluate APC and other relevant molecular alterations. NGS testing was performed as part of routine clinical practice, and results were used to guide treatment decisions. Patients were excluded if they had insufficient electronic medical record (EMR) data to assess treatment response or survival outcomes or had hereditary polyposis syndrome. Demographic information was extracted from EMR when the first-line treatment was initiated, and patient age, sex, Eastern Cooperative Oncology Group performance status (ECOG PS), tumor location, initial metastasis site and other mutation status of KRAS, BRAF and HER2 IHC were reviewed. The aberration of APC, KRAS, BRAF, tumor mutational burden (TMB) and MSI status were evaluated using an NGS with the TruSight Oncology 500 assay (Illumina, San Diego, CA, USA).

The reporting of this study conforms to the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) statement^[15] (Supplemental Material).### Next-generation sequencing

Tumor samples were obtained at the time of initial diagnosis or progression, and formalin-fixed paraffin-embedded (FFPE) material was used. The Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) was used to quantify the 40 ng of DNA. The Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) and the eight microTUBE–50 Strip AFA Fiber V2 were used for shearing. The treatment time was optimized for FFPE material, and the treatment settings were as follows: peak incident power (W): 75; duty factor: 15%; cycles per burst: 500; treatment time (s): 360; temperature (°C): 7; water level: 6. We used the TruSight™ Oncology 500 Kit (Illumina) for DNA library preparation and enrichment, following the manufacturer’s instructions. The post-enriched libraries were quantified, pooled, and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA). The quality of the NextSeq 500 (Illumina) sequencing runs was assessed with the Illumina sequencing Analysis Viewer (Illumina). TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina) was used to analyze the sequencing data. The TruSight™ Oncology 500 is a comprehensive tumor profiling assay designed to identify various tumor biomarkers, including small variants, splice variants, and fusions.### Statistical analysis

Categorical and continuous variables were summarized using descriptive statistics. Response rate (RR) was calculated as the percentage of patients experiencing a confirmed complete response (CR) or partial response (PR), and disease control rate (DCR) was calculated as RR or stable disease (SD), as per the RECIST 1.1 guidelines. The progression-free survival (PFS) was defined as the time from cetuximab plus chemotherapy to documentation of either disease progression or death. Overall survival was defined as the time from initiation of cetuximab plus chemotherapy to death from any cause. Patients with no event at the data cutoff date were censored on the last date of follow-up. Survival curves were calculated using the Kaplan–Meier method and compared using the log-rank test and Cox proportional hazards regression model. Additionally, subgroup analyses were performed according to the presence of liver metastasis to evaluate potential differences in PFS and overall survival between groups.

All p values were two-sided, and p