Metastatic colorectal cancer (mCRC) remains one of the leading causes of cancer-related mortality worldwide despite substantial therapeutic improvements over the past two decades. Advances in the understanding of colorectal tumor biology and oncogenic signaling have enabled the development of biomarker-guided therapies targeting alterations in EGFR, BRAFV600E, KRAS mutations and HER2 amplifications, improving outcomes in selected patient populations. Nevertheless, the emergence of both intrinsic and acquired resistance mechanisms continues to limit the durability of these responses. Resistance to targeted therapies in mCRC arises through multiple, often convergent mechanisms, including activation of compensatory signaling pathways, pre-existing genomic heterogeneity, and therapy-driven clonal selection. The integration of molecular profiling into clinical decision-making is essential to guide treatment selection and optimize therapeutic sequencing, ultimately enabling progress in precision oncology. Advances in genomic technologies, particularly circulating tumor DNA (ctDNA) analysis, have allowed longitudinal monitoring of tumor evolution, providing important insights into the mechanisms underlying resistance to targeted therapies. The aim of this review is to summarize the genomic landscape of mCRC and discuss current targeted therapeutic strategies in molecularly defined subgroups, with a particular focus on the mechanisms driving primary and acquired resistance.

Colorectal cancer (CRC) with mismatch-repair deficiency (dMMR) or high microsatellite instability (MSI-H) represents a distinct molecular subtype highly sensitive to immune checkpoint inhibitors (ICIs). Landmark clinical trials have established ICIs as standard-of-care in this setting, demonstrating durable responses and improved survival. However, up to one-third of patients will exhibit primary or acquired resistance, highlighting the urgent need for predictive biomarkers and novel therapeutic strategies. This review summarizes the clinical evidence supporting ICIs in dMMR/MSI-H CRC, explores mechanisms of resistance—including intrinsic and extrinsic modulators—and evaluates the role of potential predictive biomarkers of response. Finally, we discuss innovative therapeutic approaches to overcome resistance, including combination strategies, DNA repair pathway inhibitors, immune-oncology drugs beyond checkpoint inhibitors and microbiome-targeted interventions. Together, these insights aim to refine patient selection, optimize therapeutic benefit, and guide the development of next-generation therapies for dMMR/MSI-H CRC.

PURPOSE:We aim to inform the design of new diffusion MRI (dMRI) approaches for microvasculature quantification that enhance the biological specificity of imaging towards cancer. METHODS:We adopted simulation-informed modelling of the vascular dMRI signal. We synthesised signals from 1500 synthetic vascular networks, for a variety of protocols (flow-compensated [FC], non-compensated [NC], hybrid), featuring different b $$ b $$ samplings and diffusion times. We estimated the number of independent, recoverable signal degrees of freedom in presence of noise (signal-to-noise ratio of 5), and ranked 12 microvascular metrics depending on the quality of their estimation. Lastly, we demonstrated the feasibility of estimating the top-ranking metrics on 3T dMRI of a healthy volunteer and of a metastatic colorectal cancer (CRC) patient. RESULTS:Both NC and FC synthetic vascular signals exhibited complex behaviour as, for example, non-zero kurtosis and diffusion time dependence. Two independent degrees of freedom appeared recoverable from directionally-averaged vascular signals (SNR of 5). Mean volumetric flow rate q m $$ {q}_m $$ and an Apparent Network Branching (ANB) index maximised correlations between ground truth and estimated values in silico. In the patient, both q m $$ {q}_m $$ and A N B $$ ANB $$ detected re-vascularisation after 3 months of targeted therapy against liver metastases, consistently with Intra-Voxel Incoherent Motion (IVIM) metrics. CONCLUSIONS:Simulation-based modelling of the vascular dMRI signal suggests q m $$ {q}_m $$ and A N B $$ ANB $$ as the most promising metrics for tissue microvasculature characterisation. Their estimation in vivo appears feasible to capture general trends, and demonstrates contrasts that are biologically plausible, encouraging their usage in future studies.

3607 Background: Immune checkpoint inhibitor (ICI) therapy aims to instruct immune cells to efficiently recognize and eliminate cancer cells. It has shown efficacy in MSI colorectal (CRC), and, although there is no approval yet, a subset of patients with MSS CRC included in clinical trials respond to ICI, indicating a significant knowledge gap in our current understanding of immunological mechanisms in MSS CRCs. The relationship between the cellular and molecular responses to ICI in the tumor microenvironment (TME) and the clinical outcome remains poorly understood. We sought to address this gap in understanding through a series of high-throughput functional genomics analyses. Methods: Patients (pts) with refractory MSS CRC were treated with durvalumab (Dur) 1500mg plus tremelimumab (Trem) 300mg at Week 0, followed by Dur 1500 mg Q4W. We characterized the cellular makeup of the metastatic TME using single-cell RNA-seq, and cataloged T-cells clonotypes within metastases and in the blood using single-cell and deep TCR-seq. We collected data for each patient before and upon treatment initiation to explore the dynamic changes of the TME and blood in response to ICI. To facilitate direct comparisons between metastatic and primary TMEs, we integrated our dataset with an external single-cell RNA-seq dataset from primary CRC tumors. Results: 24 pts were included in the analysis (18 with no liver metastases (NLM), 6 with liver metastases (LM)). Around 200,000 single-cell profiles were collected from biopsies taken from 6 pts with LM and 10 pts with lung metastases. Disease Control Rate (DCR) was 39% for MSS CRC without LM (2 PR + 5 SD) and 0% for those with LM (p=0.13). Robust gene expression changes within the immune compartment of the TME were observed upon treatment, irrespective of overall treatment outcome, yet specific biological programs were differentially altered across patient response groups. Differences in the composition of TMEs in the lung and liver metastases were found with an enrichment of T regulatory and IFN-responder CD4 populations in the lung vs more CD8+ T cell subtypes in the liver. Tumor TCR analysis revealed that, following ICI treatment, liver metastases displayed a high diverse TCR repertoire, whereas lung metastases were less diverse and showed marked clonal expansion, suggesting greater T cell activity within the lung lesions (p=0.035). Conclusions: Prospective deep immune repertoire profiling detected profound differences in TME according to dissemination patterns, which might explain differences in treatment outcomes. Despite low overall efficacy with Dur and Trem in MSS patients, a trend to better response rate and DCR was observed in pts without LM. We propose immune cell tracking as powerful tool to quantify the efficacy of ICI in real time. Clinical trial information: NCT06680739 .

3556 Background: In BRAF wt/ MSS mCRC, the mechanisms driving distinct metastatic dissemination patterns remain unclear. Understanding them is crucial, as dissemination profiles influence therapeutic strategies, such as immunotherapy for patients (pts) without liver metastasis (mets) or locoregional approaches and liver transplantation for those with liver-limited disease. NGS advances provide genomic data, offering opportunities to identify predictive signatures that link molecular profiles to metastatic patterns. This study investigates mutational profiles to uncover correlations with organ-specific mets in pts treated at our institution. Methods: This study included pts with unresectable MSS/ BRAF wt mCRC treated at Vall d’Hebron Hospital (2010–2020). Pts were grouped into three clinical categories based on metastatic patterns: liver-limited disease (LLD), exclusively extrahepatic disease (EXTRAHEP), and hepatic and extrahepatic disease (BOTH). Molecular analyses were performed using NGS prescreening data available at our institution. Mutations were grouped in two approches: (1) by biological significance using cancer hallmark genes from published datasets (Zhang, Front Genet 2020; Sondka, Nat Rev Cancer 2018), and (2) by molecular pathways based on the Sanchez-Vega dataset ( Cell 2018). Statistical analyses were conducted using R version 4.3.2. Results: A total of 1,026 pts were included (204 LLD, 297 EXTRAHEP, and 525 BOTH), with molecular analyses performed on 360 samples (35% overall; 31.8%, 39.7%, and 33.7%, in each group, respectively). The median number of genes with pathogenic mutations per sample differed significantly between groups: 2.28 in LLD, 2.44 in EXTRAHEP, and 2.65 in BOTH (p = 0.01). BOTH showed significantly greater increase than LLD in mutated genes associated with five of the ten analyzed hallmarks: activation of invasion and mets, resistance to cell death, evasion of growth suppressors, sustaining proliferative signaling, and replicative immortality. Compared to EXTRAHEP, BOTH also had more mutations in invasion and mets activation and proliferative signaling (adjusted p-value < 0.05 for all the hallmarks mentioned). For pathway associations, WNT pathway activation was higher in BOTH than EXTRAHEP (p = 0.004), driven by more frequent APC mutations in BOTH (82% vs. 69%, adj. p = 0.049). Conclusions: This study provides evidence that pts with both hepatic and extrahepatic disease exhibit enrichment in five cancer hallmarks and the WNT pathway compared to other metastatic patterns. This suggests the tumor's potential to adapt to diverse microenvironments. Despite the statistical significance, the magnitude of the observed differences in mutated genes is not yet clinically useful. These findings highlight the need for collaborative efforts to develop mutational profiles that predict organotropism and guide therapy.

Background: The development of BRAF inhibitors has reshaped the prognosis of patients with BRAF-mutated, refractory metastatic colorectal cancer (mCRC). The BEACON trial demonstrated the superiority of encorafenib-cetuximab with or without binimetinib over standard chemotherapy. However, not all patients respond, and some responses are short-lived, highlighting the need to develop accurate biomarkers. Emerging evidence suggests that sex may play a role as a biological modifier of treatment responses to anticancer therapies, however, conclusive clinical evidence is lacking. Objective: To evaluate the impact of biological sex on efficacy outcomes in the real-world setting following treatment with encorafenib-cetuximab or encorafenib-cetuximab- binimetinib. Methods: A retrospective analysis of real-world patients treated between 2015 and 2024 at 8 sites in Spain and Italy, with 5 years follow-up was performed. Consecutive adult patients with BRAF V600E mutant mCRC treated with encorafenib-cetuximab +/- binimetinib were included. Overall response rate, progression-free survival and overall survival were analyzed based on treatment type (doublet -without binimetinib- vs triplet -including binimetinib-) and sex (male vs female). Results: Of 264 patients included, the median age at baseline was 64 years [IQR 26-91], and 153 (58%) were female. In terms of OS, males receiving the triplet had significantly inferior OS compared to those receiving the doublet (7.1 vs. 11.1 months, respectively; HR 1.91, 95% CI 1.15-3.18, P = 0.01). Females had a significantly higher overall response rate when the MEK inhibitor was added (doublet: 19.6%; triplet: 45.7%; P = 0.002). Conversely, among males, binimetinib resulted in lower response rates (doublet: 26.8%; triplet: 13.0%). Biomarker analysis showed that females with a high plasmatic BRAF allele fraction experienced longer OS from the triplet (HR 0.16, 95% CI 0.04-0.61), whereas males with a low allele fraction presented better outcomes with the doublet (HR 1.76, 95% CI 0.56-5.53). Conclusions and relevance: These results suggest that male sex may be associated with MEK inhibitor refractoriness and shorter survival. This underscores the need to better understand the effect of sex in treatment response and to integrate it as a clinical variable to guide decision-making.

Liver tumors, whether primary or metastatic, significantly impact the outcomes of patients with cancer. Accurate identification and quantification are crucial for effective patient management, including precise diagnosis, prognosis, and therapy evaluation. We present SALSA (system for automatic liver tumor segmentation and detection), a fully automated tool for liver tumor detection and delineation. Developed on 1,598 computed tomography (CT) scans and 4,908 liver tumors, SALSA demonstrates superior accuracy in tumor identification and volume quantification, outperforming state-of-the-art models and inter-reader agreement among expert radiologists. SALSA achieves a patient-wise detection precision of 99.65%, and 81.72% at lesion level, in the external validation cohorts. Additionally, it exhibits good overlap, achieving a dice similarity coefficient (DSC) of 0.760, outperforming both state-of-the-art and the inter-radiologist assessment. SALSA's automatic quantification of tumor volume proves to have prognostic value across various solid tumors (p = 0.028). SALSA's robust capabilities position it as a potential medical device for automatic cancer detection, staging, and response evaluation.

EGFR is a known oncogenic driver in colorectal cancer (CRC). The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is expressed in CRC stem-like cells and drives tumor initiation, growth and metastasis. Petosemtamab, a human, common light chain, IgG1 bispecific ADCC-enhanced antibody, targets tumor cells by degrading EGFR in LGR5+ stem-like cancer cells, blocking EGFR signaling and triggering Fc-mediated immune response. Here we report preliminary data from expansion cohorts of the phase 2 trial (NCT03526835) of petosemtamabin combination with standard of care FOLFOX or FOLFIRI in 1L, 2L metastatic CRC (mCRC) and petosemtamab monotherapy in 3L+ mCRC. Patients (pts) with left- and right-sided, KRAS, NRAS, and BRAF wildtype microsatellite stable mCRC received petosemtamab 1500 mg Q2W, in combination with FOLFOX or FOLFIRI or as monotherapy, until disease progression or unacceptable toxicity. Pts treated in 1L or 2L had no prior anti-EGFR therapy, with 2L having 1 prior chemotherapy regimen in the metastatic setting. 3L+ mCRC had at least 2 prior regimens in the metastatic setting, including a prior anti-EGFR therapy. Tumor imaging conducted Q8W. Primary endpoint is investigator-assessed overall response rate (RECIST v1.1); secondary endpoints include duration of response and safety. As of April 28, 2025, 36 pts were treated. Median age was 56 yrs (range 30-80), 58% male, 75% white, ECOG PS 0/1 in 39%/61% of pts. 7 pts were treated in 1L (6 FOLFOX and 1 FOLFIRI), with 6 ongoing. 3 pts were efficacy evaluable, median follow up 2.6 months, with 1 unconfirmed complete response and 2 partial responses (PR; 1 unconfirmed). 10 pts were treated in 2L (1 FOLFOX and 9 FOLFIRI), with 8 ongoing. 8 were efficacy evaluable, median follow up 3.4 months, with 4 PRs (2 unconfirmed), 3 stable diseases (SD; all ongoing) and 1 clinical deterioration prior to first scan. All unconfirmed responders are ongoing without disease progression. Most frequent treatment-emergent adverse events (TEAEs) regardless of causality in FOLFOX combination (all Grades [G]/G3) were dermatitis acneiform (71%/0%), constipation (43%/0%), fatigue (43%/0%), and peripheral neuropathy (43%/0%). No fatal treatment-related TEAEs observed. Most frequent TEAEs regardless of causality in FOLFIRI combination (all G/G3) were diarrhea (70%/0%), mucosal inflammation (50%/10%), and fatigue (40%/0%). 19 pts were treated in 3L+ (monotherapy), with 12 pts ongoing. 14 were efficacy evaluable, median follow up 2.5 months, with 1 unconfirmed PR ongoing without disease progression, 6 SDs (all ongoing), 6 progressive diseases and 1 death unrelated to treatment prior to first scan. Most frequent TEAEs regardless of causality (all G/G3) were rash (58%/0%), and nausea (26%/0%). No fatal treatment-related TEAEs observed. Petosemtamab demonstrates promising efficacy and manageable safety across 1L, 2L and 3L+ mCRC. Updated data will be provided; biomarker analysis is ongoing. Moh'd Khushman, Ruth Vera, Thibault Mazard, DiSean Kendall, Maria Elena. Elez, Francesc Salva, Elisa Fontana, Gregory P. Botta, Marcos Melián, Leon Pappas, Ludovic Evesque, Anastasia Murat, Chris Yan, Marina Magin, Gianluca Laus, Fabian Zohren, Josep Tabernero. Petosemtamab (MCLA-158) monotherapy or with chemotherapy in metastatic colorectal cancer: Preliminary antitumor activity and safety data from a phase 2 trial [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B033.

Background: For patients with refractory metastatic colorectal cancer (mCRC), trifluridine/tipiracil (FTD–TPI) has been associated with a significant improvement in overall survival (OS). However, data are lacking regarding the activity of FTD–TPI in patients with BRAF-mutated mCRC. Methods: This retrospective, multicenter, international cohort included patients with BRAF-mutated mCRC treated with FTD–TPI in a real-life setting in Spain and Italy. Survival analysis was performed using Kaplan–Meier methods and Cox proportional hazard models and according to established prognostic groups: good prognosis characteristics (GPC; < 3 metastatic sites and time from metastases to FTD–TPI ≥ 18 months) and poor prognosis characteristics (PPC; ≥ 3 metastatic sites or time from metastases to FTD–TPI < 18 months). Results: In the 26 patients included, the median age was 61 years, 13 (50%) were female, and 20 (77%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 1. Fourteen (56%) patients had right-sided tumors, six (23%) had microsatellite instability tumors, and thirteen (50%) had liver metastases. Median progression-free survival was 2.3 months (95% CI 2.0–3.2), and median OS (mOS) was 6.6 months (95% CI 4.4–12.0). mOS was 7.6 vs. 4.2 months (HR 1.64, 95% CI 0.65–4.10, p = 0.3) for GPC and PPC patients, respectively. Exploratory analyses identified ECOG as the only feature associated with survival. The most frequent grade 3–4 adverse events were neutropenia (8%), anemia (8%), and asthenia (4%). Conclusions: Patients with BRAF mutant mCRC achieved modest benefits with FTD–TPI; however, patients with GPC and ECOG 0 achieved longer OS compared with those with PPC or ECOG 1–2, thus warranting further exploration in prospective cohorts.

INTRODUCTION:The global prevalence of colorectal cancer highlights the need to enhance treatment strategies for improved patient outcomes. The pivotal role of epidermal growth factor receptor (EGFR) signaling in regulating cellular processes for this disease pinpoints its value as a therapeutic target, despite the emergence of resistance mechanisms over time. AREAS COVERED:This review discusses the clinical evidence supporting the use of EGFR inhibitors in molecularly-selected patients based on molecular characteristics (notably BRAF V600E and KRAS G12C) including combination approaches targeting different points in in the signaling pathway, as well as strategies such as EGFR inhibitor rechallenge. The role of HER2 inhibitors and emerging approaches such as bispecific antibodies are also reviewed. EXPERT OPINION:Recently, inhibitors targeting the KRAS G12C variant have emerged, albeit with modest monotherapy activity compared to other tumor types, emphasizing the influence of histologic origins on the EGFR signaling pathway. Integration of EGFR inhibitors into precision medicine has facilitated tailored therapies addressing resistance mechanisms. Patient selection for EGFR inhibitor rechallenge guided by ctDNA findings is crucial, with ongoing investigations exploring novel combinations to enhance EGFR blockade, highlighting the transformative potential of precision medicine in shaping the future of mCRC treatment toward personalized and targeted approaches.

Introduction: The mitogen-activated protein kinase (MAPK) signalling network aberrations in metastatic colorectal cancer (mCRC) generate intrinsic dynamic effects and temporal variations that are crucial but often overlooked in clinical trial populations. Here, we investigate the time-varying impact of MAPK pathway mutation genotype on each treatment line's contribution to the overall clinical course.Methods: The PROMETEO study focused on mCRC patients undergoing second-line treatment at 20 hospitals. We evaluated genotypes and employed flexible models to analyse the dynamic effect of each mutation.Results: We examined data derived from 1160 patients. The effects of KRAS G12C or G12V, and BRAF V600E are clearly time-varying, with unexpected consequences such as the deleterious effect of BRAF V600E vs other genotypes dissipating over time when subjects receive antiangiogenics, or KRAS G12V and G12C showing increasing aggressiveness over time. Thus, contrary to expectations, the 12-month survival rate from the second line for those who survived >6 months was 49.9% (95% CI, 32.7-67.3) for KRAS G12C and 59% (95% CI, 38.5-80.6) for BRAF V600E.Conclusions: The dynamic perspective is essential for understanding the behaviour of tumours with specific genotypes, especially from the second line onward. This may be relevant in patient monitoring and treatment decision-making, particularly in cases with distinct mutations.

The treatment of unresectable metastatic colorectal cancer has evolved over the last two decades, as knowledge of cancer biology has broadened and new targets have emerged. ‘The Hallmarks of Cancer’ illustrate the crucial capabilities acquired by cells to become malignant and represent the evolution of knowledge of tumor biology. This review integrates these novel targets and therapies into selected hallmarks: sustaining proliferative signaling, inducing vasculature, avoiding immune destruction, genome instability and mutation, reprogramming cellular metabolism, and resisting cell death. The different strategies and combinations under study are based on treatments with anti-EGFR, anti-VEGF, and anti-HER2 agents, KRAS G12C inhibitors, BRAF and MEK inhibitors, and immune checkpoint inhibitors. However, new approaches are emerging, including vaccines, WEE1 inhibitors, and PARP inhibitors, among others. The further deciphering of cancer biology will unravel new targets, develop novel therapies, and improve patients’ outcomes.

Cetuximab, a chimeric IgG1 monoclonal antibody targeting the epidermal growth factor receptor (EGFR), has revolutionized personalized treatment of metastatic colorectal cancer (mCRC) patients. This review highlights the mechanism of action, characteristics, and optimal indications for cetuximab in mCRC. Cetuximab has emerged as a pivotal partner for novel therapies in specific molecular subgroups, including BRAF V600E, KRAS G12C, and HER2-altered mCRC. Combining cetuximab with immunotherapy and other targeted agents further expands the therapeutic landscape, offering renewed hope for mCRC patients who face the development of resistance to conventional therapies. Ongoing clinical trials have continued to uncover innovative cetuximab-based treatment strategies, promising a brighter future for mCRC patients. This review provides a comprehensive overview of cetuximab's role and its evolving importance in personalized targeted therapy of mCRC patients, offering valuable insights into the evolving landscape of colorectal cancer treatment.

Colorectal cancer (CRC) is a complex and genetically heterogeneous disease presenting a specific metastatic pattern, with the liver being the most common site of metastasis. Around 20-25% of patients with CRC will develop exclusively hepatic metastatic disease throughout their disease history. With its specific characteristics and therapeutic options, liver-limited disease (LLD) should be considered as a specific entity. The identification of these patients is particularly relevant in view of the growing interest in liver transplantation in selected patients with advanced CRC. Identifying why some patients will develop only LLD remains a challenge, mainly because of a lack of a systemic understanding of this complex and interlinked phenomenon given that cancer has traditionally been investigated according to distinct physiological compartments. Recently, multidisciplinary efforts and new diagnostic tools have made it possible to study some of these complex issues in greater depth and may help identify targets and specific treatment strategies to benefit these patients. In this review we analyze the underlying biology and available tools to help clinicians better understand this increasingly common and specific disease.

Kirsten rat sarcoma virus oncogene homolog (KRAS) is the most frequently mutated oncogene in human cancer. In colorectal cancer (CRC), KRAS mutations are present in more than 50% of cases, and the KRAS glycine-to-cysteine mutation at codon 12 (KRAS G12C) occurs in up to 4% of patients. This mutation is associated with short responses to standard chemotherapy and worse overall survival compared to non-G12C mutations. In recent years, several KRAS G12C inhibitors have demonstrated clinical activity, although all patients eventually progressed. The identification of negative feedback through the EGFR receptor has led to the development of KRAS inhibitors plus an anti-EGFR combination, thus boosting antitumor activity. Currently, several KRAS G12C inhibitors are under development, and results from phase I and phase II clinical trials are promising. Moreover, the phase III CodeBreaK 300 trial demonstrates the superiority of sotorasib-panitumumab over trifluridine/tipiracil, establishing a new standard of care for patients with colorectal cancer harboring KRAS G12C mutations. Other combinations such as adagrasib-cetuximab, divarasib-cetuximab, or FOLFIRI-panitumumab-sotorasib have also shown a meaningful response rate and are currently under evaluation. Nonetheless, most of these patients will eventually relapse. In this setting, liquid biopsy emerges as a critical tool to characterize the mechanisms of resistance, consisting mainly of acquired genomic alterations in the MAPK and PI3K pathways and tyrosine kinase receptor alterations, but gene fusions, histological changes, or conformational changes in the kinase have also been described. In this paper, we review the development of KRAS G12C inhibitors in colorectal cancer as well as the main mechanisms of resistance.