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PubMed2026

Bergamottin, a bioactive component of bergamot: dual inhibition of Japanese encephalitis virus internalization and genome replication.

Japanese encephalitis virus (JEV) is associated with high mortality and severe neurological sequelae, and existing prevention and control strategies remain insufficient. Therefore, the development of novel antiviral agents is of critical public health importance. This study systematically evaluated the antiviral activity and underlying mechanism of bergamottin, a natural product. Bergamottin exhibited significant dose-dependent inhibitory effects against JEV in multiple cell lines, including BHK-21, HuH-7, and Vero cells, demonstrating potent antiviral efficacy. Mechanistic investigations revealed that bergamottin primarily targeted the internalization and replication stages of the JEV life cycle, thereby effectively suppressing viral proliferation. Additionally, adaptive mutation screening indicated that the D389G mutation in envelope protein E confers drug resistance by potentially changing E protein conformation or reducing endocytic efficiency. In vivo experiment, bergamottin significantly reduced viral loads in mouse brain tissue and effectively improved the survival rate of infected mice. Our findings indicated that bergamottin exerted antiviral activity by dual targeting of key steps in the viral life cycle, making it a highly promising candidate for anti-JEV therapy. Further exploration of the antiviral properties of bergamottin is expected to facilitate its clinical development as a treatment for JEV infection.

Animals↗

PubMed2025

Engineered histones reshape chromatin in human cells.

Histone proteins and their variants have been found to play crucial and specialized roles in chromatin organization and the regulation of downstream gene expression; however, the relationship between histone sequence and its effect on chromatin organization remains poorly understood, limiting our functional understanding of sequence variation between distinct subtypes and across evolution and frustrating efforts to rationally design synthetic histones that can be used to engineer specified cell states. Here, we make the first advance towards engineered histone-driven chromatin organization. By expressing libraries of sequence variants of core histones in human cells, we identify variants that dominantly modulate chromatin structure. We further interrogate variants using a combination of imaging, proteomics, and genomics to reveal both cis and trans-acting mechanisms of effect. Functional screening with transcription factor libraries identifies transcriptional programs that are facilitated by engineered histone expression. Double mutation screens combined with protein language models allow us to learn sequence-to-function patterns and design synthetic histone proteins optimized to drive specific chromatin states. This work establishes a foundation for the high-throughput evaluation and engineering of chromatin-associated proteins and positions histones as tunable nodes for understanding and modulating mesoscale chromatin organization.

Journal Article↗

PubMed2025

Timing Genomic Antigen Loss in Multiple Myeloma Treated with T Cell-Redirecting Immunotherapies.

UNLABELLED: Genomic antigen loss is a recurring mechanism of resistance to chimeric antigen receptor T-cell (CAR-T) and T-cell engagers (TCE) in relapsed/refractory multiple myeloma (RRMM). Yet, it remains unclear whether these events are acquired under treatment or merely selected from preexisting, undetectable clones. By leveraging chemotherapy mutational signatures as temporal barcodes within whole-genome sequencing data, we could time genomic antigen escape in 4 of 11 patients with RRMM. In all cases, the biallelic loss was driven by genomic events acquired after exposure to BCMA- and GPCR5D-targeted CAR-T/TCE and not present at baseline. Longitudinal digital PCR analysis corroborated that resistance mutations were undetectable at therapy initiation but emerged preceding relapse. Among 752 newly diagnosed patients, only 2.7% and 9% had monoallelic inactivation of TNFRSF17 and GPCR5D, respectively, with no biallelic loss. Our findings suggest limited utility of mutational screening prior to CAR-T/TCE while underscoring the importance of dynamic surveillance during therapy. SIGNIFICANCE: Multiple myeloma has been demonstrated to recurrently develop resistance to T-cell redirection via genomic antigen escape. By leveraging chemotherapy mutational signatures, we demonstrate that somatic antigen-escape mechanisms are uniformly acquired following treatment initiation and not selected from among preexisting clones, emphasizing the importance of dynamic longitudinal surveillance for their emergence. See related commentary by Kauer et al., p. 532.

Humans↗

PubMed2025

Whole-exome sequencing uncovers the genetic basis of hereditary concomitant exotropia in ten Chinese pedigrees.

PURPOSE: To explore possible pathogenic genes for concomitant exotropia using whole-exome sequencing. METHODS: In this study, 47 individuals from 10 concomitant exotropia (including intermittent exotropia and constant exotropia) pedigrees were enrolled. Whole-exome sequencing was used to screen mutational profiles in 25 affected individuals and 10 unaffected individuals. Sanger sequencing and in silico analysis were performed for all participants. Two target genes were used to capture the sequences of 220 sporadic samples. RESULTS: All 10 concomitant exotropia pedigrees presented autosomal dominant inheritance with childhood onset (3.35 ± 1.51 years old). Eleven different missense variants were identified among seven potential pathogenic genes (COL4A2, SYNE1, LOXHD1, AUTS2, GTDC2, HERC2 and CDH3) that cosegregated with pedigree members. All variants were predicted to be deleterious and had low frequencies in the general population. Distinct variants of COL4A2 were present in three pedigrees, and distinct variants of SYNE1 were present in two pedigrees. Fifteen variants in AUTS2 and four variants in GTDC2 were identified in 220 patients with sporadic concomitant exotropia using a target-capture sequencing approach. CONCLUSION: This is the first study to explore the genetic mechanism of concomitant exotropia and identify seven associated genes (COL4A2, SYNE1, LOXHD1, AUTS2, GTDC2, HERC2 and CDH3) that may be candidate genes causing concomitant exotropia. More samples and in-depth studies are needed to verify these findings.

Adult↗

PubMed2025

A base editor facilitates simultaneous purine and pyrimidine substitutions for ex vivo and in vivo mutagenesis screens.

Genetic mutations are closely linked to human diseases, yet the relationship between many mutations and their corresponding phenotypes remains poorly understood. Furthermore, tools to study the connection between nucleotide variations and phenotypes are limited. To address this issue, we developed ACGBEmax by fusing the dual-functional deaminase, engineered N-methylpurine DNA glycosylase, and evolved SOS response associated peptidase domain with nCas9(D10A). ACGBEmax enables the precise conversion of A, C, and G to other bases in mammalian cells, thereby generating an extensive range of base mutations types. We used ACGBEmax to generate HPRT variants, identifying mutations conferring resistance to 6-thioguanine. Additionally, we performed in situ mutagenesis of Ctnnb1 in mouse liver, identifying both known and potential oncogenic mutations. Our results prove that ACGBEmax is a powerful tool for generating a wide spectrum of mutation types at specific gene loci, highlighting its significant potential for applications in functional screening and the directed evolution of protein variants.

Animals↗

PubMed2026

PAT: An Image Analysis Tool for Automated Scoring of Pollen in Alexander-Stained Anthers.

Quantitative pollen viability analysis is a critical but labor-intensive step in plant reproductive biology. Existing deep-learning Segment Anything Models (SAM) fail to reliably segment viable pollen in Alexander-stained anthers. To address this, we fine-tuned an existing Cellpose-SAM model for pollen segmentation. We integrated it into PAT (Pollen Analysis Tool), a cross-platform desktop application. PAT features instance segmentation with interactive quality control, an in-app model retraining module, and publication-ready statistical outputs. We deployed PAT in an EMS suppressor screen of semi-sterile Arabidopsis smg7-6 mutants, enabling efficient candidate prioritization for whole-genome sequencing and mapping of the candidate mutation. This screen led to the identification of a point mutation in CAP-D2 (capd2-2), a Condensin I subunit, that rescues the smg7-6 meiotic phenotype. Notably, mutation in a Condensin II subunits (CAP-D3 and CAP-H2) does not confer rescue. Further characterization suggests the capd2-2 allele is hypomorphic, showing no defects in vegetative growth, chromocenter compaction, or transposable element silencing. Collectively, we demonstrate that accessible AI tools have the potential to bridge gaps in plant phenotyping and accelerate the pace of biological discovery.

Alexander staining↗

PubMed2014

Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow.

BACKGROUND: Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. METHODS: We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. RESULTS: Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. CONCLUSIONS: Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

Bone Marrow↗

PubMed2026

The emergence of putative epistatic mutations and iSNVs in SARS-CoV-2 XBB.1.16 variants linked with alteration in immunogenic determinants.

The SARS-CoV-2 XBB variants have been proposed to evolve towards immune evasion against vaccination or natural infection, which may contribute to higher transmissibility. The XBB.1.16 independently emerged due to accumulation of two important substitutions, E180V and T478R in the spike protein. Its pseudoviral infectivity and evasion of humoral immunity were similar to XBB.1 and XBB.1.5. In March 2023, XBB.1.16 had outcompeted other dominant XBB variants in India, which indicate a potential growth advantage. Here, intra-host single nucleotide variations (iSNV) and mutations were screened in SARS-CoV-2 genomes in closely related individuals at two time points: at symptoms onset, and during recovery. The prominence of putative epistatic iSNVs (E180V, G184V, G252V, D253G, and P521S/T) in XBB.1.16 variants were detected during the recovery phase. E180V exhibits mutational constellations with the G252V and P521T in a subset of samples, and this pattern was also detected in contemporary SARS-CoV-2 genomes. Higher order protein structural predictions suggested that the putative epistatic interactions among E180V, G184V, and G252V, D253G may be associated with S protein folding and structural stability. This study involving genomics and computational analyses highlights the potential role of these putative epistatic interactions in immune evasion, which may have contributed to dominance of XBB variants.

Humans↗

PubMed2025

Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice.

Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.

Animals↗

PubMed2025

BRCA1 Exon 11 Mutations in Breast Cancer: A Study From Pakistan.

Breast cancer ranks among the top causes of cancer-related deaths in women around the globe, with genetic mutations in the BRCA1 gene being a frequent cause of breast or ovarian cancer. This study investigates hotspot mutations in exon 11 of the BRCA1 gene among Pakistani women diagnosed with breast cancer. Thirty clinically diagnosed breast cancer patients, all women, were enrolled in the current study, and high-quality DNA was extracted from peripheral blood samples. Two of the twenty-five successfully sequenced samples had a homozygous missense variant (c.2312T > C: p.Leu771Ser) detected by Sanger sequencing after PCR amplification. Upon investigation in the ClinVar database, the identified variant showed conflicting interpretations of pathogenicity. Demographic data highlighted an early disease onset, showing that 56% of patients were under 50 years of age. The need for genetic screening was further supported by the fact that 24% of the patients had a positive family history of cancer. Our study emphasizes the necessity of screening BRCA1 gene mutations to better understand the pathogenic potential of the identified variants in the Pakistani population.

Humans↗

PubMed2026

Hypertensive mt. tRNAIle4263A>G mutation orchestrates vascular senescence and apoptosis by activation of mitochondria-ER interplay.

The pathogenic mechanism underlying diseases caused by mitochondrial DNA (mtDNA) mutation, including hypertension, persists as an unresolved global challenge. Although mutation-induced mitochondrial defects have been well characterized, how these mito-perturbations are converted into critical intermediary signaling cascades and contribute to diseases remain unknown. Here, using human induced pluripotent stem cell (hiPSC)-derived vascular organoids (VOs) and vascular cells, the hypertensive mt. tRNAIle4263A > G mutation was identified to induce vascular senescence, apoptosis and vascular-specific dysfunction through mitochondria-endoplasmic reticulum (ER) interaction. For the first time, this study mapped the transcriptional reprogramming landscape of human VOs carrying this mutation. Through systematic screening and functional validation, ER stress was screened out, together with downstream mitochondria-associated ER membranes-mitochondrial Ca2+ overload resulting in vascular abnormality. Pathological reactive oxygen species (ROS) elevation, driven by tRNAIle destabilization and bioenergetic failure, acts as the primary instigator of maladaptive ER stress activation in this cascade. Pharmacological targeting of this axis-using mito-Tempol (a mitochondria-targeted ROS scavenger), Tauro Ursodeoxycholic Acid (an ER stress inhibitor), or RU265 (a highly-selective mitochondrial calcium uniporter inhibitor)-rescues vascular abnormality. This study highlights mt. tRNAIle4263A > G mutation orchestrates vascular pathology through ROS induced activation of inter-organelle communication, resolving a long-standing knowledge gap between mtDNA mutations and diseases and establishing therapeutic nexuses for mtDNA mutation-related cardiovascular diseases.

Hypertension↗

PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important, promiscuous divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (~150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ~20% underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗

PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (∼150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ∼20% for AlphaMissense underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular protein trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗

PubMed2026

Innovations in microbial physical mutagenesis for food fermentation: An overview from traditional to emerging technologies.

Microbial strains serve as an important factor affecting fermentation efficiency and product quality. To obtain superior strains, mutation breeding is a classic strategy. Compared to chemical mutagenesis, physical mutagenesis directly induces genomic changes, providing notable advantages such as the elimination of chemical residues and environmental sustainability, hence rendering it a favored method for enhancing food-grade microorganisms. Conventional physical mutagenesis mostly depends on UV, rays, high pressure, or space radiation. As physical technologies advance, emerging methods such as ion implantation, plasma, microwave, ultrasound, and pulsed light are widely utilized for genetic modification. Mutagenesis technologies are progressively transitioning from single-effect to multi-effect synergy. Recent evaluations indicate that emerging technologies can enhance microbial mutation efficiency at the application level relative to established technologies. Nonetheless, the systematic clarification and comparative analysis at the mechanistic level remain inadequate, hindering intuitive comprehension of the qualities and distinctions across techniques. Furthermore, physical mutagenesis encounters several significant obstacles, such as cellular damage, limited rates of advantageous mutations, and laborious screening processes. This review carefully elucidates the mechanisms and properties of physical mutagenesis technology and delineates the distinctions among approaches through comparative analysis. Simultaneously, solutions for optimizing mutagenesis are presented to tackle the principal challenges mentioned above. This review aims to offer a theoretical foundation and practical guidance for the enhanced application of physical mutagenesis technologies in microbial breeding.

Mutagenesis↗

PubMed2026

Global Diffusion of IncC Plasmid Harboring blaNDM-1in the High-Risk Escherichia coli ST131 Clone.

AIMS: The global expansion of quinolone-resistant Escherichia coli (QR-EC) is increasingly associated with β-lactam resistance and mobile genetic elements that facilitate resistance dissemination. This study investigated the molecular mechanisms underlying fluoroquinolone and β-lactam resistance in clinical QR-EC isolates and explored the plasmid type associated. METHODS AND RESULTS: A total of 123 non-duplicate QR-EC clinical isolates responsible mainly for gastrointestinal colonization were collected between 2019 and 2021. Plasmid-mediated quinolone resistance (PMQR), extended-spectrum β-lactamase (ESBL), and carbapenemase genes were screened by PCR. Mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC were analyzed using sequencing and mismatch amplification mutation assay PCR (MAMA-PCR). Selected isolates underwent multilocus sequence typing (MLST). Whole-genome sequencing (WGS) of a representative extensively drug-resistant strain carrying multiple quinolone resistance determinants, ESBL genes, and carbapenemase genes, was performed. PMQR genes were prevalent among QR-EC, dominated by aac(6')-Ib-cr (60.9% of isolates). ESBL genes were identified in 93.5% of isolates, predominantly blaCTX-M (95.7%). Among ertapenem-resistant isolates (QCR-EC) (n=18), blaNDM-1 and blaOXA-48 were detected in 13 and 11 isolates, respectively. QRDR mutations were highly frequent, particularly gyrA83 (98.4%) and parC80 (30.9%). Major QCR-EC genotypes belonged to sequence types ST167 (n=2), ST1196, ST469, and ST410. High-risk E. coli ST131 clone harboring IncC plasmid encoding blaNDM-1 was described for the first time in Africa, following its emergence, in two continents, Asia and America. Despite the very rare description of these strains worldwide, their description in three continents sign their global diffusion. CONCLUSIONS: This finding highlights the ongoing spread of carbapenem resistance and underscores the urgent need for strengthened genomic surveillance.

Escherichia coli↗

PubMed2025

The NmpRSTU multi-component signaling system of Myxococcus xanthus regulates expression of an oxygen utilization regulon.

UNLABELLED: Myxococcus xanthus has numerous two-component signaling systems (TCSs), many of which regulate the complex social behaviors of this soil bacterium. A subset of TCSs consists of NtrC-like response regulators (RRs) and their cognate histidine sensor kinases (SKs). We have previously demonstrated that a multi-component, phosphorelay TCS named NmpRSTU plays a role in M. xanthus social motility. NmpRSTU was discovered through a screen that identified mutations in nmp genes that restored Type-IV pili-dependent motility to a nonmotile strain. The Nmp pathway begins with the SK NmpU, which is predicted to be active in the presence of oxygen. NmpU phosphorylates another SK, NmpS, a hybrid kinase containing an RR domain and a HisKA-CA domain. These two kinases work in a reciprocal fashion: when NmpU is active, NmpS is inactive, and vice versa. Finally, the phosphorelay culminates in NmpS phosphorylating the NtrC-like RR NmpR. To better understand the role of NmpRSTU in M. xanthus physiology, we determined the NmpR regulon by combining in silico predictions of the NmpR consensus binding sequence with in vitro electromobility shift assays (EMSAs) and in vivo transcriptional reporters. We identified several NmpR-dependent, upregulated genes likely to be important in oxygen utilization. Additionally, we demonstrate NmpRSTU plays a role in fruiting body development, suggesting a role for oxygen sensing in this behavior. We propose that NmpRSTU senses oxygen-limiting conditions, and NmpR upregulates genes associated with optimal utilization of that oxygen. This may be necessary for M. xanthus physiology and behaviors in the highly dynamic soil where oxygen concentrations vary dramatically. IMPORTANCE: Bacteria use two-component signaling systems (TCSs) to respond to a multitude of environmental signals and subsequently regulate complex cellular physiology and behaviors. Myxococcus xanthus is a ubiquitous soil bacterium that encodes numerous two-component systems to respond to the conditions of its soil environment and coordinate multicellular behaviors such as coordinated motility, microbial predation, fruiting body development, and sporulation. To better understand how this bacterium uses a two-component system that has been linked to the sensing of oxygen concentrations, NmpRSTU, we determined the gene regulatory network of this system. We identified several genes regulated by NmpR that are likely important in oxygen utilization and for the M. xanthus response to varied oxygen concentrations in the dynamic soil environment.

Myxococcus xanthus↗

PubMed2026

A multicenter survey on BRAF screening for the implementation of perioperative cancer genomic medicine for resectable colorectal oligometastases.

BACKGROUND: Genomic screening is an essential, but potentially time-consuming procedure, especially in neoadjuvant settings. We evaluated the preoperative screening of the BRAF V600E mutation for recruitment to a clinical trial among patients with resectable colorectal oligometastases (CRM). METHODS: In April 2022, an investigator-initiated trial was launched to investigate the efficacy and safety of perioperative use of the BEACON triplet regimen for BRAF V600E mutant resectable CRM. BRAF screening was retrospectively conducted in patients with resected colorectal liver metastases in 2019 for planning the trial and prospectively conducted in preoperative patients with resectable CRM from January 2022 to June 2025 for patient recruitment to the trial. RESULTS: BRAF V600E mutation was detected in 12 (3.2%) of 379 postoperative patients retrospectively and in 36 (1.7%) of 2140 preoperative patients prospectively, with 1840 patients (86.0%) carrying the wild-type and 264 patients (12.3%) classified as untested. The detection rate of the BRAF V600E mutation was significantly lower when the screening was performed prospectively in preoperative patients (P < 0.001). The untested rates varied across metastatic organs, with 10.3% in the liver, 18.1% in the lungs, 12.0% in the lymph nodes, 16.7% in the peritoneum, and 7.8% in other organs. The untested rates decreased consistently across semiannual comparisons: 28.5% in the first evaluation, followed by 15.0%, 12.0%, 8.4%, 9.1%, 7.3%, and 7.1% (P < 0.01 when compared with the first period). CONCLUSION: Raising physician awareness, as reflected by the untested rate, is a crucial factor in conducting clinical trials to implement perioperative cancer genomic medicine.

Humans↗

PubMed2026

primary analysis of the RANDOMIZED eortc-2139/columbus-ad trial: Adjuvant encorafenib and binimetinib versus placebo in high-risk stage II BRAF-V600E/K melanoma.

PURPOSE: Stage IIB/IIC melanoma has a high risk of recurrence after resection. Combined BRAF/MEK inhibitor therapy showed benefit in resected high-risk stage III and advanced melanoma. The objective of this study was to investigate its role in stage IIB/IIC. METHODS: Adult patients with resected stage IIB/IIC cutaneous melanoma which had a BRAF V600E/K mutation were randomized 1:1 to receive encorafenib (enco) 450 mg QD + binimetinib (bini) 45 mg BID orally for one year or placebo. The study planned to randomize 815 patients and was designed to demonstrate superiority regarding recurrence-free survival (RFS). Following a premature termination of accrual, the study was amended with safety as the primary endpoint and RFS as secondary endpoint. RESULTS: Between June 9, 2022, and October 9, 2023, 339 patients were screened for a BRAF mutation and 110 randomized. Data cutoff was 19 Nov. 2024, after the last patient discontinued study participation. Among randomized patients, 87 (79%) had a BRAF V600E mutation, and 39 (35%) AJCC8 stage IIC. Median follow-up was 12 and 7 months for enco/bini and placebo arms, respectively. Among 54 patients who initiated enco + bini, grade ≥ 3 treatment-related adverse events (AE) occurred in 13 (24%) patients, and 18 (33%) patients had an AE leading to permanent treatment discontinuation. RFS at 12 months was 86% (95% CI: 65-95%) in the enco + bini and 70% (95% CI: 46-85%) in the placebo arm, distant metastasis-free survival at 12 months was 92% (95% CI: 77-97%) for enco + bini and 82% (95% CI: 55-93%) for placebo. CONCLUSION: EORTC 2139 - Columbus-AD demonstrated a consistent and manageable safety profile and encouraging efficacy results for the combination of enco and bini in resected stage IIB/C BRAF V600E/K-mutated cutaneous melanomas.

Adult↗