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PubMed2026

Short-read genome skimming enables molecular barcoding of old myxomycete collections.

This study evaluates the effectiveness of Illumina-based genome skimming for barcoding myxomycete herbarium collections ranging from 29 to 91 years in age. We successfully retrieved partial sequences of the standard marker gene (nucSSU) in all cases, as well as additional markers (mtSSU, EF1a, and COI) for certain collections. Altogether, 28 genes were recognized in the studied material. In a 33-year-old specimen of Lindbladia tubulina, the assembly reached an N50 of 4.19 kb, enabling the recovery of extended functional loci. The input genomic DNA quantity emerges as the primary determinant of sequencing success. Samples with high DNA yields provide representative amounts of contigs coming confirmedly (matching sequences in the NCBI nucleotide database) or potentially (no-hit fraction) from myxomycetes, regardless of specimen age. In addition to target DNA, we revealed distinct signals of both anthropogenic contamination (human DNA and skin microflora) and natural substrate inhabitants, including oribatid mites and bacteria from dead wood, soil, and grass litter. Thus, even in old collections, metagenomic data still carry information regarding the substrate upon which the myxomycete developed. The results demonstrate that short-read genome skimming may help to integrate historical type material of myxomycetes into contemporary phylogenetic research. This method overcomes the length-dependent limitations of traditional Sanger sequencing, thus providing a roadmap for the future of museomics in myxomycetology.

Amoebozoa↗

PubMed2026

WWOX-related developmental and epileptic encephalopathy (WOREE): A case series of seven patients from Argentina.

PURPOSE: WWOX-related developmental and epileptic encephalopathy (WOREE) is a rare autosomal recessive disorder caused by biallelic pathogenic WWOX variants, characterized by very early-onset epilepsy, profound developmental delay, and progressive brain abnormalities. Detailed electroclinical descriptions remain limited. METHODS: We conducted a retrospective study of seven patients with pathogenic/likely pathogenic WWOX variants. Clinical features, seizure evolution, EEG findings, brain MRI, and genetic data were reviewed. Epilepsy syndromes were classified according to International League against Epilepsy (ILAE) criteria. Variants were identified through next-generation sequencing and interpreted following American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: Median seizure onset was 3 months (range 2-6). Five patients presented with focal seizures evolving to infantile epileptic spasms syndrome (IESS), while two had IESS at onset. Epilepsy was drug-resistant in all. Developmental delay was evident from birth with generalized hypotonia, acquired microcephaly, and impaired visual attention. Four patients had dysmorphic features. During the IESS period, EEG showed hypsarrhythmia in six patients and a severely disorganized encephalopathic background that did not strictly fulfill the criteria for hypsarrhythmia in one. Brain MRI revealed abnormalities in all patients, including frontotemporal atrophy and corpus callosum hypoplasia; delayed myelination was observed in one case. Eight pathogenic/likely pathogenic WWOX variants were found; including one novel variant (NM_016373.4:c.571C>T, p.(Gln191*)). The recurrent splice-site variant NM_016373.4:c.107+1G>A was identified in five patients, suggesting a possible regional founder effect. CONCLUSION: WOREE shows a recognizable electroclinical and neuroimaging profile with early drug-resistant epilepsy and profound developmental delay. Recognition of this pattern may facilitate early diagnosis and targeted genetic testing, particularly in populations with recurrent variants.

Developmental and epileptic encephalopathy↗

PubMed2026

Clinical and genomic characterization of Influenza A co-infection with SARS-CoV-2 and Influenza B: a respiratory surveillance study in Assam, India.

Influenza and SARS-CoV-2 are the primary contributors to seasonal respiratory infections and frequently co-circulate, creating significant health challenges. The present respiratory surveillance study was conducted in Dibrugarh, Assam, India from January 2025 to August 2025 to investigate the genomic characteristics of circulating viruses and identify potential co-infections. Overall, 4,948 respiratory samples were screened using multiplex real-time PCR, followed by subtyping of Influenza A and Influenza B. Next-generation sequencing (NGS) was performed in selected positives of SARS-CoV-2 and Influenza A. Genomic analysis included mutational profiling, phylogenetic analysis and N-glycosylation site prediction using bioinformatics tools. Two co-infection cases were detected: one involving Influenza A (H3N2) with SARS-CoV-2 (Omicron XFG lineage) and another involving Influenza A (H3N2) with Influenza B (Victoria lineage). Both patients experienced mild illness without hospitalisation. NGS revealed that the Influenza A (H3N2) viruses belonged to clade 3C.2a1b.2a.2a.3a.1 while SARS-CoV-2 sequence was classified under the Omicron XFG lineage. Mutational analysis of the HA gene showed several amino acid differences compared to the reference vaccine strain A/Darwin/6/2021. N-glycosylation analysis predicted conserved sites at positions 79, 181, 262, and 301 in all strains along with an additional predicted site at position 110 in both co-infection cases. Although the co-infection cases presented with mild clinical manifestations, the observed genomic variations indicate a potential role of co-infecting viruses in shaping viral evolution. Given the limited genomic data available from Northeast India, the study underscores the need for sustained large scale follow up and genomic surveillance to monitor emerging mutations and target future vaccine strategies.

Humans↗

PubMed2026

Prenatal Alcohol Exposure Produces Selective Changes in Neuroimmune Gene Expression Across Brain Regions of Adult Mice.

BACKGROUND: An overwhelming body of evidence suggests neuroimmune dysfunction as a key underlying mechanism of fetal alcohol spectrum disorder (FASD)-associated adverse central nervous system (CNS) outcomes. While few studies have highlighted the lingering effects of prenatal alcohol exposure (PAE) on producing specific immune factors, others suggest a primed neuroimmune state in adulthood, in which a proinflammatory bias is unmasked following subsequent immune activation in later life. However, the PAE-induced neuroimmune landscape in adulthood remains poorly defined. We hypothesized that PAE induces long-term changes in gene expression linked to neuroimmune function that may be brain region-specific. METHODS: Using long-read next-generation RNA sequencing of brain tissues from a previously established model of a moderate PAE in mice, we compared across six regions: medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), hypothalamus, hippocampus, midbrain, and medulla. A comprehensive bioinformatics analysis investigated PAE-induced changes, dysregulated gene pathways, and transcriptional regulators with a focus on neuroimmune function. RESULTS: Our data identified at least 60 differentially expressed genes per brain region, many of which were associated with neuroimmune function. Upregulation of multiple pro-inflammatory factors and pathways was observed, suggesting ongoing baseline neuroimmune activation, potentially involving PXR, TNF, TLR4, the complement pathway, and various cytokine and chemokine signaling. A comparative analysis identified multiple upstream transcriptional regulators across multiple brain regions, including MECP2, TCF7L2, and IL-4. Importantly, this unbiased analysis revealed heterogeneity across brain regions in the activation of canonical immune pathways and highlighted previously unprecedented roles of pathways such as PXR, matrix metalloproteases, and cytokine signaling (e.g., IL-15, IL-27, IL-17) in PAE. CONCLUSIONS: PAE creates a unique inflammatory signature in the adult brain, even in the absence of secondary injury, with novel patterns of region-specific changes in genes implicated in glial-immune function. These data identify potential immune targets to elucidate the mechanisms underlying behavioral dysfunction and provide a framework for future therapeutic interventions.

Animals↗

PubMed2025

Marine-Inspired Antimicrobial Peptides Disrupt Gene Expression at the DNA Level.

Genome mining of Streptomyces sp. H-KF8 combined with sequence engineering yielded two serum-stable, noncytotoxic, nonlytic antimicrobial peptides, L3 and L3-K. Initial studies in uropathogenic Escherichia coli suggested membrane effects and nucleoid relaxation, prompting a comprehensive investigation of their mode of action. In this study tandem mass tag (TMT)-based quantitative proteomics revealed extensive proteome remodeling, with 175 and 120 differentially expressed proteins (DEPs) after treatment with L3 and L3-K, respectively. L3 induced predominantly upregulated responses linked to metabolism, RNA processing, transport, and homeostasis, whereas L3-K mainly caused the downregulation of proteins involved in metabolism, transport, and cell structure. Both peptides disrupted ABC transporter-mediated nutrient uptake and elicited stress responses, while L3 specifically perturbed the mal regulon, indicative of broader transcriptional dysregulation. Complementary fluorescent dye displacement and in vitro transcription/translation assays demonstrated nonspecific DNA binding, stronger for L3 than L3-K, and potent inhibition of transcriptional and translational processes. Strikingly, inhibitory concentrations paralleled their minimum inhibitory concentrations, directly linking DNA binding and interference with central information processing to antimicrobial activity. These findings reveal that L3 and L3-K primarily act by targeting DNA and interfering with the transcription-translation machinery. Beyond offering mechanistic insights, this study underscores peptides' potential to act as scaffolds for next-generation antimicrobial peptides with DNA-binding and nonmembrane-lytic activity.

Antimicrobial Peptides↗

PubMed2026

AI In Leukemia Diagnostics: Complementing the Pathologist's Role.

Artificial intelligence (AI) is reshaping every stage of leukemia diagnostics, from digital morphology and multiparameter flow cytometry to next-generation sequencing, multi-omics analysis, and emerging computational frontiers such as quantum-inspired feature selection. This review outlines how contemporary AI tools can automate labor-intensive quantitation, flag diagnostically salient patterns, and standardize interpretation, while the pathologist or hematologist retains authority over validation, context-specific integration, and clinical decision-making. We present an illustrative "human-in-the-loop" workflow that embeds AI modules within current laboratory information systems, emphasizing points where expert oversight mitigates algorithmic bias and resolves discordant findings. We further map the validator-integrator role across morphology, flow cytometry, and genomic/multi-omic interpretation and provide practical training competencies and use cases for AI-assisted hematopathology. Beyond technical deployment, the article addresses the educational transformation required for sustainable adoption. Drawing on international competency frameworks, including the Digital Health Competencies in Medical Education Framework and recently proposed AI-specific Entrustable Professional Activities, we map core skills that future hematopathologists must master: data-science literacy, critical appraisal of AI outputs, and ethical governance. We highlight evaluated training models such as the Pathology Informatics Essentials for Residents curriculum, Stanford Artificial Intelligence in Machine and Imaging workshops, and College of American Pathologists bootcamps and propose integration strategies adaptable across resource settings. By pairing rigorous validation with targeted education, AI can elevate rather than eclipse the diagnostic role of the leukemia specialist, enabling more timely, reproducible, and personalized patient care.

Humans↗

PubMed2026

Proteome-wide curation of experimentally validated HPV T-cell epitopes identifies key gaps in our understanding of cellular immunity to HPV and informs vaccine design.

BACKGROUND: Human papillomavirus (HPV) drives both malignant and benign tumours. Current prophylactic vaccines are type-restricted, not optimised for T-cell induction, and lack therapeutic efficacy. Although T-cells are critical for both preventing and clearing HPV infection, experimentally validated HPV T-cell epitopes remain fragmented across the literature, limiting systematic evaluation of cellular immune targets. METHODS: We curated experimentally validated HPV T-cell epitopes from the Immune Epitope Database (IEDB). Epitopes were mapped across HPV proteins and genotypes, and analysed for response rate, sequence conservation across 454 representative HPV genomes, and HLA restriction patterns. RESULTS: 485 unique experimentally validated HPV epitopes have been described (133 studies; 1,494 functional assays). Consistent with research focus and viral biology, E6 and E7 proteins account for >60% of known HPV epitopes despite accounting for ~10% of the viral proteome. High-risk HPV types, especially HPV16 and HPV18, were the most studied (p <.001) and were enriched for CD8+ epitopes (p <.001). We identified major knowledge gaps, including: underrepresentation of structural proteins such as L2; limited epitope coverage for low-prevalence HPV genotypes; a bias towards common HLA alleles. In silico analysis indicated greater conservation of epitopes in L1/L2 and across high-risk HPV types. Conserved, commonly detected, and HLA-promiscuous epitopes were highlighted and we provide panels of candidate epitopes for consideration in immune monitoring, broad-spectrum prophylactic vaccines, and high-risk targeted therapeutic vaccines. CONCLUSION: This study provides the first comprehensive atlas of experimentally validated HPV T-cell epitopes and ranked epitope candidates for translational application. We demonstrate that our understanding of HPV T-cell immunity is constrained by biases in antigen, genotype and HLA focus and by incomplete epitope mapping. Addressing these gaps will be essential for a comprehensive assessment of cellular immunity and for utilising T-cells in next-generation vaccines.

Epitopes, T-Lymphocyte↗

PubMed2026

Epilepsy of infancy with migrating focal seizures: A scoping review of clinical features, diagnostic testing including genetics, long-term outcomes, mortality, and current and emerging therapeutic strategies.

BACKGROUND: Epilepsy of infancy with migrating focal seizures (EIMFS) is among the most severe developmental and epileptic encephalopathies (DEEs), marked by intractable multifocal seizures migrating across both hemispheres, profound developmental arrest, and high early mortality. Advances in next-generation sequencing have revealed a heterogeneous genetic architecture dominated by KCNT1 gain-of-function variants across more than 30 implicated genes, creating opportunities for precision therapeutics. OBJECTIVE: To systematically map published evidence on the clinical, electrophysiological, neuroimaging, genetic, and therapeutic landscape of EIMFS, and to delineate critical knowledge gaps and future research priorities. METHODS: A scoping review was conducted following the Arksey and O'Malley framework, searching PubMed, Ovid MEDLINE, Embase, Cochrane Library/CENTRAL, and ClinicalTrials.gov. RESULTS: Of 643 articles screened, 89 met inclusion criteria. Beyond confirmation of the canonical electroclinical phenotype, several gaps emerged: neonatal versus post-neonatal onset stratification by genetic etiology remains largely uncharacterized; genotype-specific EEG biomarkers are lacking except for a single small KCNT1 study; and the clinical significance of atypical EEG features-including burst suppression and hypsarrhythmia-is undefined. Neuroimaging literature documents progressive cerebral atrophy and myelination abnormalities without quantitative volumetry, diffusion tractography markers, or attribution to seizure burden, medication effects, or underlying etiology. Genetic diagnostic yield was 70-80%, with KCNT1 accounting for 30-50% of solved cases; however, genotype-outcome stratification is limited. Seizures were broadly refractory; potassium bromide, ketogenic diet, cannabidiol, and quinidine (in KCNT1-confirmed cases) showed partial efficacy. Emerging precision approaches include sodium channel blockers for SCN2A gain-of-function variants, novel small molecules, fluoxetine, antisense oligonucleotides, and divalent siRNA targeting KCNT1. Systemic-to-pulmonary collateral circulation causing severe cardiopulmonary complications was reported across multiple cases, yet no consensus screening protocol exists. CONCLUSIONS: EIMFS remains one of the most refractory epilepsy syndromes of infancy. Precision genetic diagnosis is essential to guide targeted therapy. International collaborative registries, standardized outcome measures, genotype-stratified biomarker studies, and rapid point-of-care genomic testing are urgently needed to advance evidence-based care for this highly vulnerable population.

Humans↗

PubMed2024

Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes.

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Humans↗

PubMed2026

The diagnostic potential of combined quantitative polymerase chain reaction and next-generation sequencing using the same primers for periprosthetic joint infection.

Next-generation sequencing (NGS) enables the detection of specific pathogens unidentifiable by conventional cultures, but its application in orthopedics remains inconsistent due to background contamination and irreproducible findings. This study evaluated the diagnostic performance of a novel workflow combining broad-range 16S rRNA gene quantitative PCR (qPCR) screening with downstream NGS, focusing on bacterial biomass thresholds. The qPCR assay demonstrated excellent intrarater reliability, with an intraclass correlation coefficient (ICC) of 0.961 (95% confidence interval, 0.881 to 0.997). Based on serially diluted positive controls, a quantitative threshold of 10⁵ CFU/mL was established as the minimum concentration required for the consistent detection of fastidious taxa, such as Escherichia coli. When evaluated against conventional cultures using 95 sonicate fluid and 276 pre/intraoperative tissue samples, the qPCR assay achieved a sensitivity of 80% and a specificity of 72%. Subsequent NGS sequencing of 26 clinical samples and 9 controls showed concordance in 4 of 6 culture-positive infected cases with NGS taxonomy, whereas the remaining discrepancies were likely attributable to culture-based phenotypic misidentification. Notably, among the qPCR-positive cases, three were culture-negative, including two hip prosthesis loosening cases exhibiting polymicrobial profiles, and one post-traumatic osteoarthritis case harboring low-level Staphylococcus. Crucially, this post-traumatic patient developed delayed periprosthetic joint infection (PJI) 2 years post-surgery, with cultures identifying Staphylococcus previously detected by the initial NGS analysis. Integrating qPCR screening with targeted NGS effectively refines pathogen identification, filters environmental artifacts, and overcomes the diagnostic limitations of culture-negative infections in orthopedic practice.IMPORTANCENext-generation sequencing (NGS) enables the detection of specific pathogens in clinical samples that are not identifiable by conventional methods. However, NGS applications in orthopedics have not been quantitatively evaluated, and findings have been inconsistent owing to contaminants and the presence of non-credible causative organisms. These factors primarily stem from the failure to evaluate low-biomass samples and the absence of proper controls, such as negative controls or mock community DNA samples. This study demonstrates that interpreting results from low-biomass samples requires careful consideration because NGS relies on relative bacterial abundances; distinguishing likely pathogens from contaminants is particularly challenging when bacterial loads are low. We demonstrated that combining NGS with quantitative PCR (qPCR) and applying a Cq cutoff can reduce false positives.

Humans↗

PubMed2026

Engineering extracellular vesicles for targeted siRNA delivery: Advances, therapeutic applications, and clinical translation.

Small interfering RNA (siRNA) therapeutics have emerged as a transformative approach for sequence-specific gene silencing, offering the potential to treat a broad spectrum of diseases by selectively suppressing disease-associated genes. However, the clinical translation of siRNA remains limited by rapid enzymatic degradation, poor cellular uptake, inadequate endosomal escape, and off-target effects, necessitating the development of efficient delivery systems. Extracellular vesicles (EVs) have gained considerable attention as natural nanocarriers owing to their excellent biocompatibility, low immunogenicity, intrinsic targeting capability, and ability to protect therapeutic cargo while traversing complex biological barriers. This review comprehensively discusses the biological characteristics of EVs, the molecular basis of RNA interference, and the major challenges associated with siRNA delivery [Fig. 1]. Recent advances in EV engineering, including cargo-loading strategies such as electroporation, sonication, extrusion, parent-cell engineering, and microfluidic approaches, together with surface functionalization using peptides, antibodies, aptamers, and hybrid nanoplatforms, are critically evaluated for improving targeting specificity and intracellular delivery. Furthermore, the therapeutic applications of engineered EV-mediated siRNA delivery in cancer, neurological disorders, liver diseases, cardiovascular diseases, inflammatory disorders, and infectious diseases are systematically summarized, highlighting their potential to enhance gene silencing while minimizing systemic toxicity. Current challenges related to large-scale manufacturing, cargo-loading efficiency, standardization, quality control, regulatory approval, and clinical translation are also discussed, together with emerging technologies involving synthetic biology, genome engineering, artificial intelligence, and multifunctional hybrid vesicles. Overall, engineered extracellular vesicles represent a highly versatile and biologically inspired platform for targeted siRNA delivery, providing a promising foundation for the development of next-generation precision RNA therapeutics and accelerating the clinical translation of gene-silencing strategies.

Extracellular vesicle engineering↗

PubMed2026

Recurrent and niche-specific functional bacteriome of maize hybrid revealed by integrated metabarcoding and culturomics.

The plant microbiome plays a pivotal role in plant survival in natural habitats by facilitating nutrient acquisition, stress adaptation, and disease suppression, while also offering opportunities to enhance crop productivity and climate resilience. However, the distribution of persistent and culturable bacteriome across maize-associated niches and their functional potential remain poorly resolved. This study integrated metagenomic next-generation sequencing (mNGS-based metabarcoding) and culturomics to characterise the maize-associated bacteriome of bulk soil, rhizoplane, phylloplane, and cob of the maize hybrid PHM-1 under contrasting cropping and tillage systems, and to identify recurrent and agriculturally promising bacteriome components. The bacteriome exhibited pronounced niche-specific structuring, whereas overall bacterial community composition did not differ significantly across cropping and tillage treatments (ANOSIM, R = 0.038, p = 0.306). Proteobacteria predominated in the culturable bacteriome (69-84%; mean, 76.2%) but accounted for only 1% of the total bacteriome, whereas Patescibacteria and Firmicutes were relatively enriched. Niche-specific dominance was evident, with Pantoea accounting for 40.79% of the total and 56.27% of the culturable phylloplane bacteriome under cereal monocropping, while Serratia represented 31.59% and 59.40% of the total and culturable cob bacteriomes, respectively. Across niches, mNGS captured substantially greater bacteriome diversity, particularly uncultured and unidentified taxa in soil-associated compartments, whereas culturomics recovered a narrower but functionally accessible fraction. Culturomics yielded 99 isolates representing 32 species across 12 genera, including six genera shared with the mNGS-derived recurrent bacteriome: Bacillus, Enterobacter, Pantoea, Pseudomonas, Serratia, and Stenotrophomonas. Functional screening identified strong biocontrol and plant-beneficial traits among core-associated isolates. Pseudomonas oryzihabitans ZM-DL-PA10 inhibited Rhizoctonia solani, Macrophomina phaseolina, and Bipolaris maydis by up to 40.6%, 43.9%, and 45.2%, respectively, through secreted and volatile metabolites; exhibited P, K, and Zn solubilisation; and produced IAA and siderophores. It also recorded the lowest B. maydis disease index (ADI) of 1.00. Pantoea ananatis ZM-BH-EA4 showed 52.4% and 68.5% inhibition of R. solani and B. maydis, respectively, through volatile metabolites. Collectively, the integration of mNGS and culturomics revealed a strongly compartmentalised maize bacteriome and identified recurrent, culturable, and functionally promising bacterial taxa, providing a targeted resource for microbiome-based crop protection and climate-resilient maize production.

Zea mays↗

PubMed2026

Comprehensive Somatic Profiling of Gastroenteropancreatic Neuroendocrine Neoplasms.

BACKGROUND: The incidence of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) is rising, yet their biological heterogeneity and variable response to treatments remain poorly understood. Comprehensive genomic characterization may uncover somatic drivers and inform biomarker-driven therapeutic strategies. METHODS: We retrospectively analyzed clinically ordered next-generation sequencing (NGS) results from tumor samples of 111 patients with confirmed GEP-NENs treated at Johns Hopkins Hospital between 2020 and 2022. Pathogenic and likely pathogenic mutations were identified using OncoKB, CHASMplus, and COSMIC databases. Mutational patterns were correlated with clinical characteristics and overall survival using univariate and multivariate analyses. RESULTS: In this retrospective study of 111 patients with gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), somatic pathogenic or likely pathogenic mutations were identified in 79% of cases. The most frequent alterations involved TP53 (19%), MEN1 (17%), and chromatin remodeling genes such as DAXX (9%) and ATRX (6%). Notably, we also identified a subset of patients (9%) patients with mutations typically associated with hematologic malignancies. Distinct co-mutation and mutual exclusivity patterns were observed between pancreatic and non-pancreatic NENs. Poorly differentiated or high-grade tumors correlated with mutations in TP53, KRAS, and CDKN2A. Mutations in KRAS, DAXX/ATRX, and hematologic malignancy-associated genes were independently associated with worse overall survival. CONCLUSIONS: This study reveals distinct somatic mutation patterns in GEP-NENs associated with tumor differentiation, grade, primary site, and survival. The identification of hematologic malignancy-associated mutations in a subset of GEP-NENs suggests possible shared molecular phenotypes with poor prognostic implications. The presence of KRAS mutations supports exploring pan-RAS inhibitors as potential therapies in select patients. These findings highlight the clinical utility of genomic profiling in GEP-NENs.

Neuroendocrine neoplasms↗

PubMed2026

Genomic profiling as an option for ovarian cancer diagnostics.

INTRODUCTION: Ovarian cancer (OC) is a highly heterogeneous and lethal gynecological malignancy. Precision oncology has shifted the management paradigm to comprehensive molecular profiling. Genomic-based diagnostics are now a clinical necessity for accurate prognostic stratification and the rational selection of targeted therapeutics, such as PARP and immune checkpoint inhibitors. AREAS COVERED: This review evaluates current literature regarding the distinct genomic landscapes defining OC histotypes to underlined the role of molecular profiling in the diagnostic field of OC. We discuss the practical implementation, technical aspect, and clinical validity of the main molecular diagnostic platforms, focusing on tissue-based Comprehensive Genomic Profiling (CGP) and Homologous Recombination Deficiency (HRD). Furthermore, we explore emerging translational data on liquid biopsy (LBx) applications. EXPERT OPINION: While current tissue-based methodologies provide critical baseline data, the OC diagnostic paradigm must pivot from static testing to proactive and longitudinal tracking. Integrating advanced LBx approaches enables a real-time monitoring of dynamic parameters as minimal residual disease (MRD) and acquired resistance. Integrating these dynamic blood-based assays with multi-omic profiling and artificial intelligence (AI)-driven tools allows a full understanding of the complex tumor behavior.

Humans↗

PubMed2026

Next-Generation Sequencing Completion and Timeliness Using a Reflex Testing Protocol for Patients with Stage II to IV Nonsquamous Non-Small Cell Lung Cancer.

BACKGROUND: Next-generation Sequencing (NGS) is critical for providing treatment recommendations across multiple stages of non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not undergo testing. This study evaluated the completion rates and timeliness of NGS in patients with stage II to IV NSCLC at a single academic institution with a reflex NGS testing protocol. METHODS: Patients with stage II to IV nonsquamous NSCLC (ns-NSCLC) diagnosed between 2015 and 2022 were identified retrospectively. A reflex, tissue-based testing protocol was initiated in 2015 using in-house NGS. Pyrosequencing was performed if NGS failed. RESULTS: 501 patients were included: 75 (15.0%) with stage II, 82 (16.4%) with stage III, and 344 (68.6%) with stage IV ns-NSCLC. Tissue NGS was completed in 380 (75.8%) patients and 465 (92.8%) completed some tissue-based genomic testing when including pyrosequencing. Median time from biopsy to NGS was 17.0 days (range, 6-61 days). 61.0% of patients had NGS results prior to a first treatment of any type and 88.4% had tissue NGS results prior to systemic therapy. Among stage IV patients with completed NGS, median overall survival was 2.27 years for patients with NGS results prior to first treatment compared to 1.08 years for patients without NGS results prior to treatment initiation (P = .04). CONCLUSIONS: Implementation of an in-house, reflex NGS testing protocol enabled rapid genomic profiling in a high proportion of patients with stage II to IV ns-NSCLC. NGS completion prior to receiving first-line therapy was associated with improved survival compared to completion after first line treatment in stage IV patients.

Humans↗

PubMed2025

Molecular features influencing clinical outcome of advanced HER2-positive gastric cancer receiving trastuzumab plus chemotherapy.

BACKGROUND: Less than half of the human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC) patients respond to trastuzumab plus chemotherapy, and the outcomes are unsatisfactory. Understanding the underlying mechanisms remains crucial for identifying patients who are more likely to benefit from treatment. PATIENTS AND METHODS: We performed targeted DNA sequencing on paired pre-treatment and progressive tumour tissues from 22 HER2-positive advanced GC patients undergoing first-line treatment with trastuzumab and chemotherapy. Clinicopathological and genomic characteristics were assessed for the correlation with clinical outcomes. RESULTS: A performance status (PS) of 0-1 was associated with improved progression-free survival (PFS) and overall survival (OS) than a PS of 2. Poorly differentiated tumours exhibited shorter PFS than moderate or moderate-poor ones. Pre-treatment amplification of MYC or TOP2A gene was association with increased PFS, and suggested a potential benefit for OS. Patients with higher tumour mutation burden (TMB) experienced significantly worse PFS, while higher chromosome instability (CIN) appeared to be correlated with longer PFS. Compared to non-responders, responders had a higher CIN but similar TMB and intratumoural heterogeneity (ITH). PS and MYC amplification emerged as independent factors related to PFS according to multivariate survival analysis. Additionally, after treatment, TMB significantly increased in non-responders, while CIN significantly decreased in responders. CONCLUSIONS: Pre-treatment MYC amplification and PS were independently associated with clinical outcomes in HER2-positive advanced GC patients treated with first-line trastuzumab plus chemotherapy. Dynamic post-treatment changes in TMB and CIS provide valuable insights into the relationship between therapeutic response and distinct evolutionary trajectories.

Humans↗

PubMed2025

Chromatin Immunoprecipitation for Standard, Rare, or Weakly Binding Proteins.

Various proteins interact with specific genome regions, playing crucial roles in gene regulation. Chromatin Immunoprecipitation (ChIP) is the most commonly used method to study protein-DNA interactions in vivo. By combining ChIP with high-throughput sequencing, ChIP-seq allows for studying the genome-wide localization of proteins. Although several ChIP protocols are available for plant tissues, they are primarily designed for histone modifications and abundant proteins with high DNA-binding affinity, which are considered as the "standard targets." Here we describe a ChIP protocol for plant tissues not only optimized for the standard targets but also adapted for proteins with low abundances or weak DNA-binding ability. Successful execution of the protocol enables reliable generation of DNA templates for quantitative PCR or libraries for next-generation sequencing, which makes it an effective tool for analyzing genomic interactions of a wide range of proteins.

Chromatin Immunoprecipitation↗

PubMed2026

Programmable enzymes for targeted gene insertion.

Genome editing technologies have advanced from nuclease-based reagents that generate programmed DNA double-strand breaks, which can cause deleterious effects, to next-generation reagents that perform controlled DNA modification through double-strand break-independent mechanisms, such as base editing and prime editing. Although these approaches enable precise small-scale sequence changes, methods for programmable insertion of large DNA cargos have been limited. The ability to write entire genes or large regions into the genome could transform the treatment of genetically heterogeneous disorders, for which numerous pathogenic variants underlie a common disease and mutation-specific editing strategies are impractical. Recent advances in computational genome mining have accelerated the discovery of naturally occurring enzymes with novel biochemical and functional properties, including recombinases and transposases capable of large-scale modifications. Moreover, directed evolution, rational engineering and expanded homologue discovery are enabling the repurposing and optimization of these systems for genome engineering. Here we review recent technology development efforts that harness diverse enzymes for kilobase-scale genome engineering, with a particular focus on CRISPR-associated transposase systems.

Journal Article↗