Pancreatic ductal adenocarcinoma (PDAC) with anaplastic features is a rare and highly aggressive subtype with an extremely poor prognosis. However, the molecular mechanisms underlying anaplastic transformation remain poorly understood. To characterize the anaplastic transformation molecular landscape, we analyzed genomic alterations in 5,997 conventional PDAC and 41 anaplastic carcinoma cases from the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) cohort. Additionally, four PDAC samples from the Kyorin cohort with both tubular and anaplastic components were subjected to spatial transcriptomics using the Visium platform. Differential gene expression and pathway enrichment analyses were performed to identify key molecular drivers. Genomic analysis revealed higher KRAS (90 versus 75%), TP53 (85 versus 60%), and CDKN2A (50 versus 30%) mutation frequencies, but notably fewer SMAD4 mutations (5 versus 20%; p = 0.022), in anaplastic carcinoma than in conventional PDAC. Spatial transcriptomics identified distinct molecular signatures, with epithelial-mesenchymal transition (EMT) pathways significantly enriched in anaplastic regions. Levels of ELF3, an epithelial transcription factor, were consistently downregulated in all anaplastic components. Importantly, we detected a strong inverse correlation between SMAD4 and ELF3 expression in both the bulk RNA sequencing cohort and spatial transcriptomic data. Functional validation using the KC-shSmad4 mouse model demonstrated that increased Smad4 suppressed Elf3 expression and promoted mesenchymal reprogramming. Our findings suggest a novel SMAD4-ELF3 regulatory axis that may contribute to anaplastic transformation in PDAC. The retention of SMAD4 functions coupled with ELF3 downregulation may promote EMT and contribute to tumor dedifferentiation. This SMAD4-ELF3 regulatory axis may provide a framework for identifying therapeutic vulnerabilities in this aggressive PDAC variant and further highlight the utility of spatial transcriptomics for understanding tumor heterogeneity. © 2026 The Pathological Society of Great Britain and Ireland.

Pretreatment prediction of neoadjuvant response remains an unresolved challenge in locally advanced rectal cancer, where management is moving towards risk-adapted and response-oriented treatment strategies. Prediction remains difficult because response reflects interacting tumour-intrinsic, immune, stromal, and spatial processes that are incompletely captured by standard clinical and radiological assessment. From a technology-centred perspective, in this Review we assess how molecular, spatial, image-based, and artificial intelligence-enabled approaches are being used to study and predict response to neoadjuvant treatment from pretreatment biopsies and how readily they may be translated into clinical workflows. Genomic profiling has its clearest current clinical role in identifying hypermutated immune-responsive subgroups, whereas broader genomic, epigenomic, and immune-repertoire markers remain largely investigational. Bulk transcriptomics has generated multiple response signatures with variable reproducibility but has consistently highlighted immune activation, interferon signalling, stromal remodelling, and epithelial-mesenchymal transition as recurrent response-associated programmes; its most feasible clinical route is likely through targeted expression panels rather than open-ended transcriptome profiling. Higher-resolution approaches such as spatial transcriptomic and proteomic platforms refine these observations by localising signals to tumour, immune, and stromal compartments and capturing therapy-induced tissue reorganisation, but they are currently best positioned for deepening biological understanding and nominating rational treatment combinations for future testing. Established platforms such as digital pathology are closer to routine deployment as they build on clinically embedded workflows, although response-prediction algorithms still require robust external validation, calibration, and regulatory-grade implementation. Patient-derived models provide functional systems for testing candidate biomarkers and resistance mechanisms. Multimodal artificial intelligence may support response prediction by identifying complementary signals across data types and distilling complex signatures into parsimonious models suitable for clinical translation. Progress will depend on harmonised, longitudinal, multi-centre studies that couple biological depth with assay standardisation, model reduction, and prospective evidence that response predictors improve patient management. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Calcium oxalate nephrolithiasis is commonly viewed as a consequence of urinary supersaturation, although crystal retention and renal injury are also influenced by gut microbiota dysbiosis and local tissue environment. In a recent study by Li et al, published in The Journal of Pathology, the authors integrated human metagenomic and metabolomic analyses with mouse and tubular cell models to identify indole-3-propionic acid (IPA) as a key gut-derived metabolite associated with kidney stone disease. Patients with calcium oxalate stones showed altered gut microbial composition and reduced serum IPA levels, which were inversely related to stone burden. In experimental models, IPA supplementation reduced renal crystal deposition, preserved kidney function, and limited tubular injury and apoptosis. Mechanistic studies showed that IPA activated aryl hydrocarbon receptor (AhR) in tubular epithelial cells, increased suppressor of cytokine signaling 3 (SOCS3) expression, and suppressed signal transducer and activator of transcription 1 (STAT1) phosphorylation and nuclear accumulation. These findings were supported by complementary activation, inhibition, knockdown, and overexpression experiments. Collectively, the data suggest that IPA protects renal epithelial cells through the AhR-SOCS3-STAT1 signaling pathway and may offer a potential therapeutic approach for calcium oxalate nephropathy. © 2026 The Pathological Society of Great Britain and Ireland.

Calcium oxalate (CaOx) nephropathy is a highly prevalent urological disease worldwide. Gut microbiota dysbiosis and host metabolic dysregulation are recognized as pivotal drivers in disease pathogenesis, yet the underlying mechanisms remain incompletely understood. In this study, by integrating metagenomics and metabolomics, we identified dysregulation of tryptophan metabolites in patients with CaOx nephrolithiasis, with microbiota-derived indole-3-propionic acid (IPA) as the most discriminatory differential metabolite. Oral IPA supplementation markedly reduced renal CaOx crystal deposition and tubular injury in a CaOx nephropathy murine model. Mechanistically, IPA activated the aryl hydrocarbon receptor (AhR), which translocated to the nucleus and transcriptionally upregulated suppressor of cytokine signaling 3 (SOCS3)-a negative regulator of signal transducer and activator of transcription 1 (STAT1)-thereby suppressing STAT1 phosphorylation, alleviating oxalate-induced tubular cell injury and apoptosis, and inhibiting CaOx crystal deposition. Our findings identify tryptophan metabolite alteration as a critical metabolic signature of CaOx nephropathy and demonstrate that microbiota-derived IPA attenuates oxalate-induced renal tubular cell injury and apoptosis via the AhR-SOCS3-STAT1 axis. © 2026 The Pathological Society of Great Britain and Ireland.

Propionic acidemia (PA) is a neurometabolic disorder caused by propionyl-CoA carboxylase deficiency with frequent neurological involvement, yet cell-type-specific mechanisms remain poorly defined. We established human induced pluripotent stem cell (iPSC)-derived astrocytes (iAs) from patients harboring PCCA or PCCB mutations and investigated functional and molecular alterations associated with PA. Both control- and patient-derived iPSCs yielded astrocyte-enriched cultures through a neural progenitor intermediate, with expression of astrocyte-associated markers. Patient iAs showed impaired mitochondrial respiration (lower basal and maximal oxygen consumption and ATP-linked respiration), together with reduced high-affinity uptake of glutamate and glycine neurotransmitters and evidence of disturbed Ca[2+] homeostasis. They also migrated faster in wound-healing assays and displayed reactive-like features, including increased GFAP and connexin 43 (Cx43) protein levels and elevated AQP4 mRNA levels. Targeted miRNA profiling revealed upregulation of miR-124-3p, miR-9-5p, and miR-125b-5p, concomitant with downregulation of miR-146a-5p, miR-155-5p, and miR-145-5p. Consistent with these changes, reported target genes associated with innate immune signaling (IRAK1, TRAF6, TAB2), astrocyte remodeling (GFAP), and water/ion homeostasis (AQP4) were upregulated. Inflammatory and proliferative markers were likewise increased, including IL1B, TNF, and MKI67. Together, these convergent alterations suggest a phenotype characterized by impaired homeostatic functions and reactive-like features, which may heighten neural network vulnerability under metabolic stress. Collectively, our data support astrocyte dysfunction as a potential contributor to PA neuropathology and highlight patient-derived iAs as a useful model for future mechanistic studies and therapeutic exploration. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

There is increasing evidence to suggest that dysregulated macrophages play an important role in the promotion and maintenance of inflammatory bowel disease (IBD), yet the underlying mechanisms remain unclear. A recent study published by Lv, Xia, Qiao et al in The Journal of Pathology investigated the role of Mas-related G protein-coupled receptor D (MrgprD) as a surrogate for IBD using a dextran sulfate sodium-induced colitis mouse model. Through a series of experiments using Mrgprd knockout mice, the authors demonstrated that MrgprD drives colitis through its expression in macrophages, promoting M1 macrophage polarization via NF-κB signaling. They also showed that colitis was primarily driven by myeloid-specific rather than neuronal-specific MrgprdD expression. Finally, analysis of genomic data from patients with IBD supported the translational relevance of these findings to human disease. However, their findings require further verification. If confirmed, MrgprD could emerge as a promising treatment target. In any case, macrophages are receiving increasing attention, and pathologists may soon need to consider their assessment in their reporting. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Inflammatory bowel disease (IBD) is characterized by aberrant immune responses in the gut. Mas-related G protein-coupled receptor D (MrgprD), which is primarily expressed in sensory neurons of the dorsal root ganglia (DRG), is known to promote neuroinflammation. However, its potential role in driving intestinal inflammation remains undefined. Here, we show that systemic Mrgprd KO alleviated dextran sodium sulfate (DSS)-induced acute colitis. We found that MrgprD was expressed in colonic macrophages and DSS treatment increased its expression. Mrgprd global deletion reduced macrophage infiltration in the colon and suppressed their pro-inflammatory M1 polarization following DSS challenge. Using conditional KO mouse lines, we further demonstrate that myeloid-specific Mrgprd deletion conferred stronger protection than its neuronal deletion. This is evidenced by preserved colon length, reduced histopathology scores, improved barrier integrity, and decreased M1 macrophage proportions. Notably, neuron-specific, but not myeloid-specific, Mrgprd KO accelerated intestinal motility under physiological conditions. MrgprD deficiency reduced M1-associated gene expression and suppressed NF-κB signaling activation in both colonic lamina propria cells and bone marrow-derived macrophages (BMDMs), whereas activation of MrgprD by its agonist enhanced these responses in BMDMs. Bioinformatic analysis further supports the involvement of the MRGPRD-NF-κB signaling axis in human IBD. Collectively, our findings identify MrgprD as a novel driver of acute colitis, acting primarily through NF-κB-mediated pro-inflammatory macrophage polarization in the colon. © 2026 The Pathological Society of Great Britain and Ireland.

TP53 is the most frequently mutated gene in oral squamous cell carcinoma (OSCC); however, its role in promoting epithelial-mesenchymal transition (EMT) and tumour progression remains unclear, particularly within the context of intra-tumour heterogeneity. We performed multi-regional whole-exome sequencing (WES) on 43 tumour regions from nine OSCC patients, including matched lymph node metastases. p53, epithelial, and mesenchymal markers were assessed by conventional and multiplexed immunofluorescence (MxIF), followed by digital image analysis using HALO software. EMT-associated gene expression was quantified by RT-qPCR. In vitro assays were performed in epithelial cell lines transiently transfected with the hotspot mutant p53R248Q. TP53 mutations were detected in 7 of 9 patients (77.8%), predominantly as missense mutations in the DNA-binding domain. Mutant tumours exhibited strong nuclear and cytoplasmic p53 accumulation, accompanied by reduced E-cadherin and increased vimentin expression. MxIF revealed a higher proportion of partial EMT-like cells (PCK[+] vimentin[+]) in mutant tumours (p < 0.05). RT-qPCR showed upregulation of VIM, SLUG, ZEB1, and ZEB2 in TP53-mutant tumours. In vitro, p53R248Q expression increased N-cadherin levels, disrupted E-cadherin membrane localisation, and altered F-actin organisation at the junctions, consistent with EMT-associated phenotypic changes. Collectively, our findings suggest that TP53 mutations, particularly p53R248Q, are associated with partial EMT activation in OSCC. These findings highlight a potential link between TP53 mutations and EMT-related changes in OSCC, suggesting that mutant p53 may contribute to tumour phenotypic plasticity. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The prognostic and predictive impact of TP53 variants in leukemia led to their inclusion in diagnostic and treatment guidelines, increasing the demand for rapid, reliable laboratory analysis, interpretation, and reporting. While most TP53 variants identified in tumor samples can be interpreted using data from large-scale functional studies, evidence regarding the impact of in-frame deletions and insertions not disturbing the reading frame remains limited. Consequently, the classification of somatic in-frame variants often relies on expert judgment rather than standardized criteria. To address this gap, we collected in-frame TP53 variants identified during routine diagnostic testing in laboratories within the TP53 Network of Education and Research Initiative on Chronic Lymphocytic Leukemia (ERIC). We analyzed 60 leukemia samples from 13 laboratories, comprising 54 distinct variants. Initial classification using existing data and interpretation guidelines assigned these variants predominantly to the category of variants of uncertain significance. Functional analyses using a yeast-based transactivation assay, complemented by testing selected variants in the TP53-knockout RPE1 cell line, showed a nonfunctional phenotype for the vast majority of in-frame TP53 variants. This indicates that most in-frame alterations detected in leukemia samples are functionally deleterious, as further supported by their association with second-allele inactivation and frequent clonal expansion in relapse. With respect to variant localization, we observed that 49 variants located within the core region of the DNA-binding domain showed complete loss of transactivation function. The five variants located downstream of codon 286 exhibited heterogeneous functional phenotypes depending on their localization and the experimental system used. In conclusion, the integration of our findings with published functional data supports the interpretation that in-frame TP53 variants within the core DNA-binding domain identified in tumor samples are likely pathogenic. Conversely, interpretation of less frequent in-frame variants outside this region requires integration of multiple evidence, including functional studies, published literature, and curated variant databases. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Prostate cancer (PCa) is typically immunologically 'cold', which limits the effectiveness of immunotherapy, and this study sought to identify key genes driving this immunosuppressive phenotype and assess their therapeutic potential. We analyzed transcriptomic data from 487 TCGA-PRAD patients and three Gene Expression Omnibus datasets; weighted gene co-expression network analysis was used to identify cancer-related genes, TIMER to evaluate their influence on the tumor microenvironment, and findings were validated via flow cytometry on 32 clinical PCa specimens. Immunoblotting measured Nrf2, p-AKT, and p-GSK-3β levels, while ChIP-qPCR assessed Nrf2 and RNA polymerase II binding to the CX3CL1 promoter, and functional studies were conducted in allograft and xenograft mouse models using GapmeR antisense oligonucleotides combined with anti-PD-1 antibodies or PSMA-targeted CAR-T cells. We found PPP1R14B (PP1 regulatory inhibitor subunit 14B) was overexpressed in PCa and associated with poor prognosis, promoting PCa cell proliferation, suppressing apoptosis, and correlating with increased tumor purity but reduced CD8[+] T-cell infiltration; mechanistically, CX3CL1 was regulated by Nrf2 and PPP1R14B elevated p-AKT/p-GSK-3β to stabilize Nrf2, which bound the CX3CL1 promoter, suppressed its expression, and impaired CD8[+] T-cell chemotaxis. GapmeR-mediated PPP1R14B knockdown suppressed tumor growth and enhanced anti-PD-1/CAR-T efficacy in mice, indicating PPP1R14B is a promising target to overcome PCa immunotherapy resistance. © 2026 The Pathological Society of Great Britain and Ireland.

Lactate has emerged from being viewed as a glycolytic byproduct to a central metabolic and signaling hub that coordinates tumor evolution and therapeutic adaptation. This review integrates current evidence on how lactate production, transport, and accumulation reshape cancer biology, with particular emphasis on the lactate-lactylation axis. In tumors, lactate is generated not only by malignant cells through aerobic glycolysis, hypoxia-driven metabolic rewiring, and glutamine-derived carbon flow, but also by stromal cells, immune cells, adipocytes, and tumor-associated microbiota. Through monocarboxylate transporter-mediated shuttling, lactate supports metabolic symbiosis, extracellular acidification, matrix remodeling, angiogenesis, invasion, and immune escape. Beyond these metabolic and signaling functions, lactate acts as a substrate for lysine lactylation, linking altered metabolism to epigenetic and post-translational regulation. Histone and nonhistone lactylation, governed by emerging writers, erasers, and putative readers, regulates transcription, DNA damage repair, cancer stemness, ferroptosis resistance, immune checkpoint expression, and resistance to chemotherapy, radiotherapy, targeted therapy, and immunotherapy. We further discuss the biomarker potential of lactate-related enzymes, transporters, site-specific lactylation marks, and therapeutic strategies targeting lactate production, transport, sensing, depletion, and lactylation machinery. A deeper understanding of lactate flux and lactylation-dependent vulnerabilities may enable biomarker-guided combinations that integrate metabolic intervention, epigenetic modulation, and immuno-oncology for precision cancer therapy. © 2026 The Pathological Society of Great Britain and Ireland.

Accurate detection of lymph node (LN) metastases is critical for prognosis and surgical decision-making in head and neck squamous cell carcinoma (HNSCC) and other epithelial cancers. Standard histopathology examines only a small fraction of LN tissue, limiting sensitivity for micrometastases and isolated tumour cells. Scalable approaches are needed to improve staging accuracy and reduce overtreatment. We adapted the FDA-approved CELLSEARCH System, originally developed for circulating tumour cell detection, for use on dissociated 50 μm formalin-fixed paraffin-embedded (FFPE) LN sections. Tumour cells were enriched via EpCAM-based immunomagnetic capture, stained with cytokeratin (CK)/DAPI and quantified using the ACCEPT (Automated CTC Classification, Enumeration and Phenotyping Technology) image analysis tool. Specificity and reproducibility were tested in 50 metastasis-positive and 50 metastasis-free LNs from patients with HNSCC. Enriched cells underwent single-cell and pooled whole-genome amplification (WGA), quality-controlled by a genomic integrity index and profiled for copy number alterations (CNA) by low-pass whole-genome sequencing. Tumour cells were detected in 100% of LN metastases (range: 37-12,479), with counts strongly correlating with histological tumour area (p < 0.0001). Rare background events in control LNs were excluded by manual CELLSEARCH review. Based on their frequency, a threshold of > 12 cells was defined to classify samples as positive, yielding an assay specificity and sensitivity of 100%. Reproducibility across adjacent sections was high. Despite FFPE preservation, WGA succeeded in 27% of single cells and > 75% of pooled fractions. CNA profiling yielded 50 high-quality tumour genomes, revealing typical HNSCC alterations and marked inter-/intra-patient heterogeneity. This adapted CELLSEARCH workflow enables sensitive detection and genomic profiling of tumour cells from FFPE LN tissue. Demonstrated in HNSCC as a proof of concept, the approach is broadly applicable to epithelial cancers where LN staging is prognostically decisive. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The cellular composition and disease susceptibilities of the distinct zones of the human prostate remain incompletely understood. Benign prostatic hyperplasia (BPH) is a common condition that causes widespread morbidity and is nearly exclusively localized to the transition zone (TZ). Through extensive single-cell RNA sequencing (scRNA-seq) of benign regions from prostatectomy specimens, we identified a basal cell population expressing WIF1, VCAN, and NRG1, among other genes, that was significantly enriched in the TZ. Analysis of previously published scRNA-seq datasets further confirmed that WIF1[+] basal cells were significantly enriched in BPH compared with normal prostate. Pathway and cell-cell communication analyses revealed that this basal subtype is associated with programs related to cell proliferation, epithelial-mesenchymal transition, immune regulation, angiogenesis, and hormone response. Together, the molecular signature, zonal distribution, and pathway enrichment suggest that TZ-enriched WIF1[+] basal cells may contribute to BPH pathogenesis by promoting epithelial and stromal remodeling. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Germline variations contribute to esophageal squamous cell carcinoma (ESCC) susceptibility. We identified a germline deletion (exons 7-8) in HIF3A in an ESCC family and investigated its functional impact using CRISPR/Cas9-engineered cells and Hif3a-eKO1 mice (heterozygous for exons 7-8 deletion). Multi-omics analysis of Hif3a-eKO1 and WT mice revealed dysregulated pathways in normal esophagus and during 4NQO-induced carcinogenesis, with key biomarkers validated by immunohistochemistry. Hif3a deficiency enhanced ESCC cell proliferation and invasion in vitro and accelerated 4NQO-induced tumorigenesis in vivo, with Hif3a-eKO1 mice developing more and larger neoplastic lesions. Multi-omics analysis revealed downregulation of cytokeratin-related genes (notably Krt17) and γδ T cells in normal esophagus of Hif3a-eKO1 compared with WT. Consistently reduced Krt17 expression in Hif3a-eKO1 was confirmed by both esophageal immunohistochemistry and cellular Western blot analyses. During 4NQO-induced carcinogenesis, Hif3a deficiency upregulated DNA damage response markers, including Krüppel-like factor 4 (Klf4) and ATR serine/threonine kinase (Atr). Notably, epithelial cells with abundant γH2AX foci lacked Krt17 expression, while Krt17-positive cells showed minimal γH2AX foci. Heterozygous germline Hif3a deletion (exons 7-8) may promote ESCC by disrupting esophageal barrier function-impairing Krt17-mediated epithelial integrity and reducing γδ T cells-while exacerbating genomic instability. These findings reveal ESCC predisposition mechanisms and therapeutic targets. © 2026 The Pathological Society of Great Britain and Ireland.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor originating from calcitonin producing C-cells and accounts for 1-5% of thyroid cancers. Total thyroidectomy is curative in localized disease (N0), whereas lymph node metastases (N1) are associated with poorer prognosis. However, the molecular mechanisms driving the metastatic shift remain poorly understood. This study aimed to identify miRNA features linked to metastatic spread in MTC, focusing on the transition from N0 to N1. Co-expression networks were constructed for N0 and N1 tumors, and differential connectivity analysis was used to identify key miRNAs acting as regulatory hubs. Functional annotation of their target genes was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Reactome pathway analyses. Validation experiments were carried out in MTC cells to evaluate the effects of selected miRNAs on cell proliferation, survival, and MAPK pathway activation. Network analysis revealed distinct miRNA co-expression patterns between N0 and N1 tumors. Differential network analysis highlighted miR-145-3p as a central regulatory hub, exhibiting 29 altered co-expression changes and a marked loss of connectivity in N1. Target enrichment identified 59 validated genes, including key oncogenic drivers such as MYC, PTEN, BCL2, PIK3CA, AKT1, and MAPK7. In MTC cells, simultaneous inhibition of miR-145-3p together with its top co-expressed miRNAs increased proliferation and survival, and enhanced ERK phosphorylation, indicating MAPK pathway activation and a shift toward a more aggressive phenotype. In conclusion, this study identifies a miRNA regulatory hub centered on miR-145-3p that is associated with metastatic progression and highlights the value of network-based approaches in uncovering mechanisms of cancer dissemination. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.