The Journal of Pathology最新文献

This study explored the complex interactions between glycosylation patterns, tumour biology, and therapeutic responses to temozolomide (TMZ) in human malignant glioma, specifically CNS WHO grade 3 oligodendroglioma (ODG) and glioblastoma (GB). Using spatial imaging of N-glycans in formalin-fixed paraffin-embedded (FFPE) tissue sections via MALDI-MSI, we analysed the N-glycome in primary and recurrent GB tissues and orthotopic xenografts of patient-derived brain tumour-initiating cells (BTIC) sensitive or resistant to TMZ. We identified unique N-glycosylation profiles, with nontumor brain (NTB) and ODG showing higher levels of bisecting and tri-antennary structures, while GB exhibited more tetra-antennary and sialylated N-glycans. Distinctive sialylation patterns were observed, with specific α2,6 and α2,3 isomeric linkages significantly altered in GB. Moreover, comparative analysis of primary and recurrent GB tissues revealed elevated high mannose N-glycans in primary GB and fucosylated bi- and tri-antennary N-glycans in recurrent GB tissues. Next, in the orthotopic xenografts of TMZ-sensitive and TMZ-resistant patient brain tumour initiating cells (BTIC), we identified potential N-glycan markers for TMZ treatment response and resistance. Finally, we found significantly altered expression of genes involved in N-glycan biosynthesis in malignant glioma, highlighting the crucial role of N-glycans in glioma and therapy resistance. This study lays the foundation for developing glycosylation-based diagnostic biomarkers and targeted therapies, potentially improving clinical outcomes for GB patients. © 2025 The Pathological Society of Great Britain and Ireland.

Over 90% of gastrointestinal stromal tumors (GISTs) harbor mutations in KIT or PDGFRA that can predict response to tyrosine kinase inhibitor (TKI) therapies, as recommended by NCCN (National Comprehensive Cancer Network) guidelines. However, gene sequencing for mutation testing is expensive and time-consuming and is susceptible to a variety of preanalytical factors. To overcome the challenges associated with genetic screening by sequencing, in the current study we developed an artificial intelligence-based deep-learning （DL） model that uses convolutional neural networks (CNN) to analyze digitized hematoxylin and eosin staining in tumor histological sections to predict potential response to imatinib or avapritinib treatment in GIST patients. Assessment with an independent testing set showed that our DL  model could predict imatinib sensitivity with an area under the curve (AUC) of 0.902 in case-wise analysis and 0.807 in slide-wise analysis. Case-level AUCs for predicting imatinib-dose-adjustment cases, avapritinib-sensitive cases, and wildtype GISTs were 0.920, 0.958, and 0.776, respectively, while slide-level AUCs for these respective groups were 0.714, 0.922, and 0.886, respectively. Our model showed comparable or better prediction of actual response to TKI than sequencing-based screening (accuracy 0.9286 versus 0.8929; DL model versus sequencing), while predictions of nonresponse to imatinib/avapritinib showed markedly higher accuracy than sequencing (0.7143 versus 0.4286). These results demonstrate the potential of a DL model to improve predictions of treatment response to TKI therapy from histology in GIST patients. © 2025 The Pathological Society of Great Britain and Ireland.

Pancreatic ductal adenocarcinoma (PDAC) tumor interpatient heterogeneity has been well described with two major prognostic subtypes (classical and basal-like). An important intrapatient heterogeneity has been reported but has not yet been studied extensively due to the lack of standardized, reproducible, and easily accessible high-throughput methods. We built an immunohistochemical (IHC) tool capable of differentiating RNA-defined classical and basal-like tumors by selecting relevant antibodies using a multistep process. The successive stages of (i) an in silico selection from a literature review and a bulk transcriptome analysis of 309 PDACs, (ii) a tumor-specific selection from 30 patient-derived xenografts and single-cell data, followed by (iii) the validation on tissue microarrays in 50 PDAC were conducted. We used our final IHC panel on two independent cohorts of resected PDAC (n = 95, whole-slide, n = 148, tissue microarrays) for external validation. After digitization and registration of pathology slides, we performed a tile-based analysis in tumor areas to identify relevant marker combinations. Sequential marker selection led to the following panel: GATA6, CLDN18, TFF1, MUC16, S100A2, KRT17, PanBasal. Four different phenotypes were identified: one classical, one intermediate (KRT17+), and two basal-like (MUC16+ versus S100A2+) with specific biological properties. The presence of a minor basal contingent drastically reduced overall survival [hazard ratio (HR) = 1.90, p = 0.03], even in classical predominant PDACs. Analysis of preneoplastic lesions suggested that pancreatic carcinogenesis might follow a progressive evolution from classical toward a basal through an early intermediate phenotype. In conclusion, our IHC panel redefined and easily assessed the high degree of intra- and intertumoral heterogeneity of PDAC. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Micropapillary colorectal adenocarcinoma is a morphologic subtype of colorectal cancer (CRC) with insufficiently characterized prognostic significance and biological features. We analyzed the histopathological, immunological, and prognostic features of micropapillary adenocarcinoma in two independent CRC cohorts (N = 1,876). We found that micropapillary adenocarcinomas accounted for 4.9% and 6.4% of CRCs in the two cohorts. A micropapillary growth pattern was associated with advanced stage and lymphovascular invasion (p < 0.001), but also with shorter overall survival independent of these factors and other prognostic parameters (Cohort 1: hazard ratio [HR] 1.76, 95% confidence interval [CI] 1.08-2.87; Cohort 2: HR 1.47, 95% CI 1.08-2.00). Multiplex immunohistochemistry and machine learning-assisted image analysis showed that the micropapillary growth pattern was associated with decreased CD3⁺ T-cell and CD14⁺HLA-DR⁺ monocytic cell densities. Molecular features of micropapillary adenocarcinoma were studied using bioinformatic analyses in The Cancer Genome Atlas (TCGA) cohort (N = 629) and validated with optical genome mapping and immunohistochemistry. These analyses revealed that micropapillary adenocarcinomas frequently present with chromosome region 8q24 copy number gain, TP53 mutation, and overexpression of UPK2, MUC16, and epithelial-mesenchymal transition involved genes, such as L1CAM. These results indicate that micropapillary colorectal adenocarcinoma is an aggressive morphologic subtype of CRC characterized by shorter overall survival, decreased antitumorigenic immune response, and unique molecular features. Our findings support the classification of micropapillary adenocarcinoma as a distinct, high-risk subtype of CRC, which should be systematically evaluated in patient care. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

This study investigated the clonal evolution of chronic myelomonocytic leukemia (CMML) progression to secondary acute myeloid leukemia (sAML) by next-generation sequencing and pyrosequencing for variant allele frequency (VAF) of gene mutations and SNP microarray for copy neutral loss of heterozygosity (CN-LOH) in 38 paired samples from CMML/sAML patients of Taiwanese origin. The median interval between CMML and sAML samples collection was 14.9 months (1.0-89.6). RUNX1 (57%), TET2 (46%), SRSF2 (37%), and ASXL1 (28%) mutations were frequent at CMML diagnosis. Baseline VAF in epigenetic regulator genes was high (>35%) in 83% of mutational events at the CMML phase, remained stable in 78% (VAF changes <10%), and increased in 20% (increased VAF > 10%) during progression to sAML. Transcription factor genes showed high VAF (>35%) in 51% at the CMML phase, and stable VAF in 60% during progression. VAF of spliceosome genes was high (>35%) in 70% at CMML phase, and stable in 61% during progression. Activated signaling genes exhibited acquisition or loss during progression. TET2 mutations were often founding clones, and SRSF2, ASXL1, DNMT3A, EZH2, or spliceosome genes also acted as ancestral mutations. RUNX1 mutations were typically later events and occasionally ancestral hits or germline mutations. Acquisition of cytogenetic changes, signaling pathways genes (PTPN11, FLT3, NRAS, CBL), or AML-defined genes (NPM1, CEBPA, CBFB::MYH11) by linear or branching evolution occurred during sAML progression. CN-LOH was noted in EZH2, CBL, TET2, and DNMT3A genes. CEBPA mutation and concurrent biallelic TET2 with NRAS mutations at CMML diagnosis were risk factors for time to AML progression and overall survival. A characteristic ASXL1^MT/RUNX1^MT/Spliceosome^MT/signaling^WT genetic profile was associated with monocyte counts of 0.5-1.0 × 10⁹/l. This study highlights the complexity and heterogeneity of dynamic changes in clonal architecture during CMML progression, emphasizing its importance in pathogenesis, phenotype, risk stratification, and therapeutic strategy. © 2025 The Pathological Society of Great Britain and Ireland.

Pancreatic ductal adenocarcinoma (PDAC) often arises from preexisting cystic lesions such as intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN). This study investigated the molecular heterogeneity and mutational landscape of MCN in relation to PDAC, highlighting the significance of KRAS mutations in tumor progression. Utilizing targeted next-generation sequencing on low-grade MCN and invasive PDAC samples, we identified a substantial overlap in mutational profiles, particularly mutations in KRAS, TP53, and FBXW7. Specifically, 69.2% of MCN exhibited somatic mutations, with KRAS mutations being a predominant oncogenic driver. The characterization of mutant versus wildtype KRAS variant allele frequencies (VAF) indicated higher mutation levels in PDAC compared to MCN, suggesting an evolutionary trajectory toward malignancy. Further histological analysis of 12 additional MCN cases revealed significant intratumor heterogeneity, with variant KRAS mutation distributions correlating with distinct cellular morphologies and dysplastic features. Additionally, we explored the potential of liquid biopsies, demonstrating a concordance rate of 71.4% for KRAS mutation detection in circulating tumor DNA (ctDNA) relative to tissue biopsies across cohorts. Our findings underscore the relevance of evaluating KRAS mutations-herein referred to as VAF per microdissected region-as they relate to histopathological markers of dysplasia, contributing to improved stratification of pancreatic lesions and facilitating personalized treatment strategies. In conclusion, this comprehensive analysis of MCN highlights the importance of KRAS as a crucial biomarker for both malignant progression and therapeutic decision-making in pancreatic pathology. Ultimately, our study suggests that characterizing the mutational landscape and histological features of MCN can enhance early detection and intervention strategies for at-risk patients. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ferroptosis has been characterised by disruption of the cell membrane through iron-related lipid peroxidation. However, regulation of iron homeostasis in lung cancer cells that are resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains unclear. Transcriptome analysis identified a significant downregulation of apoptosis-associated tyrosine kinase (AATK) mRNA expression in gefitinib-resistant PC9 (PC9-GR) cells, which were found to be more susceptible to ferroptosis inducers. An in-depth analysis of publicly available datasets revealed that downregulation of AATK mRNA was associated with lymph node metastasis and poor prognosis in patients with lung adenocarcinoma. Knockdown of AATK-sensitised PC9, HCC827, and H441 cells to the ferroptosis inducer RSL3, whereas ectopic expression of AATK reduced RSL3-induced cell death in PC9-GR and HCC827-GR cells. Compared to PC9 cells, PC9-GR cells exhibited higher transferrin uptake, endosome recycling rate, and increased intracellular iron levels. Blocking iron transport reduced RSL3-induced ferroptosis in PC9-GR cells. Mechanistic studies showed that AATK localised to both early and recycling endosomes. Knockdown of AATK facilitated endosome recycling and elevated intracellular ferrous iron (Fe²⁺) levels in PC9 cells. Conversely, ectopic expression of AATK delayed endosome recycling and reduced intracellular Fe²⁺ levels in PC9-GR cells. Inhibition of AATK downregulation-induced iron accumulation decreased RSL3-induced ferroptosis. Taken together, our study indicates that the downregulation of AATK contributes to endosome recycling and iron accumulation, leading to an increased susceptibility to ferroptosis in EGFR-TKI-resistant lung cancer cells. © 2025 The Pathological Society of Great Britain and Ireland.

The impact of high heterogeneity of cancer-associated fibroblasts (CAFs) on triple-negative breast cancer (TNBC) immunotherapy response has not been fully elucidated, restricting progress in precision immuno-oncology. We integrated single-cell transcriptomic data from 18 TNBC patients and analyzed fibroblast subpopulations. Extracellular matrix CAFs (ecmCAFs) were identified as a fibroblast subpopulation with distinct ECM-associated characteristics. The ecmCAFs were significantly enriched in TNBC patients with residual disease after neoadjuvant immunotherapy and contributed to a fibrotic tumor microenvironment and T-cell exclusion. Secreted phosphoprotein 1 (SPP1) positive macrophages (SPP1⁺ Mφs) were closely localized to ecmCAFs and produced more transforming growth factor beta (TGFB1), interleukin 1 beta (IL1B), and SPP1 under hypoxic conditions. SPP1⁺ Mφs were found to facilitate the differentiation of normal breast fibroblasts to ecmCAFs, thus promoting ECM remodeling and stromal fibrosis. Our work revealed the critical role of ecmCAFs in generating a desmoplastic architecture and driving immunosuppression in TNBC. © 2025 The Pathological Society of Great Britain and Ireland.

Spatial transcriptomics (ST) offers enormous potential to decipher the biological and pathological heterogeneity in precious archival cancer tissues. Traditionally, these tissues have rarely been used and only examined at a low throughput, most commonly by histopathological staining. ST adds thousands of times as many molecular features to histopathological images, but critical technical issues and limitations require more assessment of how ST performs on fixed archival tissues. In this work, we addressed this in a cancer-heterogeneity pipeline, starting with an exploration of the whole transcriptome by two sequencing-based ST protocols capable of measuring coding and non-coding RNAs. We optimised the two protocols to work with challenging formalin-fixed paraffin-embedded (FFPE) tissues, derived from skin. We then assessed alternative imaging methods, including multiplex RNAScope single-molecule imaging and multiplex protein imaging (CODEX). We evaluated the methods’ performance for tissues stored from 4 to 14 years ago, covering a range of RNA qualities, allowing us to assess variation. In addition to technical performance metrics, we determined the ability of these methods to quantify tumour heterogeneity. We integrated gene expression profiles with pathological information, charting a new molecular landscape on the pathologically defined tissue regions. Together, this work provides important and comprehensive experimental technical perspectives to consider the applications of ST in deciphering the cancer heterogeneity in archived tissues. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

DICER1-associated sarcoma is an emerging entity, defined by either somatic or germline dicer 1, ribonuclease III (DICER1) mutations and sharing characteristic morphologic features irrespective of the site of origin. In addition to the DICER1 driver mutation, concurrent genomic alterations, including tumor protein 53 (TP53) inactivation and RAS pathway activation, are frequently detected. Tumors that morphologically resemble malignant peripheral nerve sheath tumor (MPNST) have rarely been reported among DICER1 sarcomas and often pose diagnostic challenges. This study was prompted by a case involving morphologic features of MPNST, which harbored co-existing DICER1 and hotspot KRAS mutations. Hence, we investigated the incidence of these alterations in PNST from our molecular database compared to the genomic and morphologic spectrum of DICER1-mutant sarcomas. In total, we identified three cases diagnosed as MPNST with co-existing DICER1, ATRX chromatin remodeler (ATRX), and KRAS G12V/A alterations occurring in brain, cerebellopontine angle, and intra-abdominal sites. Two additional cases each of MPNSTs and neurofibromas were identified with hotspot KRAS mutations. All five MPNSTs lacked canonical neurofibromin 1 (NF1)/neurofibromin 2 (NF2) alterations, displaying a classic morphologic appearance with fascicular monomorphic spindle cells and followed a diverse clinical behavior. Among the 38 DICER1-associated sarcomas in our database, eight (21%) had secondary KRAS hotspot mutations, all composed of monomorphic spindle and/or round cells, including three with an MPNST-like histology. In contrast, all 10 (26%) DICER1-mutant sarcomas with TP53 mutations showed a pleomorphic phenotype. The DNA-based methylation profile of our index case clustered within the group of sarcomas with DICER1 alterations. Our results highlight a small subset of MPNST associated with DICER1 and/or KRAS mutations. However, their relationship with conventional MPNST remains to be determined in larger studies. © 2025 The Pathological Society of Great Britain and Ireland.