The Journal of Pathology最新文献

Splicing dysregulation is a relevant mechanism of pathogenicity for variants in disease susceptibility genes. Variants affecting the critical intronic +1 and +2 GT nucleotides of the 5’ splice sites (5'ss) are generally strong indicators of pathogenicity. However, some +2 T variants create functional noncanonical 5'ss that generate wildtype transcripts, hampering accurate variant interpretation and genetic counseling. We previously showed that variants PALB2 c.108+2T > C and ATM c.1898+2T > G generated significant levels of full-length (FL) transcripts by creating functional atypical GC and GG donor sites, respectively. In this study, we aimed to investigate the splicing impact of +2T variants in the breast cancer susceptibility genes ATM, BRCA1, and PALB2. For this purpose, five minigenes encompassing 29 exons of ATM, BRCA1, and PALB2 were employed. A total of 30 +2T > C/G/A variants were introduced into these constructs by site-directed mutagenesis and analyzed in MCF-7 cells. Four +2T > C variants (ATM c.6347+2T > C, BRCA1 c.5193+2T > C and c.5277+2T > C, and PALB2 c.2748+2T > C) and ATM variants c.6347+2T > A/G produced FL-transcripts (4%–81% of the overall expression). All +2T > C leaky variants conserved a central core of 6 nucleotides (AGgcaa). Variants were assessed according to the ClinGen specifications of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) interpretation guidelines. Two variants (ATM c.6347+2T > C and BRCA1 c.5193+2T > C) were classified as likely benign, consistent with predictions based on their respective ACMG/AMP-based gene specifications. Conversely, two variants (ATM c.6347+2T > G and BRCA1 c.4675+2T > C), initially predicted as likely pathogenic, were reclassified as variant of uncertain significance (VUS). In conclusion, a significant proportion of +2T variants can create functional noncanonical 5'ss, resulting in the production of FL-transcripts that may preserve gene function. Variant-splicing assays provide essential data for accurate clinical classification and for the development of effective clinical management strategies for patients and their families. © 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.

Exploiting vulnerabilities in switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complexes for cancer therapy is a promising therapeutic strategy. The SWI/SNF chromatin remodeling complex acts as a regulatory component of transcription, and our previous study found an immune-active microenvironment and better response to immunotherapy of gastric cancer with ARID1A loss. However, little is known about the clinical significance of SMARCA4, which encodes for another subunit of the SWI/SNF complex, in gastric cancer (GC) patients. This study analyzed the association of SMARCA4 status with clinicopathological features, survival outcomes, therapeutic response, and immune microenvironment characteristics in three independent cohorts: Zhongshan Hospital (ZSHS) cohort (n = 442), Zhongshan Hospital immune checkpoint blockade (ZSHS-ICB) cohort (n = 41), and Samsung Medical Center cohort (SMC, n = 51). SMARCA4-deficient GC patients exhibit clinicopathological features associated with enhanced tumor aggressiveness, including a higher prevalence of poorly differentiated disease (p = 0.034), pN3 stage at diagnosis (p = 0.059), E-cadherin negative expression (p < 0.001), and genomically stable (GS) and microsatellite stable/epithelial–mesenchymal transition molecular subtype (MSS/EMT) (p < 0.001 and p < 0.001, respectively). Kaplan–Meier analysis revealed that SMARCA4 deficiency indicated poor prognosis in GC (p < 0.001). Moreover, SMARCA4 deficiency identified a subgroup of GC patients who exhibited poor outcomes despite receiving adjuvant chemotherapy in the GS subtype (p = 0.029). In contrast, these patients demonstrated increased sensitivity to anti-PD-1 therapy in both the ZSHS-ICB (p = 0.039) and SMC (p = 0.062) cohorts. Immunological analysis revealed a distinct immune profile characterized by abundant but exhausted CD8⁺ T cells in SMARCA4-deficient GC. In conclusion, patients with SMARCA4-deficient GC patients demonstrated poor prognosis but improved response to immunotherapy. These observed clinical outcomes may be attributed to the immunosuppressive microenvironment, highlighting the potential for developing novel therapeutic approaches. © 2025 The Pathological Society of Great Britain and Ireland.

Proliferative vitreoretinopathy (PVR) represents a common and challenging complication leading to blindness following ocular injury. The prevailing view, which guides current drug development efforts, posits that the mesenchymal transition of retinal pigment epithelium (RPE) underlies the pathogenesis of PVR. However, in our study, by employing single-cell sequencing and immunofluorescence staining on surgically excised PVR membranes, we demonstrate that PVR exhibits distinct cytopathological characteristics depending on their retinal location. Specifically, epiretinal PVR predominantly comprises macrophages, whereas subretinal PVR is primarily constituted of PMEL⁺ RPE-derived cells. Both molecular pathological phenotypes are unified by retinal hypoxia following injury, yet they diverge in their downstream hypoxic pathway selection. Targeting HIF1α-regulated glycolysis selectively reduced epiretinal PVR formation, while inhibiting reactive oxygen species production specifically abrogated subretinal PVR. Furthermore, the application of hyperoxia chamber in a mouse model of dispase-induced retinal injury effectively eradicated PVR across all retinal regions and restored retinal morphology. Our findings establish hypoxia-induced regional heterogeneity as a pathological mechanism in PVR progression and advocate for anatomically targeted therapeutic strategies. © 2025 The Pathological Society of Great Britain and Ireland.

Synchronous/multifocal gastric-type lesions (SMGLs) of the female genital tract are heterogeneous diseases that are rare and challenging to diagnose. The core issue is distinguishing between multiple primaries and multifocal metastases from a single lesion. This is vital for staging, prognosis, and treatment decisions, especially when metastases mimic primary and early lesions at the relevant sites. Traditional morphological diagnosis often faces a paradoxical situation on this key issue and cannot quantitatively evaluate the correlations among multiple foci. Here, six cases of SMGL were collected, two of which exhibited pagetoid dissemination within the genital tract, with all lesions being noninvasive. A total of 24 samples were subjected to whole-exome sequencing. By inference based on overlapping genetic variations, base substitution mutation patterns, composition and similarity of COSMIC signatures, clonality indices, and the construction of evolutionary trees, it was inferred that the multiple foci in each patient were clonally related, indicating that all cases were metastatic. The follow-up duration ranged from 7 to 62 months (median: 24.5 months). Four patients died of disease (median survival time: 24.5 months, range: 8–47 months), including one patient who had no invasive lesions at initial diagnosis; two patients experienced recurrences at 17 and 40 months, respectively. These results imply that even if all foci exhibit the appearance of in situ or premalignant changes histologically, they may actually be aggressive. Hence, for SMGLs, before opting for conservative treatment, comprehensive clinical assessment, appropriate surgical extent, adequate sampling, and careful microscopic examination are crucial. Clonal analysis should also be conducted where necessary to avoid undertreatment due to understaging. The study further explored the genomic traits of SMGLs involving more than two sites. © 2025 The Pathological Society of Great Britain and Ireland.

Both Ménétrier's disease (MD) and juvenile polyposis syndrome (JPS) are rare premalignant conditions that can lead to gastric cancer. MD is an acquired disease without known causative mutations that is characterized by increased expression of an EGF receptor (EGFR) ligand, transforming growth factor-alpha (TGFα), in the stomach. JPS is inherited in an autosomal dominant pattern and is caused by BMPR1A or SMAD4 mutations. Although there are distinct clinico-pathological features that differ between the two diseases, they also share similar features that often lead to misdiagnosis. To identify diagnostic markers for MD and to better understand the pathogenesis of the disease, we performed transcriptomic profiling of stomach tissues from normal (NL), MD, and JPS patients. Comparative analysis between MD and JPS revealed both common and differential gene signatures. Common gene signatures included estrogen receptor signaling, integrin signaling, mTOR signaling, and others, which may be responsible for histopathological similarities. Among differential gene signatures, we found that Hedgehog (Hh) signaling is upregulated in MD and confirmed that protein expression of Hh signaling downstream targets, GLI1 (glioma-associated oncogene homolog 1) and HHIP (Hedgehog-interacting protein), is higher in MD than in JPS, particularly in foveolar cells by immunohistochemistry. We also demonstrated that treatment with an Hh pathway inhibitor partially rescued the histopathological phenotypes in an MD mouse model. This study provides valuable insights into the potential mechanisms underlying the similar clinico-pathological features observed in MD and JPS. We also identified GLI1 and HHIP as diagnostic markers that can help to distinguish MD from JPS. Furthermore, Hh signaling was shown to play an important role in the pathogenesis of MD and may serve as a potential therapeutic target. © 2025 The Pathological Society of Great Britain and Ireland.

Pancreatic ductal adenocarcinoma (PDAC) subtyping typically relies on immunohistochemistry (IHC) staining for critical markers like HNF1A and KRT81, a labor-intensive manual staining process that introduces variability. Virtual staining methods offer promising alternatives by generating synthetic IHC images from routine hematoxylin and eosin (H&E) slides. However, most current approaches evaluate success by image quality measures rather than assessing diagnostically relevant features. Here, we introduce a novel cycleGAN framework utilizing a contrastive-inspired approach trained on semipaired datasets derived from consecutive tissue sections. Our method significantly enhances PDAC subtyping accuracy based on synthetic IHC images generated from standard H&E inputs, improving the classification F1-score from 0.66 to 0.77 for KRT81 and from 0.61 to 0.73 for HNF1A, compared with classification directly on H&E images. This approach also substantially outperforms baseline CycleGAN models. These results underscore the clinical potential of contrastive virtual staining to streamline PDAC diagnostics and improve their robustness. © 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.

Clinical trials targeting B7-H3 (CD276), a membranous immunomodulatory molecule in the B7 superfamily, have shown promise in prostate cancer and may be expanded to additional tumor types with high expression, such as those with mTOR signaling activation. MiT/TFE-rearranged translocation renal cell carcinoma (tRCC) is a rare, aggressive subtype that is relatively immune-depleted, with high levels of mTOR activity. Thus, we assessed B7-H3 expression in preclinical tRCC models and human tRCC samples. As hypothesized, we found that induction of TFE3 fusion proteins, including SFPQ-TFE3, PRCC-TFE3, ASPSCR1-TFE3, and NONO-TFE3, is associated with upregulation of B7-H3 in multiple human preclinical tRCC cell line systems and transgenic mouse models. Pharmacologic or genetic inhibition of mTOR signaling is sufficient to downregulate B7-H3 expression in inducible and patient-derived, human cell line models of tRCC. In keeping with these preclinical results, human tRCC demonstrated significantly higher gene expression of CD276 than normal kidney, across five of the six fusions studied. At the protein level, tRCC had higher tumor cell B7-H3 intensity and proportion scores than normal kidney or clear cell RCC (ccRCC). B7-H3 expression in tumor vasculature was similar in tRCC and ccRCC, both of which showed significantly higher expression than normal kidney. Within tRCC cases, higher CD276 expression was observed in metastatic compared to localized tumors and was associated with lower tumoral CD4⁺ T-cell content by bulk RNAseq deconvolution. Taken together, tRCC fusion proteins upregulate B7-H3 expression via increased mTOR signaling, resulting in a higher tumoral B7-H3 expression compared to normal kidney or conventional RCC, suggesting that B7-H3 may be a promising therapeutic target in tRCC. © 2025 The Pathological Society of Great Britain and Ireland.

Development of ipsilateral breast carcinoma following a diagnosis of breast ductal carcinoma in situ (DCIS) has been assumed to represent recurrence of the primary tumour. However, this may not always be the case, and it is important to determine how often such recurrences represent new tumours. Ipsilateral primary–recurrence pairs (n = 78) were sequenced to test their clonal relatedness. Shared genetic events were identified from whole exome sequencing (n = 54 pairs) using haplotype-specific copy number and phylogenetic analysis. The remaining pairs were sequenced using a targeted panel or low-coverage whole genome sequencing. We included 32 non-recurrent DCIS to compare recurrent and non-recurrent disease. We found that 7% of DCIS recurrences were non-clonal by whole exome sequencing, indicative of a new breast carcinoma. Lower resolution methods detected a higher non-clonality rate (29%). By comparing primary DCIS with their recurrence, we found that the evolution of DCIS to invasive disease was associated with increased ploidy and copy number events. TP53 mutations were enriched in DCIS with clonal recurrence compared with non-recurrent DCIS. Our results verify that de novo ‘recurrent tumours’ of independent origin occur in patients who may be at high risk. © 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.

Approximately 10% of testicular Leydig cell tumors (LCTs) are clinically malignant and unresponsive to systemic treatment. Predicting their clinical behavior can be problematic because there are no biomarkers that can consistently discriminate between benign and malignant LCTs. We assessed microRNA expression profiles of LCTs to identify differentially expressed microRNAs that could potentially distinguish benign from malignant neoplasms. The study consisted of two phases. In the first (discovery) phase, we interrogated 768 microRNAs in a series of 11 LCTs (six malignant and five benign) using Taqman Low-Density Array (TLDA) microRNA profiling. In the second phase, we validated the top differentially expressed microRNA targets with real-time quantitative PCR on a series of 35 LCTs (17 malignant and 18 benign), assessing their clinical performance for distinguishing malignant from benign LCTs. Target biologic pathways were analyzed using the miRTargetLink 2.0 tool. A total of 50 microRNAs were differentially regulated in malignant LCTs (27 upregulated, 23 downregulated). The top six microRNA candidates (top three upregulated and top three downregulated) were validated, showing good performance for discriminating between malignant and benign LCTs, with an area under the curve (AUC) ranging between 0.69 and 0.87. MiR-196b-5p showed the best performance, with sensitivity, specificity, negative predictive value, positive predictive value, and accuracy of 82%, 83%, 83%, 82%, and 83%, respectively. A panel (i.e. combined) analysis reached 100% sensitivity and 83% specificity. Pathway analysis revealed significant overlap in the biological process targeted by the upregulated microRNAs in malignant LCTs, including proliferation, development, metabolism, hormone synthesis, and cell death. Our results support the idea that malignant LCTs are associated with a distinct microRNA signature. MiR-196b-5p was identified as a potentially useful biomarker to distinguish benign from malignant tumors. The shared downstream targets of the top upregulated microRNAs suggest that dysregulation of cell proliferation and apoptosis underlie aggressive biologic behavior in LCTs and may offer opportunities for targeted therapies. © 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.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent precious resources for clinical genomic profiling studies, especially when coupled with comprehensive medical records. Even though next-generation sequencing (NGS) is an effective tool to detect somatic mutations and somatic copy number alterations (sCNA), the biggest challenges in unlocking clinically translatable genomic information from FFPE tissue are low DNA yields and degraded DNA, affected by variable formalin fixation. Another issue is that the proportion of carcinoma and other noncarcinoma cells is variable and can be confounded by intratumoral heterogeneity. To explore these challenges, we isolated pure carcinoma and stromal cells using the DEPArray™ NxT system, a microchip-based digital sorter that allows isolation of pure, homogeneous subpopulations of cells from FFPE samples. We isolated pure carcinoma and stromal cell populations from 12 FFPE tissues, including tissues from nine primary and metastatic breast cancer and three primary ovarian high-grade serous carcinomas. This was followed by downstream shallow whole-genome sequencing (WGS) for copy number landscape profiling (10 samples) and/or a targeted panel for somatic mutation and sCNA analysis (seven samples), subject to cell availability. Seven out of 10 samples (even some with low tumour content or of old age) produced good-quality genomic data, detecting sCNA in all carcinoma population samples but not in the stromal populations. Mutation analysis was performed successfully in 6/7 samples and somatic mutations were detected in all of them. Our workflow enabled the identification of clinically actionable targets, including PIK3CA, ERBB2, FGFR1/2, CDK6, CCNE1, KRAS amplifications and RB, BRCA1/2 losses in patients that would direct therapy. © 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.