Search Results (1,079)

Background/Objectives: Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE); it is associated with increased morbidity and mortality, underscoring the need for new diagnostic markers and therapeutic strategies. In this context, the exostosin 1 (EXT1)/exostosin 2 (EXT2) heterodimer has emerged as a novel antigen in membranous nephropathy associated with SLE. This study evaluated EXT1 prevalence in renal biopsies from patients with lupus membranous nephropathy (LMN) and compared clinical, laboratory, and histopathological characteristics on diagnosis and renal outcomes. Methods: This retrospective study included 97 LMN patients whose renal biopsy underwent immunohistochemistry (IHC) for EXT1. EXT1-positive and EXT1-negative groups were compared using descriptive analyses and repeated measures models. Results: EXT1 positivity was observed in 35% of the cohort, and is more frequent in pure LMN (40%) than in cases with a proliferative component (32%). Regarding SLE diagnostic criteria, EXT1-positive patients showed a higher frequency of antiphospholipid antibodies, although data were available for only a subset of patients. This group also exhibited lower serum creatinine levels, but without statistical significance. EXT1-negative patients more frequently received cyclophosphamide as induction therapy (57.6% vs. 34.5%; p = 0.041). No differences in clinical outcomes were observed during follow-up. Conclusions: EXT1 prevalence was consistent with the literature, reinforcing the epidemiological reproducibility of this marker. EXT1-positive and EXT1-negative groups did not differ regarding clinical presentation, disease progression, and renal outcomes, heightening the need for prospective studies to further elucidate the diagnostic and prognostic role of EXT1 in LMN. Full article

Human epidermal growth factor receptor 2 (HER2) dysregulation contributes to tumorigenesis in gastric and gastroesophageal junction adenocarcinomas (GC/GEJ). HER2 overexpression has been associated in multiple cohorts with aggressive behavior and poor outcomes. While HER2 amplification has long guided therapy in HER2-positive disease, antibody–drug conjugates (ADCs) have shifted attention toward the HER2-low category, typically defined as immunohistochemistry (IHC) 1+ or IHC 2+ with negative in situ hybridization (ISH). This narrative review integrates evidence from the peer-reviewed literature, current testing recommendations, and registered clinical trials. It clarifies practical issues in HER2-low assessment and maps the evolving therapeutic landscape of HER2-targeted ADCs including rational combination strategies that may extend benefit beyond conventionally HER2-positive tumors. A cross-tumor perspective contrasts GC/GEJ testing and biology with the breast cancer paradigm and summarizes the importance of HER2-low expression in non-gastric malignancies. Finally, we discuss the therapeutic strategies in HER2-low GC/GEJ and highlight key safety and monitoring considerations for HER2-directed ADCs. Full article

The VHL–HIF-1α–VEGF axis regulates angiogenesis and metabolism. Beyond oncology, pVHL is essential for pancreatic β-cell function and is reduced in hypercaloric diet (HCD)-induced type 2 diabetes mellitus (T2DM). This study aimed to overexpress pVHL in pancreatic tissue and evaluate its effects on blood glucose levels and the expression of proteins related to glucose metabolism in the pancreas. HCD-induced diabetic C57BL/6 and BALB/c mice received a single intrapancreatic injection of an adenoviral vector (1 × 10¹² viral particles) encoding the murine Vhlh gene (AdVHL) to induce transient pVHL overexpression. The glycemic delta (post-load glucose minus fasting) and net incremental area under the curve (niAUC) were determined on days 3, 6, 9, 12, and 15 post-treatment, as the peak in GFP overexpression (used as a surrogate reporter of transduction efficiency) was detected between days 9 and 12. Immunohistochemistry (IHC) and immunofluorescence (IF) were used to assess the expression of pVHL, HIF-1α, GLUT-1, GLUT-2, and insulin in pancreatic tissue. AdVHL treatment significantly decreased the glycemic delta and niAUC in mice with T2DM (p < 0.01). On day 15 after treatment, HIF-1α and GLUT-1 expression were markedly reduced in AdVHL-treated mice (p < 0.01), while GLUT-2 and insulin were significantly increased (p < 0.01). These results were reproduced in both mouse strains. Transient overexpression of pVHL in pancreatic tissue of mice with T2DM was associated with decreased glucose levels and changes in the expression of proteins related to glucose metabolism in the pancreas, resembling a healthier phenotype than that of mice with T2DM. These findings support an important functional role of the pVHL–HIF-1α axis in pancreatic physiology, provide a proof-of-concept for further mechanistic and translational studies, and implicate pVHL in the altered glucose metabolism observed in T2DM. Full article

Background/Objectives: With the ongoing efforts in supporting the discovery of novel targeted drug delivery systems for the upper region of the female reproductive tract (FRT), it is imperative to understand the local drug disposition pathways. We aim to obtain a comprehensive profile of the drug transporters and drug-metabolizing enzymes in the human ectocervix, uterus, and fallopian tubes, as these factors may substantially influence mucosal penetration, tissue exposure, drug disposition, and the risk of drug–drug interactions. Methods: Gene expression of 12 drug transporters and 21 drug-metabolizing enzymes was quantified using RT-qPCR. Protein expression of highly expressed transporters was assessed using immunohistochemistry (IHC). Results: Among the 12 transporters analyzed, the efflux transporters P-gp, BCRP, and MRP4 exhibited the highest expression across the ectocervix, endometrium, myometrium, and fallopian tubes, with P-gp consistently showing the greatest abundance in all evaluated FRT tissues. Expression of these transporters was significantly higher (6–17×) in myometrium compared with ectocervix. IHC demonstrated strong localization of P-gp, BCRP, and MRP4 to epithelial layers facing the lumen, as well as to stromal and vascular endothelial cells. For drug-metabolizing enzymes, all 21 phase I and II enzymes were detectable across the FRT, and 15 were expressed at comparatively higher levels across all tissue types. These included CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C19, CYP3A4, UGT1A1, UGT1A3, UGT1A4, UGT1A7, UGT1A8, UGT1A10, UGT2B4, UGT2B15, and UGT2B17. Conclusions: The gene expression and localization data obtained from this work may improve our understanding of drug disposition in the FRT, which will inform selection, design, and optimization of drugs intended for targeted delivery within the FRT. Full article

Background: Microsatellite instability (MSI) is an important biomarker for the diagnosis of Lynch syndrome and for guiding immunotherapy in various solid tumors. Droplet digital PCR (ddPCR) has emerged as a highly sensitive method for detecting MSI, particularly in circulating tumor DNA (ctDNA). This study aimed to evaluate the analytical and clinical performance of a ddPCR assay using three MSI markers (BAT-26, ACVR2A, and DEFB105A/B) in colorectal, gastric, and endometrial cancers. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 190 patients (83 colorectal, 44 gastric, and 63 endometrial cancers) and 21 plasma samples from patients with metastatic solid tumors were analyzed. MSI status determined by ddPCR was compared with conventional PCR using a pentaplex panel and immunohistochemistry (IHC). Analytical performance, including limit of blank (LoB) and limit of detection (LoD), was evaluated using cell line DNA, and clinical cut-offs were established using receiver operating characteristic analysis. Results: The ddPCR assay demonstrated high analytical sensitivity, with LoD values of 0.075% for BAT-26, 0.1% for ACVR2A, and 0.025% for DEFB105A/B. Using optimized clinical cut-offs, the concordance rate between ddPCR and conventional PCR assays was 98.4% in tissue samples. Marker performance varied by cancer type, with reduced sensitivity observed in endometrial cancer. In plasma samples, MSI-H was detected in 1 of 21 cases (4.8%), and the overall concordance rate with tissue-based MSI status was 94.7%. Conclusions: The ddPCR assay demonstrated high concordance with conventional MSI testing methods and showed potential as a sensitive tool for MSI detection in both tissue and plasma samples. However, optimization of marker panels and establishment of sample-type-specific clinical cut-offs are required, particularly for ctDNA-based analysis. Further large-scale studies are needed to validate the clinical utility of ddPCR for MSI detection and monitoring. Full article

Post-infectious bronchiolitis obliterans (PiBO) is a chronic lung disease that develops after severe lower respiratory infections and leads to persistent inflammation and fibrotic changes in the small airways. In the present study, gene expression analysis was used to identify differentially expressed genes (DEGs) in sputum cells derived from PiBO patients and compare them to healthy controls. Clinical history, lung function parameters, and induced sputum samples were collected from nine patients with PiBO and eight healthy controls. Multiplex immunohistochemistry (mIHC) as well as mRNA sequencing (MACE-Seq) were performed. Evaluation of the biological targets was done by KEGG pathway enrichment analysis. PiBO patients showed significantly reduced lung function parameters, an increased neutrophil count, and an altered macrophage profile in sputum. Transcriptome analysis revealed significant upregulation of the TNFα-dependent NFκB signalling pathway, as well as significant downregulation of the oxidative phosphorylation (OXPHOS). Linear regression analyses and mIHC indicated a shift in macrophage polarisation that may contribute to the dysregulated gene expression. Notably, expression of these DEGs significantly correlated with FEV₁ lung function. These findings indicate a central role of macrophages in the immunopathology of PiBO and contribute to our understanding of the molecular mechanisms involved in the disease process. Full article

Background/Objectives: Atypical teratoid/rhabdoid tumor (AT/RT) is a rare and aggressive pediatric embryonal tumor of the central nervous system with marked histological and immunophenotypic heterogeneity, which can make diagnosis difficult in some cases. This study aimed to summarize the clinicopathological and molecular features of pediatric AT/RT and to evaluate an HE–IHC dual-path deep learning model as an auxiliary diagnostic approach. Methods: Clinical, histopathological, immunophenotypic, ultrastructural, and fluorescence in situ hybridization (FISH) data were retrospectively collected from 18 children with AT/RT treated at Beijing Children’s Hospital between February 2010 and April 2021. A total of 361 pathological images were used to train and test a ResNet50-based dual-path classification model with transfer learning and feature fusion. An additional independent test set of 175 histological and immunohistochemical images from six newly collected patients was used for supplementary validation. Results: The mean age at diagnosis was 2 years and 3 months. All cases showed loss of INI1 expression, positivity for CK and EMA, and a high Ki-67 index. FISH analysis identified SMARCB1 deletion in 7 of 15 tested cases. In the original image-based test set, the dual-path model achieved an accuracy of 90.91%, compared with 81.82% for the model without transfer learning, 86.36% for the single-path immunohistochemistry model, and 50.00% for the single-path histological model. In the additional independent test set, the trained model correctly classified all 175 images. Conclusions: Pediatric AT/RT shows diverse clinicopathological features and complex SMARCB1 alteration patterns. The HE–IHC dual-path model showed encouraging preliminary performance for auxiliary pathological assessment; however, larger multicenter cohorts with molecular subgroup annotation are needed for further validation before routine clinical application. Full article

Breast cancer represents a major public health problem worldwide. Despite radical surgery for localized disease, a substantial proportion of patients experience disease recurrence. The aim of this study was to evaluate the expression of the mammaglobin and CK19 genes in sentinel lymph node biopsies from patients with early breast cancer. This descriptive study included 301 sentinel lymph node biopsies from patients with stage I–II breast cancer treated at the San Carlos Clinical Hospital in Madrid, Spain. Metastases were identified using conventional histopathology (H&E), immunohistochemistry (IHC), and molecular detection of mammaglobin and CK19 using PCR-based methods. Associations between variables were assessed using Fisher’s exact test with a 95% confidence level. Statistical analyses were performed using STATA 12.0. The predictive value for metastatic involvement was 12.29% for CK19 and 16.61% for mammaglobin, increasing to 19.27% when conventional staining was combined with immunohistochemistry. The overall sensitivity was 68.9%, and the specificity was 93.42%. Mammaglobin showed slightly better diagnostic performance than CK19, and the combined molecular detection of both genes improved diagnostic accuracy when compared with individual markers. Intraoperative molecular evaluation of sentinel lymph nodes using mammaglobin and CK19 is comparable to conventional histopathological assessment combined with immunohistochemistry. The combined RT-PCR detection of both genes improves diagnostic performance and represents a clinically useful complementary tool for the detection of metastatic involvement in early breast cancer. Full article

Background: Evaluation of gene-level copy number variants (CNVs) for diagnosis and therapeutic decision making has become standard of care with next-generation sequencing (NGS), immunohistochemistry (IHC), and/or fluorescence in situ hybridization (FISH) being used to detect gene amplifications/deletions in tumor tissue. In contrast to most solid tumors, CNS cancers are challenging to evaluate by resection and/or biopsy due to the associated risks with invasive brain surgery that can also result in death or associated morbidity and therefore alternate methods are required.Methods: This study presents the analytical validation of using quantitative PCR (qPCR) to detect gene CNVs directly from cerebrospinal fluid (CSF)-derived DNA and from the Ascent^TM low-pass whole genome sequencing (LP-WGS) libraries, demonstrating concordance with the gold standard of NGS/IHC/FISH used in tumor tissue. Results: The analytical sensitivity of qPCR to detect gene amplification calls for ERBB2 (erb-b2 receptor tyrosine kinase 2) was demonstrated to be 100% and that of EGFR (epidermal growth factor receptor) was 83%, with specificities of 96% and 100%, respectively. The analytical sensitivity of qPCR to detect gene deletions for CDKN2A/2B (cyclin-dependent kinase inhibitor 2A/2B) was 60% and that for MTAP (methylthioadenosine phosphorylase) was 100% with a specificity of 100% for all three genes. Ascent^TM was demonstrated to have a higher sensitivity (100%) when compared to qPCR for the same genes evaluated and demonstrated 100% positive agreement and 100% negative agreement with known CNV status. Conclusions: The results demonstrate that given the paucity of cells in CSF limiting the use of IHC and FISH, qPCR and Ascent^TM provide highly sensitive, novel, minimally invasive methods for the evaluation of gene copy number (CN) status to inform the diagnosis and management of CNS cancers. Full article

A recently identified mesenchymal cell population, telocytes (TCs), has been found in many tissues of different animal species. Jet needle-free injection (JNFI) is a promising non-invasive drug-delivery method that can trigger effective immune responses in the skin. In this preliminary morphological study, an inactivated porcine circovirus vaccine was delivered into pig neck skin by JNFI, and untreated normal neck skin served as the control. Histopathology, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to evaluate TC distribution, ultrastructure, and selected quantitative TEM parameters 24 h after injection. TCs were widely distributed in porcine skin, located between collagen fibers and around blood vessels, adipocytes, and sweat glands. They were also observed in contact with mast cells. TCs around sweat glands were CD34⁺, Vimentin⁺, and α-SMA⁺, whereas TCs at other sites were CD34⁺, Vimentin⁺, and α-SMA⁻. After JNFI, inflammatory cell infiltration into the skin was observed; TCs were in contact with these cells, and TCs surrounding adipocytes redistributed into the adjacent loose connective tissue. Quantitative TEM analysis showed that TC profile density and visible telopod length did not differ significantly between normal skin and JNFI 24 h skin (p ≥ 0.05). In contrast, vesicle profiles per TC increased significantly in both perivascular and adipose-associated compartments (p < 0.05). These findings provide morphological evidence suggesting that TCs may participate in early cutaneous responses after JNFI vaccine delivery. Full article

Background: Historically, diverticulosis and its complications emerged with the industrial revolution and subsequent socioeconomic changes, including dietary, which went from an agrarian high-fiber to a low-fiber, processed food product. Corporeal body habitus basal metabolic index (BMI) increased exponentially, and colonic diverticulosis now involves 70% of the population at >80 years of age. The correlation of severity in diverticulosis was predictive of diverticulitis (DIV). Methods: In the individual patient, this is very difficult to predict, so we turned to a database of patients from a colorectal neoplasia database to compare parameters those who had experienced diverticulitis with those who had no documented diverticulitis. We were able to ascertain 28 patients and compare them to 2256 controls. We used an antibody (Adnab-9) that recognizes an epitope on the p87 molecule, a product of the innate immune system (InImS) and as a denominator of serum ferritin yield the FERAD ratio and directly evaluated the expression of p87 by immunohistochemistry (IHC) in six contiguous regions of the colon. Results: The FERAD ratio was significantly lower in the diverticulitis patients, 2567 versus 18,989. IHC showed significant lower p87 in the rectosigmoid regions (p < 0.01). Significant differences were also seen in blood platelets (p < 0.036); serum creatinine (p < 0.005); 65.6% DIV smokers versus 38% controls (p < 0.008). Surprisingly there were no differences in BMI, mortality, or age. Conclusions: The InImS appears not to be active in DIV patients as compared to controls, which may predispose to DIV. Smoking also appears to predispose to DIV. Platelets are lower and creatinine higher as has been described in the literature. Full article

Human unilateral cleft lip morphopathogenesis is a complex process involving multiple genes and proteins. Factors such as myosin heavy chain 3 (MYH3), interferon regulatory factor 6 (IRF6), and Gremlin 1 (GREM1) are implicated in craniofacial development; however, their precise role in unilateral cleft formation remains unclear, limiting improvements in treatment strategies. Immunohistochemistry (IHC) for MYH3, IRF6, and GREM1 proteins and chromogenic in situ hybridization (CISH) for IRF6 and GREM1 genes were used to analyze postnatal unilateral right cleft lip tissue (ten patients) and control tissue (six patients). The semi-quantitative counting method was applied, followed by statistical analysis. IHC revealed increased MYH3 expression in cleft muscle tissue and elevated IRF6 expression in the epithelium, whereas GREM1 showed low expression, with significant differences in connective tissue. CISH demonstrated increased IRF6 gene expression in the cleft epithelium, whereas GREM1 expression did not differ from controls. Multiple statistically significant correlations were identified, highlighting their potential involvement in cleft morphopathogenesis. Full article

Background/Objectives: Pediatric cancers are disorders of dysregulated development driven largely by genomic and epigenetic alterations. Precisely modeling these developmental differences is essential for understanding the unique biology of childhood cancers. Patient-derived organoids (PDOs) offer a powerful in vitro platform that recapitulates tumor heterogeneity, plasticity, microenvironment (including immune cells) and disease-relevant features. Methods: Here, we describe a step-by-step protocol for the establishment of PDOs from cells derived from pediatric brain tumors and extracranial solid tumor biopsies and bone marrow aspirates, including tumor processing, organoid culture/subculture, and cryopreservation. Results: Furthermore, we present the use of PDOs for further experimental analysis such as fluorescence imaging, Western blotting, flow cytometry, and immunohistochemistry (IHC) to investigate the underlying pathophysiology of tumorigenesis. Conclusions: Expanding the application of organoids to childhood malignancies holds exceptional promise for elucidating pediatric tumor biology and advancing therapeutic strategies, representing the long-overdue convergence of technology and clinical need. Full article

Background/Objectives: While human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) is pivotal for breast cancer management, its reliance on additional tissue processing beyond routine H&E staining remains a clinical burden. Although virtual staining offers a potential solution, current methods often fail to explicitly account for HER2 score-specific expression patterns. To address this gap, we developed a score-aware framework designed for the precise generation of virtual HER2 IHC images. Methods: We introduce the non-contrastive multi-task (NCMT) framework, which integrates negative-free patch alignment, style–content constraints, and auxiliary HER2 score supervision for high-fidelity H&E-to-IHC translation. For rigorous evaluation, the model was validated on the BCI dataset, utilizing an official split of 3896 training and 977 independent test images derived from 51 whole-slide images. Results: NCMT demonstrated superior virtual staining performance, achieving a Fréchet Inception Distance (FID) of 38.8, a Kernel Inception Distance (KID) of 5.6, and an average Perceptual Hash Value (PHV) of 0.439. In downstream HER2 scoring tasks, while virtual IHC images alone yielded an accuracy of 83.01%, the fusion of H&E and virtual IHC further elevated performance to 97.85% accuracy and a 98.23% F1 score. These findings suggest that our framework effectively preserves diagnostic features while providing complementary information to H&E-based morphological analysis. Conclusions: NCMT enables HER2 score-aware virtual IHC generation from H&E and can serve as a complementary tool for HER2 assessment in digital pathology. Full article

Automated multiplex immunohistochemistry (IHC) and in situ hybridization (ISH) require staining platforms that combine stable reagent exchange, low-volume operation, process observability, and protocol flexibility. Existing autostainers are often rigid and costly, whereas microfluidic and sensing solutions remain largely component-specific rather than system-oriented. This study proposes and partially validates a layered cyber-physical architecture for multiplex histological staining. The architecture integrates five functional layers—biochemical workflow, fluidic processing, capacitive sensing, protocol-driven control, and software-based process representation—within a unified formal framework and is supported at the subsystem level by experimental characterization of its fluidic and sensing layers. Fluidic experiments on a slot-type microfluidic chamber identified a practical operating window in which upper-feed operation, moderate calibrated flow conditions, and low chamber angles between 10° and 40° provide stable filling and acceptable drainage. The differential slot-line capacitive sensing subsystem detected liquid volumes as low as 0.5 µL, with stable threshold-based interpretation at a practical detection threshold of approximately 5 fF after digital filtering. The control and software layers are specified at the architectural and formal model level; their hardware implementation and closed-loop validation remain subjects of future work. Together, the reported results demonstrate that controlled reagent transport and sensing-based process observability are jointly feasible within the proposed modular framework, establishing a conceptual and experimental foundation for scalable, flexible, and resource-efficient multiplex IHC/ISH systems. Full article