Search Results (222)

5-Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is one of the treatment modalities for oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC). However, the role of ferroptosis in ALA-PDT for OLK and OSCC remains unclear. Therefore, this study aimed to investigate whether ALA-PDT can induce ferroptosis in OLK and OSCC. We detected relative cellular dehydrogenase activity (CCK-8 assay), long-term proliferative viability, reactive oxygen species (ROS) generation, glutathione levels, and mitochondrial morphology after ALA-PDT. The expression of ferroptosis-related proteins was detected using Western blot. A tongue OSCC model was established in male BalB/c nude mice, and then ALA-PDT was performed. Immunohistochemical staining of Ki67, GPX4 and FTH1 was conducted to evaluate the effect of ALA-PDT. Subsequently, OLK and OSCC cells were pre-treated with ferrostatin-1 (Fer-1) before ALA-PDT. Relative cellular dehydrogenase activity, ROS generation, lipid peroxidation, Fe²⁺ levels, and ferroptosis-related protein expression were measured. Finally, OLK and OSCC cells were treated with a combination of ALA-PDT and erastin, and mitochondrial function was evaluated. In vitro study showed that ALA-PDT increased ROS generation and decreased GSH/GSSG ratio in OLK and OSCC cells. After ALA-PDT, mitochondrial morphology exhibited typical characteristics of ferroptosis. In vivo experiments showed that immunohistochemistry (IHC) scores of Ki67, GPX4 and FTH1 in the tissues decreased after ALA-PDT. Moreover, pre-treatment with Fer-1 could reverse ROS levels, lipid peroxidation and intracellular Fe²⁺ accumulation in OLK and OSCC cells after ALA-PDT. Additionally, Fer-1 pre-treatment reversed the changes in protein expression induced by ALA-PDT. The combination of ALA-PDT and erastin significantly reduced mitochondrial O₂•⁻ production and decreased mitochondrial membrane potential. Above all, ALA-PDT can induce ferroptosis in OLK and OSCC. The use of ferroptosis agonists may enhance the therapeutic efficacy of ALA-PDT for OLK and OSCC. Full article

Background: Osteosarcoma (OSA) is the most common type of bone cancer in canines. Novel therapies are required to prevent the growth, survival, and metastatic progression of this cancer, to increase life expectancy of patients. Immunohistochemical (IHC) studies and RNA sequencing help us gain a deeper understanding into the molecular mechanisms of the disease. Methods: We previously compared canine OSA tissues with patient matched non-tumour tissues, revealing 442 overexpressed genes within the samples. The present research used IHC staining for four of these genes in OSA tissues: G protein-coupled receptor 64 (GPR64), TOX High Mobility Group Box Family Member 3 (TOX3), Matrix Metallopeptidase 12 (MMP-12), and Forkhead Box F1 (FOXF1). H-scoring was performed to quantitatively assess protein expression and qualitatively contextualise staining locations. Additional analyses addressed whether gender or anatomical location of lesions (axial or appendicular tumours) affected protein expression. cBioPortal was employed to analyse expression and genetic alterations in patients. Results: GPR64, TOX3, MMP-12, and FOXF1 showed high mRNA expression and genetic alterations in people with OSA. GPR64, TOX3, MMP-12, and FOXF1 were all expressed in canine OSA with novel findings regarding cellular expression. Additionally, differential sex expression was revealed for GPR64 and TOX3. Potential biomarkers or therapeutic targets were identified. Conclusions: These studies, and subsequent analysis, have provided insights into the molecular mechanisms associated with OSA progression and revealed potential biomarkers for diagnostic and prognostic purposes. A deeper understanding of genetic and protein interactions will support and progress novel pathways towards diagnostic, prognostic, and treatment interventions for OSA in both veterinary and human medicine. Full article

Background: HER2-low breast cancer (HER2 immunohistochemical [IHC] score 1+, or IHC 2+ without HER2 gene amplification) is distinct from HER2-positive and HER2-0 breast cancer (IHC 0), with a differing prognosis and specific therapeutic options. The DESTINY-Breast04 trial demonstrated notable efficacy of the HER2 antibody–drug conjugate trastuzumab deruxtecan over standard chemotherapy in patients with metastatic breast cancer (MBC) defined as HER2-low. More recently, the DESTINY-Breast06 trial confirmed this benefit in hormone receptor-positive and HER2-ultralow (less than 1+, but with ≤10% of infiltrating cancer cells showing incomplete and faint/weak membrane staining) cases, prompting re-evaluation of HER2 diagnostic thresholds and treatment strategies. Methods: Eligible patients were women with HER2-low or HER2-0 MBC evaluated at MD Anderson between January 2006 and January 2019. HER2-low was defined as either (1) IHC 1+ or (2) IHC 2+ and negative on fluorescence in situ hybridization. Multivariate logistic regression was used to evaluate distinct clinicopathologic features of patients with HER2-low status. Overall survival (OS) was estimated by the Kaplan–Meier method. Multivariate Cox proportional hazards regression was applied to assess the effects of covariates of interest on OS across different HER2 groups. Results: We included 3834 women: 2637 (69%) with recurrent and 1197 (31%) with de novo MBC; HER2-low disease was present in 1575 (60%) and 712 (59%), respectively. In de novo cases, higher nuclear grade was associated with HER2-low status (grade 2 vs. 1, OR = 2.02, p = 0.007; grade 3 vs. 1, OR = 1.87, p = 0.015), while recurrent cases were associated with ER-positivity (OR = 1.96, p < 0.001) and prior adjuvant radiotherapy (OR = 0.79, p = 0.007). Median OS was 3.2 years (95% CI 3.0–3.5). In de novo disease, Black race (HR = 1.48), metaplastic (HR = 3.15) or other non-ductal/lobular histologies (HR = 2.36), and grade 3 (HR = 1.67) predicted worse OS, whereas Hispanic ethnicity (HR = 0.74) and Other races (HR = 0.57), higher ER (HR = 0.48–0.41) and PR (HR = 0.72–0.53), and HER2-low status (HR = 0.77) conferred improved outcomes. In recurrent disease, Black race predicted worse OS (HR = 1.21, 95% CI 1.05–1.39), while Other race (HR = 0.78, 95% CI 0.62–0.97), higher ER (HR = 0.69–0.44) and PR (HR = 0.73–0.73), and HER2-low (HR = 0.89) were protective. HER2 discordance between primary and metastatic sites occurred in 38.8% of recurrent and 13.1% of de novo cases. Conclusions: HER2-low status was significantly associated with longer OS compared to HER2-0 status in both recurrent and de novo MBC cases. These real-world data help establish the prevalence of HER2-low status and its distinct outcomes. The discrepancy in HER2-low status between the primary tumor and metastatic sites highlights the potential for changes in HER2 expression over time, exploring the interaction between HER2-low breast cancer and the tumor microenvironment and emphasizing the importance of monitoring and reassessing HER2 status at various stages to guide treatment decisions effectively and the need for more quantitative and reproducible HER assays. Full article

Immunohistochemistry (IHC) is essential for diagnostic, prognostic, and predictive biomarker assessment in oncology, but manual interpretation is limited by subjectivity and inter-observer variability. Machine learning (ML), a computational subset of AI that allows algorithms to recognise patterns and learn from annotated datasets to make predictions or decisions, has led to advancements in digital pathology by supporting automated quantification of biomarker expression on whole-slide images (WSIs). This review evaluates the role of ML-assisted IHC scoring in the transition from validated biomarkers to the discovery of emerging prognostic and predictive IHC biomarkers for genitourinary (GU) tumours. Current applications include ML-based scoring of routinely used biomarkers such as ER/PR, HER2, mismatch repair (MMR) proteins, PD-L1, and Ki-67, demonstrating improved consistency and scalability. Emerging studies in GU cancers show that algorithms can quantify markers including androgen receptor (AR), PTEN, cytokeratins, Uroplakin II, Nectin-4 and immune checkpoint proteins, with early evidence indicating associations between ML-derived metrics and clinical outcomes. Important limitations remain, including limited availability of training datasets, variability in staining protocols, and regulatory challenges. Overall, ML-assisted IHC scoring is a reproducible and evolving approach that may support biomarker discovery and enhance precision GU oncology. Full article

Vaccination is a tool to control Lawsonia intracellularis (LI) in pigs. However, pigs may have co-infections that worsen clinical signs and lesions. The aim of this study was to characterize systemic and gut-mediated humoral and cell-mediated immune (CMI) responses in pigs vaccinated with a killed intramuscular LI vaccine and to analyze the impact of co-infection with Brachyspira hyodysenteriae (Bhyo) on the immune response. The study included eighty pigs and five study groups: V-CO (LI-vaccinated and co-infected with LI + Bhyo, n = 21), P-CO (placebo and co-infected with LI + Bhyo, n = 18), V-LI (LI-vaccinated and infected with LI, n = 21), P-LI (placebo and infected with LI, n = 12), and NC (negative control, placebo and non-challenged, n = 8). Parameters analyzed: fecal score and pathogen shedding), gross intestinal lesions, LI intestinal colonization (IHC), serum IgG, LI-specific IFN-γ production (ELISPOT), and immune cell subsets (flow cytometry) in blood, mesenteric lymph nodes, Peyer’s patches, and intestinal epithelium. LI vaccination significantly reduced LI fecal shedding, intestinal colonization, and macroscopic lesions—even under Bhyo co-infection. Vaccinated pigs had earlier and stronger serum IgG and IFN-γ responses. B cells seem to play an important role in the local immune response, and T regulatory cells apparently do not have a significant role in immunomodulation. This study contributes to a better understanding of LI immune response and can provide subtract for further research in the control of LI. Full article

Objective: Giant cell lesions (GCLs) share similar histopathologic features. The influence of immune involvement on the biology of giant cell lesions remains largely elusive. This study aimed to evaluate and compare lymphocyte and mast cell infiltration and distribution among three giant cell lesions. Study design: A total of 30 FFPE tissue blocks, comprising 10 PGCGs, 10 CGCGs (aggressive and nonaggressive), and 10 GCTs (aggressive and nonaggressive) of bone, were subjected to IHC staining for CD3 and CD20 lymphocyte markers and toluidine blue staining for mast cells. The mean count of positively stained cells was calculated and categorized into three scores, along with a group for negative cases. Statistical analysis was conducted to assess significance at p < 0.05. Result: Lymphocyte infiltration was observed across all lesions. CD3⁺ and CD20⁺ cell counts were significantly elevated in PGCGs, followed by CGCGs, and were lowest in GCTs of bone. In contrast, mast cell counts were high in GCTs of bone and CGCGs and low in PGCGs. Aggressive giant cell lesions of bone showed a significantly low number of CD3⁺ and CD20⁺ cells (Mann–Whitney U test; p = 0.05, 0.004) and a high number of mast cells (Mann–Whitney U test; p < 0.001) compared with nonaggressive lesions of bone. PGCGs and nonaggressive CGCGs showed comparable CD3 expression, with no significant difference between them (p = 0.59). CD20 levels were higher in nonaggressive CGCGs but did not reach statistical significance (Mann–Whitney U test; p = 0.07). Mast cell density was significantly lower in PGCGs compared with intraosseous nonaggressive CGCGs. Conclusions: The present study shows that GCTs of bone, CGCGs, and PGCGs possess distinct immune microenvironmental profiles. Aggressive lesions demonstrate reduced lymphocyte infiltration and increased mast cell density, a pattern particularly evident in GCTs of bone. This imbalance may contribute to their aggressive behavior by enabling them to escape host immune regulation. Full article

Cluster of Differentiation 47 (CD47), an innate immune checkpoint, facilitates immune escape by binding signal regulatory protein alpha (SIRPα) to inhibit macrophage phagocytosis. Its significance in colorectal cancer (CRC) has garnered heightened interest. This review summarizes five immunohistochemistry (IHC) studies and complementary transcriptomic analyses assessing CD47 in CRC. IHC results consistently indicated membrane overexpression, though positivity rates varied widely (16–91%) due to methodological heterogeneity. Transcriptomic results confirmed CD47 upregulation, especially in Consensus Molecular Subtype 1 (CMS1) and CMS4 subtypes and revealed co-expression with immune checkpoints and oncogenic pathways. Clinically, high CD47 levels were associated with advanced TNM stage, metastasis, poor differentiation, and altered immune infiltration; however, the prognostic significance varied among cohorts. Overall, CD47 appears to be a promising biomarker and therapeutic target, but clinical translation requires standardized evaluation, including harmonized antibody selection and scoring cut-offs, and prospective validation. Full article

Lupus nephritis (LN) can affect up to 60% of patients with systemic lupus erythematosus (SLE). The NLRP3 inflammasome has been implicated in the pathogenesis of LN. This study aimed to evaluate the role of the NLRP3 inflammasome as a predictor of response to immunosuppressive treatment in patients with active LN. A prospective cohort study was conducted with 20 adult patients with active LN, classes III, IV, and V, from January 2021 to September 2023. Patients were followed up at biopsy (T0) and 6 months (T6) and 12 months (T12) after treatment and classified according to the primary efficacy renal response (PERR) at 12 months. Gene expression of NLRP3, CARD8, CASP1, IL1B, and IL18 was evaluated by RT-qPCR in PBMCs. Immunohistochemistry (IHC) for NLRP3 was performed on kidney tissue. The concentration of cytokine IL-1β was measured using the BD™ Cytometric Bead Array (CBA). The mean age was 31.9 ± 8.3 years, with 19 females and 1 male. After 12 months, 65% of patients achieved PERR. The IHC intensity in inflammatory cells was higher in patients with no PERR (p = 0.0426). In the no-PERR group, the gene expression of IL1B showed a significant increase at T6 (FC = 2.22: p = 0.0037) and T12 (FC = 2.91; p = 0.0001) compared with T0. Relative expression of IL1B was higher in no-PERR patients at T12 compared to the PERR group (p = 0.0477). The no-PERR group also had higher serum IL-1β levels compared to the PERR group at 12 months (2.9 ± 0.5 vs. 2.5 ± 0.7, p = 0.0164). In conclusion, our study evidenced an increase in IL1B expression and IL-1β levels over the 12 months of treatment in no-PERR patients, suggesting a potential biomarker of disease activity. Furthermore, a strong NLRP3 IHC staining score was associated with a higher likelihood of no PERR, highlighting the potential of the NLRP3 inflammasome as a predictor of worse clinical outcomes. Full article

Background/Objectives: Immunohistochemistry (IHC) is a critical diagnostic tool in forensic pathology, enabling molecular-level assessment of wound vitality, post-mortem interval, and cause of death. However, IHC interpretation is subject to variability due to its reliance on human expertise. This study investigates whether artificial intelligence (AI), specifically a generative model, can assist in the diagnostic evaluation of IHC slides and replicate expert-level scoring, thereby improving consistency and reproducibility. Methods: A total of 225 high-resolution IHC images were classified into five immunoreactivity categories. The AI model (ChatGPT-4V) was trained on 150 labeled images and tested blindly on 75 unseen slides. Performance was assessed using confusion matrices, per-class precision/recall/F1, overall accuracy, Cohen’s κ (unweighted and weighted), and binary metrics (sensitivity, specificity, MCC). Results: Overall accuracy was 81.3% (95% CI: 71.1–88.5%), with substantial agreement (κ = 0.767 unweighted; 0.805 linear-weighted; 0.848 quadratic-weighted). Binary classification achieved a sensitivity of 98.3%, specificity of 93.3%, MCC of 0.92. Accuracy was highest in extreme categories (− and +++, 93.3%), while intermediate classes (+ and ++) showed reduced performance (error rates up to 33%). Evaluation was rapid and consistent but lacked interpretative reasoning and struggled with borderline cases. Conclusions: AI-assisted diagnostic evaluation of IHC slides demonstrates promising accuracy and consistency, particularly in well-defined staining patterns. While not a replacement for human expertise, AI can serve as a valuable adjunct in forensic pathology, supporting rapid and standardized assessments. Ethical and legal considerations must guide its implementation in medico-legal contexts. Full article

The cHER2+ breast cancer subtype is characterized by the overexpression of the HER2 oncoprotein based on immunohistochemistry (IHC)/or by ERBB2 gene amplification using in situ hybridization (ISH) techniques. Targeted therapies are significantly changing cancer treatment outcomes. However, not all patients benefit from it due to misclassification or intrinsic mechanisms of resistance. Identifying predictive factors of response to therapy is thus crucial for optimizing treatment protocol. In addition, with the development of effective antibody–drug conjugates for targeting HER2-low subtypes, enhancing the HER2 molecular classification is crucial. In this study, a comprehensive analysis of publicly available datasets (TCGA, METABRIC, I-SPY, NOAH and CHER-LOB trials) has been considered. We present a metagene expression score (HER2MtGx 31-gene assay) based on the most informative genes associated with each molecular profile. HER2MtGx scores represent three linear subspaces associated with the HER2, Luminal and Basal-like profiles (STAT). In the METABRIC cohort, the scores are useful to discriminate against the HER2-enriched phenotype and this classification is significantly associated with long-term survival in cHER2+ patients (HR = 1.76; 95%CI = 1.09–2.86). In terms of response to neoadjuvant chemo/target therapy including I-SPY, NOAH, and CHER-LOB trials, the metagene scores are associated with the pathological response to therapy (OR = 2.26; 95%CI = 1.74–2.98). The HER2MtGx assay is a reliable tool for selecting patients for HER2-targeted therapy. Full article

Glutamine addiction in human melanoma is a premier example of the cancer hallmark of metabolic reprogramming. In the present study, we investigate the presence of metabotropic glutamate receptor 1 (mGluR1/GRM1) and glutaminase (GLS1/GLS) in canine oral malignant melanoma (OMM) and those of low malignant potential, termed histologically well-differentiated melanocytic neoplasm of the lips and oral mucosa (HWDMN). We used immunohistochemistry (IHC) and qPCR to evaluate mGluR1 and GLS1 protein expression and RNA expression, respectively. Nearly 20% of OMM cases had an mGluR1 IHC score ≥ 1, while none of the HWDMN cases had any expression. Due to low IHC expression, only 10 cases were selected for determination of GRM1 RNA expression, and none were positive. GLS RNA expression did not differ between OMM and HWDMN. A GLS1 IHC score ≥ 1 was significantly higher in OMM cases and highly specific (95%) for correctly identifying tumors with a Ki67 index ≥ 19.5. These results may have been negatively impacted by use of a brown chromogen for IHC labeling among background pigment, particularly in HWDMN. Ultimately, these findings suggest that canine OMM does not heavily rely on mGluR1 for tumorigenesis or progression. Differential GLS1 protein expression warrants further investigation with protein quantification. Full article

Background: Salivary gland tumors pose a diagnostic challenge due to their histological heterogeneity and overlapping features. While immunohistochemistry (IHC) is critical for accurate classification, selecting appropriate markers can be subjective and influenced by resource availability. Artificial intelligence (AI), particularly large language models (LLMs), may support diagnostic decisions by recommending IHC panels. This study evaluated the performance of ChatGPT-4, a free and widely accessible general-purpose LLM, in recommending IHC markers for salivary gland tumors. Methods: ChatGPT-4 was prompted to generate IHC recommendations for 21 types of salivary gland tumors. A consensus of expert pathologists established reference panels. Each tumor type was queried using a standardized prompt designed to elicit IHC marker recommendations (“What IHC markers are recommended to confirm a diagnosis of [tumor type]?”). Outputs were assessed using a structured scoring rubric measuring accuracy, completeness, and relevance. Agreement was measured using Cohen’s Kappa, and diagnostic performance was evaluated via sensitivity, specificity, and F1-scores. Repeated-measures ANOVA and Bland–Altman analysis assessed consistency across three prompts. Results were compared to a rule-based system aligned with expert protocols. Results: ChatGPT-4 demonstrated moderate overall agreement with the pathologist panel (κ = 0.53). Agreement was higher for benign tumors (κ = 0.67) than for malignant ones (κ = 0.40), with pleomorphic adenoma showing the strongest concordance (κ = 0.74). Sensitivity values across tumor types ranged from 0.25 to 0.96, with benign tumors showing higher sensitivity (>0.80) and lower specificity (<0.50) observed in complex malignancies. The overall F1-score was 0.84 for benign and 0.63 for malignant tumors. Repeated prompts produced moderate variability without significant differences (p > 0.05). Compared with the rule-based system, ChatGPT included more incorrect and missed markers, indicating lower diagnostic precision. Conclusions: ChatGPT-4 shows promise as a low-cost tool for IHC panel selection but currently lacks the precision and consistency required for clinical application. Further refinement is needed before integration into diagnostic workflows. Full article

Programmed death-ligand 1 (PD-L1) biomarker testing in gastric cancer is required to identify patients suitable for immunotherapy. However, the PD-L1 testing landscape is complex, with various PD-L1 tests available and multiple algorithms that combine tumor and immune cell staining. To provide guidance on the best practices for PD-L1 testing in gastric cancer, we reviewed the literature and incorporated our extensive experience using the PD-L1 IHC PharmDx 22C3 and 28-8 assays and scoring with the combined positive score (CPS) algorithm. This review summarizes inter-reader agreement and PD-L1 assay concordance studies in gastric cancer, highlights practical challenges and pitfalls encountered in our own laboratory, and proposes solutions to address them. Accurate and consistent interpretation of PD-L1 CPS in gastric cancer is challenging, but can be improved with training, experience, and close attention to interpretation guidelines. Techniques are available that can optimize the automated staining of PharmDx PD-L1 assays using the Autostainer Link 48 to ensure consistent staining performance. The PD-L1 IHC PharmDx 22C3 and PD-L1 IHC PharmDx 28-8 assays show high concordance when used according to manufacturers’ guidelines. Full article

Pediatric seronegative immune-mediated cerebellar ataxia (IMCA) remains a poorly defined and often under-recognized diagnosis, particularly in young children, where symptoms are frequently misattributed to self-limited post-infectious processes. We report the case of a 2.5-year-old girl who presented with acute-onset ataxia (mSARA score: 14). Cerebrospinal fluid analysis revealed pleocytosis and positive oligoclonal bands, while serial brain imaging and extensive autoantibody panels were unremarkable. However, indirect immunohistochemistry (TIIF/IHC) demonstrated a positive intracellular signal in cerebellar Purkinje cells, supporting the diagnosis of isolated seronegative IMCA. The patient showed sustained clinical improvement with prolonged corticosteroid therapy (mSARA score: 1). To date, only a few similar cases have been reported in the literature. It remains unclear whether these presentations fall within the spectrum of autoimmune encephalitis (AIE) or represent a distinct pediatric phenotype, potentially expanding the age range of primary autoimmune cerebellar ataxia previously described in adults. We recommend incorporating TIIF/IHC into the diagnostic workup of both isolated and combined pediatric cerebellar ataxia syndromes to support diagnosis and guide individualized treatment. Additionally, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) are emerging as promising biomarkers in this context and warrant further investigation. Full article

Transcription factors orthodenticle homeobox 2 gene (OTX2), paired box 6 gene (PAX6), and SRY-box transcription factor 2 gene (SOX2) are key regulators of ocular morphogenesis; however, their comparative embryonic localization across species—and the correspondence between transcript and protein distributions—remain incompletely defined. Chromogenic in situ hybridization (CISH) was employed to detect OTX2, PAX6, and SOX2 transcripts, while biotin–streptavidin immunohistochemistry (IHC) was used to assess Otx2, Pax6, and Sox2 protein expression. A semi-quantitative scoring system was applied to evaluate positive structures across ocular compartments. Transcripts were predominantly localized to the retina in both species, with occasional low-level expression in the optic nerve, sclera, and eyelid. Proteins displayed broader distributions: Otx2 was abundant in the retina and eyelid, while Pax6 and Sox2 were detected in multiple tissues, including cornea and extraocular muscles. OTX2, PAX6, and SOX2 show retina-predominant transcription and wider protein expression across ocular tissues. These findings highlight spatial differences between transcript and protein localization, supporting a complex regulatory framework underlying vertebrate eye development. Full article